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Sample records for chromatin condensation assay

  1. Osmotic Challenge Drives Rapid and Reversible Chromatin Condensation in Chondrocytes

    PubMed Central

    Irianto, Jerome; Swift, Joe; Martins, Rui P.; McPhail, Graham D.; Knight, Martin M.; Discher, Dennis E.; Lee, David A.

    2013-01-01

    Changes in extracellular osmolality have been shown to alter gene expression patterns and metabolic activity of various cell types, including chondrocytes. However, mechanisms by which physiological or pathological changes in osmolality impact chondrocyte function remain unclear. Here we use quantitative image analysis, electron microscopy, and a DNase I assay to show that hyperosmotic conditions (>400 mOsm/kg) induce chromatin condensation, while hypoosmotic conditions (100 mOsm/kg) cause decondensation. Large density changes (p < 0.001) occur over a very narrow range of physiological osmolalities, which suggests that chondrocytes likely experience chromatin condensation and decondensation during a daily loading cycle. The effect of changes in osmolality on nuclear morphology (p < 0.01) and chromatin condensation (p < 0.001) also differed between chondrocytes in monolayer culture and three-dimensional agarose, suggesting a role for cell adhesion. The relationship between condensation and osmolality was accurately modeled by a polymer gel model which, along with the rapid nature of the chromatin condensation (<20 s), reveals the basic physicochemical nature of the process. Alterations in chromatin structure are expected to influence gene expression and thereby regulate chondrocyte activity in response to osmotic changes. PMID:23442954

  2. Genome size and chromatin condensation in vertebrates.

    PubMed

    Vinogradov, Alexander E

    2005-02-01

    Cell membrane-dependent chromatin condensation was studied by flow cytometry in erythrocytes of 36 species from six classes of vertebrates. A positive relationship was found between the degree of condensation and genome size. The distribution of variances among taxonomic levels is similar for both parameters. However, chromatin condensation varied relatively more at the lower taxonomic levels, which suggests that the degree of DNA packaging might serve for fine-tuning the 'skeletal' and/or 'buffering' function of noncoding DNA (although the range of this fine-tuning is smaller than the range of genome size changes). For two closely related amphibian species differing in genome size, change in chromatin condensation under the action of elevated extracellular salinity was investigated. Condensation was steadier and its reaction to changes in solvent composition was more inertial in the species with a larger genome, which is in agreement with the buffering function postulated for redundant DNA. The uppermost genome size in vertebrates (and in living beings in general) was updated using flow cytometry and was found to be about 80 pg (78,400 Mb). The widespread opinion that the largest genome occurs in unicellular organisms is rejected as being based on artifacts. PMID:15647899

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  5. The effect of salt on oligocation-induced chromatin condensation.

    PubMed

    Korolev, Nikolay; Zhao, Yongqian; Allahverdi, Abdollah; Eom, Khee Dong; Tam, James P; Nordenskiöld, Lars

    2012-02-10

    Condensation of model chromatin in the form of fully saturated 12-mer nucleosome arrays, induced by addition of cationic ligands (?-oligolysines with charge varied from +4 to +11), was studied in a range of KCl concentrations (10-500mM) using light scattering and precipitation assay titrations. The dependence of EC(50) (ligand concentration at the midpoint of the array condensation) on C(KCl) displays two regimes, a salt-independent at low C(KCl) and a salt-dependent at higher salt concentrations. In the salt-dependent regime EC(50) rises sharply with increase of C(KCl). Increase of ligand charge shifts the transition from the salt-independent to salt-dependent regime to higher salt. In the nucleosome array system, due to the partial neutralization of the DNA charge by histones, a lower oligocation concentration is needed to provoke condensation in the salt-independent regime compared to the related case of DNA condensation by the same cation. In the physiological range of salt concentrations (C(KCl)=50-300mM), K(+) ions assist array condensation by shifting EC(50) of the ?-oligolysines to lower values. At higher C(KCl), K(+) competes with the cationic ligands, which leads to increase of EC(50). Values of salt-dependent dissociation constant for the ?-oligolysine-nucleosome array interaction were obtained, by fitting to a general equation developed earlier for DNA, describing the dependence of EC(50) on dissociation constant, salt and polyelectrolyte concentrations. PMID:22227197

  6. PTEN interacts with histone H1 and controls chromatin condensation.

    PubMed

    Chen, Zhu Hong; Zhu, Minglu; Yang, Jingyi; Liang, Hui; He, Jinxue; He, Shiming; Wang, Pan; Kang, Xi; McNutt, Michael A; Yin, Yuxin; Shen, Wen H

    2014-09-25

    Chromatin organization and dynamics are integral to global gene transcription. Histone modification influences chromatin status and gene expression. PTEN plays multiple roles in tumor suppression, development, and metabolism. Here, we report on the interplay of PTEN, histone H1, and chromatin. We show that loss of PTEN leads to dissociation of histone H1 from chromatin and decondensation of chromatin. PTEN deletion also results in elevation of histone H4 acetylation at lysine 16, an epigenetic marker for chromatin activation. We found that PTEN and histone H1 physically interact through their C-terminal domains. Disruption of the PTEN C terminus promotes the chromatin association of MOF acetyltransferase and induces H4K16 acetylation. Hyperacetylation of H4K16 impairs the association of PTEN with histone H1, which constitutes regulatory feedback that may reduce chromatin stability. Our results demonstrate that PTEN controls chromatin condensation, thus influencing gene expression. We propose that PTEN regulates global gene transcription profiling through histones and chromatin remodeling. PMID:25199838

  7. METHODS IN MOLECULAR BIOLOGY: ASSAYING CHROMATIN SIRTUINS

    PubMed Central

    Silberman, Dafne M.; Sebastian, Carlos; Mostoslavsky, Raul

    2015-01-01

    Summary Most of the sirtuins’ nuclear substrates indentified so far are histones or other chromatin-associated proteins and, thus, it is of special relevance the development of good biochemical techniques to analyze the biology of these proteins in the context of chromatin. Here, we describe several of the chromatin-based techniques to identify sirtuins’ substrates, including a chromatin immunoprecipitation (ChIP) protocol, an acid-extraction protocol, and a nucleosomal immunoprecipitation protocol to analyze putative sirtuin chromatin interactors. PMID:24014405

  8. CHD5 is Required for Spermiogenesis and Chromatin Condensation

    PubMed Central

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

    2014-01-01

    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. PMID:24252660

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

    PubMed

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

    2015-12-01

    Repression of maternal Xist (Xm-Xist) during preimplantation in mouse embryos is essential for establishing imprinted X chromosome inactivation. Nuclear transplantation (NT) studies using nuclei derived from non-growing (ng) and full-grown (fg) oocytes have indicated that maternal-specific repressive modifications are imposed on Xm-Xist during oogenesis, as well as on autosomal imprinted genes. Recent studies have revealed that histone H3 lysine 9 trimethylation (H3K9me3) enrichments on Xm-Xist promoter regions are involved in silencing at the preimplantation stages. However, whether H3K9me3 is imposed on Xm-Xist during oogenesis is not known. Here, we dissected the chromatin states in ng and fg oocytes and early preimplantation stage embryos. Chromatin immunoprecipitation experiments against H3K9me3 revealed that there was no significant enrichment within the Xm-Xist region during oogenesis. However, NT embryos with ng nuclei (ngNT) showed extensive Xm-Xist derepression and H3K9me3 hypomethylation of the promoter region at the 4-cell stage, which corresponds to the onset of paternal Xist expression. We also found that the chromatin state at the Xist genomic locus became markedly condensed as oocyte growth proceeded. Although the condensed Xm-Xist genomic locus relaxed during early preimplantation phases, the extent of the relaxation across Xm-Xist loci derived from normally developed oocytes was significantly smaller than those of paternal-Xist and ngNT-Xist genomic loci. Furthermore, Xm-Xist from 2-cell metaphase nuclei became derepressed following NT. We propose that chromatin condensation is associated with imprinted Xist repression and that skipping of the condensation step by NT leads to Xist activation during the early preimplantation phase. PMID:26459223

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

    PubMed Central

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

    2015-01-01

    Repression of maternal Xist (Xm-Xist) during preimplantation in mouse embryos is essential for establishing imprinted X chromosome inactivation. Nuclear transplantation (NT) studies using nuclei derived from non-growing (ng) and full-grown (fg) oocytes have indicated that maternal-specific repressive modifications are imposed on Xm-Xist during oogenesis, as well as on autosomal imprinted genes. Recent studies have revealed that histone H3 lysine 9 trimethylation (H3K9me3) enrichments on Xm-Xist promoter regions are involved in silencing at the preimplantation stages. However, whether H3K9me3 is imposed on Xm-Xist during oogenesis is not known. Here, we dissected the chromatin states in ng and fg oocytes and early preimplantation stage embryos. Chromatin immunoprecipitation experiments against H3K9me3 revealed that there was no significant enrichment within the Xm-Xist region during oogenesis. However, NT embryos with ng nuclei (ngNT) showed extensive Xm-Xist derepression and H3K9me3 hypomethylation of the promoter region at the 4-cell stage, which corresponds to the onset of paternal Xist expression. We also found that the chromatin state at the Xist genomic locus became markedly condensed as oocyte growth proceeded. Although the condensed Xm-Xist genomic locus relaxed during early preimplantation phases, the extent of the relaxation across Xm-Xist loci derived from normally developed oocytes was significantly smaller than those of paternal-Xist and ngNT-Xist genomic loci. Furthermore, Xm-Xist from 2-cell metaphase nuclei became derepressed following NT. We propose that chromatin condensation is associated with imprinted Xist repression and that skipping of the condensation step by NT leads to Xist activation during the early preimplantation phase. PMID:26459223

  11. Axial contraction and short-range compaction of chromatin synergistically promote mitotic chromosome condensation

    PubMed Central

    Kruitwagen, Tom; Denoth-Lippuner, Annina; Wilkins, Bryan J; Neumann, Heinz; Barral, Yves

    2015-01-01

    The segregation of eukaryotic chromosomes during mitosis requires their extensive folding into units of manageable size for the mitotic spindle. Here, we report on how phosphorylation at serine 10 of histone H3 (H3 S10) contributes to this process. Using a fluorescence-based assay to study local compaction of the chromatin fiber in living yeast cells, we show that chromosome condensation entails two temporally and mechanistically distinct processes. Initially, nucleosome-nucleosome interaction triggered by H3 S10 phosphorylation and deacetylation of histone H4 promote short-range compaction of chromatin during early anaphase. Independently, condensin mediates the axial contraction of chromosome arms, a process peaking later in anaphase. Whereas defects in chromatin compaction have no observable effect on axial contraction and condensin inactivation does not affect short-range chromatin compaction, inactivation of both pathways causes synergistic defects in chromosome segregation and cell viability. Furthermore, both pathways rely at least partially on the deacetylase Hst2, suggesting that this protein helps coordinating chromatin compaction and axial contraction to properly shape mitotic chromosomes. DOI: http://dx.doi.org/10.7554/eLife.10396.001 PMID:26615018

  12. Osmotic stress alters chromatin condensation and nucleocytoplasmic transport

    SciTech Connect

    Finan, John D.; Leddy, Holly A.; Department of Biomedical Engineering, Duke University, Durham, NC ; Guilak, Farshid; Department of Biomedical Engineering, Duke University, Durham, NC

    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.

  13. Influence of chromatin condensation on the absorption spectra of nuclei stained with toluidine blue.

    PubMed

    Erenpreisa, J; Freivalds, T; Selivanova, G

    1992-01-01

    To study the influence of chromatin condensation on the absorption spectra of nuclei stained with toluidine blue, DNA staining methods--which favour or prevent dye polymerization--were applied to the imprints of rat tissues that differed greatly in the density of chromatin packing. It is stated that all factors promoting dye polymerization cause a left shift of the spectra while the factors preventing it, a right one. It was found that condensation of the chromatin can raise prerequisites that both enhance and hinder polymerization, and that the final result depends on the staining method, the manner of chromatin folding, and the density of its packing. PMID:1365771

  14. 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 ?-structure in the phosphorylated samples, concomitant to a decrease in ?-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

  15. 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

  16. Chromatin Immunoprecipitation (ChIP) Assay in Candida albicans.

    PubMed

    Mitra, Sreyoshi; Rai, Laxmi Shanker; Chatterjee, Gautam; Sanyal, Kaustuv

    2016-01-01

    Chromatin immunoprecipitation (ChIP) is a widely used technique which can determine the in vivo association of a specific protein on a particular DNA locus in the genome. In this method cross-linked chromatin is sheared and immunoprecipitated with antibodies raised against a target protein of interest. The end result of this process is the enrichment of DNA fragments associated with the desired protein. Thus, interactions between proteins and genomic loci in cellular context can be determined by this technique. Here, we are describing a ChIP protocol that is optimized for Candida albicans. The protocol requires 4-5 days for completion of the assay and has been used to produce robust ChIP results for diverse proteins in this organism and its related species including Candida dubliniensis and Candida tropicalis. PMID:26519064

  17. Effects of ethanol consumption on chromatin condensation and DNA integrity of epididymal spermatozoa in rat.

    PubMed

    Talebi, Ali Reza; Sarcheshmeh, Abolghasem Abbasi; Khalili, Mohammad Ali; Tabibnejad, Nasim

    2011-06-01

    Alcohol abuse is considered as one of the problems associated with poor semen production and sperm quality. Both acute and chronic alcohol consumption may affect spermatozoal chromatin disorders through apoptosis. Therefore, for the first time, this experimental study was performed to evaluate the effect of ethanol consumption on sperm parameters and chromatin integrity of spermatozoa aspirated from cauda epididymis of rats. Twenty adult Wistar rats were divided into ethanol consumption and control groups. Access to ethanol and water was provided ad libitum for experimental and control animals, respectively. The cauda epididymal spermatozoa were aspirated for analysis of sperm parameters and sperm chromatin integrity with aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), and acridine orange (AO) assays. Sperm progressive and nonprogressive motility of ethanol-consuming rats were significantly decreased compared with control animals (P < .05). In addition, the rates of AB-reacted spermatozoa were similar in both groups (P > .05). However, with regard to CMA3, AO, and TB stainings, there was a significant increase in ethanol group when compared with the controls (P < .05). The majority of TB+ and AO+ spermatozoa were higher than "cut-off" value in ethanol group, whereas the mean rates of CMA3+ spermatozoa was below the "cut-off" value in both groups. The results showed that ethanol consumption disturbs sperm motility, nuclear maturity and DNA integrity of spermatozoa in rat. Therefore, ethanol abuse results in the production of spermatozoa with less condensed chromatin, and this may be one possible cause of infertility following ethanol consumption. PMID:21145692

  18. Insulation of the chicken beta-globin chromosomal domain from a chromatin-condensing protein, MENT.

    PubMed

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

    2003-09-01

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

  19. The Forkhead Transcription Factor FoxI1 Remains Bound to Condensed Mitotic Chromosomes and Stably Remodels Chromatin Structure†

    PubMed Central

    Yan, Jizhou; Xu, Lisha; Crawford, Gregory; Wang, Zenfeng; Burgess, Shawn M.

    2006-01-01

    All forkhead (Fox) proteins contain a highly conserved DNA binding domain whose structure is remarkably similar to the winged-helix structures of histones H1 and H5. Little is known about Fox protein binding in the context of higher-order chromatin structure in living cells. We created a stable cell line expressing FoxI1-green fluorescent protein (GFP) or FoxI1-V5 fusion proteins under control of the reverse tetracycline-controlled transactivator doxycycline inducible system and found that unlike most transcription factors, FoxI1 remains bound to the condensed chromosomes during mitosis. To isolate DNA fragments directly bound by the FoxI1 protein within living cells, we performed chromatin immunoprecipitation assays (ChIPs) with antibodies to either enhanced GFP or the V5 epitope and subcloned the FoxI1-enriched DNA fragments. Sequence analyses indicated that 88% (106/121) of ChIP sequences contain the consensus binding sites for all Fox proteins. Testing ChIP sequences with a quantitative DNase I hypersensitivity assay showed that FoxI1 created stable DNase I sensitivity changes in condensed chromosomes. The majority of ChIP targets and random targets increased in resistance to DNase I in FoxI1-expressing cells, but a small number of targets became more accessible to DNase I. Consistently, the accessibility of micrococcal nuclease to chromatin was generally inhibited. Micrococcal nuclease partial digestion generated a ladder in which all oligonucleosomes were slightly longer than those observed with the controls. On the basis of these findings, we propose that FoxI1 is capable of remodeling chromatin higher-order structure and can stably create site-specific changes in chromatin to either stably create or remove DNase I hypersensitive sites. PMID:16354687

  20. ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide.

    PubMed

    Buenrostro, Jason D; Wu, Beijing; Chang, Howard Y; Greenleaf, William J

    2015-01-01

    This unit describes Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq), a method for mapping chromatin accessibility genome-wide. This method probes DNA accessibility with hyperactive Tn5 transposase, which inserts sequencing adapters into accessible regions of chromatin. Sequencing reads can then be used to infer regions of increased accessibility, as well as to map regions of transcription-factor binding and nucleosome position. The method is a fast and sensitive alternative to DNase-seq for assaying chromatin accessibility genome-wide, or to MNase-seq for assaying nucleosome positions in accessible regions of the genome. © 2015 by John Wiley & Sons, Inc. PMID:25559105

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

    PubMed

    Baron, Margaret H; Barminko, Jeffrey

    2016-03-01

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

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

    PubMed Central

    Spagnol, Stephen T.; Dahl, Kris Noel

    2016-01-01

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

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

    PubMed

    Spagnol, Stephen T; Dahl, Kris Noel

    2016-01-01

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

  4. The Histone Variant H2A.W Defines Heterochromatin and Promotes Chromatin Condensation in Arabidopsis

    PubMed Central

    Yelagandula, Ramesh; Stroud, Hume; Holec, Sarah; Zhou, Keda; Feng, Suhua; Zhong, Xuehua; Muthurajan, Uma M.; Nie, Xin; Kawashima, Tomokazu; Groth, Martin; Luger, Karolin; Jacobsen, Steven E.; Berger, Frédéric

    2015-01-01

    Summary Histone variants play crucial roles in gene expression, genome integrity and chromosome segregation. However, to what extent histone variants control chromatin architecture remains largely unknown. We report genome-wide profiles of all four types of H2A variants in Arabidopsis and identify that the previously uncharacterized histone variant H2A.W specifically associates with heterochromatin. Genetic analyses show that H2A.W acts in synergy with the heterochromatic marks H3K9me2 and DNA methylation to maintain genome integrity. In vitro, H2A.W enhances chromatin condensation through a higher propensity to promote fiber-to-fiber interactions via its conserved C-terminal motif. In vivo, elimination of H2A.W causes decondensation of heterochromatin and conversely, ectopic expression of H2A.W promotes heterochromatin condensation. These results demonstrate that H2A.W plays critical roles in heterochromatin by promoting higher order chromatin condensation. Since motifs similar to the H2A.W C-terminal motif are present in other histone variants in other organisms, our findings impact our understanding of heterochromatin condensation in eukaryotes. PMID:24995981

  5. 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

  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. 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. PMID:24766543

  8. Restriction Enzyme Analysis of DNA Methylation in “Condensed” Chromatin of Ha-Ras-Transformed NIH 3T3 Cells

    PubMed Central

    Mello, Maria Luiza S.; Chambers, Ann F.; Vidal, Benedicto C.; Planding, Wolfgang; Schenck, Ulrich

    2000-01-01

    Increased amounts of chromatin condensation (i.e., localized areas of high DNA density, or chromatin higher order packing state) have been described in NIH 3T3 cells transformed with the Ha?ras oncogene. The structural basis for this oncogene?mediated alteration in nuclear organization is unknown. Since DNA methylation is likely to be involved in regulating the nucleosomal level of DNA packaging, we studied the role of DNA methylation in higher?order chromatin organization induced by Ha?ras. CpG?methylated DNA content was estimated in “condensed” chromatin of Ha?ras?transformed NIH 3T3 cell lines which differ in ras expression and ras?induced metastatic ability but present approximately the same values of “condensed” chromatin areas. The question posed was that if DNA methylation were involved with the chromatin higher?order organization induced by Ha?ras in these cell lines, the methylated DNA density in the “condensed” chromatin would also be the same. The DNA evaluation was performed by video image analysis in Feulgen?stained cells previously subjected to treatment with Msp I and Hpa II restriction enzymes, which distinguish between methylated and non?methylated DNA. The amount of methylated CpG sequences not digested by Hpa II in “condensed” chromatin regions was found to vary in the studied ras?transformed cell lines. DNA CpG methylation status is thus suggested not to be involved with the higher order chromatin condensation induced by ras transformation in the mentioned NIH 3T3 cell lines. PMID:11205319

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

    PubMed

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

    2015-01-01

    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. PMID:26710245

  10. GENOTOXICITY OF TEN CIGARETTE SMOKE CONDENSATES IN FOUR TEST SYSTEMS: COMPARISONS BETWEEN ASSAYS AND CONDENSATES

    EPA Science Inventory

    What is the study?
    This the first assessment of a set of cigarette smoke condensates from a range of cigarette types in a variety (4) of short-term genotoxicity assays.
    Why was it done?
    No such comparative study of cigarette smoke condensates has been reported. H...

  11. Chromatin.

    PubMed

    Gross, David S; Chowdhary, Surabhi; Anandhakumar, Jayamani; Kainth, Amoldeep S

    2015-12-21

    Chromatin is a complex of proteins, RNA and DNA that constitutes the physiological state of the genome. Its basic structure is essentially the same in nearly all eukaryotes, from single-celled yeasts to the most complex multicellular organisms (exceptions include the chromatin of dinoflagellates and vertebrate sperm). Its fundamental role is to package the genome in a sufficiently compact form that allows comparatively very large molecules of DNA to fit inside the cell's nucleus. In human cells, the contour length of the DNA molecules comprising the largest chromosomes is nearly 10,000 times the diameter of the nucleus (typically on the order of 5-10 microns). How is this compaction accomplished? Through multiple layers of folding. PMID:26702648

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

    PubMed

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

    1995-07-14

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

  13. Caspase-6 gene disruption reveals a requirement for lamin A cleavage in apoptotic chromatin condensation.

    PubMed

    Ruchaud, Sandrine; Korfali, Nadia; Villa, Pascal; Kottke, Timothy J; Dingwall, Colin; Kaufmann, Scott H; Earnshaw, William C

    2002-04-15

    To study the role of caspase-6 during nuclear disassembly, we generated a chicken DT40 cell line in which both alleles of the caspase-6 gene were disrupted. No obvious morphological differences were observed in the apoptotic process in caspase-6- deficient cells compared with the wild type. However, examination of apoptosis in a cell-free system revealed a block in chromatin condensation and apoptotic body formation when nuclei from HeLa cells expressing lamin A or lamin A-transfected Jurkat cells were incubated in caspase-6-deficient apoptotic extracts. Transfection of exogenous caspase-6 into the clone reversed this phenotype. Lamins A and C, which are caspase-6-only substrates, were cleaved by the wild-type and heterozygous apoptotic extracts but not by the extracts lacking caspase-6. Furthermore, the caspase-6 inhibitor z-VEID-fmk mimicked the effects of caspase-6 deficiency and prevented the cleavage of lamin A. Taken together, these observations indicate that caspase-6 activity is essential for lamin A cleavage and that when lamin A is present it must be cleaved in order for the chromosomal DNA to undergo complete condensation during apoptotic execution. PMID:11953316

  14. Caspase-6 gene disruption reveals a requirement for lamin A cleavage in apoptotic chromatin condensation

    PubMed Central

    Ruchaud, Sandrine; Korfali, Nadia; Villa, Pascal; Kottke, Timothy J.; Dingwall, Colin; Kaufmann, Scott H.; Earnshaw, William C.

    2002-01-01

    To study the role of caspase-6 during nuclear disassembly, we generated a chicken DT40 cell line in which both alleles of the caspase-6 gene were disrupted. No obvious morphological differences were observed in the apoptotic process in caspase-6- deficient cells compared with the wild type. However, examination of apoptosis in a cell-free system revealed a block in chromatin condensation and apoptotic body formation when nuclei from HeLa cells expressing lamin A or lamin A-transfected Jurkat cells were incubated in caspase-6-deficient apoptotic extracts. Transfection of exogenous caspase-6 into the clone reversed this phenotype. Lamins A and C, which are caspase-6-only substrates, were cleaved by the wild-type and heterozygous apoptotic extracts but not by the extracts lacking caspase-6. Furthermore, the caspase-6 inhibitor z-VEID-fmk mimicked the effects of caspase-6 deficiency and prevented the cleavage of lamin A. Taken together, these observations indicate that caspase-6 activity is essential for lamin A cleavage and that when lamin A is present it must be cleaved in order for the chromosomal DNA to undergo complete condensation during apoptotic execution. PMID:11953316

  15. [Application of chromatin immunoprecipitation assay in deciphering DNA-protein interactions].

    PubMed

    Wang, Chun-Yu; Shi, Jian-Dang; Zhu, Yan; Zhang, Ju

    2005-09-01

    In the post-genomic era, identifying and characterizing various DNA-protein interactions are a major challenge in the research of gene transcriptional regulation. Although many techniques can be used for this purpose, chromatin immunoprecipitation assay (ChIP), by contrast, is ideally suited for studying DNA-protein interactions in vivo. In recent years, standard ChIP assay has been modified to uncover some known factors' unknown target sequences, especially when combined with DNA microarray and molecular cloning strategies. These high-throughput ChIP assays are more and more used to reveal the distribution profile of trans-acting factor binding sites throughout the genome, which may yield many new insights into the DNA-protein interaction network. This article summarized the methods of ChIP assay, and highlighted recent progress in the application of this technique. PMID:16257913

  16. Glycolytic metabolism influences global chromatin structure

    PubMed Central

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

    2015-01-01

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

  17. Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays

    PubMed Central

    2011-01-01

    Background The processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP) and methylated DNA (MeDIP) represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription. Results To allow simultaneous measurements of DNA methylation with other genomic processes, we developed and validated a simple and easy-to-use high throughput microplate-based platform for analysis of DNA methylation. Compared to the traditional beads-based MeDIP the microplate MeDIP was more sensitive and had lower non-specific binding. We integrated the MeDIP method with a microplate ChIP assay which allows measurements of both DNA methylation and histone marks at the same time, Matrix ChIP-MeDIP platform. We illustrated several applications of this platform to relate DNA methylation, with chromatin and transcription events at selected genes in cultured cells, human cancer and in a model of diabetic kidney disease. Conclusion The high throughput capacity of Matrix ChIP-MeDIP to profile tens and potentially hundreds of different genomic events at the same time as DNA methylation represents a powerful platform to explore complex genomic mechanism at selected genes in cultured cells and in whole tissues. In this regard, Matrix ChIP-MeDIP should be useful to complement genome-wide studies where the rich chromatin and transcription database resources provide fruitful foundation to pursue mechanistic, functional and diagnostic information at genes of interest in health and disease. PMID:22098709

  18. RBF1 promotes chromatin condensation through a conserved interaction with the Condensin II protein dCAP-D3

    PubMed Central

    Longworth, Michelle S.; Herr, Anabel; Ji, Jun-Yuan; Dyson, Nicholas J.

    2008-01-01

    The Drosophila retinoblastoma family of proteins (RBF1 and RBF2) and their mammalian homologs (pRB, p130, and p107) are best known for their regulation of the G1/S transition via the repression of E2F-dependent transcription. However, RB family members also possess additional functions. Here, we report that rbf1 mutant larvae have extensive defects in chromatin condensation during mitosis. We describe a novel interaction between RBF1 and dCAP-D3, a non-SMC component of the Condensin II complex that links RBF1 to the regulation of chromosome structure. RBF1 physically interacts with dCAP-D3, RBF1 and dCAP-D3 partially colocalize on polytene chromosomes, and RBF1 is required for efficient association of dCAP-D3 with chromatin. dCap-D3 mutants also exhibit chromatin condensation defects, and mutant alleles of dCap-D3 suppress cellular and developmental phenotypes induced by the overexpression of RBF1. Interestingly, this interaction is conserved between flies and humans. The re-expression of pRB into a pRB-deficient human tumor cell line promotes chromatin association of hCAP-D3 in a manner that depends on the LXCXE-binding cleft of pRB. These results uncover an unexpected link between pRB/RBF1 and chromatin condensation, providing a mechanism by which the functional inactivation of RB family members in human tumor cells may contribute to genome instability. PMID:18367646

  19. Identification of transcription factor-DNA interactions using chromatin immunoprecipitation assays.

    PubMed

    Nie, Liping; Vázquez, Ana E; Yamoah, Ebenezer N

    2009-01-01

    Expression of almost every gene is regulated at the transcription level. Therefore, transcriptional factor Transcription factors, consequently, have marked effects on the fate of a cell by establishing the gene expression patterns that determine biological processes. In the auditory and vestibular systems, transcription factors have been found to be responsible for development, cell growth, and apoptosis. It is vital to identify the transcription factor target genes and the mechanisms by which transcription factors control and guide gene expression and regulation pathways. Compared with earlier methods devised to study transcription factor-DNA interactions, the advantage of the chromatin immunoprecipitation (ChIP) assay is that the interaction of a transcription factor with its target genes is captured in the native context of chromatin in living cells. Therefore, ChIP base assays are powerful tools to identify the direct interaction of transcription factors and their target genes in vivo. More importantly, ChIP assays have been used in combination with molecular biology techniques, such as PCR and real time PCR, gene cloning, and DNA microarrays, to determine the interaction of transcription factor-DNA from a few potential individual targets to genome-wide surveys. PMID:18839356

  20. 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.

  1. Sperm DNA quality evaluated by comet assay and sperm chromatin structure assay in stallions after unilateral orchiectomy.

    PubMed

    Serafini, R; Varner, D D; Bissett, W; Blanchard, T L; Teague, S R; Love, C C

    2015-09-15

    Unilateral orchiectomy (UO) may interfere with thermoregulation of the remaining testis caused by inflammation surrounding the incision site, thus altering normal spermatogenesis and consequently sperm quality. Two measures of sperm DNA quality (neutral comet assay and the sperm chromatin structure assay [SCSA]) were compared before UO (0 days) and at 14, 30, and 60 days after UO to determine whether sperm DNA changed after a mild testis stress (i.e., UO). The percent DNA in the comet tail was higher at 14 and 60 days compared to 0 days (P < 0.05) after UO. All other comet tail measures (i.e., length, moment, migration) were higher at all time periods after UO compared to 0 days (P < 0.05). Two SCSA measures (mean-αt, mode-αt) increased at 14 days after UO (P < 0.05), whereas two measures (SD-αt and COMP-αt) did not change. This study identified a decrease in sperm DNA quality using both the neutral comet assay and the SCSA, which was not identified using traditional measures of sperm quality. PMID:26104544

  2. Chromatin immunoprecipitation assays: analyzing transcription factor binding and histone modifications in vivo.

    PubMed

    Pillai, Smitha; Dasgupta, Piyali; Chellappan, Srikumar P

    2015-01-01

    Studies in the past decade have shown that differential gene expression depends not only on the binding of specific transcription factors to discrete promoter elements but also on the epigenetic modification of the DNA as well as histones associated with the promoter. While techniques like electrophoretic mobility shift assays could detect and characterize the binding of specific transcription factors present in cell lysates to DNA sequences in in vitro binding conditions, they were not effective in assessing the binding in intact cells. Development of chromatin immunoprecipitation technique in the past decade enabled the analysis of the association of regulatory molecules with specific promoters or changes in histone modifications in vivo, without overexpressing any component. ChIP assays can provide a snapshot of how a regulatory transcription factor affects the expression of a single gene, or a variety of genes at the same time. Availability of high quality antibodies that recognizes histones modified in a specific fashion further expanded the use of ChIP assays to analyze even minute changes in histone modification and nucleosomes structure. This chapter outlines the general strategies and protocols used to carry out ChIP assays to study the differential recruitment of transcription factors as well as histone modifications. PMID:25827895

  3. Early and late effects of Ibuprofen on mouse sperm parameters, chromatin condensation, and DNA integrity in mice

    PubMed Central

    Roodbari, Fatemeh; Abedi, Nahid; Talebi, Ali Reza

    2015-01-01

    Background: There are few studies indicating the detrimental effects of ibuprofen on sperm fertility potential and DNA integrity. Objective: To determine the effects of Ibuprofen on sperm parameters, chromatin condensation and DNA integrity of mice. Materials and Methods: In this experimental study, 36 adult male mice with average weight 37 gr were divided into three groups, including control (group I, n=12), normal dosage of ibuprofen (group II, n=12) and high dosage (group III, n=12). Ibuprofen with different doses was dissolved in daily water of animals. After 35, 70 and 105 days, the cauda epididymis of mice were cut and incubated in Ham’s F10 media. Sperm samples were analyzed for parameters (motility, morphology and count), DNA integrity (SCD test) and chromatin condensation (chromomycin A3 and Aniline blue staining). Results: After 35 days, in addition to above mentioned sperm parameters, all of the treated mice showed statistically significant increase in spermatozoa with immature chromatin (P<0.05). However, after 70 days, the rate of sperm DNA fragmentation assessed by SCD was increased in group II (66.5±0.7) and the percentage of immature spermatozoa (AB+ and CMA3+) was higher in group III (77.5±0.7 and 49.5±6.3 respectively) than other groups. After 105 days, the AB+ spermatozoa were increased in both normal dose and high dose groups. Conclusion: Ibuprofen may cause a significant reduction in sperm parameters and sperm chromatin/DNA integrity in mice. It should be noted that these deleterious effects are dose-dependent and can be seen in early and late stage of drug treatments. PMID:26730245

  4. 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

  5. Akt phosphorylation of zyxin mediates its interaction with acinus-S and prevents acinus-triggered chromatin condensation.

    PubMed

    Chan, C-B; Liu, X; Tang, X; Fu, H; Ye, K

    2007-09-01

    Zyxin, a focal adhesion molecule, contains LIM domains and shuttles between the cytoplasm and the nucleus. Nuclear zyxin promotes cardiomyocyte survival, which is mediated by nuclear-activated Akt. However, the molecular mechanism of how zyxin antagonizes apoptosis remains elusive. Here, we report that zyxin binds to acinus-S, a nuclear speckle protein inducing apoptotic chromatin condensation after cleavage by caspases, and prevents its apoptotic action, which is regulated by Akt. Akt binds and phosphorylates zyxin on serine 142, leading to its association with acinus. Interestingly, 14-3-3gamma, but not zeta isoform selectively, triggers zyxin nuclear translocation, which is Akt phosphorylation dependent. Zyxin is also a substrate of caspases, but Akt phosphorylation is unable to prevent its apoptotic cleavage. Expression of zyxin S142D, a phosphorylation mimetic mutant, diminishes acinus proteolytic cleavage and chromatin condensation; by contrast, wild-type zyxin or unphosphorylated S142A mutant fails. Thus, Akt regulates zyxin/acinus complex formation in the nucleus, contributing to suppression of apoptosis. PMID:17572661

  6. Chromatin Immunoprecipitation Assay to Identify Genomic Binding Sites of Regulatory Factors.

    PubMed

    Wagner, Meike; Jung, Johannes; Koslowski, Michael; Türeci, Özlem; Tiwari, Vijay K; Sahin, Ugur

    2016-01-01

    DNA-protein interactions are vital to fundamental cellular events including transcription, replication, DNA repair, and recombination. Thus, their study holds the key to our understanding of mechanisms underlying normal development and homeostasis as well as disease. Transcriptional regulation is a highly complex process that involves recruitment of numerous factors resulting in formation of multi-protein complexes at gene promoters to regulate gene expression. The studied proteins can be, for example, transcription factors, epigenetic regulators, co-activators, co-repressors, or ligand-activated nuclear receptors as estrogen receptor-? (ER?) bound either directly to the DNA or indirectly by interaction with other DNA-bound factors. Chromatin immunoprecipitation (ChIP) assay is a powerful method to study interactions of proteins and a specific genomic DNA region. Recruitment of ER? to promoters of estrogen-dependent genes is a common mechanism to activate or enhance gene transcription in breast cancer thus promoting tumor progression. In this chapter, we demonstrate a stepwise protocol for ChIP assay using binding of ER? to its genomic targets after stimulation with 17?-estradiol (E2) in breast cancer cells as an example. PMID:26585127

  7. Isolation of Nuclei from Skeletal Muscle Satellite Cells and Myofibers for Use in Chromatin lmmunoprecipitation Assays

    PubMed Central

    Ohkawa, Yasuyuki; Mallappa, Chandrashekara; Dacwag Vallaster, Caroline S.; lmbalzano, Anthony N.

    2014-01-01

    Studies investigating mechanisms controlling gene regulation frequently examine specific DNA sequences using chromatin immunoprecipitation (ChIP) assays to determine whether specific regulatory factors or modified histones are present. While use of primary cells or cell line models for differentiating or differentiated tissue is widespread, the ability to assess factor binding and histone modification in tissue defines the events that occur in vivo and provides corroboration for studies in cultured cells. Many tissues can be analyzed with minimal modification to existing ChIP protocols that are designed for cultured cells; however, some tissues, such as skeletal muscle, are problematic in that accessibility of the cross-linking agent is limited. We describe a method to isolate skeletal muscle tissue nuclei suitable for use in ChIP protocols. Furthermore, we utilize a simple fractionation of digested skeletal muscle tissue that can separate mature myofibers from satellite cells, which are responsible for postnatal skeletal muscle regeneration, thereby allowing simultaneous preparation of nuclei from both cell types. PMID:22130858

  8. Sperm chromatin structure assay results in Nigerian men with unexplained infertility

    PubMed Central

    Kolade, Charles Oluwabukunmi

    2015-01-01

    Objective Several publications have established a relationship between sperm DNA damage and male factor infertility, based on data from America, Europe, and Asia. This study aimed to compare the extent of sperm DNA damage in sperm samples from Nigerian men with unexplained infertility and in sperm samples from a fertile group composed of sperm donors who had successfully impregnated a female partner naturally or through assisted conception. Methods A total of 404 men underwent male fertility evaluation at Androcare Laboratories and Cryobank participated in this study. Semen analysis and a sperm chromatin structure assay (SCSA) were performed on all subjects. Results The men in the unexplained infertility group were slightly older than the men in the fertile sperm group (36±10 years vs. 32±6 years, p=0.051). No significant difference was observed between the two groups in semen analysis parameters (p?0.05). Men in the unexplained infertility group with normal semen parameters had a significantly higher DNA fragmentation index (DFI) than men in the fertile sperm group (27.5%±7.0% vs. 14.1%±5.3%, p<0.05). In the unexplained infertility group, 63% of the men had a DFI greater than 20%, compared to 4% in the fertile sperm group. In the unexplained infertility group, 15.2% of the subjects had a DFI greater than 30%, compared to 1% in the fertile sperm group. Conclusion Our study showed that the SCSA may be a more reliable predictor of fertility potential than traditional semen analysis in cases of unexplained infertility. PMID:26473109

  9. Gamma irradiation-induced apoptosis in murine pre-B cells prevents the condensation of fibrillar chromatin in early S phase.

    PubMed

    Nagy, G; Gacsi, M; Rehak, M; Basnakian, A G; Klaisz, M; Banfalvi, G

    2004-11-01

    Local changes in chromatin structure leading to temporally distinct geometric forms were characterized in nuclei of reversibly permeabilized cells. Reversal of permeabilization was tested by 3H-thymidine incorporation and trypan blue dye exclusion. Apoptotic changes were visualized in a cell cycle dependent manner at the chromatin level by fluorescent microscopy in non-irradiated cells and after 400 rad Co60 irradiation. Fluorescent microscopy of chromatin structures belonging mainly to the interphase of the cell cycle confirmed the existence of specific geometric forms in nuclei of non-irradiated cells. In this control population, the following main transitory forms of condensing chromatin were distinguished: decondensed veil-like structures and fibrous structures in early and mid S phase (2.0-2.5 average C-value), chromatin bodies, semicircles later in mid S phase (3.0-3.5 C), precondensed chromosomes in late S (3.5-3.7 C) and metaphase chromosomes at the end and after S phase (3.7-4.0 C). Our results show that upon gamma-irradiation (a) the cellular and nuclear sizes were increased, (b) the DNA content was lower in each elutriated subpopulation of cells, (c) the progression of the cell cycle was arrested in the early S phase at 2.4 C value, (d) the chromatin condensation was blocked between the fibrillar chromatin and precondensed elongated chromosomal forms, and (e) the number and size of apoptotic bodies were inversely correlated with the progression of the cell cycle, with many small apoptotic bodies in early S phase and less and larger apoptotic bodies in late S phase. PMID:15505419

  10. 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

  11. Sperm chromatin structure assay in prediction of in vitro fertilization outcome.

    PubMed

    Oleszczuk, K; Giwercman, A; Bungum, M

    2016-03-01

    Sperm DNA fragmentation index (DFI) assessed by sperm chromatin structure assay is a valuable tool for prediction of fertility in vivo. Previous studies on DFI as predictor of in vitro fertilization (IVF) outcome, based on relatively small materials, gave contradictory results. The present study examines, in a large cohort, the association between sperm DFI and the outcome of IVF/ICSI procedure. The study is based on 1633 IVF or ICSI cycles performed at the Reproductive Medicine Centre, Skåne University Hospital, Malmö, Sweden, between May 2007 and March 2013. DFI values were categorized into four intervals: DFI ≤ 10% (reference group), 10% < DFI ≤ 20%, 20% < DFI ≤ 30%, DFI > 30%. For the three latter intervals, the following outcomes of IVF/ICSI procedures were analyzed in relation to the reference group: fertilization, good quality embryo, pregnancy, miscarriage, and live births. In the standard IVF group, a significant negative association between DFI and fertilization rate was found. When calculated per ovum pick-up (OPU) Odds Ratios (ORs) for at least one good quality embryo (GQE) were significantly lower in the standard IVF group if DFI > 20%. OR for live birth calculated per OPU was significantly lower in standard IVF group if DFI > 20% (OR 0.61; 95% CI: 0.38-0.97; p = 0.04). No such associations were seen in the ICSI group. OR for live birth by ICSI compared to IVF were statistically significantly higher for DFI > 20% (OR 1.7; 95% CI: 1.0-2.9; p = 0.05). OR for miscarriage was significantly increased for DFI > 40% (OR 3.8; 95% CI: 1.2-12; p = 0.02). The results suggest that ICSI might be a preferred method of in vitro treatment in cases with high DFI. Efforts should be made to find options for pharmacologically induced reduction of DFI. The study was based on retrospectively collected data and prospective studies confirming the superiority of ICSI in cases with high DFI are warranted. PMID:26757265

  12. Investigation of histone H4 hyperacetylation dynamics in the 5S rRNA genes family by chromatin immunoprecipitation assay.

    PubMed

    Burliba?a, Liliana; Suciu, Ilinca

    2015-12-01

    Oogenesis is a critical event in the formation of female gamete, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the presence of H4 acetylation of the oocyte and somatic 5S rRNA genes in Triturus cristatus, using chromatin immunoprecipitation assay (ChIP). Our findings suggest that some epigenetic mechanisms such as histone acetylation could be involved in the transcriptional regulation of 5S rRNA gene families. PMID:25315165

  13. Actin cytoskeleton differentially alters the dynamics of lamin A, HP1? and H2B core histone proteins to remodel chromatin condensation state in living cells.

    PubMed

    Toh, Kee Chua; Ramdas, Nisha M; Shivashankar, G V

    2015-10-01

    Cells in physical microenvironments regulate their functioning and geometry in response to mechanical stimuli. Recent studies have demonstrated the influence of the integrated actin cytoskeleton on nuclear integrity and chromatin organization. However, the mechanisms underlying the mechanotransduction of their physical coupling to nuclear protein dynamics are not well understood. In this study, we take advantage of micropatterned geometric substrates in NIH3T3 mouse fibroblasts to probe the functional influence of actin organization on nuclear lamina and chromatin assembly. Fluorescence correlation spectroscopy studies demonstrate that stabilization of perinuclear actin strengthens the transient interactions of lamin A with the chromatin. Correspondingly, fluorescence recovery after photobleaching studies reveal enhanced mobility of these nuclear lamina proteins when actin organization is perturbed. Combining these fluorescence dynamics assays, we also demonstrate an actin-driven differential modulation of core histone H2B and heterochromatin HP1? protein dynamics with chromatin. These altered dynamics are reflected structurally by concomitant changes in the architecture of the heterochromatin foci as seen by immunofluorescence assays. Taken together, our study provides a demonstration of the differential mechanical control of perinuclear actin on the dynamics of the nuclear lamina, euchromatin and heterochromatin regimes of the nucleus, and suggests an actin-mediated route to spatially and structurally tune chromatin organization and dynamics. PMID:26359759

  14. GENOTOXICITY OF TEN CIGARETTE SMOKE CONDENSATES IN FOUR TEST SYSTEMS: COMPARISONS AMONG ASSAYS AND CONDENSATES

    EPA Science Inventory

    The particulate fraction of cigarette smoke, cigarette smoke condensate (CSC), is genotoxic in many short-term in vitro tests and carcinogenic in rodents. However, no study has evaluatedd a set of CSCs prepared from a diverse set of cigarettes in a variety of short-term genotoxic...

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

    SciTech Connect

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

    2012-03-10

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

  16. 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 cell proliferation.

  17. Genomic and Functional Assays Demonstrate Reduced Gammaretroviral Vector Genotoxicity Associated With Use of the cHS4 Chromatin Insulator

    PubMed Central

    Li, Chang Long; Xiong, Ding; Stamatoyannopoulos, George; Emery, David W

    2009-01-01

    Interest in the use of recombinant retroviral vectors for clinical gene therapy has been tempered by evidence of vector-mediated genotoxicity involving the activation of cellular oncogenes flanking sites of vector integration. We report here that the rate of gammaretroviral vector genotoxicity can be significantly reduced by addition of the cHS4 chromatin insulator, based on two complementary approaches for assessing vector-mediated genotoxicity. One approach involves the direct, genomewide assessment of cellular gene dysregulation using panels of transduced cell clones and genomic microarrays, whereas the other involves the functional assessment of malignant transformation using a factor-dependent cell line. Both assays are robust and quantitative, and indicate the cHS4 chromatin insulator can reduce vector-mediated genotoxicity approximately sixfold (ranged three to eight fold). These approaches also provide a means for assessing various aspects of vector-mediated genotoxicity, including the overall rate of cellular gene dysregulation, the potential influence of vector provirus over large genomic distances, and the involvement of oncogenic pathways in vector-mediated malignant transformation. PMID:19240697

  18. 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.

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

    PubMed

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

    2015-11-01

    Combination of next-generation DNA sequencing, single nucleotide polymorphism array analyses and bioinformatics has revealed the striking phenomenon of chromothripsis, described as complex genomic rearrangements acquired in a single catastrophic event affecting one or a few chromosomes. Via an unproven mechanism, it is postulated that mechanical stress causes chromosome shattering into small lengths of DNA, which are then randomly reassembled by DNA repair machinery. Chromothripsis is currently examined as an alternative mechanism of oncogenesis, in contrast to the present paradigm that considers a stepwise development of cancer. While evidence for the mechanism(s) underlying chromosome shattering during cancer development remains elusive, a number of hypotheses have been proposed to explain chromothripsis, including ionizing radiation, DNA replication stress, breakage-fusion-bridge cycles, micronuclei formation and premature chromosome compaction. In the present work, we provide experimental evidence on the mechanistic basis of chromothripsis and on how chromosomes can get locally shattered in a single catastrophic event. Considering the dynamic nature of chromatin nucleoprotein complex, capable of rapid unfolding, disassembling, assembling and refolding, we first show that chromatin condensation at repairing or replicating DNA sites induces the mechanical stress needed for chromosome shattering to ensue. Premature chromosome condensation is then used to visualize the dynamic nature of interphase chromatin and demonstrate that such mechanical stress and chromosome shattering can also occur in chromosomes within micronuclei or asynchronous multinucleate cells when primary nuclei enter mitosis. Following an aberrant mitosis, chromosomes could find themselves in the wrong place at the wrong time so that they may undergo massive DNA breakage and rearrangement in a single catastrophic event. Specifically, our results support the hypothesis that premature chromosome condensation induces mechanical stress and triggers shattering and chromothripsis in chromosomes or chromosome arms still undergoing DNA replication or repair in micronuclei or asynchronous multinucleate cells, when primary nuclei enter mitosis. PMID:26520389

  20. A simplified chromatin dispersion (nuclear halo) assay for detecting DNA breakage induced by ionizing radiation and chemical agents.

    PubMed

    Galaz-Leiva, S; Pérez-Rodríguez, G; Blázquez-Castro, A; Stockert, J C

    2012-04-01

    Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H(2)O(2). After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H(2)O(2) concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested. PMID:21916782

  1. 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...

  2. The effects of sodium and magnesium-ion interactions on chromatin structure and solubility.

    PubMed

    Dixon, D K; Burkholder, G D

    1985-03-01

    The effects of sodium and magnesium-ion interactions on chromatin structure and solubility were examined in isolated mouse liver nuclei. To facilitate this study, a simple assay of chromatin structure was developed, based on the absorbances at 260 nm (A260) and 320 nm (A320) of nuclei in test solutions. By subtracting the A320 from the A260, a single "spectral index" was obtained which served as a useful, but not absolute, indicator of chromatin structure. Electron microscopy verified the validity of this approach. The results indicate that either 200 mM NaCl or 0.5 mM MgCl2 were capable of preserving the native 20 to 30 nm chromatin fiber structure. Below 200 mM NaCl, the native fiber progressively uncoiled to the 10 nm unit fiber. The presence of 0.5 mM MgCl2 inhibited this uncoiling. Only divalent cations stabilized condensed chromatin (heterochromatin) within the nucleus. Monovalent and divalent cations interacted with one another at critical concentrations and modified their individual effects on chromatin structure; e.g., 10 to 25 mM NaCl interfered with the action of 0.5 to 1.5 mM MgCl2, causing a complete loss of condensed chromatin. Maximum solubility of micrococcal nuclease-digested chromatin occurred at 10 mM NaCl, which treatment allowed the chromatin to unfold to the 10 nm fiber. However, ionic conditions that disrupted condensed chromatin but maintained the native chromatin fiber morphology still resulted in relatively high yields of soluble chromatin. Minimum solubility occurred under conditions which preserved the structure of condensed chromatin. PMID:3996434

  3. Assessing the applicability of FISH-based prematurely condensed dicentric chromosome assay in triage biodosimetry.

    PubMed

    Suto, Yumiko; Gotoh, Takaya; Noda, Takashi; Akiyama, Miho; Owaki, Makiko; Darroudi, Firouz; Hirai, Momoki

    2015-03-01

    The dicentric chromosome assay (DCA) has been regarded as the gold standard of radiation biodosimetry. The assay, however, requires a 2-d peripheral blood lymphocyte culture before starting metaphase chromosome analyses to estimate biological doses. Other biological assays also have drawbacks with respect to the time needed to obtain dose estimates for rapid decision on the correct line of medical treatment. Therefore, alternative technologies that suit requirements for triage biodosimetry are needed. Radiation-induced DNA double strand breaks in G0 lymphocytes can be detected as interphase chromosome aberrations by the cell fusion-mediated premature chromosome condensation (PCC) method. The method, in combination with fluorescence in situ hybridization (FISH) techniques, has been proposed in early studies as a powerful tool for obtaining biological dose estimates without 2-d lymphocyte culture procedures. The present work assesses the applicability of FISH-based PCC techniques using pan-centromeric and telomeric peptide nucleic acid (PNA) probes in triage mode biodosimetry and demonstrates that an improved rapid procedure of the prematurely condensed dicentric chromosome (PCDC) assay has the potential for evaluating exposed radiation doses in as short as 6 h after the collection of peripheral blood specimens. PMID:25627950

  4. Validation of a field based chromatin dispersion assay to assess sperm DNA fragmentation in the bottlenose dolphin (Tursiops truncatus).

    PubMed

    Sánchez-Calabuig, M-J; López-Fernández, C; Martínez-Nevado, E; Pérez-Gutiérrez, J F; de la Fuente, J; Johnston, S D; Blyde, D; Harrison, K; Gosálvez, J

    2014-10-01

    Over the last two decades, there have been significant advances in the use of assisted reproductive technology for genetic and reproductive management of captive dolphin populations, including evaluation of sperm DNA quality. This study validated a customized sperm chromatin dispersion test (SCDt) for the bottlenose dolphin (Tursiops truncatus) as a means of assessing sperm DNA damage both in the field and in the laboratory. After performing the SCDt, two different sperm morphotypes were identified: (i) sperm with fragmented DNA showed large haloes of dispersed DNA fragments emerging from a compact sperm nucleoid core and (ii) sperm containing non-fragmented DNA displayed small compact haloes surrounded by a dense core of non-dispersed DNA and protein complex. Estimates of sperm DNA fragmentation by means of SCDt were directly comparable to results obtained following a two-tailed comet assay and showed a significant degree of correlation (r = 0.961; p < 0.001). This investigation also revealed that the SCDt, with minor modifications to the standard protocol, can be successfully conducted in the field using a LED florescence microscopy obtaining a high correlation (r = 0.993; p = 0.01) between the data obtained in the laboratory and in the field. PMID:25130370

  5. 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.

  6. Premature chromosome condensation assay for biodosimetry: studies with fission-neutrons.

    PubMed

    Prasanna, P G; Kolanko, C J; Gerstenberg, H M; Blakely, W F

    1997-04-01

    Characterization of the premature chromosome condensation assay for radiation quality is needed. To that end, human lymphocytes were exposed in vitro to various doses of 250-kVp x rays (Y(D) = 4 keV microm(-1), Y(D) is the dose-mean lineal energy of the absorbed dose distribution, D(y), where y is defined as the energy deposited in a volume by a single event divided by the mean chord length of the volume) and to fission neutrons (Y(D) = 65 keV microm(-1)). The distribution of prematurely condensed chromosome and fragments following exposure to x rays or to neutrons were non-Poisson after repair at 37 degrees C for 24 h. Dose-response curves were constructed for the yield of excess prematurely condensed chromosome fragments as necessary for biodosimetry applications. The curves were fitted to a weighted linear model by the least-squares regression method. The neutron relative biological effectiveness (RBE) value was estimated to be 2.4 +/- 0.39. PMID:9119684

  7. LONG-TERM EFFECTS OF TRIETHYLENEMELAMINE EXPOSURE ON MOUSE TESTIS CELLS AND SPERM CHROMATIN STRUCTURE ASSAYED BY FLOW CYTOMETRY

    EPA Science Inventory

    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. he first experiment examined effects of fo...

  8. 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

  9. Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA[reg])

    SciTech Connect

    Evenson, Donald P. . E-mail: scsa@brookings.net; Wixon, Regina

    2005-09-01

    Studies over the past two decades have clearly shown that reproductive toxicants cause sperm DNA fragmentation. This DNA fragmentation can usually be detected prior to observing alterations of metaphase chromosomes in embryos. Thus, Sperm Chromatin Structure Assay (SCSA)-detected DNA damage is viewed as the molecular precursor to later gross chromosome damage observed under the light microscope. SCSA measurements of animal or human sperm consist of first obtaining a fresh or flash frozen neat semen sample in LN2 or dry ice. Samples are then sent to a SCSA diagnostic laboratory where the samples are thawed, diluted to {approx}1-2 x 106 sperm/ml, treated for 30 s with a pH 1.2 detergent buffer and then stained with acridine orange (AO). The low pH partially denatures DNA at the sites of DNA strand breaks and the AO-ssDNA fluoresces red while the AO-dsDNA fluoresces green. Flow cytometry measurements of 5000 sperm/sample provide statistically robust data on the ratio of red to green sperm, the extent of the DNA fragmentation and the standard deviations of measures. Numerous experiments on rodents treated with reproductive toxicants clearly showed that SCSA measures are highly dose responsive and have a very low CV. Different agents that act on germ cells at various stages of development usually showed sperm DNA fragmentation when that germ cell fraction arrived in the epididymis or ejaculate. Some of these treated samples were capable of successful in vitro fertilization but with frequent embryo failure. A 2-year longitudinal study of men living a valley town with a reported abnormal level of infertility and spontaneous miscarriages and also a seasonal atmospheric smog pollution, showed, for the first time, that SCSA measurements of human sperm DNA fragmentation were detectable and correlated with dosage of air pollution while the classical semen measures were not correlated. Also, young men spraying pesticides without protective gear are at an increased risk for elevated sperm DNA fragmentation. Extensive DNA fragmentation probably cannot be repaired by the egg and the spontaneous abortion rate is {approx}2x higher if a man has more than 30% of sperm showing DNA fragmentation. DNA fragmentation is an excellent marker for exposure to potential reproductive toxicants and a diagnostic/prognostic tool for potential male infertility.

  10. Phenotypic and metabolic aspects of prostatic epithelial cells in aged gerbils after antisteroidal therapy: turnover in the state of chromatin condensation and androgen-independent cell replacement.

    PubMed

    Campos, Silvana G P; Gonçalves, Bianca F; Scarano, Wellerson R; Góes, Rejane M; Taboga, Sebastião R

    2014-01-01

    The gerbil is a rodent considered a good model for studies of prostatic morphophysiology under different experimental conditions. Studies involving castration and steroidal blockers of aged gerbils showed that the glandular epithelium persists after long-term therapy, preventing the organ atrophy. Thus, the objective of this study was to evaluate the phenotypic characteristics and behavior of prostatic epithelial cells that remained after different periods of hormone ablation in aged gerbils. The identification of elements that influenced the survival of this cell type was performed by morphometric, nuclear phenotypes, ultrastructural and immune histochemical analysis. The most significant responses to treatment, by analyzing morphometric features, were observed during the first three time points (day 1, day 3, and day 7), after which there appeared to be an adjustment of the gland to the hormone ablation. All treatments led to changes in the state of chromatin condensation, DNA methylation pattern and phenotypic changes indicated cell senescence. Additionally, an increase in the basal cells seemed to guarantee self-renewal properties to the epithelium. These data indicate that changes occur at many levels, including gene expression and nuclear architecture in the epithelial cells, when aging and steroidal blockade are associated. These aspects are important when considering castration-resistant prostate cancer, a malignant tumor posing difficult therapeutic intervention. PMID:23942056

  11. Acetone enhances the direct analysis of total condensed tannins in plant tissues by the butanol-HCl-iron assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The butanol-HCl spectrophotometric assay is widely used to quantify extractable and insoluble forms of condensed tannin (CT, syn. proanthocyanidin) in foods, feeds, and foliage of herbaceous and woody plants. However, this method underestimates total CT content when applied directly to plant materia...

  12. Evaluation of Direct Phloroglucinolysis and Colorimetric Depolymerization Assays and Their Applicability for Determining Condensed Tannins in Grape Marc.

    PubMed

    Hixson, Josh L; Bindon, Keren A; Smith, Paul A

    2015-11-18

    To determine the optimum methods for determining condensed tannin (CT) content in grape marc, butanol-hydrochloric acid assays and phloroglucinolysis were adapted for use, applied to a range of grape marc types, and the methods compared. Porter's assay (butanol-HCl) was found to give unreliable results due to nonlinear color responses to grape skin and seed tannin concentrations, whereas the modification to include acetone (Grabber's assay) overcame this. Differences between skin and seed tannin responses highlighted the need to adequately select the correct grape tannin standard, and the formation of pH-dependent color was accounted for through acidification of blank samples. For phloroglucinolysis, the inability to remove highly bound tannins from cell wall material was highlighted, although a measure of tannins remaining post-phloroglucinolysis (Grabber's assay) showed a trend with the level of exposure to oxidative storage or processing conditions. The comparison of CT concentrations from phloroglucinolysis and Grabber's assay gave poor correlation coefficients. PMID:26551987

  13. 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

  14. 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.

  15. Germline chromatin.

    PubMed Central

    Schaner, Christine E; Kelly, William G

    2006-01-01

    The DNA in eukaryotes is wrapped around a histone octamer core, together comprising the main subunit of chromatin, the nucleosome. Modifications of the nucleosomal histones in the genome correlate with the ability or inability of chromatin to form higher order structures, that in turn influence gene activity. The genome in primordial germ cells in early C. elegans germ cells carries a unique pattern of histone modifications that correlate with transcriptional repression in these cells, and aspects of this chromatin regulation are conserved in Drosophila. Loss of repression causes sterility in the adults, suggesting that chromatin-based repression is essential for germ line maintenance. The post-embryonic germ line also exhibits unique and dynamic aspects of chromatin regulation, with chromosome-wide regulation particularly evident on the X chromosome. Several properties of X-specific chromatin assembly are also sex-specific. These properties appear to be responding to the meiotic pairing status of the X chromosome, rather than the sex of the germ cells. Finally, gamete-specific chromatin regulation during gametogenesis impacts on X chromatin assembly in the offspring, leading to an apparent sperm-imprinted X inactivation in the early embryo. Other potential roles for germline-specific modes of chromatin assembly in genome regulation and protection are discussed. PMID:18050477

  16. Chromatin computation.

    PubMed

    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

  17. 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

  18. Chromatin Hydrodynamics

    PubMed Central

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

    2014-01-01

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

  19. 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

  20. Acetone enhances the direct analysis of Procyanidin- and Prodelphinidin-based condensed tannins in lotus species by the butanol-HCl-iron assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The butanol-HCl spectrophotometric assay is widely used for quantifying extractable and insoluble condensed tannins (CT, syn. proanthocyanidins) in foods, feeds, and foliage of herbaceous and woody plants, but the method underestimates total CT content when applied directly to plant material. To imp...

  1. 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.

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

    PubMed

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

    1998-01-01

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

  3. DNA methylation affects nuclear organization, histone modifications, and linker histone binding but not chromatin compaction.

    PubMed

    Gilbert, Nick; Thomson, Inga; Boyle, Shelagh; Allan, James; Ramsahoye, Bernard; Bickmore, Wendy A

    2007-05-01

    DNA methylation has been implicated in chromatin condensation and nuclear organization, especially at sites of constitutive heterochromatin. How this is mediated has not been clear. In this study, using mutant mouse embryonic stem cells completely lacking in DNA methylation, we show that DNA methylation affects nuclear organization and nucleosome structure but not chromatin compaction. In the absence of DNA methylation, there is increased nuclear clustering of pericentric heterochromatin and extensive changes in primary chromatin structure. Global levels of histone H3 methylation and acetylation are altered, and there is a decrease in the mobility of linker histones. However, the compaction of both bulk chromatin and heterochromatin, as assayed by nuclease digestion and sucrose gradient sedimentation, is unaltered by the loss of DNA methylation. This study shows how the complete loss of a major epigenetic mark can have an impact on unexpected levels of chromatin structure and nuclear organization and provides evidence for a novel link between DNA methylation and linker histones in the regulation of chromatin structure. PMID:17485486

  4. Sensitive and selective chemiluminescence assay for hydrogen peroxide in exhaled breath condensate using nanoparticle-based catalysis

    NASA Astrophysics Data System (ADS)

    Li, Xiaohua; Zhang, Zhujun; Tao, Liang; Gao, Miao

    2013-04-01

    The catalytic properties of cubiform Co3O4 nanoparticles, α-Fe2O3 nanorods, and NiO nanoparticles were studied using both microarray method and FI-CL method. These nanoarticles exhibit high specific catalytic effects on the chemiluminescence (CL) reaction of the luminol-H2O2 system in alkaline solution compared with other common catalysts. A reaction mechanism is described. It provides new insights into the application of nanoparticle materials. The CL method based on the use of the Co3O4 nanoparticles is ultrasensitive and particularly selective. Therefore, it was applied to the analysis of H2O2 which can be determined in the concentration range from 1.0 nM to 1000 nM, with a detection limit of 0.3 nM. The relative standard deviation is 2.1% at 0.1 μM of H2O2 (for n = 11). The method was successfully applied to the determination of trace quantities of H2O2 in exhaled breath condensate (EBC) where it is a mediator of oxidative stress and a promising biomarker for diagnosing. The assay requires a small sample and no incubation time, and has an analytical runtime of <1 min. It is timesaving and suitable for larger studies. The levels of H2O2 in EBC are found to be elevated in healthy subjects (average = 0.54 nM), rheum subjects (average = 0.24 nM), and feverish subjects (average = 0.16 nM). Our data suggested that the average H2O2 concentration of EBC from feverish subjects was significantly higher than healthy subjects and rheumatic subjects.

  5. 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

  6. MUTAGENICITY OF THE FRACTIONATED ORGANIC EMISSIONS FROM DIESEL, CIGARETTE SMOKE CONDENSATE, COKE OVEN, AND ROOFING TAR IN THE AMES ASSAY

    EPA Science Inventory

    Mobile and stationary sources emit particle-bound organics that have demonstrated mutagenicity. The objective of this study was to measure the mutagenicity of the fractionated organic emissions from diesel, cigarette smoke condensate (CSC), coke oven and roofing tar in the Ames a...

  7. Acetone Enhances the Direct Analysis of Total Condensed Tannins in Forage Legumes by the Butanol-HCl Assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Depending on concentration, condensed tannins (CT) in forages have no effect, enhance, or impede protein utilization and performance of ruminants. Defining optimal forage CT levels has been elusive, partly because current methods for estimating total soluble plus insoluble CT are laborious or inaccu...

  8. 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

  9. 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

  10. 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

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

    PubMed

    Castillo, Judit; Estanyol, Josep Maria; Ballescá, 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

  12. Chromatin enrichment for proteomics

    PubMed Central

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

    2015-01-01

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

  13. Bull sperm head morphometry related to abnormal chromatin structure and fertility.

    PubMed

    Sailer, B L; Jost, L K; Evenson, D P

    1996-06-01

    This study investigated the relationship between morphologically abnormal sperm, sperm chromatin structure, and fertility. Semen samples were obtained from 13 bulls. The sperm chromatin structure assay (SCSA), a sensitive measure of denaturability of sperm nuclear DNA in situ following acid treatment, was performed on each sample to quantitate abnormal chromatin structure. Feulgenstained sperm head morphology was measured by computerized image analysis (ONCOR-Image): Sixteen parameters were measured for each of 200 nuclei per sample. Fertility estimates were available for nine of the bulls. The SCSA variable SD alpha t was correlated with fertility ranking (r = 0.617, P < 0.01). No correlations were seen between means of the imaging variables and SCSA variables % COMP alpha t or SD alpha t. Significant correlations (P < 0.05) were seen between SD alpha t and the variation of imaging variables eccentricity, width, and light blobs. Significant correlations (P < 0.05) were seen between % COMP alpha t and the variation of imaging variables area, perimeter, p2a, bending energy, nmac, sphericity, eccentricity, length, and width. A regression model for fertility rankings incorporating the standard deviation of the imaging variables area, bending energy, nmac, eccentricity, condensity, light blobs, and dark blobs was highly significant (r2 = 0.999, P < 0.05). These results indicate that variation of morphometry measurements is likely a sensitive biomarker related to fertility potential and abnormal chromatin structure. PMID:8725666

  14. Application of the premature chromosome condensation assay in simulated partial-body radiation exposures: evaluation of the use of an automated metaphase-finder.

    PubMed

    Blakely, W F; Prasanna, P G; Kolanko, C J; Pyle, M D; Mosbrook, D M; Loats, A S; Rippeon, T L; Loats, H

    1995-05-01

    The premature chromosome condensation (PCC) assay has been proposed as a useful and rapid end point for biological dosimetry following accidental high-dose radiation overexposures. A major benefit of the PCC assay is that it does not require cells to divide for evaluation of cytogenetic damage. The PCC assay was performed on isolated human peripheral lymphocytes exposed in vitro to doses from 1 to 9 Gy of 250 kVp x-rays. The dose-response relationships of the frequency distribution and the yield of PCC fragments in cells were determined after one day of repair at 37 degrees C. A Qpcc approach, which involves the analysis of the yield of excess PCC fragments in damaged cells, was used to establish a dose-response calibration curve. This method is identical in concept to the Qdr technique introduced by Sasaki for partial-body exposure dose-estimates using asymmetrical chromosome aberrations (i.e., dicentrics and rings) in metaphase spreads of human lymphocytes. A simulated in vitro test of a partial-body exposure to a 6-Gy dose was performed. The results from this test provided dose estimates of 5.3 +/- 0.6, 4.7 +/- 0.6, 5.0 +/- 0.6 and 4.7 +/- 0.8 Gy for the 20, 30, 50 and 75 percent component of 6-Gy irradiated cells, respectively. An automated metaphase-finding system was evaluated for use with the PCC assay. This system helped to locate PCC spreads among the mitotic inducer Chinese hamster ovary (CHO) metaphase spreads, thereby facilitating rapid scoring of samples.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7488950

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

    PubMed

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

    2015-12-24

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

  16. In Vivo Chromatin Organization of Mouse Rod Photoreceptors Correlates with Histone Modifications

    PubMed Central

    Kizilyaprak, Caroline; Spehner, Danièle; Devys, Didier; Schultz, Patrick

    2010-01-01

    Background The folding of genetic information into chromatin plays important regulatory roles in many nuclear processes and particularly in gene transcription. Post translational histone modifications are associated with specific chromatin condensation states and with distinct transcriptional activities. The peculiar chromatin organization of rod photoreceptor nuclei, with a large central domain of condensed chromatin surrounded by a thin border of extended chromatin was used as a model to correlate in vivo chromatin structure, histone modifications and transcriptional activity. Methodology We investigated the functional relationships between chromatin compaction, distribution of histone modifications and location of RNA polymerase II in intact murine rod photoreceptors using cryo-preparation methods, electron tomography and immunogold labeling. Our results show that the characteristic central heterochromatin of rod nuclei is organized into concentric domains characterized by a progressive loosening of the chromatin architecture from inside towards outside and by specific combinations of silencing histone marks. The peripheral heterochromatin is formed by closely packed 30nm fibers as revealed by a characteristic optical diffraction signal. Unexpectedly, the still highly condensed most external heterochromatin domain contains acetylated histones, which are usually associated with active transcription and decondensed chromatin. Histone acetylation is thus not sufficient in vivo for complete chromatin decondensation. The euchromatin domain contains several degrees of chromatin compaction and the histone tails are hyperacetylated, enriched in H3K4 monomethylation and hypo trimethylated on H3K9, H3K27 and H4K20. The transcriptionally active RNA polymerases II molecules are confined in the euchromatin domain and are preferentially located at the vicinity of the interface with heterochromatin. Conclusions Our results show that transcription is located in the most decondensed and highly acetylated chromatin regions, but since acetylation is found associated with compact chromatin it is not sufficient to decondense chromatin in vivo. We also show that a combination of histone marks defines distinct concentric heterochromatin domains. PMID:20543957

  17. 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 after IR and {sup 111}In-DTPA-hEGF compared to controls but did not significantly alter clonogenic survival. Conclusions: Chromatin structure affects DNA damage and cell survival after exposure to Auger electron radiation.

  18. 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,…

  19. 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

  20. Chromatin deregulation in disease.

    PubMed

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

    2016-03-01

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

  1. CTCF Binding Polarity Determines Chromatin Looping.

    PubMed

    de Wit, Elzo; Vos, Erica S M; Holwerda, Sjoerd J B; Valdes-Quezada, Christian; Verstegen, Marjon J A M; Teunissen, Hans; Splinter, Erik; Wijchers, Patrick J; Krijger, Peter H L; de Laat, Wouter

    2015-11-19

    CCCTC-binding factor (CTCF) is an architectural protein involved in the three-dimensional (3D) organization of chromatin. In this study, we assayed the 3D genomic contact profiles of a large number of CTCF binding sites with high-resolution 4C-seq. As recently reported, our data also suggest that chromatin loops preferentially form between CTCF binding sites oriented in a convergent manner. To directly test this, we used CRISPR/Cas9 genome editing to delete core CTCF binding sites in three loci, including the CTCF site in the Sox2 super-enhancer. In all instances, CTCF and cohesin recruitment were lost, and chromatin loops with distal, convergent CTCF sites were disrupted or destabilized. Re-insertion of oppositely oriented CTCF recognition sequences restored CTCF and cohesin recruitment, but did not re-establish chromatin loops. We conclude that CTCF binding polarity plays a functional role in the formation of higher-order chromatin structure. PMID:26527277

  2. Aurora B kinase maintains chromatin organization during the MI to MII transition in surf clam oocytes.

    PubMed

    George, Olivia; Johnston, Mantissa A; Shuster, Charles B

    2006-11-01

    Meiosis represents a specialized cell cycle whereby cells undergo two reductive divisions without an intervening S phase. In oocytes, the transition from meiosis I to II is brief, with paired sister chromatids remaining condensed throughout the interkinesis period. This stands in contrast to mitotic divisions where cytokinesis and the return to interphase is always accompanied by chromatin decondensation and nuclear envelope reformation. Because other aspects of M phase exit are normal, we probed the mechanisms that allow for polar body extrusion while retaining chromatin condensation in Spisula solidissima oocytes. If oocytes were activated in the presence of protein synthesis inhibitors, oocytes progressed normally through MI, but arrested in interkinesis with condensed chromatin, phosphorylated histone H3 and a disorganized MII spindle. Neither inhibition of CDK1- nor MAPK activity in arrested oocytes was sufficient to drive chromatin decondensation or nuclear envelope reformation, suggesting that these kinases were not responsible for the maintenance of chromatin condensation. However, inhibition of Aurora B kinase activity resulted in chromatin decondensation, loss of histone H3 phosphorylation and reformation of the nuclear envelope. Inhibition of Aurora B activity following MI also resulted in chromosome segregation defects during MII and blocked polar body formation, consistent with Aurora B's well-established role in cytokinesis. Together, these results suggest that extended Aurora B activity between meiotic divisions maintains chromatin condensation, thus allowing for the rapid reassembly of the MII spindle and progression through meiosis. PMID:17172833

  3. Direct Measurement of Local Chromatin Fluidity Using Optical Trap Modulation Force Spectroscopy

    PubMed Central

    Roopa, T.; Shivashankar, G. V.

    2006-01-01

    Chromatin assembly is condensed by histone tail-tail interactions and other nuclear proteins into a highly compact structure. Using an optical trap modulation force spectroscopy, we probe the effect of tail interactions on local chromatin fluidity. Chromatin fibers, purified from mammalian cells, are tethered between a microscope coverslip and a glass micropipette. Mechanical unzipping of tail interactions, using the micropipette, lead to the enhancement of local fluidity. This is measured using an intensity-modulated optically trapped bead positioned as a force sensor on the chromatin fiber. Enzymatic digestion of the histone tail interactions of tethered chromatin fiber also leads to a similar increase in fluidity. Our experiments show that an initial increase in the local fluidity precedes chromatin decompaction, suggesting possible mechanisms by which chromatin-remodeling machines access regulatory sites. PMID:17012315

  4. 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. PMID:12888554

  5. Chromatin remodelling during male gametophyte development.

    PubMed

    Borg, Michael; Berger, Frédéric

    2015-07-01

    The plant life cycle alternates between a diploid sporophytic phase and haploid gametophytic phase, with the latter giving rise to the gametes. Male gametophyte development encompasses two mitotic divisions that results in a simple three-celled structure knows as the pollen grain, in which two sperm cells are encased within a larger vegetative cell. Both cell types exhibit a very different type of chromatin organization - highly condensed in sperm cell nuclei and highly diffuse in the vegetative cell. Distinct classes of histone variants have dynamic and differential expression in the two cell lineages of the male gametophyte. Here we review how the dynamics of histone variants are linked to reprogramming of chromatin activities in the male gametophyte, compaction of the sperm cell genome and zygotic transitions post-fertilization. PMID:25892182

  6. Mitotic checkpoint control and chromatin remodeling.

    PubMed

    Yao, Yixin; Dai, Wei

    2012-01-01

    In order to maintain chromosomal stability during cell division, eukaryotic cells have evolved a number of surveillance mechanisms termed checkpoints. These checkpoints monitor the completion of essential molecular and cellular processes of one stage before entering another. The spindle checkpoint watches the bi-orientation attachment of spindle microtubules to all condensed chromosomes before initiation of nuclear division during mitosis. Histones are subject to a number of post-translational modifications during the cell cycle, which may in turn modify or facilitate cell cycle progression. Recent studies suggest that mitotic proteins including Bub1 and Sgo1 that are involved in the spindle checkpoint also play a major role in the regulation of histone modifications and chromatin remodeling. This mini-review summarizes emerging information about the new role of spindle checkpoint proteins in chromatin remodeling. PMID:22201785

  7. 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

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

    PubMed Central

    Cognetti, G; Shaw, B R

    1981-01-01

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

  9. Chromatin immunoprecipitation using microarrays.

    PubMed

    Durand-Dubief, Mickaël; Ekwall, Karl

    2009-01-01

    Chromatin immunoprecipitation (ChIP) is a powerful procedure to investigate the interactions between proteins and DNA. ChIP-chip combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in vivo. This genome-wide analysis of protein-DNA association is carried out in several steps including chemical cross-linking, cell lysis, DNA fragmentation and immunoaffinity purification that allow the identification of DNA interactions and provide a powerful tool for genome-wide investigations. Immunoprecipitated DNA fragments associated with the desired protein are amplified, labelled and hybridized to DNA microarrays to detect enriched signals compared to a labelled reference sample. PMID:19381973

  10. Epigenomics and chromatin dynamics

    PubMed Central

    2012-01-01

    A report of the 'Joint Keystone Symposium on Epigenomics and Chromatin Dynamics', Keystone, Colorado, 17-22 January 2012. This year's Joint Keystone Symposium on Epigenomics and Chromatin Dynamics was one of the largest Keystone meetings to date, reflecting the excitement and many developments in this area. Richard Young opened the meeting by giving a historic overview before sharing more detailed insights from his recent work in describing the role of the lysine demethylase Lsd1 in mouse embryonic stem (ES) cell differentiation. He also set the broader stage and highlighted the excitement concerning recent advances in epigenetic drugs such as the new bromodomain inhibitors. PMID:22364154

  11. Chromatin organization during spermiogenesis in Octopus vulgaris. I: Morphological structures.

    PubMed

    Ribes, Enric; Giménez-Bonafé, Pepita; Martínez-Soler, Fina; Gonzalez, Angel; Saperas, Núria; Kasinsky, Harold E; Chiva, Manel

    2004-06-01

    In the process of the chromatin remodeling that occurs during spermiogenesis in some animal species, it is possible to distinguish between two separate aspects: the chromatin condensation pattern itself (granular, fibrillar, or lamellar), and the architecture of this pattern, that is to say, its arrangement within the nucleus. In the cephalopod Octopus vulgaris these two aspects are clearly differentiated. The condensation pattern develops from 25 nm fibers to fibers with a tubular aspect and with a progressively increasing diameter (40-60 nm and then to 80 nm), to end finally in the form of very thin fibers (3-5 nm) product of the coalescence and dissolution of the major fibers. The main directive force that governs this process lies in the global change that occurs in the proteins that interact with all (or the major part) of the genomic DNA. The condensation pattern by itself in this species does not present a fixed order: most of the fibers appear without any predominant spatial direction in the spermiogenic nuclei. However, as the nuclei elongate, the chromatin fibers arrange in parallel following the elongation axis. This parallel disposition of the chromatin fibers appears to be mediated by two specific areas, each of which we call a "polar nuclear matrix" (PNM). These matrices differentiate in the basal and apical nuclear poles adjacent to the centriolar implantation fosse and the acrosome, respectively. The areas that constitute the PNM have the following characteristics: (a) they are the only areas where DNA is found anchored to the nuclear membrane; (b) they are the zones from which the chromatin condensation pattern (fibers/tubules) begins; and (c) they are most probably the points through which the mechanical forces originating from nuclear elongation are transmitted to chromatin, causing the chromatin fibers/tubules to adopt an almost perfectly parallel disposition. Finally, we discuss the importance of the architecture of the chromatin condensation pattern, as it is one of the determining factors of the spatial organization of the mature sperm genome and chromosome positioning. PMID:15095344

  12. 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,…

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

    PubMed Central

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

    2015-01-01

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

  14. Nucleosome Shape Dictates Chromatin Fiber Structure

    PubMed Central

    Depken, Martin; Schiessel, Helmut

    2009-01-01

    In addition to being the gateway for all access to the eukaryotic genome, chromatin has in recent years been identified as carrying an epigenetic code regulating transcriptional activity. Though much is known about the biochemistry of this code, little is understood regarding the different fiber structures through which the regulation is mediated. Over the last three decades many fiber models have been suggested, but none are able to predict even the basic characteristics of the fiber. In this work, we characterize the set of all possible dense fibers, which includes, but is not limited to, all previously suggested structures. To guide future experimental efforts, we show which fiber characteristics depend on the underlying structure and, crucially, which do not. Addressing the predictive power of these models, we suggest a simple geometric criterion based on the nucleosome shape alone. This enables us to predict the observed characteristics of the condensed chromatin fiber, and how these change with varying nucleosome repeat length. Our approach sheds light on how the in vivo observed heterogeneity in linker lengths can be accommodated within the 30 nm fiber, and suggest an important role for nucleosome surface interactions in the regulation of chromatin structure and function. PMID:19186120

  15. Chromatin fiber allostery and the epigenetic code

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  16. Facilitation of base excision repair by chromatin remodeling.

    PubMed

    Hinz, John M; Czaja, Wioletta

    2015-12-01

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

  17. 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. PMID:21289049

  18. Alternative Lengthening of Telomeres is characterized by reduced compaction of telomeric chromatin

    PubMed Central

    Episkopou, Harikleia; Draskovic, Irena; Van Beneden, Amandine; Tilman, Gaëlle; Mattiussi, Marina; Gobin, Matthieu; Arnoult, Nausica; Londoño-Vallejo, Arturo; Decottignies, Anabelle

    2014-01-01

    Proper telomeric chromatin configuration is thought to be essential for telomere homeostasis and stability. Previous studies in mouse suggested that loss of heterochromatin marks at telomeres might favor onset of Alternative Lengthening of Telomeres (ALT) pathway, by promoting homologous recombination. However, analysis of chromatin status at human ALT telomeres has never been reported. Here, using isogenic human cell lines and cellular hybrids, which rely either on telomerase or ALT to maintain telomeres, we show that chromatin compaction is reduced at ALT telomeres and this is associated with a global decrease in telomeric H3K9me3. This, subsequently, leads to upregulation of telomere transcription. Accordingly, restoration of a more condensed telomeric chromatin through telomerase-dependent elongation of short ALT telomeres reduces telomere transcription. We further show that loss of ATRX chromatin remodeler function, a frequent characteristic of ALT cells, is not sufficient to decrease chromatin condensation at telomeres nor to increase the expression of telomeric RNA species. These results offer new insight on telomeric chromatin properties in ALT cells and support the hypothesis that telomeric chromatin decondensation is important for ALT pathway. PMID:24500201

  19. Mapping chromatin modifications in nanochannels

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

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

    PubMed

    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 IE1-CTD specifically interacts with the H2A-H2B acidic patch and impairs the compaction of higher-order chromatin structure. Our results suggest that IE1 loosens up the folding of host chromatin during hCMV infections. PMID:26812545

  1. 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 IE1-CTD specifically interacts with the H2A-H2B acidic patch and 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

  2. 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

  3. 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. PMID:16790270

  4. How chromatin is remodelled during DNA repair of UV-induced DNA damage in Saccharomyces cerevisiae.

    PubMed

    Yu, Shirong; Teng, Yumin; Waters, Raymond; Reed, Simon H

    2011-06-01

    Global genome nucleotide excision repair removes DNA damage from transcriptionally silent regions of the genome. Relatively little is known about the molecular events that initiate and regulate this process in the context of chromatin. We've shown that, in response to UV radiation-induced DNA damage, increased histone H3 acetylation at lysine 9 and 14 correlates with changes in chromatin structure, and these alterations are associated with efficient global genome nucleotide excision repair in yeast. These changes depend on the presence of the Rad16 protein. Remarkably, constitutive hyperacetylation of histone H3 can suppress the requirement for Rad7 and Rad16, two components of a global genome repair complex, during repair. This reveals the connection between histone H3 acetylation and DNA repair. Here, we investigate how chromatin structure is modified following UV irradiation to facilitate DNA repair in yeast. Using a combination of chromatin immunoprecipitation to measure histone acetylation levels, histone acetylase occupancy in chromatin, MNase digestion, or restriction enzyme endonuclease accessibility assays to analyse chromatin structure, and finally nucleotide excision repair assays to examine DNA repair, we demonstrate that global genome nucleotide excision repair drives UV-induced chromatin remodelling by controlling histone H3 acetylation levels in chromatin. The concerted action of the ATPase and C3HC4 RING domains of Rad16 combine to regulate the occupancy of the histone acetyl transferase Gcn5 on chromatin in response to UV damage. We conclude that the global genome repair complex in yeast regulates UV-induced histone H3 acetylation by controlling the accessibility of the histone acetyl transferase Gcn5 in chromatin. The resultant changes in histone H3 acetylation promote chromatin remodelling necessary for efficient repair of DNA damage. Recent evidence suggests that GCN5 plays a role in NER in human cells. Our work provides important insight into how GG-NER operates in chromatin. PMID:21698136

  5. How Chromatin Is Remodelled during DNA Repair of UV-Induced DNA Damage in Saccharomyces cerevisiae

    PubMed Central

    Yu, Shirong; Teng, Yumin; Waters, Raymond; Reed, Simon H.

    2011-01-01

    Global genome nucleotide excision repair removes DNA damage from transcriptionally silent regions of the genome. Relatively little is known about the molecular events that initiate and regulate this process in the context of chromatin. We've shown that, in response to UV radiation–induced DNA damage, increased histone H3 acetylation at lysine 9 and 14 correlates with changes in chromatin structure, and these alterations are associated with efficient global genome nucleotide excision repair in yeast. These changes depend on the presence of the Rad16 protein. Remarkably, constitutive hyperacetylation of histone H3 can suppress the requirement for Rad7 and Rad16, two components of a global genome repair complex, during repair. This reveals the connection between histone H3 acetylation and DNA repair. Here, we investigate how chromatin structure is modified following UV irradiation to facilitate DNA repair in yeast. Using a combination of chromatin immunoprecipitation to measure histone acetylation levels, histone acetylase occupancy in chromatin, MNase digestion, or restriction enzyme endonuclease accessibility assays to analyse chromatin structure, and finally nucleotide excision repair assays to examine DNA repair, we demonstrate that global genome nucleotide excision repair drives UV-induced chromatin remodelling by controlling histone H3 acetylation levels in chromatin. The concerted action of the ATPase and C3HC4 RING domains of Rad16 combine to regulate the occupancy of the histone acetyl transferase Gcn5 on chromatin in response to UV damage. We conclude that the global genome repair complex in yeast regulates UV-induced histone H3 acetylation by controlling the accessibility of the histone acetyl transferase Gcn5 in chromatin. The resultant changes in histone H3 acetylation promote chromatin remodelling necessary for efficient repair of DNA damage. Recent evidence suggests that GCN5 plays a role in NER in human cells. Our work provides important insight into how GG-NER operates in chromatin. PMID:21698136

  6. Archaeal chromatin and transcription.

    PubMed

    Reeve, John N

    2003-05-01

    Archaea contain a variety of sequence-independent DNA binding proteins consistent with the evolution of several different, sometimes overlapping and exchangeable solutions to the problem of genome compaction. Some of these proteins undergo residue-specific post-translational lysine acetylation or methylation, hinting at analogues of the histone modifications that regulate eukaryotic chromatin structure and transcription. Archaeal transcription initiation most closely resembles the eukaryotic RNA polymerase II (RNAPII) system, but Archaea do not appear to have homologues of the multisubunit complexes that remodel eukaryotic chromatin and activate RNAPII initiation. In contrast, they have sequence-specific regulators that repress and perhaps activate archaeal transcription by mechanisms superficially similar to the bacterial paradigm of regulating promoter binding by RNAP. Repressors compete with archaeal TATA-box binding protein (TBP) and TFB for the TATA-box and TFB-recognition elements (BRE) of the archaeal promoter, or with archaeal RNAP for the site of transcription initiation. Transcript-specific regulation by repressors binding to sites of transcript initiation is consistent with such sites having very little sequence conservation. However, most Archaea have only one TBP and/or TFB that presumably must therefore bind to similar TATA-box and BRE sequences upstream of most genes. Repressors that function by competing with TBP and/or TFB binding must therefore also make additional contacts with transcript-specific regulatory sites adjacent or remote from the TATA-box/BRE region. The fate of the archaeal TBP and TFB following transcription initiation remains to be determined. Based on functional homology with their eukaryotic RNAPII-system counterparts, archaeal TBP and possibly also TFB should remain bound to the TATA-box/BRE region after transcription initiation. However, this seems unlikely as it might limit repressor competition at this site to only the first round of transcription initiation. PMID:12694606

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

    PubMed

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

    2016-03-01

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

  8. 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. Microsc. Res. Tech. 78:1117-1120, 2015. © 2015 Wiley Periodicals, Inc. PMID:26515508

  9. 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.

  10. 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. PMID:26055943

  11. Chromatin and Transcription in Yeast

    PubMed Central

    Rando, Oliver J.; Winston, Fred

    2012-01-01

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

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

    PubMed

    Postnikov, Yuri V; Bustin, Michael

    2016-03-01

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

  13. 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

  14. DNA Looping Facilitates Targeting of a Chromatin Remodeling Enzyme

    PubMed Central

    Yadon, Adam N; Singh, Badri Nath; Hampsey, Michael; Tsukiyama, Toshio

    2013-01-01

    Summary ATP-dependent chromatin remodeling enzymes are highly abundant and play pivotal roles regulating DNA-dependent processes. The mechanisms by which they are targeted to specific loci have not been well understood on a genome-wide scale. Here we present evidence that a major targeting mechanism for the Isw2 chromatin remodeling enzyme to specific genomic loci is through sequence-specific transcription factor (TF)-dependent recruitment. Unexpectedly, Isw2 is recruited in a TF-dependent fashion to a large number of loci without TF binding sites. Using the 3C assay, we show that Isw2 can be targeted by Ume6- and TFIIB-dependent DNA looping. These results identify DNA looping as a previously unknown mechanism for the recruitment of a chromatin remodeling enzyme and defines a novel function for DNA looping. We also present evidence suggesting that Ume6-dependent DNA looping is involved in chromatin remodeling and transcriptional repression, revealing a mechanism by which the three-dimensional folding of chromatin affects DNA-dependent processes. PMID:23478442

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

    PubMed

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

    2002-12-10

    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 beta-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

  16. Sudemycin E influences alternative splicing and changes chromatin modifications.

    PubMed

    Convertini, Paolo; Shen, Manli; Potter, Philip M; Palacios, Gustavo; Lagisetti, Chandraiah; de la Grange, Pierre; Horbinski, Craig; Fondufe-Mittendorf, Yvonne N; Webb, Thomas R; Stamm, Stefan

    2014-04-01

    Sudemycin E is an analog of the pre-messenger RNA splicing modulator FR901464 and its derivative spliceostatin A. Sudemycin E causes the death of cancer cells through an unknown mechanism. We found that similar to spliceostatin A, sudemycin E binds to the U2 small nuclear ribonucleoprotein (snRNP) component SF3B1. Native chromatin immunoprecipitations showed that U2 snRNPs physically interact with nucleosomes. Sudemycin E induces a dissociation of the U2 snRNPs and decreases their interaction with nucleosomes. To determine the effect on gene expression, we performed genome-wide array analysis. Sudemycin E first causes a rapid change in alternative pre-messenger RNA splicing, which is later followed by changes in overall gene expression and arrest in the G2 phase of the cell cycle. The changes in alternative exon usage correlate with a loss of the H3K36me3 modification in chromatin encoding these exons. We propose that sudemycin E interferes with the ability of U2 snRNP to maintain an H3K36me3 modification in actively transcribed genes. Thus, in addition to the reversible changes in alternative splicing, sudemycin E causes changes in chromatin modifications that result in chromatin condensation, which is a likely contributing factor to cancer cell death. PMID:24623796

  17. 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

  18. The Spectrum of Anti-Chromatin/Nucleosome Autoantibodies: Independent and Interdependent Biomarkers of Disease

    PubMed Central

    Mehra, Sonal; Fritzler, Marvin J.

    2014-01-01

    Autoantibodies directed to chromatin components date back to the discovery of the LE cell and the LE cell phenomenon circa 1950, and subsequent evidence that major components of that reaction were chromatin components and histones in particular. Over time, immunoassays ranging from ELISA and line immunoassays to more modern bead-based assays incorporated histone and DNA mixtures, purified histones, and purified nucleosomes leading to a more thorough understanding of the genesis and pathogenetic relationships of antibodies to chromatin components in systemic lupus erythematosus and other autoimmune conditions. More recently, interest has focussed on other components of chromatin such as high mobility group (HMG) proteins both as targets of B cell responses and pro-inflammatory mediators. This review will focus on immunoassays that utilize chromatin components, their clinical relationships, and newer evidence implicating HMG proteins and DNA neutrophil extracellular traps (NETs) as important players in systemic autoimmune rheumatic diseases. PMID:24804269

  19. Chromatin shapes the mitotic spindle.

    PubMed

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

    2009-08-01

    In animal and plant cells, mitotic chromatin locally generates microtubules that self-organize into a mitotic spindle, and its dimensions and bipolar symmetry are essential for accurate chromosome segregation. By immobilizing microscopic chromatin-coated beads on slide surfaces using a microprinting technique, we have examined the effect of chromatin on the dimensions and symmetry of spindles in Xenopus laevis cytoplasmic extracts. While circular spots with diameters around 14-18 microm trigger bipolar spindle formation, larger spots generate an incorrect number of poles. We also examined lines of chromatin with various dimensions. Their length determined the number of poles that formed, with a 6 x 18 microm rectangular patch generating normal spindle morphology. Around longer lines, multiple poles formed and the structures were disorganized. While lines thinner than 10 mum generated symmetric structures, thicker lines induced the formation of asymmetric structures where all microtubules are on the same side of the line. Our results show that chromatin defines spindle shape and orientation. For a video summary of this article, see the PaperFlick file available with the online Supplemental Data. PMID:19665972

  20. 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

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

    PubMed

    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

  2. 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 HP1? on heterochromatic satellite 2 and ?-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

  3. Effect of the fungal mycotoxin patulin on the chromatin structure of fission yeast Schizosaccharomyces pombe.

    PubMed

    Horvath, Eszter; Nagy, Gabor; Turani, Melinda; Balogh, Eniko; Papp, Gabor; Pollak, Edit; Pocsi, Istvan; Pesti, Miklos; Banfalvi, Gaspar

    2012-12-01

    The fungal mycotoxin patulin is produced by several molds, especially by Aspergillus and Penicillium. The aim of this study was to clarify whether patulin causes alterations in plasma membrane permeability of Schizosaccharomyces pombe lead to cellular shrinkage charateristic to apoptosis or increases cell size indicating necrosis in cells. Transmission and scanning electronmicroscopy revealed that lower concentrations of patulin induced cellular shrinkage and blebbing, higher concentration caused expansion without cellular disruption. Large-scale morphological changes of individual cells were followed by time lapse video microscopy. Patulin caused the elongation and stickiness of cells or rounded up their shapes. To visualize chromatin structures of S. pombe nuclei upon patulin treatment, protoplasts were isolated from S. pombe and subjected to fluorescent microscopy. Chromatin changes in the presence of 50 ?M patulin concentration were characterized by elongated nuclei containing sticky fibrillary chromatin and enlarged round shaped nuclei trapped at the fibrillary stage of chromatin condensation. Short (60 min) incubation of S. pombe cells in the presence of high (500 ?M) patulin concentration generated patches of condensed chromatin bodies inside the nucleus and caused nuclear expansion, with the rest of chromatin remaining in fibrillary form. Longer (90 min, 500 ?M) incubation resulted in fewer highly condensed chromatin patches and in nuclear fragmentation. Although, high patulin concentration increased the size of S. pombe size, it did not lead to necrotic explosion of cells, neither did the fragmented nuclei resemble apoptotic bodies that would have indicated programmed cell death. All these morphological changes and the high rate of cell survival point to rapid adaptation and mixed type of fungistatic effects. PMID:22359238

  4. Snapshots: Chromatin Control of Viral Infection

    PubMed Central

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

    2012-01-01

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

  5. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state.

    PubMed

    Rotem, Assaf; Ram, Oren; Shoresh, Noam; Sperling, Ralph A; Goren, Alon; Weitz, David A; Bernstein, Bradley E

    2015-11-01

    Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell are sparse, comprising on the order of 1,000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of subpopulations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone. PMID:26458175

  6. 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.

  7. Correlated spatio-temporal fluctuations in chromatin compaction states characterize stem cells.

    PubMed

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

    2013-02-01

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

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

    PubMed Central

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

    2013-01-01

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

  9. 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

  10. Prothymosin alpha associates with the oncoprotein SET and is involved in chromatin decondensation.

    PubMed

    Karetsou, Zoe; Martic, Goran; Tavoulari, Sotiria; Christoforidis, Savvas; Wilm, Matthias; Gruss, Claudia; Papamarcaki, Thomais

    2004-11-19

    Prothymosin alpha (ProTalpha) is a histone H1-binding protein that interacts with the transcription coactivator CREB-binding protein and potentiates transcription. Based on coimmunoprecipitation and mammalian two-hybrid assays, we show here that ProTalpha forms a complex with the oncoprotein SET. ProTalpha efficiently decondenses human sperm chromatin, while overexpression of GFP-ProTalpha in mammalian cells results in global chromatin decondensation. These results indicate that decondensation of compacted chromatin fibers is an important step in the mechanism of ProTalpha function. PMID:15556635

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

    PubMed

    Panday, Arvind; Xiao, LiJuan; Grove, Anne

    2015-07-13

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    1981-01-01

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

  14. Quantitative analysis of chromatin compaction in living cells using FLIM–FRET

    PubMed Central

    Llères, David; James, John; Swift, Sam; Norman, David G.

    2009-01-01

    We present a quantitative Förster resonance energy transfer (FRET)–based assay using multiphoton fluorescence lifetime imaging microscopy (FLIM) to measure chromatin compaction at the scale of nucleosomal arrays in live cells. The assay uses a human cell line coexpressing histone H2B tagged to either enhanced green fluorescent protein (FP) or mCherry FPs (HeLaH2B-2FP). FRET occurs between FP-tagged histones on separate nucleosomes and is increased when chromatin compacts. Interphase cells consistently show three populations of chromatin with low, medium, or high FRET efficiency, reflecting spatially distinct regions with different levels of chromatin compaction. Treatment with inhibitors that either increase chromatin compaction (i.e., depletion of adenosine triphosphate) or decrease chromosome compaction (trichostatin A) results in a parallel increase or decrease in the FLIM–FRET signal. In mitosis, the assay showed variation in compaction level, as reflected by different FRET efficiency populations, throughout the length of all chromosomes, increasing to a maximum in late anaphase. These data are consistent with extensive higher order folding of chromatin fibers taking place during anaphase. PMID:19948497

  15. Profiling of the Chromatin-associated Proteome Identifies HP1BP3 as a Novel Regulator of Cell Cycle Progression *

    PubMed Central

    Dutta, Bamaprasad; Ren, Yan; Hao, Piliang; Sim, Kae Hwan; Cheow, Esther; Adav, Sunil; Tam, James P.; Sze, Siu Kwan

    2014-01-01

    The chromatin-associated proteome (chromatome) regulates cellular gene expression by restricting access of transcriptional machinery to template DNA, and dynamic re-modeling of chromatin structure is required to regulate critical cell functions including growth and replication, DNA repair and recombination, and oncogenic transformation in progression to cancer. Central to the control of these processes is efficient regulation of the host cell cycle, which is maintained by rapid changes in chromatin conformation during normal cycle progression. A global overview of chromatin protein organization is therefore essential to fully understand cell cycle regulation, but the influence of the chromatome and chromatin binding topology on host cell cycle progression remains poorly defined. Here we used partial MNase digestion together with iTRAQ-based high-throughput quantitative proteomics to quantify chromatin-associated proteins during interphase progression. We identified a total of 481 proteins with high confidence that were involved in chromatin-dependent events including transcriptional regulation, chromatin re-organization, and DNA replication and repair, whereas the quantitative data revealed the temporal interactions of these proteins with chromatin during interphase progression. When combined with biochemical and functional assays, these data revealed a strikingly dynamic association of protein HP1BP3 with the chromatin complex during different stages of interphase, and uncovered a novel regulatory role for this molecule in transcriptional regulation. We report that HP1BP3 protein maintains heterochromatin integrity during G1–S progression and regulates the duration of G1 phase to critically influence cell proliferative capacity. PMID:24830416

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

    PubMed Central

    Orrego, Mary; Ponte, Imma; Roque, Alicia; Buschati, Natascha; Mora, Xavier; Suau, Pedro

    2007-01-01

    Background Histone H1 is involved in the formation and maintenance of chromatin higher order structure. H1 has multiple isoforms; the subtypes differ in timing of expression, extent of phosphorylation and turnover rate. In vertebrates, the amino acid substitution rates differ among subtypes by almost one order of magnitude, suggesting that each subtype might have acquired a unique function. We have devised a competitive assay to estimate the relative binding affinities of histone H1 mammalian somatic subtypes H1a-e and H1° for long chromatin fragments (30–35 nucleosomes) in physiological salt (0.14 M NaCl) at constant stoichiometry. Results The H1 complement of native chromatin was perturbed by adding an additional amount of one of the subtypes. A certain amount of SAR (scaffold-associated region) DNA was present in the mixture to avoid precipitation of chromatin by excess H1. SAR DNA also provided a set of reference relative affinities, which were needed to estimate the relative affinities of the subtypes for chromatin from the distribution of the subtypes between the SAR and the chromatin. The amounts of chromatin, SAR and additional H1 were adjusted so as to keep the stoichiometry of perturbed chromatin similar to that of native chromatin. H1 molecules freely exchanged between the chromatin and SAR binding sites. In conditions of free exchange, H1a was the subtype of lowest affinity, H1b and H1c had intermediate affinities and H1d, H1e and H1° the highest affinities. Subtype affinities for chromatin differed by up to 19-fold. The relative affinities of the subtypes for chromatin were equivalent to those estimated for a SAR DNA fragment and a pUC19 fragment of similar length. Avian H5 had an affinity ~12-fold higher than H1e for both DNA and chromatin. Conclusion H1 subtypes freely exchange in vitro between chromatin binding sites in physiological salt (0.14 M NaCl). The large differences in relative affinity of the H1 subtypes for chromatin suggest that differential affinity could be functionally relevant and thus contribute to the functional differentiation of the subtypes. The conservation of the relative affinities for SAR and non-SAR DNA, in spite of a strong preference for SAR sequences, indicates that differential affinity alone cannot be responsible for the heterogeneous distribution of some subtypes in cell nuclei. PMID:17498293

  17. 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.

  18. Coming to terms with chromatin structure.

    PubMed

    Even-Faitelson, Liron; Hassan-Zadeh, Vahideh; Baghestani, Zahra; Bazett-Jones, David P

    2016-03-01

    Chromatin, once thought to serve only as a means to package DNA, is now recognized as a major regulator of gene activity. As a result of the wide range of methods used to describe the numerous levels of chromatin organization, the terminology that has emerged to describe these organizational states is often imprecise and sometimes misleading. In this review, we discuss our current understanding of chromatin architecture and propose terms to describe the various biochemical and structural states of chromatin. PMID:26223534

  19. Impact of Chromatin on HIV Replication

    PubMed Central

    Agosto, Luis M.; Gagne, Matthew; Henderson, Andrew J.

    2015-01-01

    Chromatin influences Human Immunodeficiency Virus (HIV) integration and replication. This review highlights critical host factors that influence chromatin structure and organization and that also impact HIV integration, transcriptional regulation and latency. Furthermore, recent attempts to target chromatin associated factors to reduce the HIV proviral load are discussed. PMID:26437430

  20. The Use of Flow Cytometry to Assess the State of Chromatin in T Cells.

    PubMed

    Bingham, Kellie N; Lee, Megan D; Rawlings, Jason S

    2015-01-01

    During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in naïve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI. PMID:26709948

  1. The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

    PubMed Central

    Rawlings, Jason S.

    2015-01-01

    During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in naïve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI PMID:26709948

  2. Dropwise condensation

    PubMed Central

    Leach, R. N.; Stevens, F.; Langford, S. C.; Dickinson, J. T.

    2008-01-01

    Dropwise condensation of water vapor from a naturally cooling, hot water reservoir onto a hydrophobic polymer film and a silanized glass slide was studied by direct observation and simulations. The observed drop growth kinetics suggest that smallest drops grow principally by the diffusion of water adsorbed on the substrate to the drop perimeter, while drops larger than 50 μm in diameter grow principally by direct deposition from the vapor onto the drop surface. Drop coalescence plays a critical role in determining the drop size distribution, and stimulates the nucleation of new, small drops on the substrates. Simulations of drop growth incorporating these growth mechanisms provide a good description of the observed drop size distribution. Because of the large role played by coalescence, details of individual drop growth make little difference to the final drop size distribution. The rate of condensation per unit substrate area is especially high for the smallest drops, and may help account for the high heat transfer rates associated with dropwise condensation relative to filmwise condensation in heat exchange applications. PMID:17014129

  3. CENH3 distribution and differential chromatin modifications during pollen development in rye (Secale cereale L.).

    PubMed

    Houben, Andreas; Kumke, Katrin; Nagaki, Kiyotaka; Hause, Gerd

    2011-05-01

    Microgametogenesis in angiosperms results in two structurally and functionally different cells, one generative cell, which subsequently forms the sperm cells, and the vegetative cell. We analysed the chromatin properties of both types of nuclei after first and second pollen mitosis in rye (Secale cereale). The condensed chromatin of generative nuclei is earmarked by an enhanced level of histone H3K4/K9 dimethylation and H3K9 acetylation. The less condensed vegetative nuclei are RNA polymerase II positive. Trimethylation of H3K27 is not involved in transcriptional downregulation of genes located in generative nuclei as H3K27me3 was exclusively detected in the vegetative nuclei. The global level of DNA methylation does not differ between both types of pollen nuclei. In rye, unlike in Arabidopsis thaliana (Ingouff et al. Curr Biol 17:1032-1037 2007; Schoft et al. EMBO Rep 10:1015-1021 2009), centromeric histone H3 is not excluded from the chromatin of the vegetative nucleus and the condensation degree of centromeric and subtelomeric regions did not differ between the generative and vegetative nuclei. Differences between rye and A. thaliana data suggest that the chromatin organization in mature nuclei of pollen grains is not universal across angiosperms. PMID:21503764

  4. Chromatin immunoprecipitation for human monocyte derived macrophages.

    PubMed

    Wooden, Jessica; Ciborowski, Pawel

    2014-12-01

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

  5. Detecting ATM-Dependent Chromatin Modification in DNA Damage Response

    PubMed Central

    Udayakumar, Durga; Horikoshi, Nobuo; Mishra, Lope; Hunt, Clayton; Pandita, Tej K.

    2015-01-01

    Loss of function or mutation of the ataxia–telangiectasia mutated gene product (ATM) results in inherited genetic disorders characterized by neurodegeneration, immunodeficiency, and cancer. Ataxia-telangiectasia mutated (ATM) gene product belongs to the PI3K-like protein kinase (PIKKs) family and is functionally implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, cell-cycle control, and telomere maintenance. The ATM protein kinase is primarily activated in response to DNA double strand breaks (DSBs), the most deleterious form of DNA damage produced by ionizing radiation (IR) or radiomimetic drugs. It is detected at DNA damage sites, where ATM autophosphorylation causes dissociation of the inactive homodimeric form to the activated monomeric form. Interestingly, heat shock can activate ATM independent of the presence of DNA strand breaks. ATM is an integral part of the sensory machinery that detects DSBs during meiosis, mitosis, or DNA breaks mediated by free radicals. These DNA lesions can trigger higher order chromatin reorganization fuelled by posttranslational modifications of histones and histone binding proteins. Our group, and others, have shown that ATM activation is tightly regulated by chromatin modifications. This review summarizes the multiple approaches used to discern the role of ATM and other associated proteins in chromatin modification in response to DNA damage. PMID:25827888

  6. Plant chromatin warms up in Madrid

    PubMed Central

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

    2014-01-01

    The 3rd European Workshop on Plant Chromatin (EWPC) was held on August 2013 in Madrid, Spain. A number of different topics on plant chromatin were presented during the meeting, including new factors mediating Polycomb Group protein function in plants, chromatin-mediated reprogramming in plant developmental transitions, the role of histone variants, and newly identified chromatin remodeling factors. The function of interactions between chromatin and transcription factors in the modulation of gene expression, the role of chromatin dynamics in the control of nuclear processes and the influence of environmental factors on chromatin organization were also reported. In this report, we highlight some of the new insights emerging in this growing area of research, presented at the 3rd EWPC. PMID:24504145

  7. Histone lysine methylation and chromatin replication.

    PubMed

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

    2014-12-01

    In eukaryotic organisms, the replication of the DNA sequence and its organization into chromatin are critical to maintain genome integrity. Chromatin components, such as histone variants and histone post-translational modifications, along with the higher-order chromatin structure, impact several DNA metabolic processes, including replication, transcription, and repair. In this review we focus on lysine methylation and the relationships between this histone mark and chromatin replication. We first describe studies implicating lysine methylation in regulating early steps in the replication process. We then discuss chromatin reassembly following replication fork passage, where the incorporation of a combination of newly synthesized histones and parental histones can impact the inheritance of lysine methylation marks on the daughter strands. Finally, we elaborate on how the inheritance of lysine methylation can impact maintenance of the chromatin landscape, using heterochromatin as a model chromatin domain, and we discuss the potential mechanisms involved in this process. PMID:24686120

  8. Chromatin without the 30-nm fiber

    PubMed Central

    Razin, Sergey V; Gavrilov, Alexey A

    2014-01-01

    Several hierarchical levels of DNA packaging are believed to exist in chromatin, starting from a 10-nm chromatin fiber that is further packed into a 30-nm fiber. Transitions between the 30-nm and 10-nm fibers are thought to be essential for the control of chromatin transcriptional status. However, recent studies demonstrate that in the nuclei, DNA is packed in tightly associated 10-nm fibers that are not compacted into 30-nm fibers. Additionally, the accessibility of DNA in chromatin depends on the local mobility of nucleosomes rather than on decompaction of chromosome regions. These findings argue for reconsidering the hierarchical model of chromatin packaging and some of the basic definitions of chromatin. In particular, chromatin domains should be considered as three-dimensional objects, which may include genomic regions that do not necessarily constitute a continuous domain on the DNA chain. PMID:24561903

  9. Concentration of elements in mitotic chromatin as measured by x-ray microanalysis

    PubMed Central

    1977-01-01

    Unfixed frozen-dried and uncoated tissue sections of the mouse duodenum were placed on carbon planchets and analyzed in a scanning electron microscope fitted with energy dispersive X-ray equipment. Computer analysis of the X-ray spectra allowed elemental microanalysis of the nucleus, cytoplasm, and mitotic chromatin regions in the cryptal and villus enterocytes. The peak to continuum ratio of S, Cl, K, and Ca were higher in mitotic chromatin than any of the other sites measured. The redistribution of Ca at mitosis is postulated to help explain both chromosome condensation and assembly of the mitotic spindle apparatus. PMID:856831

  10. 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. PMID:25028181

  11. 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

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

    SciTech Connect

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

    2007-10-01

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

  13. Chromatin structure and gene expression changes associated with loss of MOP1 activity in Zea mays

    PubMed Central

    Madzima, Thelma F; Huang, Ji; McGinnis, Karen M

    2014-01-01

    Though the mechanisms governing nuclear organization are not well understood, it is apparent that epigenetic modifications coordinately modulate chromatin organization as well as transcription. In maize, MEDIATOR OF PARAMUTATION1 (MOP1) is required for 24 nt siRNA-mediated epigenetic regulation and transcriptional gene silencing via a putative Pol IV- RdDM pathway. To elucidate the mechanisms of nuclear chromatin organization, we investigated the relationship between chromatin structure and transcription in response to loss of MOP1 function. We used a microarray based micrococcal nuclease sensitivity assay to identify genome-wide changes in chromatin structure in mop1-1 immature ears and observed an increase in chromatin accessibility at chromosome arms associated with loss of MOP1 function. Within the many genes misregulated in mop1 mutants, we identified one subset likely to be direct targets of epigenetic transcriptional silencing via Pol-IV RdDM. We found that target specificity for MOP1-mediated RdDM activity is governed by multiple signals that include accumulation of 24 nt siRNAs and the presence of specific classes of gene-proximal transposons, but neither of these attributes alone is sufficient to predict transcriptional misregulation in mop1-1 homozygous mutants. Our results suggest a role for MOP1 in regulation of higher-order chromatin organization where loss of MOP1 activity at a subset of loci triggers a broader cascade of transcriptional consequences and genome-wide changes in chromatin structure. PMID:24786611

  14. Neutron scatter studies of chromatin structures related to functions

    SciTech Connect

    Bradbury, E.M.

    1992-01-01

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

  15. Neutron scatter studies of chromatin structures related to functions

    SciTech Connect

    Bradbury, E.M.

    1992-01-01

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

  16. Analysis of chromatin boundary activity in Drosophila cells

    PubMed Central

    Li, Mo; Belozerov, Vladimir E; Cai, Haini N

    2008-01-01

    Background Chromatin boundaries, also known as insulators, regulate gene activity by organizing active and repressive chromatin domains and modulate enhancer-promoter interactions. However, the mechanisms of boundary action are poorly understood, in part due to our limited knowledge about insulator proteins, and a shortage of standard assays by which diverse boundaries could be compared. Results We report here the development of an enhancer-blocking assay for studying insulator activity in Drosophila cultured cells. We show that the activities of diverse Drosophila insulators including suHw, SF1, SF1b, Fab7 and Fab8 are supported in these cells. We further show that double stranded RNA (dsRNA)-mediated knockdown of SuHw and dCTCF factors disrupts the enhancer-blocking function of suHw and Fab8, respectively, thereby establishing the effectiveness of using RNA interference in our cell-based assay for probing insulator function. Conclusion The novel boundary assay provides a quantitative and efficient method for analyzing insulator mechanism and can be further exploited in genome-wide RNAi screens for insulator components. It provides a useful tool that complements the transgenic and genetic approaches for studying this important class of regulatory elements. PMID:19077248

  17. Fast kinetics of chromatin assembly revealed by single-molecule videomicroscopy and scanning force microscopy

    PubMed Central

    Ladoux, Benoit; Quivy, Jean-Pierre; Doyle, Patrick; Roure, Olivia du; Almouzni, Geneviève; Viovy, Jean-Louis

    2000-01-01

    Fluorescence videomicroscopy and scanning force microscopy were used to follow, in real time, chromatin assembly on individual DNA molecules immersed in cell-free systems competent for physiological chromatin assembly. Within a few seconds, molecules are already compacted into a form exhibiting strong similarities to native chromatin fibers. In these extracts, the compaction rate is more than 100 times faster than expected from standard biochemical assays. Our data provide definite information on the forces involved (a few piconewtons) and on the reaction path. DNA compaction as a function of time revealed unique features of the assembly reaction in these extracts. They imply a sequential process with at least three steps, involving DNA wrapping as the final event. An absolute and quantitative measure of the kinetic parameters of the early steps in chromatin assembly under physiological conditions could thus be obtained. PMID:11114182

  18. 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.

  19. Chromatin signatures of the Drosophila replication program

    PubMed Central

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

    2011-01-01

    DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events. PMID:21177973

  20. 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.

  1. 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

  2. Characterization of the RNA content of chromatin

    PubMed Central

    Mondal, Tanmoy; Rasmussen, Markus; Pandey, Gaurav Kumar; Isaksson, Anders; Kanduri, Chandrasekhar

    2010-01-01

    Noncoding RNA (ncRNA) constitutes a significant portion of the mammalian transcriptome. Emerging evidence suggests that it regulates gene expression in cis or trans by modulating the chromatin structure. To uncover the functional role of ncRNA in chromatin organization, we deep sequenced chromatin-associated RNAs (CARs) from human fibroblast (HF) cells. This resulted in the identification of 141 intronic regions and 74 intergenic regions harboring CARs. The intronic and intergenic CARs show significant conservation across 44 species of placental mammals. Functional characterization of one of the intergenic CARs, Intergenic10, revealed that it regulates gene expression of neighboring genes through modulating the chromatin structure in cis. Our data suggest that ncRNA is an integral component of chromatin and that it may regulate various biological functions through fine-tuning of the chromatin architecture. PMID:20404130

  3. Chromatin state dynamics during blood formation

    PubMed Central

    Lara-Astiaso, David; Weiner, Assaf; Lorenzo-Vivas, Erika; Zaretsky, Irina; Jaitin, Diego Adhemar; David, Eyal; Keren-Shaul, Hadas; Mildner, Alexander; Winter, Deborah; Jung, Steffen; Friedman, Nir; Amit, Ido

    2015-01-01

    Chromatin modifications are crucial for development, yet little is known about their dynamics during differentiation. Hematopoiesis provides a well-defined model to study chromatin state dynamics, however technical limitations impede profiling of homogeneous differentiation intermediates. We developed a high sensitivity indexing-first chromatin immunoprecipitation approach (iChIP) to profile the dynamics of four chromatin modifications across 16 stages of hematopoietic differentiation. We identify 48,415 enhancer regions and characterize their dynamics. We find that lineage commitment involves de novo establishment of 17,035 lineage-specific enhancers. These enhancer repertoire expansions foreshadow transcriptional programs in differentiated cells. Combining our enhancer catalog with gene expression profiles, we elucidate the transcription factor network controlling chromatin dynamics and lineage specification in hematopoiesis. Together, our results provide a comprehensive model of chromatin dynamics during development. PMID:25103404

  4. 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

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

    PubMed

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

    2016-01-01

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

  6. Single cell correlation fractal dimension of chromatin

    PubMed Central

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

    2014-01-01

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

  7. Chromatin domain boundary element search tool for Drosophila

    PubMed Central

    Srinivasan, Arumugam; Mishra, Rakesh K.

    2012-01-01

    Chromatin domain boundary elements prevent inappropriate interaction between distant or closely spaced regulatory elements and restrict enhancers and silencers to correct target promoters. In spite of having such a general role and expected frequent occurrence genome wide, there is no DNA sequence analysis based tool to identify boundary elements. Here, we report chromatin domain Boundary Element Search Tool (cdBEST), to identify boundary elements. cdBEST uses known recognition sequences of boundary interacting proteins and looks for ‘motif clusters’. Using cdBEST, we identified boundary sequences across 12 Drosophila species. Of the 4576 boundary sequences identified in Drosophila melanogaster genome, >170 sequences are repetitive in nature and have sequence homology to transposable elements. Analysis of such sequences across 12 Drosophila genomes showed that the occurrence of repetitive sequences in the context of boundaries is a common feature of drosophilids. We use a variety of genome organization criteria and also experimental test on a subset of the cdBEST boundaries in an enhancer-blocking assay and show that 80% of them indeed function as boundaries in vivo. These observations highlight the role of cdBEST in better understanding of chromatin domain boundaries in Drosophila and setting the stage for comparative analysis of boundaries across closely related species. PMID:22287636

  8. Human tRNA genes function as chromatin insulators

    PubMed Central

    Raab, Jesse R; Chiu, Jonathan; Zhu, Jingchun; Katzman, Sol; Kurukuti, Sreenivasulu; Wade, Paul A; Haussler, David; Kamakaka, Rohinton T

    2012-01-01

    Insulators help separate active chromatin domains from silenced ones. In yeast, gene promoters act as insulators to block the spread of Sir and HP1 mediated silencing while in metazoans most insulators are multipartite autonomous entities. tDNAs are repetitive sequences dispersed throughout the human genome and we now show that some of these tDNAs can function as insulators in human cells. Using computational methods, we identified putative human tDNA insulators. Using silencer blocking, transgene protection and repressor blocking assays we show that some of these tDNA-containing fragments can function as barrier insulators in human cells. We find that these elements also have the ability to block enhancers from activating RNA pol II transcribed promoters. Characterization of a putative tDNA insulator in human cells reveals that the site possesses chromatin signatures similar to those observed at other better-characterized eukaryotic insulators. Enhanced 4C analysis demonstrates that the tDNA insulator makes long-range chromatin contacts with other tDNAs and ETC sites but not with intervening or flanking RNA pol II transcribed genes. PMID:22085927

  9. Tissue-Specific Regulation of Chromatin Insulator Function

    PubMed Central

    Matzat, Leah H.; Dale, Ryan K.; Moshkovich, Nellie; Lei, Elissa P.

    2012-01-01

    Chromatin insulators organize the genome into distinct transcriptional domains and contribute to cell type–specific chromatin organization. However, factors regulating tissue-specific insulator function have not yet been discovered. Here we identify the RNA recognition motif-containing protein Shep as a direct interactor of two individual components of the gypsy insulator complex in Drosophila. Mutation of shep improves gypsy-dependent enhancer blocking, indicating a role as a negative regulator of insulator activity. Unlike ubiquitously expressed core gypsy insulator proteins, Shep is highly expressed in the central nervous system (CNS) with lower expression in other tissues. We developed a novel, quantitative tissue-specific barrier assay to demonstrate that Shep functions as a negative regulator of insulator activity in the CNS but not in muscle tissue. Additionally, mutation of shep alters insulator complex nuclear localization in the CNS but has no effect in other tissues. Consistent with negative regulatory activity, ChIP–seq analysis of Shep in a CNS-derived cell line indicates substantial genome-wide colocalization with a single gypsy insulator component but limited overlap with intact insulator complexes. Taken together, these data reveal a novel, tissue-specific mode of regulation of a chromatin insulator. PMID:23209434

  10. Chromatin Regulatory Mechanisms in Pluripotency

    PubMed Central

    Lessard, Julie A.; Crabtree, Gerald R.

    2011-01-01

    Stem cells of all types are characterized by a stable, heritable state permissive of multiple developmental pathways. The past five years have seen remarkable advances in understanding these heritable states and the ways that they are initiated or terminated. Transcription factors that bind directly to DNA and have sufficiency roles have been most easy to investigate and, perhaps for this reason, are most solidly implicated in pluripotency. In addition, large complexes of ATP-dependent chromatin-remodeling and histone-modification enzymes that have specialized functions have also been implicated by genetic studies in initiating and/or maintaining pluripotency or multipotency. Several of these ATP-dependent remodeling complexes play non-redundant roles, and the esBAF complex facilitates reprogramming of induced pluripotent stem cells. The recent finding that virtually all histone modifications can be rapidly reversed and are often highly dynamic has raised new questions about how histone modifications come to play a role in the steady state of pluripotency. Another surprise from genetic studies has been the frequency with which the global effects of mutations in chromatin regulators can be largely reversed by a single target gene. These genetic studies help define the arena for future mechanistic studies that might be helpful to harness pluripotency for therapeutic goals. PMID:20624054

  11. Both Chromosome Decondensation and Condensation Are Dependent on DNA Replication in C. elegans Embryos

    PubMed Central

    Sonneville, Remi; Craig, Gillian; Labib, Karim; Gartner, Anton; Blow, J. Julian

    2015-01-01

    Summary During cell division, chromatin alternates between a condensed state to facilitate chromosome segregation and a decondensed form when DNA replicates. In most tissues, S phase and mitosis are separated by defined G1 and G2 gap phases, but early embryogenesis involves rapid oscillations between replication and mitosis. Using Caenorhabditis elegans embryos as a model system, we show that chromosome condensation and condensin II concentration on chromosomal axes require replicated DNA. In addition, we found that, during late telophase, replication initiates on condensed chromosomes and promotes the rapid decondensation of the chromatin. Upon replication initiation, the CDC-45-MCM-GINS (CMG) DNA helicase drives the release of condensin I complexes from chromatin and the activation or displacement of inactive MCM-2–7 complexes, which together with the nucleoporin MEL-28/ELYS tethers condensed chromatin to the nuclear envelope, thereby promoting chromatin decondensation. Our results show how, in an early embryo, the chromosome-condensation cycle is functionally linked with DNA replication. PMID:26166571

  12. In vitro histone demethylase assays.

    PubMed

    Kokura, Kenji; Fang, Jia

    2009-01-01

    Histone methylation plays important roles in chromatin structure, transcription, and epigenetic state of the cell. Tremendous discoveries recently demonstrated that methylation mark is not static but is dynamically regulated by both histone methyltransferases and the histone demethylases. Two families of histone demethylases have been identified to remove methyl groups from lysine side chain through different reaction mechanisms in presence of distinct cofactors. Amine oxidase LSD1 family requires flavin adenine dinucleotide (FAD) whereas dioxygenase Jmjc domain-containing proteins family relies on Fe(II) and alpha-ketoglutarate. Identification of these enzymes opened a new era in understanding how chromatin dynamic is regulated and further understanding the regulation of these enzymes will provide significant insights into fundamental mechanisms of many biological processes and human diseases. This chapter describes different assay conditions and detection methods for different family of histone demethylases. We also summarize step-by-step protocols for purification and preparation of various histone substrates for histone demethylase assays. PMID:19381934

  13. Analysis of Histones and Chromatin in Xenopus laevis Egg and Oocyte Extracts

    PubMed Central

    Banaszynski, Laura A.; Allis, C. David; Shechter, David

    2010-01-01

    Histones are the major protein components of chromatin, the physiological form of the genome in all eukaryotic cells. Chromatin is the substrate of information-directed biological processes, such as gene regulation and transcription, replication, and mitosis. A long-standing experimental model system to study many of these processes is the extract made from the eggs of the anuran Xenopus laevis. Since work in recent years has solidified the importance of post-translational modification of histones in directing biological processes, the study of histones in a biochemically dissectible model such as Xenopus is crucial for the understanding of their biological significance. Here we present a rationale and methods for isolating and studying histones and chromatin in different Xenopus egg and oocyte extracts. In particular, we present protocols for the preparation of: cell-free egg and oocyte extract; nucleoplasmic extract (“NPE”); biochemical purification of maternally-deposited, stored histones in the oocyte and the egg; assembly of pronuclei in egg extract and the isolation of pronuclear chromatin and histones; and an extract chromatin assembly assay. We also demonstrate aspects of the variability of the system to be mindful of when working with extract and the importance of proper laboratory temperature in preparing quality extracts. We expect that these methods will be of use in promoting further understanding of embryonic chromatin in a unique experimental system. PMID:20051265

  14. Mechanisms for ATP-dependent chromatin remodelling.

    PubMed

    Whitehouse, I; Flaus, A; Havas, K; Owen-Hughes, T

    2000-01-01

    Gene regulation involves the generation of a local chromatin topology that is conducive to transcription. Several classes of chromatin remodelling activity have been shown to play a role in this process. ATP-dependent chromatin-remodelling activities use energy derived from the hydrolysis of ATP to alter the structure of chromatin, making it more accessible for transcription factor binding. The yeast SWI-SWF complex is the founding member of this family of ATP-dependent chromatin-remodelling activities. We have developed a model system to study the ability of the SWI-SWF complex to alter chromatin structure. Using this system, we find that SWI-SWF is able to alter the position of nucleosomes along the DNA. This is consistent with recent reports that other ATP-dependent chromatin-remodelling activities can alter the positions of nucleosomes along DNA. This suggests that nucleosome mobilization may be a general feature of the activity of ATP-dependent chromatin-remodelling activities. Some of the mechanisms by which nucleosomes may be moved along DNA are discussed. PMID:10961923

  15. Comparative analysis of metazoan chromatin organization.

    PubMed

    Ho, Joshua W K; Jung, Youngsook L; Liu, Tao; Alver, Burak H; Lee, Soohyun; Ikegami, Kohta; Sohn, Kyung-Ah; Minoda, Aki; Tolstorukov, Michael Y; Appert, Alex; Parker, Stephen C J; Gu, Tingting; Kundaje, Anshul; Riddle, Nicole C; Bishop, Eric; Egelhofer, Thea A; Hu, Sheng'en Shawn; Alekseyenko, Artyom A; Rechtsteiner, Andreas; Asker, Dalal; Belsky, Jason A; Bowman, Sarah K; Chen, Q Brent; Chen, Ron A-J; Day, Daniel S; Dong, Yan; Dose, Andrea C; Duan, Xikun; Epstein, Charles B; Ercan, Sevinc; Feingold, Elise A; Ferrari, Francesco; Garrigues, Jacob M; Gehlenborg, Nils; Good, Peter J; Haseley, Psalm; He, Daniel; Herrmann, Moritz; Hoffman, Michael M; Jeffers, Tess E; Kharchenko, Peter V; Kolasinska-Zwierz, Paulina; Kotwaliwale, Chitra V; Kumar, Nischay; Langley, Sasha A; Larschan, Erica N; Latorre, Isabel; Libbrecht, Maxwell W; Lin, Xueqiu; Park, Richard; Pazin, Michael J; Pham, Hoang N; Plachetka, Annette; Qin, Bo; Schwartz, Yuri B; Shoresh, Noam; Stempor, Przemyslaw; Vielle, Anne; Wang, Chengyang; Whittle, Christina M; Xue, Huiling; Kingston, Robert E; Kim, Ju Han; Bernstein, Bradley E; Dernburg, Abby F; Pirrotta, Vincenzo; Kuroda, Mitzi I; Noble, William S; Tullius, Thomas D; Kellis, Manolis; MacAlpine, David M; Strome, Susan; Elgin, Sarah C R; Liu, Xiaole Shirley; Lieb, Jason D; Ahringer, Julie; Karpen, Gary H; Park, Peter J

    2014-08-28

    Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal 'arms', and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function. PMID:25164756

  16. Chromatin Remodelers: From Function to Dysfunction

    PubMed Central

    Längst, Gernot; Manelyte, Laura

    2015-01-01

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

  17. A Conserved Chromatin Architecture Marks and Maintains the Restricted Germ Cell Lineage in Worms and Flies

    PubMed Central

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

    2014-01-01

    Summary 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. PMID:14602075

  18. Loosened nucleosome linker folding in transcriptionally active chromatin of chicken embryo erythrocyte nuclei.

    PubMed Central

    Grigoryev, S A; Spirin, K S; Krasheninnikov, I A

    1990-01-01

    We have investigated the mechanism of the electrophoresis-driven chromatin aggregation which had been described by Weintraub (1984, Cell 38, 17-27) as a putative mean for propagation of genetic repression in eukaryotes. We show that the oligonucleosome aggregates are assembled de novo at the starting zone of DNP electrophoresis. A new system of native two-dimensional DNP electrophoresis has been worked out to separate the oligonucleosome aggregates ('A' particles) and the freely-migrating oligonucleosomes ('B' particles). The 'B' particle fraction which is derived from transcriptionally-active chromatin regions undergoes an extensive nuclease degradation of its DNA termini during the nuclease digestion. This fraction is partially depleted of histones H1 and H5 and is enriched in HMG nonhistone proteins. 'A' particles comprise the repressed chromatin DNA fragments which are about 60 b.p. longer than the corresponding DNA oligomers of 'B' particles. An oligonucleosome preparation containing the elongated DNA oligomers has been also isolated by means of sucrose gradient ultracentrifugation. Exonuclease III mapping reveals that the two chromatin fractions differ by an extent of terminal linker DNA trimming during the Micrococcal nuclease digestion rather than by the nucleosome repeat length. The complex character of nuclease digestion is not observed when the chromatin is digested in solution after the nuclear lysis. We argue that the protection of terminal oligonucleosome linkers is due to selective condensation of inactive chromatin in chicken erythrocyte nuclei and that the terminal DNA tails together with linker histones bound to them mediate the aggregation of repressed chromatin fragments. Images PMID:2259630

  19. An in vitro reconstitution system for the assessment of chromatin protein fluidity during Xenopus development

    SciTech Connect

    Aoki, Ryuta; Inui, Masafumi; Hayashi, Yohei; Sedohara, Ayako; Okabayashi, Koji; ICORP Organ Regeneration Project, Japan Science and Technology Agency , 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 ; Ohnuma, Kiyoshi; Murata, Masayuki; Asashima, Makoto; ICORP Organ Regeneration Project, Japan Science and Technology Agency , 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902; Organ Development Research Laboratory, National Institute of Advanced Industrial Science and Technology , Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562

    2010-09-17

    Research highlights: {yields} An in vitro reconstitution system was established with isolated nuclei and cytoplasm. {yields} Chromatin fluidities were measured in the system using FRAP. {yields} Chromatin fluidities were higher in the cytoplasm of earlier-stage embryos. {yields} Chromatin fluidities were higher in the earlier-stage nuclei with egg-extract. {yields} Chromatin fluidity may decrease during embryonic development. -- Abstract: Chromatin fluidity, which is one of the indicators of higher-order structures in chromatin, is associated with cell differentiation. However, little is known about the relationships between chromatin fluidity and cell differentiation status in embryonic development. We established an in vitro reconstitution system that uses isolated nuclei and cytoplasmic extracts of Xenopus embryos and a fluorescence recovery after photobleaching assay to measure the fluidities of heterochromatin protein 1 (HP1) and histone H1 during development. The HP1 and H1 fluidities of nuclei isolated from the tailbuds of early tadpole stage (stage 32) embryos in the cytoplasmic extracts of eggs and of late blastula stage (stage 9) embryos were higher than those in the cytoplasmic extracts of mid-neurula stage (stage 15) embryos. The HP1 fluidities of nuclei isolated from animal cap cells of early gastrula stage (stage 10) embryos and from the neural plates of neural stage (stage 20) embryos were higher than those isolated from the tailbuds of stage 32 embryos in egg extracts, whereas the HP1 fluidities of these nuclei were the same in the cytoplasmic extracts of stage 15 embryos. These results suggest that chromatin fluidity is dependent upon both cytoplasmic and nuclear factors and decreases during development.

  20. A universal description for the experimental behavior of salt-(in)dependent oligocation-induced DNA condensation

    PubMed Central

    Korolev, Nikolay; Berezhnoy, Nikolay V.; Eom, Khee Dong; Tam, James P.; Nordenskiöld, Lars

    2009-01-01

    We report a systematic study of the condensation of plasmid DNA by oligocations with variation of the charge, Z, from +3 to +31. The oligocations include a series of synthetic linear ?-oligo(l-lysines), (denoted ?Kn, n = 3–10, 31; n is the number of lysines equal to the ligand charge) and branched ?-substituted homologues of ?K10: ?YK10, ?LK10 (Z = +10); ?RK10, ?YRK10 and ?LYRK10 (Z = +20). Data were obtained by light scattering, UV absorption monitored precipitation assay and isothermal titration calorimetry in a wide range concentrations of DNA and monovalent salt (KCl, CKCl). The dependence of EC50 (ligand concentration at the midpoint of DNA condensation) on CKCl shows the existence of a salt-independent regime at low CKCl and a salt-dependent regime with a steep rise of EC50 with increase of CKCl. Increase of the ligand charge shifts the transition from the salt-independent to salt-dependent regime to higher CKCl. A novel and simple relationship describing the EC50 dependence on DNA concentration, charge of the ligand and the salt-dependent dissociation constant of the ligand–DNA complex is derived. For the ?-oligolysines ?K3–?K10, the experimental dependencies of EC50 on CKCl and Z are well-described by an equation with a common set of parameters. Implications from our findings for understanding DNA condensation in chromatin are discussed. PMID:19773427

  1. Chromatin Dynamics of Circadian Transcription

    PubMed Central

    Aguilar-Arnal, Lorena; Sassone-Corsi, Paolo

    2015-01-01

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

  2. Three-dimensional modeling of chromatin structure from interaction frequency data using Markov chain Monte Carlo sampling

    PubMed Central

    2011-01-01

    Background Long-range interactions between regulatory DNA elements such as enhancers, insulators and promoters play an important role in regulating transcription. As chromatin contacts have been found throughout the human genome and in different cell types, spatial transcriptional control is now viewed as a general mechanism of gene expression regulation. Chromosome Conformation Capture Carbon Copy (5C) and its variant Hi-C are techniques used to measure the interaction frequency (IF) between specific regions of the genome. Our goal is to use the IF data generated by these experiments to computationally model and analyze three-dimensional chromatin organization. Results We formulate a probabilistic model linking 5C/Hi-C data to physical distances and describe a Markov chain Monte Carlo (MCMC) approach called MCMC5C to generate a representative sample from the posterior distribution over structures from IF data. Structures produced from parallel MCMC runs on the same dataset demonstrate that our MCMC method mixes quickly and is able to sample from the posterior distribution of structures and find subclasses of structures. Structural properties (base looping, condensation, and local density) were defined and their distribution measured across the ensembles of structures generated. We applied these methods to a biological model of human myelomonocyte cellular differentiation and identified distinct chromatin conformation signatures (CCSs) corresponding to each of the cellular states. We also demonstrate the ability of our method to run on Hi-C data and produce a model of human chromosome 14 at 1Mb resolution that is consistent with previously observed structural properties as measured by 3D-FISH. Conclusions We believe that tools like MCMC5C are essential for the reliable analysis of data from the 3C-derived techniques such as 5C and Hi-C. By integrating complex, high-dimensional and noisy datasets into an easy to interpret ensemble of three-dimensional conformations, MCMC5C allows researchers to reliably interpret the result of their assay and contrast conformations under different conditions. Availability http://Dostielab.biochem.mcgill.ca PMID:22026390

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

    PubMed Central

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

    2014-01-01

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

  4. Perspectives on the assessment of human sperm chromatin integrity.

    PubMed

    Palermo, Gianpiero D; Neri, Queenie V; Cozzubbo, Tyler; Rosenwaks, Zev

    2014-12-01

    Apoptosis plays a significant role in regulating germ cell development by removing damaged germ cells from seminiferous tubules, thereby safeguarding the genome of a given species. The unique chromatin-packing process of the spermatozoon has important implications for both the development of male infertility screening tests and understanding of sperm chromatin characteristics, which may affect assisted reproductive technology outcomes. Sperm deoxyribonucleic acid (DNA) integrity tests have been proposed as a means to assess male gamete competence. Although these assays are currently gaining popularity, and are more often used as a supplement to traditional semen analysis, the point at which DNA damage occurs during spermiogenesis, and to what degree, remains to be elucidated. Here, we examined current studies of DNA fragmentation, to understand its origin and import, as well as its impact on pre- and post-implantation development. As the DNA fragmentation index is strongly correlated with the motility characteristics of a semen specimen, controlling for this factor may be helpful. Utilization of more sensitive assays, possibly on the actual spermatozoa used for insemination, may generate healthier conceptuses. PMID:25456796

  5. Tousled kinase TLK1B mediates chromatin assembly in conjunction with Asf1 regardless of its kinase activity

    PubMed Central

    2010-01-01

    Background The Tousled Like Kinases (TLKs) are involved in chromatin dynamics, including DNA replication and repair, transcription, and chromosome segregation. Indeed, the first two TLK1 substrates were identified as the histone H3 and Asf1 (a histone H3/H4 chaperone), which immediately suggested a function in chromatin remodeling. However, despite the straightforward assumption that TLK1 acts simply by phosphorylating its substrates and hence modifying their activity, TLK1 also acts as a chaperone. In fact, a kinase-dead (KD) mutant of TLK1B is functional in stimulating chromatin assembly in vitro. However, subtle effects of Asf1 phosphorylation are more difficult to probe in chromatin assembly assays. Not until very recently was the Asf1 site phosphorylated by TLK1 identified. This has allowed for probing directly the functionality of a site-directed mutant of Asf1 in chromatin assembly assays. Findings Addition of either wt or non-phosphorylatable mutant Asf1 to nuclear extract stimulates chromatin assembly on a plasmid. Similarly, TLK1B-KD stimulates chromatin assembly and it synergizes in reactions with supplemental Asf1 (wt or non-phosphorylatable mutant). Conclusions Although the actual function of TLKs as mediators of Asf1 activity cannot be easily studied in vivo, particularly since in mammalian cells there are two TLK genes and two Asf1 genes, we were able to study specifically the stimulation of chromatin assembly in vitro. In such assays, clearly the TLK1 kinase activity was not critical, as neither a non-phosphorylatable Asf1 nor use of the TLK1B-KD impaired the stimulation of nucleosome formation. PMID:20222959

  6. Chromatin Disassembly and Reassembly During DNA Repair

    PubMed Central

    Linger, Jeffrey G.; Tyler, Jessica K.

    2008-01-01

    Current research is demonstrating that the packaging of the eukaryotic genome together with histone proteins into chromatin is playing a fundamental role in DNA repair and the maintenance of genomic integrity. As is well established to be the case for transcription, the chromatin structure dynamically changes during DNA repair. Recent studies indicate that the complete removal of histones from DNA and their subsequent reassembly onto DNA accompanies DNA repair. This review will present evidence indicating that chromatin disassembly and reassembly occur during DNA repair and that these are critical processes for cell survival after DNA repair. Concomitantly, candidate proteins utilized for these processes will be highlighted. PMID:17303193

  7. Nucleosome structure in chromatin from heated cells

    SciTech Connect

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

    1980-12-01

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

  8. 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.

  9. 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)

  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. PMID:25012699

  12. Chromatin freeze fracture electron microscopy: a comparative study of core particles, chromatin, metaphase chromosomes, and nuclei.

    PubMed Central

    Lepault, J; Bram, S; Escaig, J; Wray, W

    1980-01-01

    Chromatin gels, metaphase chromosomes, and intact nuclei were studied by freeze fracturing followed by electron microscopy. The results complement and extend those obtained by classical electron microscopy techniques as they are obtained without fixation or dehydration. The freeze fracturing technique permits a determination of the hydrated diameters of nucleosomes in chromatin and in nuclei to be 13 nm by comparing to simultaneously studied test objects. Nucleosomes in chromatin fibers are closely spaced but are discrete particles in all conditions studied. In the presence of divalent ions, most chromatin in solution, chromosomes, and nuclei is organized into fibers whose thickness is larger than 40 nm. The images are not at all compatible with a super bead organization of the nucleofilament. Freeze fractures of intact nuclei provides information on the distribution of chromatin in a hydrated unfixed state. The images suggest that most of the chromatin is localized in large domains in contact with the inner nuclear membrane. Images PMID:7191563

  13. Effects of the chemotherapy cocktail used to treat testicular cancer on sperm chromatin integrity.

    PubMed

    Delbes, Géraldine; Hales, Barbara F; Robaire, Bernard

    2007-01-01

    The incidence of testicular cancer has increased dramatically over the past 50 years. Advances in treatment, which include the coadministration of bleomycin, etoposide, and cis-platinum (BEP), have brought the cure rate to over 90%. After treatment, most patients go through a temporary period of azoo/oligozoospermia. Although the sperm concentration in approximately 80% of the patients returns to at least 10 million/mL, little is known about the integrity of the chromatin of their germ cells. Using an animal model, we assessed DNA integrity in the spermatozoa of male rats treated for 3, 6 or 9 weeks with BEP at doses, adjusted for surface area, equivalent to 0X, 1/3X, 2/3X, or 1X of the human dose. We did not observe any difference in protamination content, as assessed by the chromomycin A3 (CMA3) assay. After 9 weeks of 1X treatment, the susceptibility of DNA to denaturation evaluated by the sperm chromatin structure assay (SCSA/acridine orange assay (AO) was increased, as well as the number of single and double DNA strand breaks measured by the TUNEL and COMET assays. Parameters obtained from the AO and TUNEL assays were highly correlated with the motility of the spermatozoa, suggesting that conventional sperm analysis parameters can serve as a good indicator of chromatin integrity and vice versa. Correlation studies also suggested that the parameters obtained with the different assays do not overlap, but complement each other. Thus, BEP treatment altered spermatozoal chromatin quality, and these alterations may impact adversely on progeny outcome. PMID:17021337

  14. On the chromatin structure of eukaryotic telomeres

    PubMed Central

    Vaquero-Sedas, María I

    2011-01-01

    Telomeres prevent chromosome fusions and degradation by exonucleases and are implicated in DNA repair, homologous recombination, chromosome pairing and segregation. All these functions of telomeres require the integrity of their chromatin structure, which has been traditionally considered as heterochromatic. In agreement with this idea, different studies have reported that telomeres associate with heterochromatic marks. However, these studies addressed simultaneously the chromatin structures of telomeres and subtelomeric regions or the chromatin structure of telomeres and Interstitial Telomeric Sequences (ITSs). The independent analysis of Arabidopsis telomeres, subtelomeric regions and ITSs has allowed the discovery of euchromatic telomeres. In Arabidopsis, whereas subtelomeric regions and ITSs associate with heterochromatic marks, telomeres exhibit euchromatic features. We think that this scenario could be found in other model systems if the chromatin organizations of telomeres, subtelomeric regions and ITSs are independently analyzed. PMID:21822057

  15. Chromatin modifiers: regulators of cellular differentiation

    PubMed Central

    Chen, Taiping; Dent, Sharon Y. R.

    2014-01-01

    Cellular differentiation, by definition, is epigenetic. Genome-wide profiling of pluripotent cells and differentiated cells suggests global chromatin remodeling during differentiation, resulting in progressive transition from a relatively open chromatin configuration to a more compact state. Genetic studies in mouse models demonstrate major roles for a variety of histone modifiers and chromatin remodelers in key developmental transitions, such as the segregation of embryonic and extraembryonic lineages in blastocyst stage embryos, the formation of the three germ layers during gastrulation, and differentiation of adult stem cells. Furthermore, rather than merely stabilizing the gene expression changes driven by developmental transcription factors, evidence is emerging that chromatin regulators have multifaceted roles in cell fate decisions. PMID:24366184

  16. In vivo binding of retinol to chromatin

    SciTech Connect

    Ferrari, N.; Pfeffer, U.; Vidali, G.

    1988-01-05

    The authors have previously shown that exposure of responding cells to vitamin A leads to profound modifications of chromatin structure as revealed by an increased susceptibility to DNase I digestion, modified patterns of histone acetylation, and impaired synthesis of a nonhistone chromosomal protein. The present results show that these effects are most probably due to the direct interaction between retinol and chromatin, and analysis of mononucleosomes and higher oligomers obtained from retinol-treated cells shows that retinol is indeed tightly bound to chromatin. Enzymatic digestions of vitamin A containing nucleosomes with proteinase K, phospholipase C, and phospholipase A/sub 2/ support a model where the final binding of retinol to chromatin is mediated by a lipoprotein: the recognition of the binding sites on DNA being dictated by the proteic component while the hydrophobic retinol is solubilized in the fatty acid moiety.

  17. Analysis of Mcm2-7 chromatin binding during anaphase and in the transition to quiescence in fission yeast

    SciTech Connect

    Namdar, Mandana; Kearsey, Stephen E. . E-mail: stephen.kearsey@zoo.ox.ac.uk

    2006-10-15

    Mcm2-7 proteins are generally considered to function as a heterohexameric complex, providing helicase activity for the elongation step of DNA replication. These proteins are loaded onto replication origins in M-G1 phase in a process termed licensing or pre-replicative complex formation. It is likely that Mcm2-7 proteins are loaded onto chromatin simultaneously as a pre-formed hexamer although some studies suggest that subcomplexes are recruited sequentially. To analyze this process in fission yeast, we have compared the levels and chromatin binding of Mcm2-7 proteins during the fission yeast cell cycle. Mcm subunits are present at approximately 1 x 10{sup 4} molecules/cell and are bound with approximately equal stoichiometry on chromatin in G1/S phase cells. Using a single cell assay, we have correlated the timing of chromatin association of individual Mcm subunits with progression through mitosis. This showed that Mcm2, 4 and 7 associate with chromatin at about the same stage of anaphase, suggesting that licensing involves the simultaneous binding of these subunits. We also examined Mcm2-7 chromatin association when cells enter a G0-like quiescent state. Chromatin binding is lost in this transition in a process that does not require DNA replication or the selective degradation of specific subunits.

  18. 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

  19. 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

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

    PubMed

    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

  1. Chromatin roadblocks to reprogramming 50 years on.

    PubMed

    Skene, Peter J; Henikoff, Steven

    2012-01-01

    A half century after John Gurdon demonstrated nuclear reprogramming, for which he was awarded the 2012 Nobel Prize in Physiology or Medicine, his group provides insights into the molecular mechanisms whereby chromatin remodeling is required for nuclear reprogramming. Among the issues addressed in Gurdon's latest work are the chromatin impediments to artificially induced reprogramming, discovered by Shinya Yamanaka, who shared the award with Gurdon. PMID:23107587

  2. Inactivating mutations in SWI/SNF chromatin remodeling genes in human cancer.

    PubMed

    Oike, Takahiro; Ogiwara, Hideaki; Nakano, Takashi; Yokota, Jun; Kohno, Takashi

    2013-09-01

    Chromosomal deoxyribonucleic acid and histone proteins form a highly condensed structure known as chromatin. Chromatin remodeling proteins regulate deoxyribonucleic acid transcription, synthesis and repair by changing nucleosomal composition in an adenosine triphosphate-dependent manner and mediate access of deoxyribonucleic acid-binding proteins to deoxyribonucleic acid double strands. Recently, large-scale genome sequencing studies identified somatic mutations in genes encoding chromatin remodeling proteins in a variety of human solid cancers. Notably, inactivating mutations in genes encoding the catalytic and regulatory subunits of the switch/sucrose non-fermenting chromatin remodeling complex have been detected in several solid cancers: sucrose non-fermenting/switch/sucrose non-fermenting-related, matrix-associated, actin-dependent regulator of chromatin, subfamily b, member 1/Brahma-related gene 1-associated factor 47/integrase interactor 1 mutations in rhabdoid tumors; AT-rich interactive domain-containing protein 1 A/Brahma-related gene 1-associated factor 250a mutations in ovarian clear cell carcinoma, hepatocellular carcinoma and gastric adenocarcinoma; polybromo 1/Brahma-related gene 1-associated factor 180 mutations in renal clear cell carcinoma; Brahma-related gene 1/switch/sucrose non-fermenting-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 mutations in non-small-cell lung carcinoma and AT-rich interactive domain-containing protein 2/Brahma-related gene 1-associated factor 200 mutations in hepatocellular carcinoma and malignant melanoma. This suggests that the switch/sucrose non-fermenting complex has a tumor-suppressive function, and that switch/sucrose non-fermenting gene deficiencies may affect the properties of cancer cells, which could be of value for the development of novel therapeutic strategies. PMID:23904343

  3. 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 investigated. PMID:26042422

  4. 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. PMID:25847096

  5. Macroautophagy-aided elimination of chromatin

    PubMed Central

    Erenpreisa, Jekaterina; Huna, Anda; Salmina, Kristine; Jackson, Thomas R.; Cragg, Mark S.

    2012-01-01

    How tumor cells process damaged or unwanted DNA is a matter of much interest. Recently, Rello-Varona et al. (Cell Cycle 2012; 11:170–76) reported the involvement of macroautophagy (hereon autophagy) in the elimination of micronuclei (MN) from osteosarcoma cells. Prior to that, diminution of whole nuclei from multinucleated TP53-mutant tumor cells was described. Here, we discuss these two kinds of chromatin autophagy evoked after genotoxic stress in the context of the various biological processes involved: (1) endopolyploidy and the ploidy cycle; (2) the timing of DNA synthesis; (3) DNA repair; (4) chromatin:nuclear envelope interactions; and (5) cytoplasmic autophagy. We suggest that whereas some MN can be reunited with the main nucleus (through interactions with envelope-limited chromatin sheets) and participate in DNA repair, failure of repair serves as a signal for the chromatin autophagy of MN. In turn, autophagy of whole sub-nuclei in multi-nucleated cells appears to favor de-polyploidization, mitigation of aneuploidy with its adverse effects, thereby promoting the survival fitness of descendents and treatment resistance. Thus, both kinds of chromatin autophagy provide tumor cells with the opportunity to repair DNA, sort and resort chromatin, reduce DNA content, and enhance survival. PMID:22935563

  6. Chromatin remodeling agents for cancer therapy.

    PubMed

    Dario, La Sala; Rosa, Magnano Anna; Mariela, Estenoz; Roberto, Giordano; Caterina, Cinti

    2008-09-01

    Alterations in chromatin structure profoundly influence gene expression during normal cellular homeostasis and malignant transformation. Methylation of cytosines within CpG islands located in promoter and proximal coding regions facilitates recruitment of chromatin-remodeling proteins, which inhibits gene expression. Posttranslational modifications, such as acetylation, methylation, and phosphorylation, of core histone proteins 'mark' regions of chromatin for recognition by multiprotein complexes, which promote either chromatin relaxation and gene expression or chromatin compaction and repression of gene expression. Many genes become transcriptionally silenced during the development of cancer. Covalent epigenetic modifications such as DNA hypermethylation and histone post-translational modifications are an important early event during carcinogenesis and tumor development. Genes involved in key DNA damage responses pathways, apoptosis signaling and DNA repair, can frequently become methylated and epigenetically silenced in tumors. This may lead to differences in intrinsic sensitivity of tumors to chemotherapy, depending on the specific function of the gene inactivated. The fact that cancer can have an epigenetic etiology has encouraged the development of a new therapeutic option that might be termed "epigenetic therapy". The DNA methylation paradox, manifested as derepression of cancer-testis antigens and silencing of tumor suppressors during malignant transformation, provides rationale for the utilization of chromatin remodeling agents for cancer therapy. In this review, the recent advances in the understanding and clinical development of DNA methyltransferase and Histone deacetylase inhibitors, as well as their current role in cancer therapy, will be discussed. PMID:18782077

  7. Mass Spectrometry-Based Proteomics for the Analysis of Chromatin Structure and Dynamics

    PubMed Central

    Soldi, Monica; Cuomo, Alessandro; Bremang, Michael; Bonaldi, Tiziana

    2013-01-01

    Chromatin is a highly structured nucleoprotein complex made of histone proteins and DNA that controls nearly all DNA-dependent processes. Chromatin plasticity is regulated by different associated proteins, post-translational modifications on histones (hPTMs) and DNA methylation, which act in a concerted manner to enforce a specific “chromatin landscape”, with a regulatory effect on gene expression. Mass Spectrometry (MS) has emerged as a powerful analytical strategy to detect histone PTMs, revealing interplays between neighbouring PTMs and enabling screens for their readers in a comprehensive and quantitative fashion. Here we provide an overview of the recent achievements of state-of-the-art mass spectrometry-based proteomics for the detailed qualitative and quantitative characterization of histone post-translational modifications, histone variants, and global interactomes at specific chromatin regions. This synopsis emphasizes how the advances in high resolution MS, from “Bottom Up” to “Top Down” analysis, together with the uptake of quantitative proteomics methods by chromatin biologists, have made MS a well-established method in the epigenetics field, enabling the acquisition of original information, highly complementary to that offered by more conventional, antibody-based, assays. PMID:23466885

  8. Sumoylated Human Histone H4 Prevents Chromatin Compaction by Inhibiting Long-range Internucleosomal Interactions*

    PubMed Central

    Dhall, Abhinav; Wei, Sijie; Fierz, Beat; Woodcock, Christopher L.; Lee, Tae-Hee; Chatterjee, Champak

    2014-01-01

    The structure of eukaryotic chromatin directly influences gene function, and is regulated by chemical modifications of the core histone proteins. Modification of the human histone H4 N-terminal tail region by the small ubiquitin-like modifier protein, SUMO-3, is associated with transcription repression. However, the direct effect of sumoylation on chromatin structure and function remains unknown. Therefore, we employed a disulfide-directed strategy to generate H4 homogenously and site-specifically sumoylated at Lys-12 (suH4ss). Chromatin compaction and oligomerization assays with nucleosomal arrays containing suH4ss established that SUMO-3 inhibits array folding and higher order oligomerization, which underlie chromatin fiber formation. Moreover, the effect of sumoylation differed from that of acetylation, and could be recapitulated with the structurally similar protein ubiquitin. Mechanistic studies at the level of single nucleosomes revealed that, unlike acetylation, the effect of SUMO-3 arises from the attenuation of long-range internucleosomal interactions more than from the destabilization of a compacted dinucleosome state. Altogether, our results present the first insight on the direct structural effects of histone H4 sumoylation and reveal a novel mechanism by which SUMO-3 inhibits chromatin compaction. PMID:25294883

  9. Independent and complementary methods for large-scale structural analysis of mammalian chromatin

    PubMed Central

    Dennis, Jonathan H.; Fan, Hua-Ying; Reynolds, Sheila M.; Yuan, Guocheng; Meldrim, James C.; Richter, Daniel J.; Peterson, Daniel G.; Rando, Oliver J.; Noble, William S.; Kingston, Robert E.

    2007-01-01

    The fundamental building block of chromatin, the nucleosome, occupies 150 bp of DNA in a spaced arrangement that is a primary determinant in regulation of the genome. The nucleosomal organization of some regions of the human genome has been described, but mapping of these regions has been limited to a few kilobases. We have explored two independent and complementary methods for the high-throughput analysis of mammalian chromatin structure. Through adaptations to a protocol used to map yeast chromatin structure, we determined sites of nucleosomal protection over large regions of the mammalian genome using a tiling microarray. By modifying classical primer extension methods, we localized specific internucleosomally cleaved mammalian genomic sequences using a capillary electrophoresis sequencer in a manner that allows high-throughput nucleotide-resolution characterization of nucleosome protection patterns. We developed algorithms for the automated and unbiased analysis of the resulting data, a necessary step toward large-scale analysis. We validated these assays using the known positions of nucleosomes on the mouse mammary tumor virus LTR, and additionally, we characterized the previously unreported chromatin structure of the LCMT2 gene. These results demonstrate the effectiveness of the combined methods for reliable analysis of mammalian chromatin structure in a high-throughput manner. PMID:17568008

  10. Hyperacetylation in prostate cancer induces cell cycle aberrations, chromatin reorganization and altered gene expression profiles

    PubMed Central

    Watson, Jenny A; McKenna, Declan J; Maxwell, Perry; Diamond, James; Arthur, Ken; McKelvey-Martin, Valerie J; Hamilton, Peter W

    2010-01-01

    Abstract Histone acetylation is a fundamental mechanism in the regulation of local chromatin conformation and gene expression. Research has focused on the impact of altered epigenetic environments on the expression of specific genes and their pathways. However, changes in histone acetylation also have a global impact on the cell. In this study we used digital texture analysis to assess global chromatin patterns following treatment with trichostatin A (TSA) and have observed significant alterations in the condensation and distribution of higher-order chromatin, which were associated with altered gene expression profiles in both immortalised normal PNT1A prostate cell line and androgen-dependent prostate cancer cell line LNCaP. Furthermore, the extent of TSA-induced disruption was both cell cycle and cell line dependent. This was illustrated by the identification of sub-populations of prostate cancer cells expressing high levels of H3K9 acetylation in the G2/M phase of the cell cycle that were absent in normal cell populations. In addition, the analysis of enriched populations of G1 cells showed a global decondensation of chromatin exclusively in normal cells. PMID:19583812

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

    PubMed

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

    2015-11-24

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

  12. Dynamics of Chromatin Decondensation Reveals the Structural Integrity of a Mechanically Prestressed Nucleus

    PubMed Central

    Mazumder, Aprotim; Roopa, T.; Basu, Aakash; Mahadevan, L.; Shivashankar, G. V.

    2008-01-01

    Genome organization within the cell nucleus is a result of chromatin condensation achieved by histone tail-tail interactions and other nuclear proteins that counter the outward entropic pressure of the polymeric DNA. We probed the entropic swelling of chromatin driven by enzymatic disruption of these interactions in isolated mammalian cell nuclei. The large-scale decondensation of chromatin and the eventual rupture of the nuclear membrane and lamin network due to this entropic pressure were observed by fluorescence imaging. This swelling was accompanied by nuclear softening, an effect that we quantified by measuring the fluctuations of an optically trapped bead adhered onto the nucleus. We also measured the pressure at which the nuclear scaffold ruptured using an atomic force microscope cantilever. A simple theory based on a balance of forces in a swelling porous gel quantitatively explains the diffusive dynamics of swelling. Our experiments on decondensation of chromatin in nuclei suggest that its compaction is a critical parameter in controlling nuclear stability. PMID:18556763

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

    PubMed Central

    Daban, Joan-Ramon

    2015-01-01

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

  14. Chromatin-dependent cooperativity between constitutive and inducible activation domains in CREB.

    TOXLINE Toxicology Bibliographic Information

    Asahara H; Santoso B; Guzman E; Du K; Cole PA; Davidson I; Montminy M

    2001-12-01

    The cyclic AMP (cAMP)-responsive factor CREB induces target gene expression via constitutive (Q2) and inducible (KID, for kinase-inducible domain) activation domains that function synergistically in response to cellular signals. KID stimulates transcription via a phospho (Ser133)-dependent interaction with the coactivator paralogs CREB binding protein and p300, whereas Q2 recruits the TFIID complex via a direct association with hTAF(II)130. Here we investigate the mechanism underlying cooperativity between the Q2 domain and KID in CREB by in vitro transcription assay with naked DNA and chromatin templates containing the cAMP-responsive somatostatin promoter. The Q2 domain was highly active on a naked DNA template, and Ser133 phosphorylation had no additional effect on transcriptional initiation in crude extracts. Q2 activity was repressed on a chromatin template, however, and this repression was relieved by the phospho (Ser133) KID-dependent recruitment of p300 histone acetyltransferase activity to the promoter. In chromatin immunoprecipitation assays of NIH 3T3 cells, cAMP-dependent recruitment of p300 to the somatostatin promoter stimulated acetylation of histone H4. Correspondingly, overexpression of hTAFII130 potentiated CREB activity in cells exposed to cAMP, but had no effect on reporter gene expression in unstimulated cells. We propose that cooperativity between the KID and Q2 domains proceeds via a chromatin-dependent mechanism in which recruitment of p300 facilitates subsequent interaction of CREB with TFIID.

  15. Development of a novel flow cytometric approach to evaluate fish sperm chromatin using fixed samples

    USGS Publications Warehouse

    Jenkins, Jill A.

    2013-01-01

    The integrity of the paternal DNA is essential for the accurate transmission of genetic information, yet fertilization is not inhibited by chromatin breakage. Some methods are available for the sensitive detection of DNA damage and can be applied in studies of environmental toxicology, carcinogenesis, aging, and assisted reproduction techniques in both clinical and experimental settings. Because semen samples obtained from remote locations undergo chromatin damage prior to laboratory assessment, the present study was undertaken to evaluate treatments for effective chromatin staining in the development of a DNA fragmentation assay using fixed milt from yellow perch (Perca flavescens). Similar to the sperm chromatin structure assay (SCSA), susceptibility of nuclear DNA to acid-induced denaturation was measured by flow cytometry (FCM). Use of 10% buffered formalin for milt fixation allowed easier peak discrimination than 4% paraformaldehyde. The effects of time and temperature of incubation in 0.08 N HCl were evaluated in order to determine the ideal conditions for promoting DNA decondensation and making strand breaks more available for staining and detection by FCM. The best results were obtained with incubation at 37°C for 1 minute, followed by cold propidium iodide staining for 30 minutes.

  16. Chromatin supraorganization, DNA fragmentation, and cell death in snake erythrocytes.

    PubMed

    Miyamoto, Maristela; Vidal, Benedicto C; Mello, Maria Luiza S

    2005-02-01

    In nucleate erythrocytes of several vertebrate groups, the frequency and intensity of DNA fragmentation associated with programmed cell death vary considerably. Although hemoglobin efficiency may be related to erythrocyte life span, and hemoglobin types and erythrocyte life spans are assumed to vary in reptiles, no data on DNA fragmentation and chromatin organization as related to cell death exist for snakes. In the present study, chromatin supraorganization, DNA fragmentation, and cell death were investigated in four snake species (Crotalus durissus terrificus, Bothrops jararaca, Bothrops alternatus, and Bothrops neuwiedii), which differ in their geographical distribution and habitats, by using image analysis of Feulgen hydrolysis kinetics, the TUNEL assay, single-cell gel electrophoresis, and transmission electron microscopy. Relatively few circulating erythrocytes were found to be simultaneously committed to cell death, although there was some variation among the snake species. Conspicuous nuclear and cytoplasmic organelles suggestive of metabolic activity were seen ultrastructurally in most snake erythrocytes. The DNA of the snake erythrocyte chromatin was much more resistant to Feulgen acid hydrolysis (DNA depurination and breakdown) than that of young adult bullfrog erythrocytes, which had a high frequency and intensity of DNA fragmentation. Of the species studied, B. neuwiedii and C. d. terrificus showed the greatest resistance to Feulgen acid hydrolysis and to the DNA fragmentation, revealed by the TUNEL assay. Although B. neuwiedii also showed the lowest frequency of cells with more damaged DNA in the single-cell gel electrophoresis assay, C. d. terrificus had the highest frequency of damaged cells, possibly because of the abundance of alkaline-sensitive DNA sites. The results for DNA fragmentation and cell death in erythrocytes of B. jararaca and B. alternatus generally differed from those for C. d. terrificus and B. neuwiedii and may reflect differences in the biology of these species selected under different geographical habitats. The differences in erythrocyte cell biology reported here may be related to hemoglobin variants selected in the mentioned snake species and that would lead the cells to different resistances to unfavorable environmental conditions. PMID:15746963

  17. 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…

  18. 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…

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

    PubMed

    Eirín-López, José M

    2013-01-01

    The study of chromatin constitutes one of the most active research fields in life sciences, being subject to constant revisions that continuously redefine the state of the art in its knowledge. As every other rapidly changing field, chromatin biology requires clear and straightforward educational strategies able to efficiently translate such a vast body of knowledge to the classroom. With this aim, the present work describes a multidisciplinary computer lab designed to introduce undergraduate students to the dynamic nature of chromatin, within the context of the one semester course "Chromatin: Structure, Function and Evolution." This exercise is organized in three parts including (a) molecular evolutionary biology of histone families (using the H1 family as example), (b) histone structure and variation across different animal groups, and (c) effect of histone diversity on nucleosome structure and chromatin dynamics. By using freely available bioinformatic tools that can be run on common computers, the concept of chromatin dynamics is interactively illustrated from a comparative/evolutionary perspective. At the end of this computer lab, students are able to translate the bioinformatic information into a biochemical context in which the relevance of histone primary structure on chromatin dynamics is exposed. During the last 8 years this exercise has proven to be a powerful approach for teaching chromatin structure and dynamics, allowing students a higher degree of independence during the processes of learning and self-assessment. PMID:23401152

  20. Changing Chromatin Fiber Conformation by Nucleosome Repositioning

    PubMed Central

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

    2014-01-01

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

  1. DNA mediated chromatin pull-down for the study of chromatin replication

    PubMed Central

    Kliszczak, Anna E.; Rainey, Michael D.; Harhen, Brendan; Boisvert, Francois M.; Santocanale, Corrado

    2011-01-01

    Chromatin replication involves duplicating DNA while maintaining epigenetic information. These processes are critical for genome stability and for preserving cell-type identity. Here we describe a simple experimental approach that allows chromatin to be captured and its content analysed after in vivo replication and labeling of DNA by cellular DNA polymerases. We show that this technique is highly specific and that proteins bound to the replicated DNA can be analyzed by both immunological techniques and large scale mass spectrometry. As proof of concept we have used this novel procedure to begin investigating the relationship between chromatin protein composition and the temporal programme of DNA replication in human cells. It is expected that this technique will become a widely used tool to address how chromatin proteins assemble onto newly replicated DNA after passage of a replication fork and how chromatin maturation is coupled to DNA synthesis. PMID:22355613

  2. 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

  3. Targeting chromatin to improve radiation response

    PubMed Central

    Olcina, M M; O'Dell, S

    2015-01-01

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

  4. 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

  5. Chromatin organization marks exon-intron structure.

    PubMed

    Schwartz, Schraga; Meshorer, Eran; Ast, Gil

    2009-09-01

    An increasing body of evidence indicates that transcription and splicing are coupled, and it is accepted that chromatin organization regulates transcription. Little is known about the cross-talk between chromatin structure and exon-intron architecture. By analysis of genome-wide nucleosome-positioning data sets from humans, flies and worms, we found that exons show increased nucleosome-occupancy levels with respect to introns, a finding that we link to differential GC content and nucleosome-disfavoring elements between exons and introns. Analysis of genome-wide chromatin immunoprecipitation data in humans and mice revealed four specific post-translational histone modifications enriched in exons. Our findings indicate that previously described enrichment of H3K36me3 modifications in exons reflects a more fundamental phenomenon, namely increased nucleosome occupancy along exons. Our results suggest an RNA polymerase II-mediated cross-talk between chromatin structure and exon-intron architecture, implying that exon selection may be modulated by chromatin structure. PMID:19684600

  6. 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

  7. 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.

  8. DNA methylation topology: potential of a chromatin landmark for epigenetic drug toxicology

    PubMed Central

    Tajbakhsh, Jian

    2011-01-01

    Targeting chromatin and its basic components through epigenetic drug therapy has become an increased focus in the treatment of complex diseases. This boost calls for the implementation of high-throughput cell-based assays that exploit the increasing knowledge about epigenetic mechanisms and their interventions for genotoxicity testing of epigenetic drugs. 3D quantitative DNA methylation imaging is a novel approach for detecting drug-induced DNA demethylation and concurrent heterochromatin decondensation/reorganization in cells through the analysis of differential nuclear distribution patterns of methylcytosine and gDNA visualized by fluorescence and processed by machine-learning algorithms. Utilizing 3D DNA methylation patterns is a powerful precursor to a series of fully automatable assays that employ chromatin structure and higher organization as novel pharmacodynamic biomarkers for various epigenetic drug actions. PMID:22126294

  9. Complexation of DNA with positive spheres: Phase diagram of charge inversion and reentrant condensation

    NASA Astrophysics Data System (ADS)

    Nguyen, T. T.; Shklovskii, B. I.

    2001-10-01

    The phase diagram of a water solution of DNA and oppositely charged spherical macroions is studied. DNA winds around spheres to form beads-on-a-string complexes resembling the chromatin 10 nm fiber. At small enough concentration of spheres these "artificial chromatin" complexes are negative, while at large enough concentrations of spheres the charge of DNA is inverted by the adsorbed spheres. Charges of complexes stabilize their solutions. In the plane of concentrations of DNA and spheres the phases with positive and negative complexes are separated by another phase, which contains the condensate of neutral DNA-spheres complexes. Thus, when the concentration of spheres grows, DNA-spheres complexes experience condensation and resolubilization (or reentrant condensation). Phenomenological theory of the phase diagram of reentrant condensation and charge inversion is suggested. Parameters of this theory are calculated by microscopic theory. It is shown that an important part of the effect of a monovalent salt on the phase diagram can be described by the nontrivial renormalization of the effective linear charge density of DNA wound around a sphere, due to the Onsager-Manning condensation. We argue that our phenomenological phase diagram or reentrant condensation is generic to a large class of strongly asymmetric electrolytes. Possible implications of these results for the natural chromatin are discussed.

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

    PubMed

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

    2004-06-01

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

  11. 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

  12. Chromatin Regulators in Pancreas Development and Diabetes.

    PubMed

    Campbell, Stephanie A; Hoffman, Brad G

    2016-03-01

    The chromatin landscape of a cell is dynamic and can be altered by chromatin regulators that control nucleosome placement and DNA or histone modifications. Together with transcription factors, these complexes help dictate the transcriptional output of a cell and, thus, balance cell proliferation and differentiation while restricting tissue-specific gene expression. In this review, we describe current research on chromatin regulators and their roles in pancreas development and the maintenance of mature ? cell function, which, once elucidated, will help us better understand how ? cell differentiation occurs and is maintained. These studies have so far implicated proteins from several complexes that regulate DNA methylation, nucleosome remodeling, and histone acetylation and methylation that could become promising targets for diabetes therapy and stem cell differentiation. PMID:26783078

  13. Chromatin dynamics during differentiation of myeloid cells.

    PubMed

    Schönheit, Jörg; Leutz, Achim; Rosenbauer, Frank

    2015-02-13

    Cellular commitment to differentiation requires a tightly synchronized, spatial-temporal interaction of regulatory proteins with the basic DNA and chromatin. A complex network of mechanisms involving induction of lineage instructive transcription factors, installation or removal of histone modifications and changes in the DNA methylation pattern locally orchestrate the three-dimensional chromatin structure and determine cell fate. Maturation of myeloid lineages from hematopoietic stem cells has emerged as a powerful model to study those principles of chromatin mechanisms in cellular differentiation and lineage fate selection. This review summarizes recent knowledge and puts forward novel ideas on how dynamics in the epigenetic landscape of myeloid cells shape the development, immune activation and leukemic transformation outcome. PMID:25172539

  14. 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

  15. Chromatin Immunoprecipitation in Adult Zebrafish Red Cells

    PubMed Central

    Trompouki, Eirini; Bowman, Teresa V.; DiBiase, Anthony; Zhou, Yi; Zon, Leonard I.

    2016-01-01

    Zebrafish has been used for many years as a model to study development and disease. The ability of zebrafish to produce thousand of embryos in a synchronous manner has made zebrafish an invaluable tool for genetic and chemical screens. Since its emergence as an important model organism the molecular tools for studying zebrafish have been limited. In this chapter, we describe a simple method to identify DNA binding sites and chromatin architecture in erythrocytes from adult zebrafish using chromatin immunoprecipitation coupled with next generation sequencing. This technique has been used extensively and successfully in other systems and it will be a useful tool for studying epigenetics in zebrafish. PMID:21924172

  16. Chromatin's thread to alternative splicing regulation.

    PubMed

    Iannone, Camilla; Valcárcel, Juan

    2013-12-01

    Intron removal (pre-mRNA splicing) is a necessary step for expression of most genes in higher eukaryotes. Alternative splice site selection is a prevalent mechanism that diversifies genome outputs and offers ample opportunities for gene regulation in these organisms. Pre-mRNA splicing occurs co-transcriptionally and is influenced by features in chromatin structure, including nucleosome density and epigenetic modifications. We review here the molecular mechanisms by which the reciprocal interplay between chromatin and RNA processing can contribute to alternative splicing regulation. PMID:23912688

  17. Dynamics of Histone Tails within Chromatin

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  18. Insights into Role of Bromodomain, Testis-specific (Brdt) in Acetylated Histone H4-dependent Chromatin Remodeling in Mammalian Spermiogenesis*

    PubMed Central

    Dhar, Surbhi; Thota, Anusha; Rao, Manchanahalli Rangaswamy Satyanarayana

    2012-01-01

    Mammalian spermiogenesis is of considerable biological interest especially due to the unique chromatin remodeling events that take place during spermatid maturation. Here, we have studied the expression of chromatin remodeling factors in different spermatogenic stages and narrowed it down to bromodomain, testis-specific (Brdt) as a key molecule participating in chromatin remodeling during rat spermiogenesis. Our immunocytochemistry experiments reveal that Brdt colocalizes with acetylated H4 in elongating spermatids. Remodeling assays showed an acetylation-dependent but ATP-independent chromatin reorganization property of Brdt in haploid round spermatids. Furthermore, Brdt interacts with Smarce1, a member of the SWI/SNF family. We have studied the genomic organization of smarce1 and identified that it has two splice variants expressed during spermatogenesis. The N terminus of Brdt is involved in the recognition of Smarce1 as well as in the reorganization of hyperacetylated round spermatid chromatin. Interestingly, the interaction between Smarce1 and Brdt increases dramatically upon histone hyperacetylation both in vitro and in vivo. Thus, our results indicate this interaction to be a vital step in the chromatin remodeling process during mammalian spermiogenesis. PMID:22215678

  19. Chromatin configuration within the germinal vesicle of horse oocytes: changes post mortem and relationship to meiotic and developmental competence.

    PubMed

    Hinrichs, K; Choi, Y H; Love, L B; Varner, D D; Love, C C; Walckenaer, B E

    2005-05-01

    We evaluated the relationship of initial chromatin configuration to time of oocyte recovery and to nuclear maturation after culture in horse oocytes having compact (Cp) and expanded (Ex) cumuli. In addition, we evaluated the effect of oocyte type, time of recovery, and duration of culture on blastocyst development after intracytoplasmic sperm injection. In oocytes collected within 1 h of slaughter, fibrillar and intermediate chromatin configurations were more prevalent in Cp than in Ex oocytes (68% and 12%, respectively). In Cp oocytes collected after a 5- to 9-h delay, the proportions in the fibrillar and intermediate configurations decreased significantly, and the proportions of degenerating and homogeneously fluorescent configurations increased. When cultured, 20% of oocytes classified as having fibrillar chromatin resumed meiosis, whereas 82% of intermediate and 81% to 86% of condensed chromatin oocytes did so. Meiotic resumption was higher in oocytes recovered immediately after slaughter, but these oocytes took longer to mature. Duration of maturation significantly affected blastocyst development rates in Cp oocytes recovered after a delay (13% and 38% for oocytes matured 24 and 36 h, respectively). Oocytes recovered after a delay had higher blastocyst development rates than did those collected immediately after slaughter. We conclude that the fibrillar and intermediate chromatin configurations may degenerate during ovary storage, resulting in decreased maturation rates, especially of Cp oocytes. Time of oocyte recovery and duration of maturation significantly affect the rate of blastocyst development. Oocytes with Cp and Ex cumuli have similar developmental competence to the blastocyst stage. PMID:15647456

  20. Regulation of spindle and chromatin dynamics during early and late stages of oocyte maturation by aurora kinases.

    PubMed

    Swain, Jason E; Ding, Jun; Wu, Jingwen; Smith, Gary D

    2008-05-01

    Examination of factors regulating oocyte chromatin remodeling is crucial to circumvent embryonic aneuploidy and resulting defects. Aurora kinases (AURK) are involved in regulation of chromatin remodeling, however, little attention has been paid to AURKs in regard to oocyte maturation. Meiotically incompetent mouse oocytes contain transcripts for all three Aurk isoforms: A, B and C. Upon achieving meiotic competence, oocytes showed significant increases in transcript levels of all three Aurk isoforms and transcript levels remained unchanged as oocytes progressed through meiosis, with AurkA being the predominant isoform. Inhibition of oocyte AURKs during the prophase-metaphase I (MI) transition via inhibitor ZM447439 (ZM) had no effect on germinal vesicle breakdown. However, meiotic spindles were malformed, and microtubule organizing centers and chromatin were scattered. Chromosomal spreads of MI oocytes indicated AURK inhibition resulted in abnormal chromosome condensation. Furthermore, inhibition of AURK during prophase I-MII prevented completion of MII and extrusion of the polar body. Inhibition of AURKs during the MI-MII transition resulted in significantly fewer cells progressing to MII and induced aberrant chromatin remodeling. Further analysis indicated that inhibition of AURKs resulted in absence of histone-H3 phosphorylation at serine 10 and 28. These data suggest a ZM-sensitive AURK may be an oocyte histone-H3 kinase capable of regulating chromatin remodeling throughout oocyte meiosis, both pre- and post-MI. PMID:18353803

  1. HDAC Up-Regulation in Early Colon Field Carcinogenesis Is Involved in Cell Tumorigenicity through Regulation of Chromatin Structure

    PubMed Central

    Stypula-Cyrus, Yolanda; Damania, Dhwanil; Kunte, Dhananjay P.; Cruz, Mart Dela; Subramanian, Hariharan; Roy, Hemant K.; Backman, Vadim

    2013-01-01

    Normal cell function is dependent on the proper maintenance of chromatin structure. Regulation of chromatin structure is controlled by histone modifications that directly influence chromatin architecture and genome function. Specifically, the histone deacetylase (HDAC) family of proteins modulate chromatin compaction and are commonly dysregulated in many tumors, including colorectal cancer (CRC). However, the role of HDAC proteins in early colorectal carcinogenesis has not been previously reported. We found HDAC1, HDAC2, HDAC3, HDAC5, and HDAC7 all to be up-regulated in the field of human CRC. Furthermore, we observed that HDAC2 up-regulation is one of the earliest events in CRC carcinogenesis and observed this in human field carcinogenesis, the azoxymethane-treated rat model, and in more aggressive colon cancer cell lines. The universality of HDAC2 up-regulation suggests that HDAC2 up-regulation is a novel and important early event in CRC, which may serve as a biomarker. HDAC inhibitors (HDACIs) interfere with tumorigenic HDAC activity; however, the precise mechanisms involved in this process remain to be elucidated. We confirmed that HDAC inhibition by valproic acid (VPA) targeted the more aggressive cell line. Using nuclease digestion assays and transmission electron microscopy imaging, we observed that VPA treatment induced greater changes in chromatin structure in the more aggressive cell line. Furthermore, we used the novel imaging technique partial wave spectroscopy (PWS) to quantify nanoscale alterations in chromatin. We noted that the PWS results are consistent with the biological assays, indicating a greater effect of VPA treatment in the more aggressive cell type. Together, these results demonstrate the importance of HDAC activity in early carcinogenic events and the unique role of higher-order chromatin structure in determining cell tumorigenicity. PMID:23724067

  2. Painting by Numbers: Increasing the Parts List for Chromatin Domains

    PubMed Central

    Chen, Hsiuyi V.; Rando, Oliver J.

    2014-01-01

    In this issue of Molecular Cell, van Bemmel and colleagues (2013) report the genome-wide mapping of 42 novel chromatin factors, systematically identifying new components of the various chromatin domains present in fly cells. PMID:23438859

  3. Roles of chromatin factors in C. elegans development.

    PubMed Central

    Cui, Mingxue; Han, Min

    2007-01-01

    It is now well established that cells modify chromatin to establish transcriptionally active or inactive chromosomal regions. Such regulation of the chromatin structure is essential for the proper development of organisms. C. elegans is a powerful organism for exploring the developmental role of chromatin factors and their regulation. This chapter presents an overview of recent studies on chromatin factors in C. elegans with a description of their key roles in a variety of cellular and developmental processes. PMID:18050494

  4. Monte Carlo simulation of chromatin stretching

    NASA Astrophysics Data System (ADS)

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

    2006-04-01

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

  5. Chromatin Modifications Associated With Diabetes and Obesity.

    PubMed

    Schones, Dustin E; Leung, Amy; Natarajan, Rama

    2015-07-01

    The incidence of obesity across the globe has doubled over the past several decades, leading to escalating rates of diabetes mellitus, cardiovascular disease, and other complications. Given this dramatic rise in disease incidence, understanding the cause of these diseases is therefore of paramount importance. Metabolic diseases, such as obesity and diabetes mellitus, result from a multitude of genetic and environmental factors. Although the genetic basis of these diseases has been extensively studied, the molecular pathways whereby environmental factors influence disease progression are only beginning to be understood. One manner by which environmental factors can contribute to disease progression is through modifications to chromatin. The highly structured packaging of the genome into the nucleus through chromatin has been shown to be fundamental to tissue-specific gene regulation. Modifications to chromatin can regulate gene expression and are involved in a myriad of biological functions, and hence, disruption of these modifications is central to many human diseases. These modifications can furthermore be epigenetic in nature, thereby contributing to prolonged disease risk. Recent work has demonstrated that modifications to chromatin are associated with the progression of both diabetes mellitus and obesity, which is the subject of this review. PMID:26044585

  6. 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

  7. Oncogene-mediated alterations in chromatin conformation

    PubMed Central

    Rickman, David S.; Soong, T. David; Moss, Benjamin; Mosquera, Juan Miguel; Dlabal, Jan; Terry, Stéphane; MacDonald, Theresa Y.; Tripodi, Joseph; Bunting, Karen; Najfeld, Vesna; Demichelis, Francesca; Melnick, Ari M.; Elemento, Olivier; Rubin, Mark A.

    2012-01-01

    Emerging evidence suggests that chromatin adopts a nonrandom 3D topology and that the organization of genes into structural hubs and domains affects their transcriptional status. How chromatin conformation changes in diseases such as cancer is poorly understood. Moreover, how oncogenic transcription factors, which bind to thousands of sites across the genome, influence gene regulation by globally altering the topology of chromatin requires further investigation. To address these questions, we performed unbiased high-resolution mapping of intra- and interchromosome interactions upon overexpression of ERG, an oncogenic transcription factor frequently overexpressed in prostate cancer as a result of a gene fusion. By integrating data from genome-wide chromosome conformation capture (Hi-C), ERG binding, and gene expression, we demonstrate that oncogenic transcription factor overexpression is associated with global, reproducible, and functionally coherent changes in chromatin organization. The results presented here have broader implications, as genomic alterations in other cancer types frequently give rise to aberrant transcription factor expression, e.g., EWS-FLI1, c-Myc, n-Myc, and PML-RARα. PMID:22615383

  8. Inferential modeling of 3D chromatin structure

    PubMed Central

    Wang, Siyu; Xu, Jinbo; Zeng, Jianyang

    2015-01-01

    For eukaryotic cells, the biological processes involving regulatory DNA elements play an important role in cell cycle. Understanding 3D spatial arrangements of chromosomes and revealing long-range chromatin interactions are critical to decipher these biological processes. In recent years, chromosome conformation capture (3C) related techniques have been developed to measure the interaction frequencies between long-range genome loci, which have provided a great opportunity to decode the 3D organization of the genome. In this paper, we develop a new Bayesian framework to derive the 3D architecture of a chromosome from 3C-based data. By modeling each chromosome as a polymer chain, we define the conformational energy based on our current knowledge on polymer physics and use it as prior information in the Bayesian framework. We also propose an expectation-maximization (EM) based algorithm to estimate the unknown parameters of the Bayesian model and infer an ensemble of chromatin structures based on interaction frequency data. We have validated our Bayesian inference approach through cross-validation and verified the computed chromatin conformations using the geometric constraints derived from fluorescence in situ hybridization (FISH) experiments. We have further confirmed the inferred chromatin structures using the known genetic interactions derived from other studies in the literature. Our test results have indicated that our Bayesian framework can compute an accurate ensemble of 3D chromatin conformations that best interpret the distance constraints derived from 3C-based data and also agree with other sources of geometric constraints derived from experimental evidence in the previous studies. The source code of our approach can be found in https://github.com/wangsy11/InfMod3DGen. PMID:25690896

  9. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

  10. An autonomous chromatin/DNA-PK mechanism for localized DNA damage signaling in mammalian cells

    PubMed Central

    Muñoz, Denise P.; Kawahara, Misako; Yannone, Steven M.

    2013-01-01

    Rapid phosphorylation of histone variant H2AX proximal to DNA breaks is an initiating event and a hallmark of eukaryotic DNA damage responses. Three mammalian kinases are known to phosphorylate H2AX in response to DNA damage. However, the mechanism(s) for damage-localized phosphorylation remains incompletely understood. The DNA-dependent protein kinase (DNA-PK) is the most abundant H2AX-modifying kinases and uniquely activated by binding DNA termini. Here, we have developed a novel approach to examine enzyme activity and substrate properties by executing biochemical assays on intact cellular structures. We apply this approach to examine the mechanisms of localized protein modification in chromatin within fixed cells. DNA-PK retains substrate specificity and independently generates break-localized ?H2AX foci in chromatin. In situ DNA-PK activity recapitulates localization and intensity of in vivo H2AX phosphorylation and requires no active cellular processes. Nuclease treatments or addition of exogenous DNA resulted in genome-wide H2AX phosphorylation, showing that DNA termini dictated the locality of H2AX phosphorylation in situ. DNA-PK also reconstituted focal phosphorylation of structural maintenance of chromatin protein 1, but not activating transcription factor 2. Allosteric regulation of DNA-PK by DNA termini protruding from chromatin constitutes an autonomous mechanism for break-localized protein phosphorylation that generates sub-nuclear foci. We discuss generalized implications of this mechanism in localizing mammalian DNA damage responses. PMID:23325849

  11. Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells

    PubMed Central

    Ram, Oren; Goren, Alon; Amit, Ido; Shoresh, Noam; Yosef, Nir; Ernst, Jason; Kellis, Manolis; Gymrek, Melissa; Issner, Robbyn; Coyne, Michael; Durham, Timothy; Zhang, Xiaolan; Donaghey, Julie; Epstein, Charles B.; Regev, Aviv; Bernstein, Bradley E.

    2012-01-01

    SUMMARY Hundreds of Chromatin Regulators (CRs) control chromatin structure and function by catalyzing and binding histone modifications, yet the rules governing these key processes remain obscure. Here, we present a systematic approach to infer CR function. We developed ChIP-string, a meso-scale assay that combines chromatin immunoprecipitation with a signature readout of 487 representative loci. We applied ChIP-string to screen 145 antibodies, thereby identifying effective reagents, which we used to map the genome-wide binding of 29 CRs in two cell types. We found that specific combinations of CRs co-localize in characteristic patterns at distinct chromatin environments, genes of coherent functions and distal regulatory elements. When comparing between cell types, CRs redistribute to different loci, but maintain their modular and combinatorial associations. Our work provides a multiplex method that substantially enhances the ability to monitor CR binding, presents a large resource of CR maps, and reveals common principles for combinatorial CR function. PMID:22196736

  12. Drosophila domino Exhibits Genetic Interactions with a Wide Spectrum of Chromatin Protein-Encoding Loci

    PubMed Central

    Ellis, Kaitlyn; Friedman, Chloe; Yedvobnick, Barry

    2015-01-01

    The Drosophila domino gene encodes protein of the SWI2/SNF2 family that has widespread roles in transcription, replication, recombination and DNA repair. Here, the potential relationship of Domino protein to other chromatin-associated proteins has been investigated through a genetic interaction analysis. We scored for genetic modification of a domino wing margin phenotype through coexpression of RNAi directed against a set of previously characterized and more newly characterized chromatin-encoding loci. A set of other SWI2/SNF2 loci were also assayed for interaction with domino. Our results show that the majority of tested loci exhibit synergistic enhancement or suppression of the domino wing phenotype. Therefore, depression in domino function sensitizes the wing margin to alterations in the activity of numerous chromatin components. In several cases the genetic interactions are associated with changes in the level of cell death measured across the dorsal-ventral margin of the wing imaginal disc. These results highlight the broad realms of action of many chromatin proteins and suggest significant overlap with Domino function in fundamental cell processes, including cell proliferation, cell death and cell signaling. PMID:26555684

  13. Increased chromatin fragmentation and reduced acrosome integrity in spermatozoa of red deer from lead polluted sites.

    PubMed

    Castellanos, Pilar; del Olmo, Enrique; Fernández-Santos, M Rocío; Rodríguez-Estival, Jaime; Garde, J Julián; Mateo, Rafael

    2015-02-01

    Vertebrates are constantly exposed to a diffuse pollution of heavy metals existing in the environment, but in some cases, the proximity to emission sources like mining activity increases the risk of developing adverse effects of these pollutants. Here we have studied lead (Pb) levels in spermatozoa and testis, and chromatin damage and levels of endogenous antioxidant activity in spermatozoa of red deer (Cervus elaphus) from a Pb mining area (n=37) and a control area (n=26). Deer from the Pb-polluted area showed higher Pb levels in testis parenchyma, epididymal cauda and spermatozoa, lower values of acrosome integrity, higher activity of glutathione peroxidase (GPx) and higher values of DNA fragmentation (X-DFI) and stainability (HDS) in sperm than in the control area. These results indicate that mining pollution can produce damage on chromatin and membrane spermatozoa in wildlife. The study of chromatin fragmentation has not been studied before in spermatozoa of wildlife species, and the sperm chromatin structure assay (SCSA) has been revealed as a successful tool for this purpose in species in which the amount of sperm that can be collected is very limited. PMID:25306093

  14. 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.

  15. Ensuring condensate recovery efficiency.

    PubMed

    Mayoh, Paul

    2012-09-01

    According to steam system specialist, Spirax Sarco, 'condensate contains about a quarter of the energy of the steam from which it came--a significant amount of heat available to an energy centre'. Ensuring that existing condensate recovery systems are as efficient as possible is therefore 'key' to reducing energy centre costs, the company says. Paul Mayoh, product manager, Spirax Sarco, considers ways to ensure that as much condensate as possible is re-used. PMID:23009016

  16. 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.

  17. O-GlcNAc Transferase Regulates Mitotic Chromatin Dynamics*

    PubMed Central

    Sakabe, Kaoru; Hart, Gerald W.

    2010-01-01

    Mitosis must faithfully divide the genome such that each progeny inherits the same genetic material. DNA condensation is crucial in ensuring that chromosomes are correctly attached to the mitotic spindle for segregation, preventing DNA breaks or constrictions from the contractile ring. Histones form an octameric complex of basic proteins important in regulating DNA organization and accessibility. Histone post-translational modifications are altered during mitosis, although the roles of these post-translational modifications remain poorly characterized. Here, we report that N-acetylglucosamine (O-GlcNAc) transferase (OGT), the enzyme catalyzing the addition of O-GlcNAc moieties to nuclear and cytoplasmic proteins at serine and threonine residues, regulates some aspects of mitotic chromatin dynamics. OGT protein amounts decrease during M phase. Modest overexpression of OGT alters mitotic histone post-translational modifications at Lys-9, Ser-10, Arg-17, and Lys-27 of histone H3. Overexpression of OGT also prevents mitotic phosphorylation of coactivator-associated arginine methyltransferase 1 (CARM1) and prevents its correct cellular localization during mitosis. Moreover, OGT overexpression results in an increase in abnormal chromosomal bridge formation. Together, these results show that regulating the amount of OGT during mitosis is important in ensuring correct chromosomal segregation during mitosis. PMID:20805223

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

    PubMed

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

    2015-12-17

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

  19. 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, ? -phage (16? 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 2-4? m in contrast to larger aggregates of size 10-50? 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.

  20. Regulating the Chromatin Landscape: Structural and Mechanistic Perspectives

    PubMed Central

    Bartholomew, Blaine

    2014-01-01

    A large family of chromatin remodelers that noncovalently modify chromatin are crucial in cell development and differentiation. They are often the targets of cancer, neurological disorders, and other human diseases. These complexes alter nucleosome positioning, higher-order chromatin structure, and nuclear organization. They also assemble chromatin, exchange out histone variants, and disassemble chromatin at defined locations. We review aspects of the structural organization of these complexes, the functional properties of their protein domains, and variation between complexes. We also address the mechanistic details of these complexes in mobilizing nucleosomes and altering chromatin structure. A better understanding of these issues will be vital for further analyses of subunits of these chromatin remodelers, which are being identified as targets in human diseases by NGS (next-generation sequencing). PMID:24606138

  1. RIGS (repeat-induced gene silencing) in Arabidopsis is transcriptional and alters chromatin configuration.

    PubMed Central

    Ye, F; Signer, E R

    1996-01-01

    We have previously reported repeat-induced gene silencing (RIGS) in Arabidopsis, in which transgene expression may be silenced epigenetically when repeated sequences are present. Among an allelic series of lines comprising a primary transformant and various recombinant progeny carrying different numbers of drug resistance gene copies at the same locus, silencing was found to depend strictly on repeated sequences and to correlate with an absence of steady-state mRNA. We now report characterization, in nuclei isolated from the same transgenic lines, of gene expression by nuclear run-on assay and of chromatin structure by nuclease protection assay. We find that silencing is correlated with absence of run-on transcripts, indicating that expression is silenced at the level of transcription. We find further that silencing is also correlated with increased resistance to both DNase I and micrococcal nuclease, indicating that the silenced state reflects a change in chromatin configuration. We propose that silencing results when a locally paired region of homologous repeated nucleotide sequences is flanked by unpaired heterologous DNA, which leads chromatin to adopt a local configuration that is difficult to transcribe, and possibly akin to heterochromatin. Images Fig. 2 Fig. 3 Fig. 3 Fig. 4 PMID:8855276

  2. Chromatin structure and DNA damage repair

    PubMed Central

    Dinant, Christoffel; Houtsmuller, Adriaan B; Vermeulen, Wim

    2008-01-01

    The integrity of the genome is continuously challenged by both endogenous and exogenous DNA damaging agents. These damaging agents can induce a wide variety of lesions in the DNA, such as double strand breaks, single strand breaks, oxidative lesions and pyrimidine dimers. The cell has evolved intricate DNA damage response mechanisms to counteract the genotoxic effects of these lesions. The two main features of the DNA damage response mechanisms are cell-cycle checkpoint activation and, at the heart of the response, DNA repair. For both damage signalling and repair, chromatin remodelling is most likely a prerequisite. Here, we discuss current knowledge on chromatin remodelling with respect to the cellular response to DNA damage, with emphasis on the response to lesions resolved by nucleotide excision repair. We will discuss the role of histone modifications as well as their displacement or exchange in nucleotide excision repair and make a comparison with their requirement in transcription and double strand break repair. PMID:19014481

  3. Advanced microscopy methods for visualizing chromatin structure.

    PubMed

    Lakadamyali, Melike; Cosma, Maria Pia

    2015-10-01

    In the recent years it has become clear that our genome is not randomly organized and its architecture is tightly linked to its function. While genomic studies have given much insight into genome organization, they mostly rely on averaging over large populations of cells, are not compatible with living cells and have limited resolution. For studying genome organization in single living cells, microscopy is indispensable. In addition, the visualization of biological structures helps to understand their function. Up to now, fluorescence microscopy has allowed us to probe the larger scale organization of chromosome territories in the micron length scales, however, the smaller length scales remained invisible due to the diffraction limited spatial resolution of fluorescence microscopy. Thanks to the advent of super-resolution microscopy methods, we are finally starting to be able to probe the nanoscale organization of chromatin in vivo and these methods have the potential to greatly advance our knowledge about chromatin structure and function relationship. PMID:25896023

  4. Distinct features of lamin A-interacting chromatin domains mapped by ChIP-sequencing from sonicated or micrococcal nuclease-digested chromatin.

    PubMed

    Lund, Eivind G; Duband-Goulet, Isabelle; Oldenburg, Anja; Buendia, Brigitte; Collas, Philippe

    2015-01-01

    The nuclear lamina has been shown to interact with the genome through lamina-associated domains (LADs). LADs have been identified by DamID, a proximity labeling assay, and more recently by chromatin immunoprecipitation-sequencing (ChIP-seq) of A- and B-type lamins. LADs form megabase-size domains at the nuclear periphery, they are gene-poor and mostly heterochromatic. Here, we show that the mode of chromatin fragmentation for ChIP, namely bath sonication or digestion with micrococcal nuclease (MNase), leads to the discovery of common but also distinct sets of lamin-interacting domains, or LiDs. Using ChIP-seq, we show the existence of lamin A/C (LMNA) LiDs with distinct gene contents, histone composition enrichment and relationships to lamin B1-interacting domains. The extent of genome coverage of lamin A/C (LMNA) LiDs in sonicated or MNase-digested chromatin is similar (∼730 megabases); however over half of these domains are uniquely detected in sonicated or MNase-digested chromatin. Sonication-specific LMNA LiDs are gene-poor and devoid of a broad panel of histone modifications, while MNase-specific LMNA LiDs are of higher gene density and are enriched in H3K9me3, H3K27me3 and in histone variant H2A.Z. LMNB1 LiDs are gene-poor and show no or little enrichment in these marks. Comparison of published LMNB1 DamID LADs with LMNB1 and LMNA LiDs identified here by ChIP-seq further shows that LMNA can associate with 'open' chromatin domains displaying euchromatin characteristics, and which are not associated with LMNB1. The differential genomic and epigenetic properties of lamin-interacting domains reflect the existence of distinct LiD populations identifiable in different chromatin contexts, including nuclease-accessible regions presumably localized in the nuclear interior. PMID:25602132

  5. 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.

  6. Single-molecule and population probing of chromatin structure using DNA methyltransferases

    PubMed Central

    Kilgore, Jessica A.; Hoose, Scott A.; Gustafson, Tanya L.; Porter, Weston; Kladde, Michael P.

    2007-01-01

    Probing chromatin structure with DNA methyltransferases offers advantages over more commonly used nuclease-based and chromatin immunoprecipitation methods for detection of nucleosomes and non-histone protein-DNA interactions. Here we describe two related methods in which the readout of MTase accessibility is obtained by assaying 5-methylcytosine in DNA through the PCR-based technique of bisulfite genomic sequencing. The methyltransferase accessibility protocol (MAP) determines the relative frequency at which the enzyme accesses each of its target sites over an entire population of PCR amplified product. While MAP yields much quantitative information about relative accessibility of a region of chromatin, a complementary single-molecule view of methyltransferase accessibility, termed MAP for individual templates (MAP-IT), is provided by analysis of cloned PCR products. Absolute rather than relative methylation frequencies in a region are obtained by summing the methylation status at each site over a cohort of clones. Moreover, as the integrity of individual molecules is maintained in MAP-IT, unique information about the distribution of multiple footprints along continuous regions is gleaned. In principle, the population MAP and single-molecule MAP-IT strategies can be used to analyze chromatin structure in a variety of model systems. Here we describe the application of MAP in living S. cerevisiae cells and MAP-IT in the analysis of a mammalian tumor suppressor gene in nuclei. This application of MAP-IT provides the first means to simultaneously determine CpG methylation of mammalian genes and their overlying chromatin structure in the same single DNA molecule. PMID:17309843

  7. Default assembly of early adenovirus chromatin

    SciTech Connect

    Spector, David J. . E-mail: dspector@psu.edu

    2007-03-01

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

  8. Titration and hysteresis in epigenetic chromatin silencing

    NASA Astrophysics Data System (ADS)

    Dayarian, Adel; Sengupta, Anirvan M.

    2013-06-01

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

  9. Chromatin signature of widespread monoallelic expression

    PubMed Central

    Nag, Anwesha; Savova, Virginia; Fung, Ho-Lim; Miron, Alexander; Yuan, Guo-Cheng; Zhang, Kun; Gimelbrant, Alexander A

    2013-01-01

    In mammals, numerous autosomal genes are subject to mitotically stable monoallelic expression (MAE), including genes that play critical roles in a variety of human diseases. Due to challenges posed by the clonal nature of MAE, very little is known about its regulation; in particular, no molecular features have been specifically linked to MAE. In this study, we report an approach that distinguishes MAE genes in human cells with great accuracy: a chromatin signature consisting of chromatin marks associated with active transcription (H3K36me3) and silencing (H3K27me3) simultaneously occurring in the gene body. The MAE signature is present in ?20% of ubiquitously expressed genes and over 30% of tissue-specific genes across cell types. Notably, it is enriched among key developmental genes that have bivalent chromatin structure in pluripotent cells. Our results open a new approach to the study of MAE that is independent of polymorphisms, and suggest that MAE is linked to cell differentiation. DOI: http://dx.doi.org/10.7554/eLife.01256.001 PMID:24381246

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. ROLE OF CHROMATIN ACCESSIBILITY IN THE OCCUPANCY AND TRANSCRIPTION OF THE INSULIN GENE BY THE PANCREATIC TRANSCRIPTION FACTOR PDX-1

    PubMed Central

    Francis, Joshua; Babu, Daniella A.; Deering, Tye G.; Chakrabarti, Swarup K.; Garmey, James C.; Evans-Molina, Carmella; Taylor, David G.; Mirmira, Raghavendra G.

    2010-01-01

    Pdx-1 is a hox-like transcription factor that is responsible for the activation of the insulin gene. Prior studies have demonstrated the interaction in vitro of Pdx-1 with short (20-40 nucleotide) DNA fragments corresponding to “A boxes” of the insulin promoter. Precisely how Pdx-1 binds to DNA in the complex milieu of chromatin, however, has never been studied. Here, we explored how Pdx-1-DNA interactions might be influenced by chromatin accessibility at the insulin gene in β cells (βTC3) vs. pancreatic ductal cells (mPAC). We demonstrate that Pdx-1 occupies the endogenous insulin promoter in βTC3 cells but not in mPAC cells, a finding that is independent of the intracellular Pdx-1 protein concentration. Based on micrococcal nuclease protection assays, the difference in promoter binding between the two cell types appears to be secondary to chromatin accessibility at predicted Pdx-1 binding sites between bp −126 to −296 (relative to the transcriptional start site) of the insulin promoter. Binding studies using pruified Pdx-1 and reconsituted chromatin in vitro suggest that the positioning of a nucleosome(s) within this crucial region of the promoter might account for differences in chromatin accessibility. Consistent with these observations, fluorescence colocalization studies show that Pdx-1 does not occupy regions of compacted, nucleosome-rich chromatin within the nucleus. Our findings suggest a model whereby insulin transcription in the β cell is at least partially facilitated by enahanced chromatin accessibility within a crucial regulatory region between bp −126 to −296, thereby permitting occupancy by transactivators such as Pdx-1. PMID:16901969

  15. The chromatin remodeling complex NoRC targets HDAC1 to the ribosomal gene promoter and represses RNA polymerase I transcription

    PubMed Central

    Zhou, Yonggang; Santoro, Raffaella; Grummt, Ingrid

    2002-01-01

    Mammalian chromatin remodeling complexes are involved in both activation and repression of transcription. Here, we show that NoRC, a SNF2h- containing nucleolar chromatin remodeling complex, represses ribosomal gene transcription. NoRC-mediated rDNA silencing was alleviated by trichostatin A, indicating that histone deacetylation is causally involved in silencing. Chromatin immunoprecipitation experiments demonstrate that overexpression of TIP5, the large subunit of NoRC, mediates deacetylation of nucleosomes in the vicinity of the rDNA promoter. Protein–protein interaction assays reveal association of TIP5 with the histone deacetylase HDAC1 in vivo and in vitro. Deletion of the C-terminal PHD finger and bromodomain abolishes the interaction of TIP5 and HDAC1, and abrogates transcriptional repression. The results suggest that NoRC silences the rDNA locus by targeting the SIN3 corepressor complex to the rDNA promoter, thereby establishing a repressed chromatin structure. PMID:12198165

  16. The chromatin remodeling complex NoRC targets HDAC1 to the ribosomal gene promoter and represses RNA polymerase I transcription.

    PubMed

    Zhou, Yonggang; Santoro, Raffaella; Grummt, Ingrid

    2002-09-01

    Mammalian chromatin remodeling complexes are involved in both activation and repression of transcription. Here, we show that NoRC, a SNF2h- containing nucleolar chromatin remodeling complex, represses ribosomal gene transcription. NoRC-mediated rDNA silencing was alleviated by trichostatin A, indicating that histone deacetylation is causally involved in silencing. Chromatin immunoprecipitation experiments demonstrate that overexpression of TIP5, the large subunit of NoRC, mediates deacetylation of nucleosomes in the vicinity of the rDNA promoter. Protein-protein interaction assays reveal association of TIP5 with the histone deacetylase HDAC1 in vivo and in vitro. Deletion of the C-terminal PHD finger and bromodomain abolishes the interaction of TIP5 and HDAC1, and abrogates transcriptional repression. The results suggest that NoRC silences the rDNA locus by targeting the SIN3 corepressor complex to the rDNA promoter, thereby establishing a repressed chromatin structure. PMID:12198165

  17. Chromatin-associated protein phosphokinases of rat ventral prostate. Characteristics and effects of androgenic status.

    PubMed

    Ahmed, K; Wilson, M J

    1975-03-25

    Protein phosphokinase activity endogenous to rat ventral prostate chromatin was assayed by using edphosphophosvitin as an exogenous substrate. For maximal activity of the kinase reaction, the presence of 200 mM NaCl, 5 mM MgCl2, and 1 mM dithiothreitol was essential. Two apparent pH optima were observed, a broad one between pH 7 and 7.4, and one at pH 7.89. At pH 7.4 the apparent Km for 31% dephosphophosvitin was 0.3 mg per ml. With respect to ATP, two apparent Km values (0.04 and 0.41 mM) were found. The kinase activity was minimal toward exogenous histones when used as substrates (3% for lysine-rich and 0.3% for arginine-rich (f3) histones, compared with dephosphophosvitin controls). The protein phosphokinases were not significantly stimulated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) when histones used as substrate. With dephosphophosvitin as substrate, cyclic AMP produced a small inhibition (5 to 15%). Orchiectomy of adult rats resulted in a rapid decline in the chromatin-associated protein phosphokinase activity assayed using optimal experimental condition described above. At 9 hours postorchiectomy, a 30% decline in the activity was observed; this was further reduced to about 50% of the control by 18 hours. This decrease in the kinase activity (e.g. at 9 hours postorchiectomy) appears to precede measurable changes in the protein and RNA complements of chromatin. Testosterone replacement following orchiectomy abolished this decline in the chromatin-associated activity. The chromatin-associated protein phosphokinase activity toward lysine-rich and arginine-rich histones was also sensitive to androgenic status of the animals and declined rapidly postorchiectomy. The results suggest the presence of multiple and androgen-sensitive protien phosphokinases associated with rat ventral prostate chromatin, which may modulate the phosphorylation of nuclear nonhistone phosphoproteins with changing gene action mediated by testosterone in this target tissue. PMID:234969

  18. 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.

  19. RAS Assays

    Cancer.gov

    The proportion of oncogenic mutants of KRAS proteins that are in the "active" (GTP-bound) form is far higher than that of wild-type RAS proteins. Scientists at the National Lab are developing high-throughput in vitro assays to measure interactions of GTP-loaded KRAS and effectors, such as CRAF and calmodulin, as well as imaging assays that can detect oncogenic KRAS interactions inside cells.

  20. Role of chromatin structure in integration of helper-dependent adenoviral vectors containing the beta-globin locus control region.

    PubMed

    Sova, Pavel; Wang, Hongjie; Bomsztyk, Karol; Stamatoyannopoulos, George; Lieber, André

    2008-02-01

    We constructed helper-dependent, fiber-chimeric adenoviral vectors that efficiently transduce human hematopoietic stem cells. We found that vectors carrying a 23-kb fragment of the beta-globin locus control region (LCR) flanked by adeno-associated virus inverted terminal repeats (Ad.LCR) preferentially integrated into the chromosomal beta-globin LCR of human erythroid Mo7e cells. We hypothesized that this targeted integration involves beta-globin LCR-specific chromatin structures. Chromatin immunoprecipitation assays of the beta-globin LCR revealed active chromatin within, and immediately downstream of, DNase hypersensitivity region 2 (HS2) in erythroid Mo7e cells, but not in nonerythroid cells. Importantly, most of the Ad.LCR integrations in Mo7e cells were found within this area. We provide further data indicating tethering of incoming Ad.LCR genomes to the chromosomal LCR. We also provide data that suggest a role for active chromatin in AAV Rep78-mediated Ad.LCR integration. Our findings support a new strategy for achieving targeted integration through chromatin tethering of vector DNA. PMID:18177253

  1. A Method to Study the Epigenetic Chromatin States of Rare Hematopoietic Stem and Progenitor Cells; MiniChIP–Chip

    PubMed Central

    2010-01-01

    Dynamic chromatin structure is a fundamental property of gene transcriptional regulation, and has emerged as a critical modulator of physiological processes during cellular differentiation and development. Analysis of chromatin structure using molecular biology and biochemical assays in rare somatic stem and progenitor cells is key for understanding these processes but poses a great challenge because of their reliance on millions of cells. Through the development of a miniaturized genome-scale chromatin immunoprecipitation method (miniChIP–chip), we have documented the genome-wide chromatin states of low abundant populations that comprise hematopoietic stem cells and immediate progeny residing in murine bone marrow. In this report, we describe the miniChIP methodology that can be used for increasing an understanding of the epigenetic mechanisms underlying hematopoietic stem and progenitor cell function. Application of this method will reveal the contribution of dynamic chromatin structure in regulating the function of other somatic stem cell populations, and how this process becomes perturbed in pathological conditions. PMID:21406121

  2. Direct Evidence for Pitavastatin Induced Chromatin Structure Change in the KLF4 Gene in Endothelial Cells

    PubMed Central

    Kanki, Yasuharu; Kohro, Takahide; Li, Guoliang; Ohta, Yoshihiro; Kimura, Hiroshi; Kobayashi, Mika; Taguchi, Akashi; Tsutsumi, Shuichi; Iwanari, Hiroko; Yamamoto, Shogo; Aruga, Hirofumi; Dong, Shoulian; Stevens, Junko F.; Poh, Huay Mei; Yamamoto, Kazuki; Kawamura, Takeshi; Mimura, Imari; Suehiro, Jun-ichi; Sugiyama, Akira; Kaneki, Kiyomi; Shibata, Haruki; Yoshinaka, Yasunobu; Doi, Takeshi; Asanuma, Akimune; Tanabe, Sohei; Tanaka, Toshiya; Minami, Takashi; Hamakubo, Takao; Sakai, Juro; Nozaki, Naohito; Aburatani, Hiroyuki; Nangaku, Masaomi; Ruan, Xiaoan; Tanabe, Hideyuki; Ruan, Yijun; Ihara, Sigeo; Endo, Akira; Kodama, Tatsuhiko; Wada, Youichiro

    2014-01-01

    Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells. PMID:24797675

  3. Condensation Temperature in Non-Equilibrium Condensation.

    NASA Astrophysics Data System (ADS)

    Tanaka, K. K.; Tanaka, H.; Nakazawa, K.

    1999-09-01

    In investigation of the origins of the presolar grains, it is important to clear the formation process of grains in ejecta of AGB stars or supernovae, where most presolar grains are suggested to be formed. The grain formation has been investigated based on the classical nucleation theory in many previous studies. On the other hand it has been pointed out that the classical nucleation rate is significantly different from that obtained by experiments, and should not be applied to grain formation in astrophysical environments (Donn and Nuth, 1985, ApJ 288, 187-190). Recently Dillmann and Meier (1991, J. Chem. Phys. 94, 3872-3884) proposed new semi-phenomological nucleation model, which achieved excellent agreements with experiments. In this study we applied the nucleation rate in the semi-phenomological model to the grain formation in astrophysical environment in order to make it clear how the grain formation changes due to the new nucleation rate. For various parameters determined by surface energy of grain and cooling time of vapor, we solved equations describing the grain formation. From the comparison between the results obtained by new nucleation rate and that by classical one we found that there is no significant difference in grain number density and grain size, but the condensation temperature is considerably different from the previous one. For example in carbon rich AGB star the condensation temperature of graphite is lower than that obtained by classical one by a few hundreds Kelvin: this means the condensation temperature is lower than the equilibrium condensation temperature by about 500 Kelvin. Furthermore we investigated the condensation of vapor in which grain impurities are already present. We obtained the condition for formation of core-mantle type grains. Our obtained condition would give constraint on the formation of core-mantle type presolar grains.

  4. Analysis of chromatin-nuclear receptor interactions by laser-chromatin immunoprecipitation.

    PubMed

    Benedetti, Rosaria; Conte, Mariarosaria; Carafa, Vincenzo; Della Ventura, Bartolomeo; Altucci, Carlo; Velotta, Raffaele; Stunnenberg, Hendrik G; Altucci, Lucia; Nebbioso, Angela

    2014-01-01

    Better defining the dynamics of biomolecular interactions is an important step in understanding molecular biology and cellular processes. DNA-protein interactions, and specifically hormone-triggered DNA-nuclear receptor interactions, are key events which are still poorly understood. To date, the most commonly used approach in studying chromatin interactions is the immunoprecipitation of chemically cross-linked chromatin (ChIP) coupled with single gene or global genomic analyses. Currently, establishing a stable interplay between nucleic acids and proteins (DNA-protein cross-link) is mainly obtained through conventional, diffusion-triggered, chemical methods using formaldehyde. Here we describe an alternative method, called Laser-ChIP (LChIP), for the specific analysis of interactions between chromatin and nuclear receptors driven by a UV laser energy source. Photo-induced cross-linking in LChIP is achieved very rapidly, allowing the study of transient interactions, depending on laser source parameters. PMID:25182758

  5. 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.

  6. SLAC synchronous condenser

    SciTech Connect

    Corvin, C.

    1995-06-01

    A synchronous condenser is a synchronous machine that generates reactive power that leads real power by 90{degrees} in phase. The leading reactive power generated by the condenser offsets or cancels the normal lagging reactive power consumed by inductive and nonlinear loads at the accelerator complex. The quality of SLAC`s utility power is improved with the addition of the condenser. The inertia of the condenser`s 35,000 pound rotor damps and smoothes voltage excursions on two 12 kilovolt master substation buses, improving voltage regulation site wide. The condenser absorbs high frequency transients and noise in effect ``scrubbing`` the electric system power at its primary distribution source. In addition, the condenser produces a substantial savings in power costs. Federal and investor owned utilities that supply electric power to SLAC levy a monthly penalty for lagging reactive power delivered to the site. For the 1993 fiscal year this totaled over $285,000 in added costs for the year. By generating leading reactive power on site, thereby reducing total lagging reactive power requirements, a substantial savings in electric utility bills is achieved. Actual savings of $150,000 or more a year are possible depending on experimental operations.

  7. Relaxed Chromatin Formation and Weak Suppression of Homologous Pairing by the Testis-Specific Linker Histone H1T.

    PubMed

    Machida, Shinichi; Hayashida, Ryota; Takaku, Motoki; Fukuto, Atsuhiko; Sun, Jiying; Kinomura, Aiko; Tashiro, Satoshi; Kurumizaka, Hitoshi

    2016-02-01

    Linker histones bind to nucleosomes and compact polynucleosomes into a higher-order chromatin configuration. Somatic and germ cell-specific linker histone subtypes have been identified and may have distinct functions. In this study, we reconstituted polynucleosomes containing human histones H1.2 and H1T, as representative somatic and germ cell-specific linker histones, respectively, and found that H1T forms less compacted chromatin, as compared to H1.2. An in vitro homologous pairing assay revealed that H1T weakly inhibited RAD51/RAD54-mediated homologous pairing in chromatin, although the somatic H1 subtypes, H1.0, H1.1, H1.2, H1.3, H1.4, and H1.5, substantially suppressed it. An in vivo recombination assay revealed that H1T overproduction minimally affected the recombination frequency, but significant suppression was observed when H1.2 was overproduced in human cells. These results suggested that the testis-specific linker histone, H1T, possesses a specific function to produce the chromatin architecture required for proper chromosome regulation, such as homologous recombination. PMID:26757249

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

    PubMed

    Rybaczek, Dorota; Musia?ek, 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

  9. 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

  10. 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.

  11. Electrolyte vapor condenser

    DOEpatents

    Sederquist, Richard A. (Newington, CT); Szydlowski, Donald F. (East Hartford, CT); Sawyer, Richard D. (Canton, CT)

    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.

  12. Dynamic Chromatin Regulation from a Single Molecule Perspective.

    PubMed

    Fierz, Beat

    2016-03-18

    Chromatin regulatory processes, like all biological reactions, are dynamic and stochastic in nature but can give rise to stable and inheritable changes in gene expression patterns. A molecular understanding of those processes is key for fundamental biological insight into gene regulation, epigenetic inheritance, lineage determination, and therapeutic intervention in the case of disease. In recent years, great progress has been made in identifying important molecular players involved in key chromatin regulatory pathways. Conversely, we are only beginning to understand the dynamic interplay between protein effectors, transcription factors, and the chromatin substrate itself. Single-molecule approaches employing both highly defined chromatin substrates in vitro, as well as direct observation of complex regulatory processes in vivo, open new avenues for a molecular view of chromatin regulation. This review highlights recent applications of single-molecule methods and related techniques to investigate fundamental chromatin regulatory processes. PMID:26565113

  13. 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. PMID:26222733

  14. Methods for identifying higher-order chromatin structure.

    PubMed

    Sajan, Samin A; Hawkins, R David

    2012-01-01

    Eukaryotic genomic DNA is combined with histones, nonhistone proteins, and RNA to form chromatin, which is extensively packaged hierarchically to fit inside a cell's nucleus. The nucleosome-comprising a histone octamer with 147 base pairs of DNA wrapped around it-is the initial level and the repeating unit of chromatin packaging, which electron microscopy first made visible to the human eye as "beads on a string" nearly four decades ago. The mechanism and nature of chromatin packaging are still under intense research. Recently, classic methods like chromatin immunoprecipitation and digestion with deoxyribonuclease and micrococcal nuclease have been combined with high-throughput sequencing to provide detailed nucleosome occupancy maps, and chromosome conformation capture and its variants have revealed that higher-order chromatin structure involves long-range loop formation between distant genomic elements. This review discusses the methods for identifying higher-order chromatin structure and the information they have provided on this important topic. PMID:22703176

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. Condenser macrofouling control technologies

    SciTech Connect

    Mussalli, Y.G.

    1984-06-01

    Condenser macrofouling is a major source of problems causing poor power plant availability and efficiency. Condenser macrofouling control technologies discussed include mechanical controls (intake screening, debris filters, and condenser backwash), thermal backwash, hydraulic control (velocity), materials (antifouling coatings and sheeting), chlorination, and manual cleaning. The latter two are discussed only briefly, since they are not within the scope of work of this project. A cost-benefit evaluation is presented together with a cost effective condenser cleaning schedule. Detailed evaluation, installation, and monitoring programs of six antifouling coatings at 19 sites and of several mechanical controls at 19 sites are presented. A total of 28 utilities are participating in this project. The antifouling coating panels were installed in the spring of 1983 and will be inspected in 1984, 1985, and 1986. The mechanical controls will be monitored and evaluated in 1984.

  1. Fibronectin matrix assembly is essential for cell condensation during chondrogenesis

    PubMed Central

    Singh, Purva; Schwarzbauer, Jean E.

    2014-01-01

    ABSTRACT Mesenchymal cell condensation is the initiating event in endochondral bone formation. Cell condensation is followed by differentiation into chondrocytes, which is accompanied by induction of chondrogenic gene expression. Gene mutations involved in chondrogenesis cause chondrodysplasias and other skeletal defects. Using mesenchymal stem cells (MSCs) in an in vitro chondrogenesis assay, we found that knockdown of the diastrophic dysplasia (DTD) sulfate transporter (DTDST, also known as SLC26A2), which is required for normal cartilage development, blocked cell condensation and caused a significant reduction in fibronectin matrix. Knockdown of fibronectin with small interfering RNAs (siRNAs) also blocked condensation. Fibrillar fibronectin matrix was detected prior to cell condensation, and its levels increased during and after condensation. Inhibition of fibronectin matrix assembly by use of the functional upstream domain (FUD) of adhesin F1 from Streptococcus pyogenes prevented cell condensation by MSCs and also by the chondrogenic cell line ATDC5. Our data show that cell condensation and induction of chondrogenesis depend on fibronectin matrix assembly and DTDST, and indicate that this transporter is required earlier in chondrogenesis than previously appreciated. They also raise the possibility that certain of the skeletal defects in DTD patients might derive from the link between DTDST, fibronectin matrix and condensation. PMID:25146392

  2. Chromatin regulation in drug addiction and depression

    PubMed Central

    Renthal, William; Nestler, Eric J.

    2009-01-01

    Alterations in gene expression are implicated in the pathogenesis of several neuropsychiatrie disorders, including drug addiction and depression, increasing evidence indicates that changes in gene expression in neurons, in the context of animal models of addiction and depression, are mediated in part by epigenetic mechanisms that alter chromatin structure on specific gene promoters. This review discusses recent findings from behavioral, molecular, and bioinformatic approaches that are being used to understand the complex epigenetic regulation of gene expression in brain by drugs of abuse and by stress. These advances promise to open up new avenues for improved treatments of these disorders. PMID:19877494

  3. Chromatin, DNA structure and alternative splicing.

    PubMed

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

    2015-11-14

    Coupling of transcription and alternative splicing via regulation of the transcriptional elongation rate is a well-studied phenomenon. Template features that act as roadblocks for the progression of RNA polymerase II comprise histone modifications and variants, DNA-interacting proteins and chromatin compaction. These may affect alternative splicing decisions by inducing pauses or decreasing elongation rate that change the time-window for splicing regulatory sequences to be recognized. Herein we discuss the evidence supporting the influence of template structural modifications on transcription and splicing, and provide insights about possible roles of non-B DNA conformations on the regulation of alternative splicing. PMID:26296319

  4. Assessment of sera for chromatin-immunoprecipitation.

    PubMed

    Nguyen, Juliette; Yamada, Daisuke; Schultz, David C; Defossez, Pierre-Antoine

    2008-01-01

    Chromatin-immunoprecipitation (ChIP) is a powerful technique for mapping the protein-DNA interactions that occur in living cells. The critical technical determinant for successful ChIP is the availability of an appropriate, "ChIP-grade" serum. Here we present a technique designed to assess whether sera are suitable for ChIP, and to quantify their efficiency relative to a positive internal reference. This approach is useful as a first step toward ChIP-on-chip or ChIP-sequencing, especially in the case of recently identified proteins for which no binding sites are yet known. PMID:18254381

  5. Role of chromatin states in transcriptional memory

    PubMed Central

    Kundu, Sharmistha; Peterson, Craig L.

    2009-01-01

    Establishment of cellular memory and its faithful propagation is critical for successful development of multicellular organisms. As pluripotent cells differentiate, choices in cell fate are inherited and maintained by their progeny throughout the lifetime of the organism. A major factor in this process is the epigenetic inheritance of specific transcriptional states or transcriptional memory. In this review, we discuss chromatin transitions and mechanisms by which they are inherited by subsequent generations. We also discuss illuminating cases of cellular memory in budding yeast and evaluate whether transcriptional memory in yeast is nuclear or cytoplasmically inherited. PMID:19236904

  6. Stem cell factors in plants: chromatin connections.

    PubMed

    Kornet, N; Scheres, B

    2008-01-01

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

  7. Chromatin insulators: regulatory mechanisms and epigenetic inheritance

    PubMed Central

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

    2008-01-01

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

  8. Histone demethylases in chromatin biology and beyond

    PubMed Central

    Dimitrova, Emilia; Turberfield, Anne H; Klose, Robert J

    2015-01-01

    Histone methylation plays fundamental roles in regulating chromatin-based processes. With the discovery of histone demethylases over a decade ago, it is now clear that histone methylation is dynamically regulated to shape the epigenome and regulate important nuclear processes including transcription, cell cycle control and DNA repair. In addition, recent observations suggest that these enzymes could also have functions beyond their originally proposed role as histone demethylases. In this review, we focus on recent advances in our understanding of the molecular mechanisms that underpin the role of histone demethylases in a wide variety of normal cellular processes. PMID:26564907

  9. 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.

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

    PubMed Central

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

    2015-01-01

    In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3∆ cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and the nuclear envelope. PMID:25798942

  11. Inference of interactions between chromatin modifiers and histone modifications: from ChIP-Seq data to chromatin-signaling

    PubMed Central

    Perner, Juliane; Lasserre, Julia; Kinkley, Sarah; Vingron, Martin; Chung, Ho-Ryun

    2014-01-01

    Chromatin modifiers and histone modifications are components of a chromatin-signaling network involved in transcription and its regulation. The interactions between chromatin modifiers and histone modifications are often unknown, are based on the analysis of few genes or are studied in vitro. Here, we apply computational methods to recover interactions between chromatin modifiers and histone modifications from genome-wide ChIP-Seq data. These interactions provide a high-confidence backbone of the chromatin-signaling network. Many recovered interactions have literature support; others provide hypotheses about yet unknown interactions. We experimentally verified two of these predicted interactions, leading to a link between H4K20me1 and members of the Polycomb Repressive Complexes 1 and 2. Our results suggest that our computationally derived interactions are likely to lead to novel biological insights required to establish the connectivity of the chromatin-signaling network involved in transcription and its regulation. PMID:25414326

  12. The INO80 chromatin remodeling complex prevents polyploidy and maintains normal chromatin structure at centromeres

    PubMed Central

    Chambers, Anna L.; Ormerod, Georgina; Durley, Samuel C.; Sing, Tina L.; Brown, Grant W.; Kent, Nicholas A.; Downs, Jessica A.

    2012-01-01

    The INO80 chromatin remodeling complex functions in transcriptional regulation, DNA repair, and replication. Here we uncover a novel role for INO80 in regulating chromosome segregation. First, we show that the conserved Ies6 subunit is critical for INO80 function in vivo. Strikingly, we found that loss of either Ies6 or the Ino80 catalytic subunit results in rapid increase in ploidy. One route to polyploidy is through chromosome missegregation due to aberrant centromere structure, and we found that loss of either Ies6 or Ino80 leads to defective chromosome segregation. Importantly, we show that chromatin structure flanking centromeres is altered in cells lacking these subunits and that these alterations occur not in the Cse4-containing centromeric nucleosome, but in pericentric chromatin. We provide evidence that these effects are mediated through misincorporation of H2A.Z, and these findings indicate that H2A.Z-containing pericentric chromatin, as in higher eukaryotes with regional centromeres, is important for centromere function in budding yeast. These data reveal an important additional mechanism by which INO80 maintains genome stability. PMID:23207916

  13. Genome-wide association of Yorkie with chromatin and chromatin remodeling complexes

    PubMed Central

    Oh, Hyangyee; Slattery, Matthew; Ma, Lijia; Crofts, Alex; White, Kevin P.; Mann, Richard; Irvine, Kenneth D.

    2013-01-01

    SUMMARY The Hippo pathway regulates growth through the transcriptional co-activator Yorkie, but how Yorkie promotes transcription remains poorly understood. We address this by characterizing Yorkie’s association with chromatin, and by identifying nuclear partners that effect transcriptional activation. Co-immunoprecipitation and mass spectrometry identify GAGA Factor (GAF), Brahma complex, and Mediator complex as Yorkie-associated nuclear protein complexes. All three are required for Yorkie’s transcriptional activation of downstream genes, and GAF and the Brahma complex subunit Moira interact directly with Yorkie. Genome-wide chromatin binding experiments identify thousands of Yorkie sites, most of which are associated with elevated transcription, based on genome-wide analysis of mRNA and histone H3K4Me3 modification. Chromatin binding also supports extensive functional overlap between Yorkie and GAF. Our studies suggest a widespread role for Yorkie as a regulator of transcription, and identify recruitment of the chromatin modifying GAF protein and BRM complex as a molecular mechanism for transcriptional activation by Yorkie. PMID:23395637

  14. 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.

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

    PubMed Central

    Linnemann, Amelia K.; Krawetz, Stephen A.

    2010-01-01

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

  16. c-Myb Binding Sites in Haematopoietic Chromatin Landscapes

    PubMed Central

    Bengtsen, Mads; Klepper, Kjetil; Gundersen, Sveinung; Cuervo, Ignacio; Drabløs, Finn; Hovig, Eivind; Sandve, Geir Kjetil; Gabrielsen, Odd Stokke; Eskeland, Ragnhild

    2015-01-01

    Strict control of tissue-specific gene expression plays a pivotal role during lineage commitment. The transcription factor c-Myb has an essential role in adult haematopoiesis and functions as an oncogene when rearranged in human cancers. Here we have exploited digital genomic footprinting analysis to obtain a global picture of c-Myb occupancy in the genome of six different haematopoietic cell-types. We have biologically validated several c-Myb footprints using c-Myb knockdown data, reporter assays and DamID analysis. We show that our predicted conserved c-Myb footprints are highly dependent on the haematopoietic cell type, but that there is a group of gene targets common to all cell-types analysed. Furthermore, we find that c-Myb footprints co-localise with active histone mark H3K4me3 and are significantly enriched at exons. We analysed co-localisation of c-Myb footprints with 104 chromatin regulatory factors in K562 cells, and identified nine proteins that are enriched together with c-Myb footprints on genes positively regulated by c-Myb and one protein enriched on negatively regulated genes. Our data suggest that c-Myb is a transcription factor with multifaceted target regulation depending on cell type. PMID:26208222

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

    PubMed

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

    2008-08-01

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

  18. A quantitative investigation of linker histone interactions with nucleosomes and chromatin.

    PubMed

    White, Alison E; Hieb, Aaron R; Luger, Karolin

    2016-01-01

    Linker histones such as H1 are abundant basic proteins that bind tightly to nucleosomes, thereby acting as key organizers of chromatin structure. The molecular details of linker histone interactions with the nucleosome, and in particular the contributions of linker DNA and of the basic C-terminal tail of H1, are controversial. Here we combine rigorous solution-state binding assays with native gel electrophoresis and Atomic Force Microscopy, to quantify the interaction of H1 with chromatin. We find that H1 binds nucleosomes and nucleosomal arrays with very tight affinity by recognizing a specific DNA geometry minimally consisting of a solitary nucleosome with a single ~18 base pair DNA linker arm. The association of H1 alters the conformation of trinucleosomes so that only one H1 can bind to the two available linker DNA regions. Neither incorporation of the histone variant H2A.Z, nor the presence of neighboring nucleosomes affects H1 affinity. Our data provide a comprehensive thermodynamic framework for this ubiquitous chromatin architectural protein. PMID:26750377

  19. The ING1b tumor suppressor facilitates nucleotide excision repair by promoting chromatin accessibility to XPA

    SciTech Connect

    Kuo, Wei-Hung W.; Wang Yemin; Wong, Ronald P.C.; Campos, Eric I.; Li Gang . E-mail: gangli@interchange.ubc.ca

    2007-05-01

    ING1b is the most studied ING family protein and perhaps the most ubiquitously and abundantly expressed. This protein is involved in the regulation of various biological functions ranging from senescence, cell cycle arrest, apoptosis, to DNA repair. ING1b is upregulated by UV irradiation and enhances the removal of bulky nucleic acid photoproducts. In this study, we provide evidence that ING1b mediates nucleotide excision repair by facilitating the access to damaged nucleosomal DNA. We demonstrate that ING1b is not recruited to UV-induced DNA lesions but enhances nucleotide excision repair only in XPC-proficient cells, implying an essential role in early steps of the 'access, repair, restore' model. We also find that ING1b alters histone acetylation dynamics upon exposure to UV radiation and induces chromatin relaxation in microccocal nuclease digestion assay, revealing that ING1b may allow better access to nucleotide excision repair machinery. More importantly, ING1b associates with chromatin in a UV-inducible manner and facilitates DNA access to nucleotide excision repair factor XPA. Furthermore, depletion of the endogenous ING1b results to the sensitization of cells at S-phase to UV irradiation. Taken together, these observations establish a role of ING1b acting as a chromatin accessibility factor for DNA damage recognition proteins upon genotoxic injury.

  20. Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions.

    PubMed

    Schep, Alicia N; Buenrostro, Jason D; Denny, Sarah K; Schwartz, Katja; Sherlock, Gavin; Greenleaf, William J

    2015-11-01

    Transcription factors canonically bind nucleosome-free DNA, making the positioning of nucleosomes within regulatory regions crucial to the regulation of gene expression. Using the assay of transposase accessible chromatin (ATAC-seq), we observe a highly structured pattern of DNA fragment lengths and positions around nucleosomes in Saccharomyces cerevisiae, and use this distinctive two-dimensional nucleosomal "fingerprint" as the basis for a new nucleosome-positioning algorithm called NucleoATAC. We show that NucleoATAC can identify the rotational and translational positions of nucleosomes with up to base-pair resolution and provide quantitative measures of nucleosome occupancy in S. cerevisiae, Schizosaccharomyces pombe, and human cells. We demonstrate the application of NucleoATAC to a number of outstanding problems in chromatin biology, including analysis of sequence features underlying nucleosome positioning, promoter chromatin architecture across species, identification of transient changes in nucleosome occupancy and positioning during a dynamic cellular response, and integrated analysis of nucleosome occupancy and transcription factor binding. PMID:26314830

  1. A quantitative investigation of linker histone interactions with nucleosomes and chromatin

    PubMed Central

    White, Alison E.; Hieb, Aaron R.; Luger, Karolin

    2016-01-01

    Linker histones such as H1 are abundant basic proteins that bind tightly to nucleosomes, thereby acting as key organizers of chromatin structure. The molecular details of linker histone interactions with the nucleosome, and in particular the contributions of linker DNA and of the basic C-terminal tail of H1, are controversial. Here we combine rigorous solution-state binding assays with native gel electrophoresis and Atomic Force Microscopy, to quantify the interaction of H1 with chromatin. We find that H1 binds nucleosomes and nucleosomal arrays with very tight affinity by recognizing a specific DNA geometry minimally consisting of a solitary nucleosome with a single ~18 base pair DNA linker arm. The association of H1 alters the conformation of trinucleosomes so that only one H1 can bind to the two available linker DNA regions. Neither incorporation of the histone variant H2A.Z, nor the presence of neighboring nucleosomes affects H1 affinity. Our data provide a comprehensive thermodynamic framework for this ubiquitous chromatin architectural protein. PMID:26750377

  2. Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions

    PubMed Central

    Schep, Alicia N.; Buenrostro, Jason D.; Denny, Sarah K.; Schwartz, Katja; Sherlock, Gavin; Greenleaf, William J.

    2015-01-01

    Transcription factors canonically bind nucleosome-free DNA, making the positioning of nucleosomes within regulatory regions crucial to the regulation of gene expression. Using the assay of transposase accessible chromatin (ATAC-seq), we observe a highly structured pattern of DNA fragment lengths and positions around nucleosomes in Saccharomyces cerevisiae, and use this distinctive two-dimensional nucleosomal “fingerprint” as the basis for a new nucleosome-positioning algorithm called NucleoATAC. We show that NucleoATAC can identify the rotational and translational positions of nucleosomes with up to base-pair resolution and provide quantitative measures of nucleosome occupancy in S. cerevisiae, Schizosaccharomyces pombe, and human cells. We demonstrate the application of NucleoATAC to a number of outstanding problems in chromatin biology, including analysis of sequence features underlying nucleosome positioning, promoter chromatin architecture across species, identification of transient changes in nucleosome occupancy and positioning during a dynamic cellular response, and integrated analysis of nucleosome occupancy and transcription factor binding. PMID:26314830

  3. A RNA-dependent RNA polymerase activity: implications for chromatin transcription experiments.

    PubMed Central

    Giesecke, K; Sippel, A E; Nguyen-Huu, M C; Groner, B; Hynes, N E; Wurtz, T; Schütz, G

    1977-01-01

    Mercurated nucleoside triphosphates have been used for transcription of chicken oviduct chromatin with E. coli RNA polymerase. The newly synthesized RNA was purified from preexisting RNA by SH-agarose chromatography and analyzed for the content of specific mRNA sequences. The apparent preferential production of ovalbumin mRNA sequences was not inhibited by actinomycin D, although total RNA synthesis was reduced by more than 90%. Furthermore, when globin mRNA alone, or added to oviduct chromatin, was incubated in the transcription assay, a significant fraction of this mRNA was retained on SH-agarose. The copurification of chromatin associated RNA with in vitro synthesized mercurated RNA was mainly due to a RNA-dependent synthesis of complementary sequences by the bacterial enzyme. Although denaturation of the transcripts prior to SH-agarose chromatography leads to a reduced contamination with endogenous ovalbumin specific RNA, we are unable to show that the messenger-specific RNA sequences purified with the newly mercurated RNA results from a DNA-dependent reaction. PMID:339205

  4. 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.

  5. Chromatin structure of hormono-dependent promoters.

    PubMed

    Adom, J; Carr, K D; Gouilleux, F; Marsaud, V; Richard-Foy, H

    1991-01-01

    Transient transfections of mutated MMTV LTRs, driving the luciferase reporter gene, have shown the presence of at least one cis-acting element cooperating with the GREs. Studies of the chromatin structure of two glucocorticoid-regulated promoters, the mouse mammary tumor virus (MMTV) long terminal repeat (LTR), a retroviral promoter, and the rat tyrosine aminotransferase (TAT) promoter, demonstrate that both DNAs are organized into precisely positioned nucleosomes. Hormonal activation of transcription is accompanied by structural changes of one (MMTV LTR) or two (TAT promoter) nucleosomes associated with the hormone-response elements (HREs). These changes can be visualized by the appearance of DNasel hypersensitive sites. Association of the hormone-receptor complex with the nucleus is necessary to induce the DNasel hypersensitive site and to maintain transcription, but is not necessary to maintain DNasel hypersensitivity. Anti-hormones, even when able to promote a strong binding of the receptor to the nucleus, are unable to induce the chromatin structural change. Using cell lines containing approx. 200 copies of a MMTV LTR/Hv-ras chimeric construct, we have demonstrated a strong, hormono-independent nuclear matrix interaction of sequences located just upstream and downstream of the ras coding sequences. PMID:1683563

  6. 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

  7. Sperm chromatin heterogeneity as an infertility factor.

    PubMed

    Ibrahim, M E; Moussa, M A; Pedersen, H

    1988-01-01

    Semen samples from husbands with a history of unexplained infertility (n = 33), of women with habitual abortion (n = 36), or normal fertile donors (n = 20) were subjected to conventional semen analysis (SA), Acridine orange test (AOT), and zona-free hamster egg penetration test (HEPT). The three tests operate independently. The most discriminatory test was AOT (p = 0.0001) followed by HEPT (p = 0.019). The frequency of sperm chromatin heterogeneity as detected by AOT red fluorescence was highest in habitual abortion (39.4%), followed by unexplained infertility (16.4%), and, last, donors (9.4%). However the percentage of penetration was highest in habitual abortion (50.7%), followed by donors (43.1%), and least in unexplained infertility (33.9%). Conventional semen parameters (sperm density, motility, abnormality, and vitality) were the least to discriminate between the three groups. The presence of abnormal sperm chromatin may lead to infertility as a result of early pregnancy loss. PMID:3223787

  8. Histone modification and chromatin remodeling during NER.

    PubMed

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

    2015-12-01

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

  9. Global chromatin fibre compaction in response to DNA damage

    SciTech Connect

    Hamilton, Charlotte; Hayward, Richard L.; Breakthrough Research Unit, The University of Edinburgh, Edinburgh EH4 2XR ; Gilbert, Nick; Breakthrough Research Unit, The University of Edinburgh, Edinburgh EH4 2XR

    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 linker histones. We suggest that following DSB formation, although there is localised chromatin unfolding to facilitate repair, the bulk genome becomes rapidly compacted protecting cells from further damage.

  10. Effects of Myogenin on Expression of Late Muscle Genes through MyoD-Dependent Chromatin Remodeling Ability of Myogenin

    PubMed Central

    Du, Chao; Jin, Ya-Qiong; Qi, Jun-Juan; Ji, Zhen-Xing; Li, Shu-Yan; An, Guo-Shun; Jia, Hong-Ti; Ni, Ju-Hua

    2012-01-01

    MyoD and myogenin (Myog) recognize sets of distinct but overlapping target genes and play different roles in skeletal muscle differentiation. MyoD is sufficient for near-full expression of early targets, while Myog can only partially enhance expression of MyoD-initiated late muscle genes. However, the way in which Myog enhances the expression of MyoD-initiated late muscle genes remains unclear. Here, we examine the effects of Myog on chromatin remodeling at late muscle gene promoters and their activation within chromatin environment. Chromatin immunoprecipitation (ChIP) assay showed that Myog selectively bound to the regulatory sequences of late muscle genes. Overexpression of Myog was found to overcome sodium butyrate-inhibited chromatin at late muscle genes in differentiating C2C12 myoblasts, shifting the transcriptional activation of these genes to an earlier time period. Furthermore, overexpression of Myog led to increased hyperacetylation of core histone H4 in differentiating C2C12 myoblasts but not NIH3T3 fibroblasts, and hyperacetylated H4 was associated directly with the late muscle genes in differentiating C2C12, indicating that Myog can induce chromatin remodeling in the presence of MyoD. In addition, co-immunoprecipitation (CoIP) revealed that Myog was associated with the nuclear protein Brd4 in differentiating C2C12 myoblasts. Together, these results suggest that Myog enhances the expression of MyoD-initiated late muscle genes through MyoD-dependent ability of Myog to induce chromatin remodeling, in which Myog-Brd4 interaction may be involved. PMID:22814845

  11. A Quantitative Proteomic Analysis of In Vitro Assembled Chromatin.

    PubMed

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

    2016-03-01

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

  12. ATP-Dependent Chromatin Remodeling Factors and DNA Damage Repair

    PubMed Central

    Osley, Mary Ann; Tsukuda, Toyoko; Nickoloff, Jac A.

    2007-01-01

    The organization of eukaryotic DNA into chromatin poses a barrier to all processes that require access of enzymes and regulatory factors to their sites of action. While the majority of studies in this area have concentrated on the role of chromatin in the regulation of transcription, there has been a recent emphasis on the relationship of chromatin to DNA damage repair. In this review, we focus on the role of chromatin in nucleotide excision repair (NER) and double-strand break (DSB) repair. NER and DSB repair use very different enzymatic machineries, and these two modes of DNA damage repair are also differentially affected by chromatin. Only a small number of nucleosomes are likely to be involved in NER, while a more extensive region of chromatin is involved in DSB repair. However, a key feature of both NER and DSB repair pathways is the participation of ATP-dependent chromatin remodeling factors at various points in the repair process. We discuss recent data that have identified roles for SWI/SNF-related chromatin remodeling factors in the two repair pathways. PMID:17291544

  13. Steam condensate leakage

    SciTech Connect

    Midlock, E.B.; Thuot, J.R.

    1996-07-01

    Argonne National Laboratory (ANL) is a multi-program research and development center owned by the United States Department of Energy and operated by the University of Chicago. The majority of the buildings on site use steam for heating and other purposes. Steam is generated from liquid water at the site`s central boiler house and distributed around the site by means of large pipes both above and below the ground. Steam comes into each building where it is converted to liquid condensate, giving off heat which can be used by the building. The condensate is then pumped back to the boiler house where it will be reheated to steam again. The process is continual but is not perfectly efficient. A substantial amount of condensate is being lost somewhere on site. The lost condensate has both economic and environmental significance. To compensate for lost condensate, makeup water must be added to the returned condensate at the boiler house. The water cost itself will become significant in the future when ANL begins purchasing Lake Michigan water. In addition to the water cost, there is also the cost of chemically treating the water to remove impurities, and there is the cost of energy required to heat the water, as it enters the boiler house 1000 F colder than the condensate return. It has been estimated that only approximately 60% of ANL`s steam is being returned as condensate, thus 40% is being wasted. This is quite costly to ANL and will become significantly more costly in the future when ANL begins purchasing water from Lake Michigan. This study locates where condensate loss is occurring and shows how much money would be saved by repairing the areas of loss. Shortly after completion of the study, one of the major areas of loss was repaired. This paper discusses the basis for the study, the areas where losses are occurring, the potential savings of repairing the losses, and a hypothesis as to where the unaccounted for loss is occurring.

  14. Normal sperm morphology and chromatin packaging: comparison between aniline blue and chromomycin A3 staining.

    PubMed

    Franken, D R; Franken, C J; de la Guerre, H; de Villiers, A

    1999-12-01

    The successful implementation of ICSI has provided a unique means of allowing couples suffering from severe male infertility to achieve their reproductive goals. However, despite the great therapeutic advantages of the technique, ICSI often provides solutions to clinicians in the absence of an aetiological or pathophysiological diagnosis. The development of a sequential diagnostic schedule for patients consulting for fertility disturbances would be an ideal method of approach. Since sperm morphology recorded by strict criteria has often been correlated with fertilization failure, the present study aimed to evaluate the relationship between normal morphology and chromatin staining among fertile and subfertile men. Both chromomycin A3 (CMA3) and acidic aniline blue (AAB) were employed to record chromatin packaging quality among 58 men visiting the andrology laboratory. Intra- and interassay variations were initially recorded for fertile sperm donors. The coefficients of variation (CV) for all intra- and inter-assay assessments were < 12%. Chromatin packaging was significantly and negatively correlated with normal sperm morphology, namely r = 0.40 (P = 0.001) and r = 0.33 (P = 0.001) for CMA3 and AAB, respectively. Receiver operator characteristics illustrated sensitivity and specificity values of 75% and 82% for CMA3 and 60% and 91% for AAB, respectively. Significantly different CMA3 and AAB staining was recorded among men with severe teratozoospermia (< 4% normal forms) when compared with normozoospermic men (> 14% normal forms), namely 49% vs. 29% for CMA3 and 51% vs. 26% for AAB staining, respectively. Chromatin packaging assessments should be a valuable addition to the sequential diagnostic programme in an assisted reproduction arena. PMID:10643511

  15. Chromatin dynamics at the hTERT promoter during transcriptional activation and repression by c-Myc and Mnt in Xenopus leavis oocytes.

    PubMed

    Wahlström, Therese; Belikov, Sergey; Arsenian Henriksson, Marie

    2013-12-10

    The transcription factors c-Myc and Mnt regulate gene expression through dimerization with Max and binding to E-boxes in target genes. While c-Myc activates gene expression via recruitment of histone modifying complexes, Mnt acts as a transcriptional repressor. Here, we used the Xenopus leavis oocyte system to address the effect of c-Myc and Mnt on transcription and chromatin remodeling over the E-box region in the human telomerase reverse transcriptase (hTERT) promoter. As expected we found elevated and decreased levels of hTERT transcription upon exogenously expressed c-Myc/Max and Mnt/Max, respectively. In addition, we confirmed binding of these heterodimers to both E-boxes already enriched with H3K9ac and H4K16ac. These chromatin marks were further enhanced upon c-Myc/Max binding followed by increased DNA accessibility in the E-box region. In contrast, Mnt/Max inhibited Myc-induced transcription and mediated repression through complete chromatin condensation and deacetylation of H3K9 and H4K16 across the E-box region. Importantly, Mnt was able to counteract c-Myc mediated activation even when expressed at low levels, suggesting Mnt to act as a strong repressor by closing the chromatin structure. Collectively our data demonstrate that the balance between c-Myc and Mnt activity determines the transcriptional outcome of the hTERT promoter by modulation of the chromatin architecture. PMID:23860446

  16. Unwind and transcribe: chromatin reprogramming in the early mammalian embryo.

    PubMed

    Biechele, Steffen; Lin, Chih-Jen; Rinaudo, Paolo F; Ramalho-Santos, Miguel

    2015-10-01

    Within the first few days of life, the unipotent gametic genomes are rapidly reprogrammed to support emergence of pluripotent cells in the early mammalian embryo. It is now appreciated that this crucial stage of development involves dramatic changes to chromatin at multiple levels, such as DNA methylation, histone modifications, histone mobility, and higher-order chromatin organization. Technological advances are beginning to allow genome-wide views of this chromatin reprogramming, and provide new approaches to functionally dissect its regulation. Here we review recent insights into the dynamic chromatin environment of the early mouse embryo. New data challenge long-held assumptions, for example, with regards to the asymmetry of DNA methylation of the parental genomes or the onset of functional zygotic genome activation. We discuss how impaired chromatin reprogramming can lead to early embryonic lethality, but might also have delayed effects that only manifest later in embryogenesis or postnatally, potentially influencing the propensity for adult-onset diseases. PMID:26183187

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

    PubMed

    Donà, Mattia; Mittelsten Scheid, Ortrun

    2015-08-01

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

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

    PubMed Central

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

    2014-01-01

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

  19. Chromatin modifications: The driving force of senescence and aging?

    PubMed Central

    DiMauro, Teresa; David, Gregory

    2009-01-01

    An emerging field of investigation in the search for treatment of human disease is the modulation of chromatin modifications. Chromatin modifications impart virtually all processes occurring in the mammalian nucleus, from regulation of transcription to genomic stability and nuclear high order organization. It has been well recognized that, as the mammalian cell ages, its chromatin structure evolves, both at a global level and at specific loci. While these observations are mostly correlative, recent technical developments allowing loss-of-function experiments and genome-wide approaches have permitted the identification of a causal relationship between specific changes in chromatin structure and the aging phenotype. Here we review the evidence pointing to the modulation of chromatin structure as a potential driving force of cellular aging in mammals. PMID:20157508

  20. Chromatin regulators of the AID induced DNA damage response

    PubMed Central

    Daniel, Jeremy A.; Nussenzweig, André

    2013-01-01

    Chemical modifications to the DNA and histone protein components of chromatin can modulate gene expression and genome stability. Understanding the physiological impact of changes in chromatin structure remains an important question in biology. As one example, in order to generate antibody diversity with somatic hypermutation and class switch recombination, chromatin must be made accessible for Activation-induced cytidine deaminase (AID)-mediated deamination of cytosines in DNA. These lesions are recognized and removed by various DNA repair pathways, but if not handled properly, can lead to formation of oncogenic chromosomal translocations. In this review, we focus the discussion on how chromatin-modifying activities and -binding proteins contribute to the native chromatin environment for which AID-induced DNA damage is targeted and repaired. Outstanding questions remain regarding the direct roles of histone post-translational modifications and the significance of AID function outside of antibody diversity. PMID:23664375

  1. 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

  2. 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

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

    PubMed Central

    Harmston, Nathan; Lenhard, Boris

    2013-01-01

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

  4. First order kaon condensate

    SciTech Connect

    Glendenning, N.K.; Schaffner-Bielich, J.; Schaffner-Bielich, J.

    1999-08-01

    First order Bose condensation in asymmetric nuclear matter and in neutron stars is studied, with particular reference to kaon condensation. We demonstrate explicitly why the Maxwell construction fails to assure equilibrium in multicomponent substances. Gibbs conditions and conservation laws require that for phase equilibrium, the charge density must have opposite sign in the two phases of isospin asymmetric nuclear matter. The mixed phase will therefore form a Coulomb lattice with the rare phase occupying lattice sites in the dominant phase. Moreover, the kaon condensed phase differs from the normal phase, not by the mere presence of kaons in the first, but also by a difference in the nucleon effective masses. The mixed phase region, which occupies a large radial extent amounting to some kilometers in our model neutron stars, is thus highly heterogeneous. It should be particularly interesting in connection with the pulsar glitch phenomenon as well as transport properties. {copyright} {ital 1999} {ital The American Physical Society}

  5. First order kaon condensate

    NASA Astrophysics Data System (ADS)

    Glendenning, Norman K.; Schaffner-Bielich, Jürgen

    1999-08-01

    First order Bose condensation in asymmetric nuclear matter and in neutron stars is studied, with particular reference to kaon condensation. We demonstrate explicitly why the Maxwell construction fails to assure equilibrium in multicomponent substances. Gibbs conditions and conservation laws require that for phase equilibrium, the charge density must have opposite sign in the two phases of isospin asymmetric nuclear matter. The mixed phase will therefore form a Coulomb lattice with the rare phase occupying lattice sites in the dominant phase. Moreover, the kaon condensed phase differs from the normal phase, not by the mere presence of kaons in the first, but also by a difference in the nucleon effective masses. The mixed phase region, which occupies a large radial extent amounting to some kilometers in our model neutron stars, is thus highly heterogeneous. It should be particularly interesting in connection with the pulsar glitch phenomenon as well as transport properties.

  6. Epigenetic modification of centromeric chromatin: hypomethylation of DNA sequences in the CENH3-associated chromatin in Arabidopsis thaliana and maize.

    PubMed

    Zhang, Wenli; Lee, Hye-Ran; Koo, Dal-Hoe; Jiang, Jiming

    2008-01-01

    The centromere in eukaryotes is defined by the presence of a special histone H3 variant, CENH3. Centromeric chromatin consists of blocks of CENH3-containing nucleosomes interspersed with blocks of canonical H3-containing nucleosomes. However, it is not known how CENH3 is precisely deposited in the centromeres. It has been suggested that epigenetic modifications of the centromeric chromatin may play a role in centromere identity. The centromeres of Arabidopsis thaliana are composed of megabase-sized arrays of a 178-bp satellite repeat. Here, we report that the 178-bp repeats associated with the CENH3-containing chromatin (CEN chromatin) are hypomethylated compared with the same repeats located in the flanking pericentromeric regions. A similar hypomethylation of DNA in CEN chromatin was also revealed in maize (Zea mays). Hypomethylation of the DNA in CEN chromatin is correlated with a significantly reduced level of H3K9me2 in Arabidopsis. We demonstrate that the 178-bp repeats from CEN chromatin display a distinct distribution pattern of the CG and CNG sites, which may provide a foundation for the differential methylation of these repeats. Our results suggest that DNA methylation plays an important role in epigenetic demarcation of the CEN chromatin. PMID:18239133

  7. Oligodendroglial Development: New Roles for Chromatin Accessibility.

    PubMed

    Huang, Nanxin; Niu, Jianqin; Feng, Yue; Xiao, Lan

    2015-12-01

    In the central nervous system, the generation of mature oligodendrocytes from their progenitors is a critical step in myelination, which is essential for normal nervous system function. Thus, understanding the regulatory mechanism underlying oligodendroglial development is of great importance, especially for the development of new therapeutic strategies that promote remyelination in demyelinating diseases, such as multiple sclerosis. Previous studies have focused on genetic patterns and revealed a network of cell signaling pathways and related transcription factors involved in oligodendroglial lineage development. Recently, epigenetic regulation, which refers to regulation of gene expression by adjusting the environment of the genes has been shown to play a profound role during oligodendroglial development. In this review, we summarize the recent data demonstrating the effects of chromatin modification and remodeling in regulating oligodendroglial development and discuss the use of high-throughput analysis and bio-informatics in future studies. PMID:25564030

  8. MYST-family histone acetyltransferases: beyond chromatin.

    PubMed

    Sapountzi, Vasileia; Côté, Jacques

    2011-04-01

    Covalently modifying a protein has proven to be a powerful mechanism of functional regulation. N-epsilon acetylation of lysine residues was initially discovered on histones and has been studied extensively in the context of chromatin and DNA metabolism, such as transcription, replication and repair. However, recent research shows that acetylation is more widespread than initially thought and that it regulates various nuclear as well as cytoplasmic and mitochondrial processes. In this review, we present the multitude of non-histone proteins targeted by lysine acetyltransferases of the large and conserved MYST family, and known functional consequences of this acetylation. Substrates of MYST enzymes include factors involved in transcription, heterochromatin formation and cell cycle, DNA repair proteins, gluconeogenesis enzymes and finally subunits of MYST protein complexes themselves. Discovering novel substrates of MYST proteins is pivotal for the understanding of the diverse functions of these essential acetyltransferases in nuclear processes, signaling, stress response and metabolism. PMID:21132344

  9. Identification of alternative topological domains in chromatin

    PubMed Central

    2014-01-01

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

  10. On the mechanochemical machinery underlying chromatin remodeling

    NASA Astrophysics Data System (ADS)

    Yusufaly, Tahir I.

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

  11. Chromatin dynamics in kidney development and function.

    PubMed

    Bechtel-Walz, Wibke; Huber, Tobias B

    2014-06-01

    Epigenetic mechanisms are fundamental key features of developing cells connecting developmental regulatory factors to chromatin modification. Changes in the environment during renal development can have long-lasting effects on the permanent tissue structure and the level of expression of important functional genes. These changes are believed to contribute to kidney disease occurrence and progression. Although the mechanisms of early patterning and cell fate have been well described for renal development, little is known about associated epigenetic modifications and their impact on how genes interact to specify the renal epithelial cells of nephrons and how this specification is relevant to maintaining normal renal function. A better understanding of the renal cell-specific epigenetic modifications and the interaction of different cell types to form this highly complex organ will not only help to better understand developmental defects and early loss of kidney function in children, but also help to understand and improve chronic disease progression, cell regeneration and renal aging. PMID:24817101

  12. Induction of condensed determination

    SciTech Connect

    Langley, P.

    1996-12-31

    In this paper we suggest determinations as a representation of knowledge that should be easy to understand. We briefly review determinations, which can be displayed in a tabular format, and their use in prediction, which involves a simple matching process. We describe CONDET, an algorithm that uses feature selection to construct determinations from training data, augmented by a condensation process that collapses rows to produce simpler structures. We report experiments that show condensation reduces complexity with no loss of accuracy, then discuss CONDET`s relation to other work and outline directions for future studies.

  13. 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.

  14. Integrated Model of Chemical Perturbations of a Biological PathwayUsing 18 In Vitro High Throughput Screening Assays for the Estrogen Receptor

    EPA Science Inventory

    We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity pa...

  15. Assaying mechanosensation*

    PubMed Central

    Chalfie, Martin; Hart, Anne C.; Rankin, Catharine H.; Goodman, Miriam B.

    2015-01-01

    C. elegans detect and respond to diverse mechanical stimuli using neuronal circuitry that has been defined by decades of work by C. elegans researchers. In this WormMethods chapter, we review and comment on the techniques currently used to assess mechanosensory response. This methods review is intended both as an introduction for those new to the field and a convenient compendium for the expert. A brief discussion of commonly used mechanosensory assays is provided, along with a discussion of the neural circuits involved, consideration of critical protocol details, and references to the primary literature. PMID:25093996

  16. Nuclear DNA Content and Chromatin Pattern of Rat Rhabdomyosarcoma Cell Sublines with Different Metastatic Potentials

    PubMed Central

    Dufer, Jean; Poupon, Marie-France; Yatouji, Sonia

    2000-01-01

    There is a constant need of features able to characterize potentially metastatic cells among the heterogeneous cell subpopulations which constitute a tumor. Image cytometry of metastatic tumor cells give rise to variable results, partly because of a heterogeneous origin of cells, or potential drug effects. The aim of this work was to characterize nuclear changes observed in metastatic cell clones issued in vitro from the same parental cell population The nuclear phenotypes of 6 cell sublines isolated from a rat rhabdomyosarcoma cell line and differing in their metastatic ability were evaluated by image cytometry on Feulgen?stained preparations. Densitometric [5], geometric [3] and textural [9] features were computed from each nuclear image. For each cell subline, a metastatic score, ranging from 0 to 10, was calculated on the basis of in vitro invasivity data, by measuring the number of pulmonary metastases observed after s.c. graft of tumor cells in rats. Data obtained were compared to karyotype, growth characteristics, and oncogene expressions of cell lines. The nuclear DNA content, the chromosome numbers, the cell sublines doubling times, and the distribution of cells within the cell cycle appear unrelated with this score. On the contrary, increase in metastatic ability is accompanied by changes in chromatin pattern as assessed by textural features. Progressive increase in chromatin condensation can be observed in cell sublines with increasing metastatic score. These results were confirmed by an unsupervised multivariate partitioning of rhabdomyosarcoma cells which identified two separate subsets whose distributions within the analyzed cell lines correlate with their metastatic ability. These data suggest that, in rat rhabdomyosarcoma cell sublines, metastatic ability could be associated with nuclear morphological changes at the level of chromatin texture. PMID:11007437

  17. The preRC protein ORCA organizes heterochromatin by assembling histone H3 lysine 9 methyltransferases on chromatin.

    PubMed

    Giri, Sumanprava; Aggarwal, Vasudha; Pontis, Julien; Shen, Zhen; Chakraborty, Arindam; Khan, Abid; Mizzen, Craig; Prasanth, Kannanganattu V; Ait-Si-Ali, Slimane; Ha, Taekjip; Prasanth, Supriya G

    2015-01-01

    Heterochromatic domains are enriched with repressive histone marks, including histone H3 lysine 9 methylation, written by lysine methyltransferases (KMTs). The pre-replication complex protein, origin recognition complex-associated (ORCA/LRWD1), preferentially localizes to heterochromatic regions in post-replicated cells. Its role in heterochromatin organization remained elusive. ORCA recognizes methylated H3K9 marks and interacts with repressive KMTs, including G9a/GLP and Suv39H1 in a chromatin context-dependent manner. Single-molecule pull-down assays demonstrate that ORCA-ORC (Origin Recognition Complex) and multiple H3K9 KMTs exist in a single complex and that ORCA stabilizes H3K9 KMT complex. Cells lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites. Changes in heterochromatin structure due to loss of ORCA affect replication timing, preferentially at the late-replicating regions. We demonstrate that ORCA acts as a scaffold for the establishment of H3K9 KMT complex and its association and activity at specific chromatin sites is crucial for the organization of heterochromatin structure. PMID:25922909

  18. Purkinje Cell Degeneration in pcd Mice Reveals Large Scale Chromatin Reorganization and Gene Silencing Linked to Defective DNA Repair*

    PubMed Central

    Baltanás, Fernando C.; Casafont, Iñigo; Lafarga, Vanesa; Weruaga, Eduardo; Alonso, José R.; Berciano, María T.; Lafarga, Miguel

    2011-01-01

    DNA repair protects neurons against spontaneous or disease-associated DNA damage. Dysfunctions of this mechanism underlie a growing list of neurodegenerative disorders. The Purkinje cell (PC) degeneration mutation causes the loss of nna1 expression and is associated with the postnatal degeneration of PCs. This PC degeneration dramatically affects nuclear architecture and provides an excellent model to elucidate the nuclear mechanisms involved in a whole array of neurodegenerative disorders. We used immunocytochemistry for histone variants and components of the DNA damage response, an in situ transcription assay, and in situ hybridization for telomeres to analyze changes in chromatin architecture and function. We demonstrate that the phosphorylation of H2AX, a DNA damage signal, and the trimethylation of the histone H4K20, a repressive mark, in extensive domains of genome are epigenetic hallmarks of chromatin in degenerating PCs. These histone modifications are associated with a large scale reorganization of chromatin, telomere clustering, and heterochromatin-induced gene silencing, all of them key factors in PC degeneration. Furthermore, ataxia telangiectasia mutated and 53BP1, two components of the DNA repair pathway, fail to be concentrated in the damaged chromatin compartments, even though the expression levels of their coding genes were slightly up-regulated. Although the mechanism by which Nna1 loss of function leads to PC neurodegeneration is undefined, the progressive accumulation of DNA damage in chromosome territories irreversibly compromises global gene transcription and seems to trigger PC degeneration and death. PMID:21700704

  19. 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.

  20. Transcriptional response to stress in the dynamic chromatin environment of cycling and mitotic cells

    PubMed Central

    Vihervaara, Anniina; Sergelius, Christian; Vasara, Jenni; Blom, Malin A. H.; Elsing, Alexandra N.; Roos-Mattjus, Pia; Sistonen, Lea

    2013-01-01

    Heat shock factors (HSFs) are the master regulators of transcription under protein-damaging conditions, acting in an environment where the overall transcription is silenced. We determined the genomewide transcriptional program that is rapidly provoked by HSF1 and HSF2 under acute stress in human cells. Our results revealed the molecular mechanisms that maintain cellular homeostasis, including HSF1-driven induction of polyubiquitin genes, as well as HSF1- and HSF2-mediated expression patterns of cochaperones, transcriptional regulators, and signaling molecules. We characterized the genomewide transcriptional response to stress also in mitotic cells where the chromatin is tightly compacted. We found a radically limited binding and transactivating capacity of HSF1, leaving mitotic cells highly susceptible to proteotoxicity. In contrast, HSF2 occupied hundreds of loci in the mitotic cells and localized to the condensed chromatin also in meiosis. These results highlight the importance of the cell cycle phase in transcriptional responses and identify the specific mechanisms for HSF1 and HSF2 in transcriptional orchestration. Moreover, we propose that HSF2 is an epigenetic regulator directing transcription throughout cell cycle progression. PMID:23959860

  1. 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

  2. Diffusible factors are responsible for differences in nuclease sensitivity among chromatins originating from different cell types.

    PubMed

    Chambers, S A; Cognetti, G; Shaw, B R

    1984-09-01

    We have examined the kinetics of nuclease digestion of chromatin from committed and uncommitted cells in experiments where the nuclei are mixed and co-digested. Cultures of the sea urchin, Arbacia punctulata, were grown to the 16-cell stage in either [3H]thymidine or [14C]thymidine and the macromere, mesomere, and micromere cell types separated. After isolation, sets of nuclei with two different blastomere types (each having different radionucleotide tagging) were mixed and co-digested with micrococcal nuclease or DNase. I. The extent of digestion was monitored by solubility in 5% perchloric acid (PCA). We find no significant differences in initial digestion rates or limit digests among the different cell types when co-digested with either nuclease. Differences in nuclease sensitivity observed when nuclei are digested separately are abolished when nuclei are probed in a mixing experiment. The results support the hypothesis that phenotypic differences in digestibility among different cell types in vitro reflect differences in chromatin-condensing factors which can diffuse between nuclei. PMID:6236093

  3. Angiogenesis Assays.

    PubMed

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P

    2016-01-01

    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis. PMID:26608294

  4. 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

  5. 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.

  6. MUNICIPAL LANDFILL GAS CONDENSATE

    EPA Science Inventory

    New regulations relative to air emissions from municipal landfills may require the installation of gas collection systems at landfills. As landfill gas (LFG) is collected, water and other vapors in the gas condense in the system or are purposely removed in the normal treatment of...

  7. Exciton-polariton condensates

    NASA Astrophysics Data System (ADS)

    Byrnes, Tim; Kim, Na Young; Yamamoto, Yoshihisa

    2014-11-01

    Recently a new type of system exhibiting spontaneous coherence has emerged--the exciton-polariton condensate. Exciton-polaritons (or polaritons for short) are bosonic quasiparticles that exist inside semiconductor microcavities, consisting of a superposition of an exciton and a cavity photon. Above a threshold density the polaritons macroscopically occupy the same quantum state, forming a condensate. The polaritons have a lifetime that is typically comparable to or shorter than thermalization times, giving them an inherently non-equilibrium nature. Nevertheless, they exhibit many of the features that would be expected of equilibrium Bose-Einstein condensates (BECs). The non-equilibrium nature of the system raises fundamental questions as to what it means for a system to be a BEC, and introduces new physics beyond that seen in other macroscopically coherent systems. In this review we focus on several physical phenomena exhibited by exciton-polariton condensates. In particular, we examine topics such as the difference between a polariton BEC, a polariton laser and a photon laser, as well as physical phenomena such as superfluidity, vortex formation, and Berezinskii-Kosterlitz-Thouless and Bardeen-Cooper-Schrieffer physics. We also discuss the physics and applications of engineered polariton structures.

  8. Depletion Effects Massively Change Chromatin Properties and Influence Genome Folding

    PubMed Central

    Diesinger, Philipp M.; Heermann, Dieter W.

    2009-01-01

    Abstract We present a Monte Carlo model for genome folding at the 30-nm scale with focus on linker-histone and nucleosome depletion effects. We find that parameter distributions from experimental data do not lead to one specific chromatin fiber structure, but instead to a distribution of structures in the chromatin phase diagram. Depletion of linker histones and nucleosomes affects, massively, the flexibility and the extension of chromatin fibers. Increasing the amount of nucleosome skips (i.e., nucleosome depletion) can lead either to a collapse or to a swelling of chromatin fibers. These opposing effects are discussed and we show that depletion effects may even contribute to chromatin compaction. Furthermore, we find that predictions from experimental data for the average nucleosome skip rate lie exactly in the regime of maximum chromatin compaction. Finally, we determine the pair distribution function of chromatin. This function reflects the structure of the fiber, and its Fourier-transform can be measured experimentally. Our calculations show that even in the case of fibers with depletion effects, the main dominant peaks (characterizing the structure and the length scales) can still be identified. PMID:19843447

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

    PubMed Central

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

    2015-01-01

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

  10. Evidence for torsional stress in transcriptionally activated chromatin.

    PubMed Central

    Leonard, M W; Patient, R K

    1991-01-01

    The existence of torsional stress in eukaryotic chromatin has been controversial. To determine whether it could be detected, we probed the structure of an alternating AT tract. These sequences adopt cruciform geometry when the DNA helix is torsionally strained by negative supercoiling. The single-strand-specific nuclease P1 was used to determine the structure of an alternating AT sequence upstream of the Xenopus beta-globin gene when assembled into chromatin in microinjected Xenopus oocytes. The pattern of cleavage by P1 nuclease strongly suggests that the DNA in this chromatin template is under torsional stress. The cruciform was detected specifically in the most fully reconstituted templates at later stages of chromatin assembly, suggesting that negative supercoiling is associated with chromatin maturation. Furthermore, the number of torsionally strained templates increased dramatically at the time when transcription of assembled chromatin templates began. Transcription itself has been shown to induce supercoiling, but the requisite negative supercoiling for cruciform extrusion by (AT)n in oocytes was not generated in this way since the characteristic P1 cutting pattern was retained even when RNA polymerase elongation was blocked with alpha-amanitin. Thus, torsional stress is associated with transcriptional activation of chromatin templates in the absence of ongoing transcription. Images PMID:1944280

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

    PubMed Central

    2012-01-01

    Background A plastic chromatin structure has emerged as fundamental to the self-renewal and pluripotent capacity of embryonic stem (ES) cells. Direct measurement of chromatin dynamics in vivo is, however, challenging as high spatiotemporal resolution is required. Here, we present a new tracking-based method which can detect high frequency chromatin movement and quantify the mechanical dynamics of chromatin in live cells. Results We use this method to study how the mechanical properties of chromatin movement in human embryonic stem cells (hESCs) are modulated spatiotemporally during differentiation into cardiomyocytes (CM). Notably, we find that pluripotency is associated with a highly discrete, energy-dependent frequency of chromatin movement that we refer to as a ‘breathing’ state. We find that this ‘breathing’ state is strictly dependent on the metabolic state of the cell and is progressively silenced during differentiation. Conclusions We thus propose that the measured chromatin high frequency movements in hESCs may represent a hallmark of pluripotency and serve as a mechanism to maintain the genome in a transcriptionally accessible state. This is a result that could not have been observed without the high spatial and temporal resolution provided by this novel tracking method. PMID:23259580

  12. High throughput automated chromatin immunoprecipitation as a platform for drug screening and antibody validation†,‡

    PubMed Central

    Wu, Angela R.; Kawahara, Tiara L.A.; Rapicavoli, Nicole A.; van Riggelen, Jan; Shroff, Emelyn H.; Xu, Liwen; Felsher, Dean W.; Chang, Howard Y.; Quake, Stephen R.

    2014-01-01

    Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein–DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large scale screening purposes. To address these challenges, we report HTChIP, an automated microfluidic-based platform for performing high-throughput ChIP screening measurements of 16 different targets simultaneously, with potential for further scale-up. From chromatin to analyzable PCR results only takes one day using HTChIP, as compared to several days up to one week for conventional protocols. HTChIP can also be used to test multiple antibodies and select the best performer for downstream ChIP applications, saving time and reagent costs of unsuccessful ChIP assays as a result of poor antibody quality. We performed a series of characterization assays to demonstrate that HTChIP can rapidly and accurately evaluate the epigenetic states of a cell, and that it is sensitive enough to detect the changes in the epigenetic state induced by a cytokine stimulant over a fine temporal resolution. With these results, we believe that HTChIP can introduce large improvements in routine ChIP, antibody screening, and drug screening efficiency, and further facilitate the use of ChIP as a valuable tool for research and discovery. PMID:22566096

  13. 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 positioning, and that care must be taken in the choice of models used to interpret the experimental properties of long chromatin fibers.

  14. 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 positioning, and that care must be taken in the choice of models used to interpret the experimental properties of long chromatin fibers. PMID:25564155

  15. Global chromatin fibre compaction in response to DNA damage

    PubMed Central

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

    2011-01-01

    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 (?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 ?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 linker histones. We suggest that following DSB formation, although there is localised chromatin unfolding to facilitate repair, the bulk genome becomes rapidly compacted protecting cells from further damage. PMID:22020103

  16. Changeability of sperm chromatin structure during liquid storage of ovine semen in milk-egg yolk- and soybean lecithin-based extenders and their relationships to field-fertility.

    TOXLINE Toxicology Bibliographic Information

    Khalifa T; Lymberopoulos A

    2013-12-01

    The aim of this experiment was to study the effect of semen extender on sperm chromatin structure and to correlate chromatin integrity with field-fertility of preserved ram semen. Ejaculates of at least 2 × 10(9) sperm/ml and 70 % progressive motility were collected using an artificial vagina from Chios rams (n = 11, 4-6 years old), split-diluted to 1 × 10(9) sperm/ml with milk-egg yolk- and soybean lecithin (Ovixcell®)-based extenders, packaged in 0.5-ml straws and examined after 6, 24 and 48 h of storage at 5 ± 1 °C. Evaluation endpoints were computer-assisted sperm motion analysis, fluorescence-based analysis of chromatin structure by chromomycin A3 and acridine orange assays, and 65-day pregnancy rate (PR) of 34- to 36-h preserved semen after intra-cervical insemination of ewes (n = 154) in progestagen-synchronized estrus. Neither extender nor storage time had any influence on incidence of decondensed chromatin. Unlike Ovixcell® extender, deterioration of sperm motility (P < 0.01) and chromatin stability (P < 0.005) was detected after 48 h of storage in milk-egg yolk extender. Sperm motility accounted for 14.4-18.5 % of variations in chromatin integrity (P < 0.001). No significant difference was found in PR of Ovixcell®- and milk-egg yolk-stored semen. Nevertheless, PR differed between rams (14.3-71.4 %; P < 0.025). Chromatin integrity explained 10.2-56.3 % of variations in PR (P < 0.05-0.01). A pronounced decline in PR (19.1 %) was observed when percentages of decondensed and destabilized chromatin have reached thresholds of 10.5-30 % and 4-9 %, respectively. In conclusion, Ovixcell® is superior to milk-egg yolk extender in preserving chromatin stability and motility. Chromatin defects are negatively associated with sperm fertility.

  17. Chromatin remodeling at DNA double-strand breaks.

    PubMed

    Price, Brendan D; D'Andrea, Alan D

    2013-03-14

    DNA double-strand breaks (DSBs) can arise from multiple sources, including exposure to ionizing radiation. The repair of DSBs involves both posttranslational modification of nucleosomes and concentration of DNA-repair proteins at the site of damage. Consequently, nucleosome packing and chromatin architecture surrounding the DSB may limit the ability of the DNA-damage response to access and repair the break. Here, we review early chromatin-based events that promote the formation of open, relaxed chromatin structures at DSBs and that allow the DNA-repair machinery to access the spatially confined region surrounding the DSB, thereby facilitating mammalian DSB repair. PMID:23498941

  18. The Necessity of Chromatin: A View in Perspective.

    PubMed

    Pirrotta, Vincenzo

    2016-01-01

    SUMMARYEpigenomics has grown exponentially, providing a better understanding of the mechanistic aspects of new and old phenomena originally described through genetics, as well as providing unexpected insights into the way chromatin modulates the genomic information. In this overview, some of the advances are selected for discussion and comment under six topics: (1) histone modifications, (2) weak interactions, (3) interplay with external inputs, (4) the role of RNA molecules, (5) chromatin folding and architecture, and, finally, (6) a view of the essential role of chromatin transactions in regulating the access to genomic DNA. PMID:26729649

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

    PubMed

    Witkowski, Leora; Foulkes, William D

    2015-12-01

    Over the past decade, chromatin remodelling emerged as one of the most important causes of both abnormal development and cancer. Although much has been written about one or another of the complexes, no recent concise summary of the chromatin remodelling families as a whole is available. In this short review, we introduce the family members, briefly summarize their role in developmental abnormalities and neoplasia, and outline the different ways in which these families remodel chromatin. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:26174723

  20. Multivalent engagement of chromatin modifications by linked binding modules

    PubMed Central

    Ruthenburg, Alexander J.; Li, Haitao; Patel, Dinshaw J.; Allis, C. David

    2015-01-01

    Various chemical modifications on histones and regions of associated DNA play crucial roles in genome management by binding specific factors that, in turn, serve to alter the structural properties of chromatin. These so-called effector proteins have typically been studied with the biochemist's paring knife — the capacity to recognize specific chromatin modifications has been mapped to an increasing number of domains that frequently appear in the nuclear subset of the proteome, often present in large, multisubunit complexes that bristle with modification-dependent binding potential. We propose that multivalent interactions on a single histone tail and beyond may have a significant, if not dominant, role in chromatin transactions. PMID:18037899

  1. Stress-induced structural changes in plant chromatin.

    PubMed

    Probst, Aline V; Mittelsten Scheid, Ortrun

    2015-10-01

    Stress defense in plants is elaborated at the level of protection and adaptation. Dynamic changes in sophisticated chromatin substructures and concomitant transcriptional changes play an important role in response to stress, as illustrated by the transient rearrangement of compact heterochromatin structures or the modulation of chromatin composition and modification upon stress exposure. To connect cytological, developmental, and molecular data around stress and chromatin is currently an interesting, multifaceted, and sometimes controversial field of research. This review highlights some of the most recent findings on nuclear reorganization, histone variants, histone chaperones, DNA- and histone modifications, and somatic and meiotic heritability in connection with stress. PMID:26042538

  2. Sperm DNA assays and their relationship to sperm motility and morphology in bulls (Bos Taurus).

    PubMed

    Serafini, Rosanna; Romano, Juan E; Varner, Dickson D; Di Palo, Rossella; Love, Charles C

    2015-08-01

    The relationship among sperm DNA assays in bulls with different sperm motility and morphology measures has not been reported. The objectives of the present study were to (1) describe Comet assay measures and examine their repeatability (inter- and intra-assay); (2) compare sperm DNA quality assays (i.e., Sperm Chromatin Structure Assay-SCSA; alkaline and neutral Comet assays and Sperm Bos Halomax assay-SBH) in two groups of bulls selected on either greater and lesser sperm motility and morphology (greater compared with lesser); (3) determine the relationship among DNA assays and sperm motility and morphology values. Inter-assay repeatability was greater for the neutral Comet assay as compared to the alkaline Comet assay. Intra-assay repeatability was greater than inter-assay repeatability for both Comet assays. Comet assay dimension measures and percentage tail DNA were the most repeatable for both Comet assays. Among sperm DNA quality assays, only SCSA measures and neutral Comet assay Ghosts (% Ghosts), head diameter and area, and comet area were different between greater and lesser sperm quality groups (P<0.05). The SCSA measures were inversely correlated with neutral Comet head measures (diameter, area, and intensity) and positively with percentage Ghosts (P<0.05). The % Ghosts and COMP-αt were correlated with some measures of sperm morphology and sperm motility. The neutral Comet assay was more appropriate for sperm evaluation than the alkaline Comet assay for distinguishing among groups with different sperm quality. PMID:26070911

  3. 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.

  4. Gravitational vacuum condensate stars

    PubMed Central

    Mazur, Pawel O.; Mottola, Emil

    2004-01-01

    A new final state of gravitational collapse is proposed. By extending the concept of Bose–Einstein condensation to gravitational systems, a cold, dark, compact object with an interior de Sitter condensate pv = -?v and an exterior Schwarzschild geometry of arbitrary total mass M is constructed. These regions are separated by a shell with a small but finite proper thickness ? of fluid with equation of state p = +?, replacing both the Schwarzschild and de Sitter classical horizons. The new solution has no singularities, no event horizons, and a global time. Its entropy is maximized under small fluctuations and is given by the standard hydrodynamic entropy of the thin shell, which is of the order kB?Mc/, instead of the Bekenstein–Hawking entropy formula, SBH = 4?kBGM2/c. Hence, unlike black holes, the new solution is thermodynamically stable and has no information paradox. PMID:15210982

  5. 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.

  6. Polycomb-mediated chromatin compaction weathers the STORM.

    PubMed

    Williamson, Iain; Bickmore, Wendy A; Illingworth, Robert S

    2016-01-01

    A recent super-resolution imaging study by Boettiger et al. elegantly demonstrates that three epigenetically defined, and functionally disparate, chromatin states have distinct folding characteristics in Drosophila nuclei. PMID:26917033

  7. Reshaping the chromatin landscape after spinal cord injury

    PubMed Central

    WONG, Jamie K.; ZOU, Hongyan

    2014-01-01

    The pathophysiology underlying spinal cord injury is complex. Mechanistic understanding of the adaptive responses to injury is critical for targeted therapy aimed at reestablishing lost connections between proximal and distal neurons. After injury, cell-type specific gene transcription programs govern distinct cellular behaviors, and chromatin regulators play a central role in shaping the chromatin landscape to adjust transcriptional profiles in a context-dependent manner. In this review, we summarize recent progress on the pleiotropic roles of chromatin regulators in mediating the diverse adaptive behaviors of neurons and glial cells after spinal cord injury, and wherever possible, discuss the underlying mechanisms and genomic targets. We specifically draw attention to the perspective that takes into consideration the impact of epigenetic modulation on axon growth potential, together with its effect on wound-healing properties of glial cells. Epigenetic modulation of chromatin state represents an emerging therapeutic direction to promote neural repair and axon regeneration after spinal cord injury. PMID:25554728

  8. 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

  9. Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin.

    PubMed

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

    2016-02-11

    Telomeres, repetitive DNA sequences at chromosome ends, are shielded against the DNA damage response (DDR) by the shelterin complex. To understand how shelterin protects telomere ends, we investigated the structural organization of telomeric chromatin in human cells using super-resolution microscopy. We found that telomeres form compact globular structures through a complex network of interactions between shelterin subunits and telomeric DNA, but not by DNA methylation, histone deacetylation, or histone trimethylation at telomeres and subtelomeric regions. Mutations that abrogate shelterin assembly or removal of individual subunits from telomeres cause up to a 10-fold increase in telomere volume. Decompacted telomeres accumulate DDR signals and become more accessible to telomere-associated proteins. Recompaction of telomeric chromatin using an orthogonal method displaces DDR signals from telomeres. These results reveal the chromatin remodeling activity of shelterin and demonstrate that shelterin-mediated compaction of telomeric chromatin provides robust protection of chromosome ends against the DDR machinery. PMID:26871633

  10. 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

  11. Chromatin modifiers and remodellers: regulators of cellular differentiation.

    PubMed

    Chen, Taiping; Dent, Sharon Y R

    2014-02-01

    Cellular differentiation is, by definition, epigenetic. Genome-wide profiling of pluripotent cells and differentiated cells suggests global chromatin remodelling during differentiation, which results in a progressive transition from a fairly open chromatin configuration to a more compact state. Genetic studies in mouse models show major roles for a variety of histone modifiers and chromatin remodellers in key developmental transitions, such as the segregation of embryonic and extra-embryonic lineages in blastocyst stage embryos, the formation of the three germ layers during gastrulation and the differentiation of adult stem cells. Furthermore, rather than merely stabilizing the gene expression changes that are driven by developmental transcription factors, there is emerging evidence that chromatin regulators have multifaceted roles in cell fate decisions. PMID:24366184

  12. DNA damage signalling targets the kinetochore to promote chromatin mobility.

    PubMed

    Strecker, Jonathan; Gupta, Gagan D; Zhang, Wei; Bashkurov, Mikhail; Landry, Marie-Claude; Pelletier, Laurence; Durocher, Daniel

    2016-03-01

    In budding yeast, chromatin mobility increases after a DNA double-strand break (DSB). This increase is dependent on Mec1, the yeast ATR kinase, but the targets responsible for this phenomenon are unknown. Here we report that the Mec1-dependent phosphorylation of Cep3, a kinetochore component, is required to stimulate chromatin mobility after DNA breaks. Cep3 phosphorylation counteracts a constraint on chromosome movement imposed by the attachment of centromeres to the spindle pole body. A second constraint, imposed by the tethering of telomeres to the nuclear periphery, is also relieved after chromosome breakage. A non-phosphorylatable Cep3 mutant that impairs DSB-induced chromatin mobility is proficient in DSB repair, suggesting that break-induced chromatin mobility may be dispensable for homology search. Rather, we propose that the relief of centromeric constraint promotes cell cycle arrest and faithful chromosome segregation through the engagement of the spindle assembly checkpoint. PMID:26829389

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

    PubMed

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

    2016-01-01

    Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here, we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of p300, EZH2, or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions, and drug treatments. PMID:26687680

  14. Nuclear morphometry and chromatin textural characteristics of basal cell carcinoma*

    PubMed Central

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

    2015-01-01

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

  15. Computational analysis of promoter elements and chromatin features in yeast.

    PubMed

    Wyrick, John J

    2012-01-01

    Regulatory elements in promoter sequences typically function as binding sites for transcription factor proteins and thus are critical determinants of gene transcription. There is growing evidence that chromatin features, such as histone modifications or nucleosome positions, also have important roles in transcriptional regulation. Recent functional genomics and computational studies have yielded extensive datasets cataloging transcription factor binding sites (TFBS) and chromatin features, such as nucleosome positions, throughout the yeast genome. However, much of this data can be difficult to navigate or analyze efficiently. This chapter describes practical methods for the visualization, data mining, and statistical analysis of yeast promoter elements and chromatin features using two Web-accessible bioinformatics databases: ChromatinDB and Ceres. PMID:22113279

  16. Biased chromatin signatures around polyadenylation sites and exons.

    PubMed

    Spies, Noah; Nielsen, Cydney B; Padgett, Richard A; Burge, Christopher B

    2009-10-23

    Core RNA-processing reactions in eukaryotic cells occur cotranscriptionally in a chromatin context, but the relationship between chromatin structure and pre-mRNA processing is poorly understood. We observed strong nucleosome depletion around human polyadenylation sites (PAS) and nucleosome enrichment just downstream of PAS. In genes with multiple alternative PAS, higher downstream nucleosome affinity was associated with higher PAS usage, independently of known PAS motifs that function at the RNA level. Conversely, exons were associated with distinct peaks in nucleosome density. Exons flanked by long introns or weak splice sites exhibited stronger nucleosome enrichment, and incorporation of nucleosome density data improved splicing simulation accuracy. Certain histone modifications, including H3K36me3 and H3K27me2, were specifically enriched on exons, suggesting active marking of exon locations at the chromatin level. Together, these findings provide evidence for extensive functional connections between chromatin structure and RNA processing. PMID:19854133

  17. Chromatin and extracellular vesicle associated sperm RNAs

    PubMed Central

    Johnson, Graham D.; Mackie, Paula; Jodar, Meritxell; Moskovtsev, Sergey; Krawetz, Stephen A.

    2015-01-01

    A diverse pool of RNAs remain encapsulated within the transcriptionally silent spermatozoon despite the dramatic reduction in cellular and nuclear volume following cytoplasm/nucleoplasm expulsion. The impact of this pronounced restructuring on the distribution of transcripts inside the sperm essentially remains unknown. To define their compartmentalization, total RNA >100 nt was extracted from sonicated (SS) mouse spermatozoa and detergent demembranated sucrose gradient fractionated (Cs/Tx) sperm heads. Sperm RNAs predominately localized toward the periphery. The corresponding distribution of transcripts and thus localization and complexity were then inferred by RNA-seq. Interestingly, the number of annotated RNAs in the CsTx sperm heads exhibiting reduced peripheral enrichment was restricted. However this included Cabyr, the calcium-binding tyrosine phosphorylation-regulated protein encoded transcript. It is present in murine zygotes prior to the maternal to the zygotic transition yet absent in oocytes, consistent with the delivery of internally positioned sperm-borne RNAs to the embryo. In comparison, transcripts enriched in sonicated sperm contributed to the mitochondria and exosomes along with several nuclear transcripts including the metastasis associated lung adenocarcinoma transcript 1 (Malat1) and several small nucleolar RNAs. Their preferential peripheral localization suggests that chromatin remodeling during spermiogenesis is not limited to nucleoproteins as part of the nucleoprotein exchange. PMID:26071953

  18. Chromatin remodeling complex in Treg function.

    PubMed

    Jani, Anant; Chi, Tian; Wan, Yisong Y

    2009-05-01

    Regulatory T cells (Treg), formerly known as suppressor T cells, are essential for maintaining self-tolerance as well as immune homeostasis. Lack of Treg or normal function of Treg often leads to lymphoproliferative syndrome and autoimmunity in human and mouse. The chromatin remodeling BAF complex regulates gene expression through the activity of Brg. Genetic ablation of Brg gene in mouse resulted in early embryonic lethality. T cell failed to develop in the thymus when Brg is deleted at DN stage. Using a Brg conditional KO mouse model, we deleted Brg at the DP stage in the thymus. Unexpectedly, T cells developed and matured normally. However, these mice displayed lympho-proliferative syndrome 2-4 months of age with enlarged peripheral lymphoid organs and leukocyte infiltration in non-lymphoid organs. T cells from these mice turned into effector cells producing increased amounts of effector cytokines as early as 4 weeks after birth. Further analysis revealed that the Treg population was specifically affected by Brg deletion. In this mini-review, we will discuss in detail the properties of Tregs controlled by Brg and the potential underlying mechanisms for an unanticipated, specific role of the Brg-containing BAF complex in controlling Treg functions. PMID:19539570

  19. The quantitative architecture of centromeric chromatin

    PubMed Central

    Bodor, Dani L; Mata, João F; Sergeev, Mikhail; David, Ana Filipa; Salimian, Kevan J; Panchenko, Tanya; Cleveland, Don W; Black, Ben E; Shah, Jagesh V; Jansen, Lars ET

    2014-01-01

    The centromere, responsible for chromosome segregation during mitosis, is epigenetically defined by CENP-A containing chromatin. The amount of centromeric CENP-A has direct implications for both the architecture and epigenetic inheritance of centromeres. Using complementary strategies, we determined that typical human centromeres contain ?400 molecules of CENP-A, which is controlled by a mass-action mechanism. This number, despite representing only ?4% of all centromeric nucleosomes, forms a ?50-fold enrichment to the overall genome. In addition, although pre-assembled CENP-A is randomly segregated during cell division, this amount of CENP-A is sufficient to prevent stochastic loss of centromere function and identity. Finally, we produced a statistical map of CENP-A occupancy at a human neocentromere and identified nucleosome positions that feature CENP-A in a majority of cells. In summary, we present a quantitative view of the centromere that provides a mechanistic framework for both robust epigenetic inheritance of centromeres and the paucity of neocentromere formation. DOI: http://dx.doi.org/10.7554/eLife.02137.001 PMID:25027692

  20. Engineered apoptotic nucleases for chromatin research.

    PubMed

    Xiao, Fei; Widlak, Piotr; Garrard, William T

    2007-01-01

    We have created new genomics tools for chromatin research by genetically engineering the human and mouse major apoptotic nucleases that are responsible for internucleosomal DNA cleavage, DNA fragmentation factor (DFF). Normally, in its inactive form, DFF is a heterodimer composed of a 45-kDa chaperone inhibitor subunit (DFF45 or ICAD), and a 40-kDa latent endonuclease subunit (DFF40 or CAD). Upon caspase-3 cleavage of DFF45, DFF40 forms active endonuclease homo-oligomers. Although Saccharomyces cerevisiae lacks DFF, expression of caspase-3 is lethal in this organism, but expression of the highly sequence-specific tobacco etch virus protease (TEVP) is harmless. Therefore, we inserted TEVP cleavage sites immediately downstream of the two caspase-3 cleavage sites within DFF45, generating a novel form of DFF (DFF-T) whose nuclease activity proved to be exclusively under the control of TEVP. We demonstrate that co-expression of TEVP and DFF-T under galactose control results in nucleosomal DNA laddering and cell death in S. cerevisiae. We also created synthetic DFF genes with optimized codons for high-level expression in Eschericia coli or S. cerevisiae. We further demonstrate the excellence of the synthetic gene products for in vitro mapping of the nucleosome positions and hypersensitive sites in specific genes such as the yeast PHO5. PMID:17626049

  1. Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells.

    PubMed

    van Rensburg, R; Beyer, I; Yao, X-Y; Wang, H; Denisenko, O; Li, Z-Y; Russell, D W; Miller, D G; Gregory, P; Holmes, M; Bomsztyk, K; Lieber, A

    2013-02-01

    Achieving transgene integration into preselected genomic sites is currently one of the central tasks in stem cell gene therapy. A strategy to mediate such targeted integration involves site-specific endonucleases. Two genomic sites within the MBS85 and chemokine (C-C motif) receptor 5 (CCR5) genes (AAVS1 and CCR5 zinc-finger nuclease (CCR5-ZFN) sites, respectively) have recently been suggested as potential target regions for integration as their disruption has no functional consequence. We hypothesized that efficient transgene integration maybe affected by DNA accessibility of endonucleases and therefore studied the transcriptional and chromatin status of the AAVS1 and CCR5 sites in eight human induced pluripotent stem (iPS) cell lines and pooled CD34+ hematopoietic stem cells (HSCs). Matrix chromatin immunoprecipitation (ChIP) assays demonstrated that the CCR5 site and surrounding regions possessed a predominantly closed chromatin configuration consistent with its transcriptional inactivity in these cell types. In contrast, the AAVS1 site was located within a transcriptionally active region and exhibited an open chromatin configuration in both iPS cells and HSCs. To show that the AAVS1 site is readily amendable to genome modification, we expressed Rep78, an AAV2-derived protein with AAVS1-specific endonuclease activity, in iPS cells after adenoviral gene transfer. We showed that Rep78 efficiently associated with the AAVS1 site and triggered genome modifications within this site. On the other hand, binding to and modification of the CCR5-ZFN site by a ZFN was relatively inefficient. Our data suggest a critical influence of chromatin structure on efficacy of site-specific endonucleases used for genome editing. PMID:22436965

  2. 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.

  3. ChIP-less analysis of chromatin states

    PubMed Central

    2014-01-01

    Background Histone post-translational modifications (PTMs) are key epigenetic regulators in chromatin-based processes. Increasing evidence suggests that vast combinations of PTMs exist within chromatin histones. These complex patterns, rather than individual PTMs, are thought to define functional chromatin states. However, the ability to interrogate combinatorial histone PTM patterns at the nucleosome level has been limited by the lack of direct molecular tools. Results Here we demonstrate an efficient, quantitative, antibody-free, chromatin immunoprecipitation-less (ChIP-less) method for interrogating diverse epigenetic states. At the heart of the workflow are recombinant chromatin reader domains, which target distinct chromatin states with combinatorial PTM patterns. Utilizing a newly designed combinatorial histone peptide microarray, we showed that three reader domains (ATRX-ADD, ING2-PHD and AIRE-PHD) displayed greater specificity towards combinatorial PTM patterns than corresponding commercial histone antibodies. Such specific recognitions were employed to develop a chromatin reader-based affinity enrichment platform (matrix-assisted reader chromatin capture, or MARCC). We successfully applied the reader-based platform to capture unique chromatin states, which were quantitatively profiled by mass spectrometry to reveal interconnections between nucleosomal histone PTMs. Specifically, a highly enriched signature that harbored H3K4me0, H3K9me2/3, H3K79me0 and H4K20me2/3 within the same nucleosome was identified from chromatin enriched by ATRX-ADD. This newly reported PTM combination was enriched in heterochromatin, as revealed by the associated DNA. Conclusions Our results suggest the broad utility of recombinant reader domains as an enrichment tool specific to combinatorial PTM patterns, which are difficult to probe directly by antibody-based approaches. The reader affinity platform is compatible with several downstream analyses to investigate the physical coexistence of nucleosomal PTM states associated with specific genomic loci. Collectively, the reader-based workflow will greatly facilitate our understanding of how distinct chromatin states and reader domains function in gene regulatory mechanisms. PMID:24872844

  4. Mutagenicity testing of condensates of smoke from titanium dioxide/hexachloroethane and zinc/hexachloroethane pyrotechnic mixtures.

    PubMed

    Karlsson, N; Fängmark, I; Häggqvist, I; Karlsson, B; Rittfeldt, L; Marchner, H

    1991-05-01

    Condensates of smoke from titanium dioxide/hexachloroethane and zinc/hexachloroethane pyrotechnic mixtures were investigated for their potential to produce genetic damage in the tester strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium and in the mouse bone marrow micronucleus assay. Both smoke condensates contained several chlorinated hydrocarbons among which tetrachloroethylene, hexachloroethane, hexachlorobutadiene and hexachlorobenzene were identified by GC/MS. Condensate of smoke from titanium dioxide/hexachloroethane showed a dose-related positive response in the Salmonella assay with strains TA98 and TA100 in the absence of metabolic activation from rat liver S9 fraction. Both smoke condensates were negative in the micronucleus assay but produced a small but significant depression of erythropoietic activity. The results indicate that smoke condensate from titanium dioxide/hexachloroethane mixtures contains unidentified compound(s) that may be considered mutagenic in the Salmonella assay. PMID:2027339

  5. Neutrophil extracellular traps: Is immunity the second function of chromatin?

    PubMed Central

    2012-01-01

    Neutrophil extracellular traps (NETs) are made of processed chromatin bound to granular and selected cytoplasmic proteins. NETs are released by white blood cells called neutrophils, maybe as a last resort, to control microbial infections. This release of chromatin is the result of a unique form of cell death, dubbed “NETosis.” Here we review our understanding of how NETs are made, their function in infections and as danger signals, and their emerging importance in autoimmunity and coagulation. PMID:22945932

  6. Chromatin Insulators: Linking genome organization to cellular function

    PubMed Central

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

    2013-01-01

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

  7. Mechanisms and Functions of ATP-Dependent Chromatin-Remodeling Enzymes

    PubMed Central

    Narlikar, Geeta J.; Sundaramoorthy, Ramasubramanian; Owen-Hughes, Tom

    2013-01-01

    Chromatin provides both a means to accommodate a large amount of genetic material in a small space and a means to package the same genetic material in different chromatin states. Transitions between chromatin states are enabled by chromatin-remodeling ATPases, which catalyze a diverse range of structural transformations. Biochemical evidence over the last two decades suggests that chromatin-remodeling activities may have emerged by adaptation of ancient DNA translocases to respond to specific features of chromatin. Here, we discuss such evidence and also relate mechanistic insights to our understanding of how chromatin-remodeling enzymes enable different in vivo processes. PMID:23911317

  8. Condenser performance monitoring and cleaning

    SciTech Connect

    Walden, J.V.

    1998-12-31

    The main condenser at Ginna Station was retubed from admiralty brass to 316 stainless steel. A condenser performance monitoring spreadsheet was developed using EPRI guidelines after fouling was discovered. PEPSE computer models were used to determine the power loss and confirm the spreadsheet results. Cleaning of the condenser was performed using plastic scrubbers. Condenser performance improved dramatically following the cleaning. PEPSE, condenser spreadsheet performance, and actual observed plant data correlated well together. The fouling mechanism was determined to be a common lake bacteria and fungus growth which was combined with silt. Chlorination of the circulating water system at the allowable limits is keeping the biofouling under control.

  9. An RNAi-Based Candidate Screen for Modifiers of the CHD1 Chromatin Remodeler and Assembly Factor in Drosophila melanogaster

    PubMed Central

    Kim, Sharon; Bugga, Lakshmi; Hong, Eugenie S.; Zabinsky, Rebecca; Edwards, Rebecca G.; Deodhar, Parimal A.; Armstrong, Jennifer A.

    2015-01-01

    The conserved chromatin remodeling and assembly factor CHD1 (chromodomains, helicase, DNA-binding domain) is present at active genes where it participates in histone turnover and recycling during transcription. In order to gain a more complete understanding of the mechanism of action of CHD1 during development, we created a novel genetic assay in Drosophila melanogaster to evaluate potential functional interactions between CHD1 and other chromatin factors. We found that overexpression of CHD1 results in defects in wing development and utilized this fully penetrant and reliable phenotype to conduct a small-scale RNAi-based candidate screen to identify genes that functionally interact with chd1 in vivo. Our results indicate that CHD1 may act in opposition to other remodeling factors, including INO80, and that the recruitment of CHD1 to active genes by RTF1 is conserved in flies. PMID:26596648

  10. Neutron scatter studies of chromatin structures related to functions. Technical progress report, November 1, 1991--May 15, 1992

    SciTech Connect

    Bradbury, E.M.

    1992-11-01

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

  11. Neutron scatter studies of chromatin structures related to functions. Technical progress report, November 1, 1991--May 15, 1992

    SciTech Connect

    Bradbury, E.M.

    1992-06-01

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

  12. FXR mediates a chromatin looping in the GR promoter thus promoting the resolution of colitis in rodents.

    PubMed

    Renga, Barbara; D'Amore, Claudio; Cipriani, Sabrina; Mencarelli, Andrea; Carino, Adriana; Sepe, Valentina; Zampella, Angela; Distrutti, Eleonora; Fiorucci, Stefano

    2013-11-01

    Glucocorticoids (GCs) are important endocrine regulators of a wide range of physiological processes ranging from immune function to glucose and lipid metabolism. For decades, synthetic glucocorticoids such as dexamethasone have been the cornerstone for the clinical treatment of inflammatory bowel diseases (IBD). A previous study has shown that farnesoid X receptor (FXR) enhances the transcription of NR3C1 gene, which encodes for human GR, by binding to a conserved FXR response element (FXRE) in the distal promoter of this gene. In the present study we demonstrate that FXR promotes the resolution of colitis in rodents by enhancing Gr gene transcription. We used the chromatin conformation capture (3C) assay to demonstrate that this FXRE is functional in mediating a head-to-tail chromatin looping, thus increasing Gr transcription efficiency. These findings underscore the importance of FXR/GR axis in the control of intestinal inflammation. PMID:24004655

  13. Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries.

    PubMed

    Nguyen, Uyen T T; Bittova, Lenka; Müller, Manuel M; Fierz, Beat; David, Yael; Houck-Loomis, Brian; Feng, Vanessa; Dann, Geoffrey P; Muir, Tom W

    2014-08-01

    Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone post-translational modification (PTM) signatures remains a daunting task in the epigenetics field. We introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semisynthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries, once they have been 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 preexisting PTMs, alone or synergistically, affect further PTM deposition via cross-talk 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

  14. Histone Depletion Facilitates Chromatin Loops on the Kilobasepair Scale

    PubMed Central

    Diesinger, Philipp M.; Kunkel, Susanne; Langowski, Jörg; Heermann, Dieter W.

    2010-01-01

    The packing of eukaryotic DNA in the nucleus is decisive for its function; for instance, contact between remote genome sites constitutes a basic feature of gene regulation. Interactions among regulatory proteins, DNA binding, and transcription activation are facilitated by looping of the intervening chromatin. Such long-range interactions depend on the bending flexibility of chromatin, i.e., the ring-closure probability is a directly measurable indicator of polymer flexibility. The applicability of a wormlike chain model to naked DNA has been widely accepted. However, whether this model also suffices to describe the flexibility of eukaryotic interphase chromatin is still a matter of discussion. Here we compare both 5C data from a gene desert and data from fluorescence in situ hybridization with the results of a Monte Carlo simulation of chromatin fibers with and without histone depletion. We then estimate the ring-closure probabilities of simulated fibers with estimates from analytical calculations and show that the wormlike chain model grossly underestimates chromatin flexibility for sharp bends. Most importantly, we find that only fibers with random depletion of linker histones or nucleosomes can explain the probability of random chromatin contacts on small length scales that play an important role in gene regulation. It is possible that missing linker histones and nucleosomes are not just simple, unavoidable, randomly occurring defects, but instead play a regulatory role in gene expression. PMID:21044597

  15. Modeling co-occupancy of transcription factors using chromatin features

    PubMed Central

    Liu, Liang; Zhao, Weiling; Zhou, Xiaobo

    2016-01-01

    Regulation of gene expression requires both transcription factor (TFs) and epigenetic modifications, and interplays between the two types of factors have been discovered. However study of relationships between chromatin features and TF–TF co-occupancy remains limited. Here, we revealed the relationship by first illustrating distinct profile patterns of chromatin features related to different binding events, including single TF binding and TF–TF co-occupancy of 71 TFs from five human cell lines. We further implemented statistical analyses to demonstrate the relationship by accurately predicting co-occupancy genome-widely using chromatin features including DNase I hypersensitivity, 11 histone modifications (HMs) and GC content. Remarkably, our results showed that the combination of chromatin features enables accurate predictions across the five cells. For individual chromatin features, DNase I enables high and consistent predictions. H3K27ac, H3K4me 2, H3K4me3 and H3K9ac are more reliable predictors than other HMs. Although the combination of 11 HMs achieves accurate predictions, their predictive ability varies considerably when a model obtained from one cell is applied to others, indicating relationship between HMs and TF–TF co-occupancy is cell type dependent. GC content is not a reliable predictor, but the addition of GC content to any other features enhances their predictive ability. Together, our results elucidate a strong relationship between TF–TF co-occupancy and chromatin features. PMID:26590261

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

    PubMed 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

  17. Chd5 orchestrates chromatin remodeling during sperm development

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

    The fusion of the gametes upon fertilization results in the formation of a totipotent cell. Embryonic chromatin is expected to be able to support a large degree of plasticity. However, whether this plasticity relies on a particular conformation of the embryonic chromatin is unknown. Moreover, whether chromatin plasticity is functionally linked to cellular potency has not been addressed. Here, we adapted fluorescence recovery after photobleaching (FRAP) in the developing mouse embryo and show that mobility of the core histones H2A, H3.1, and H3.2 is unusually high in two-cell stage embryos and decreases as development proceeds. The transition toward pluripotency is accompanied by a decrease in histone mobility, and, upon lineage allocation, pluripotent cells retain higher mobility than the differentiated trophectoderm. Importantly, totipotent two-cell-like embryonic stem cells also display high core histone mobility, implying that reprogramming toward totipotency entails changes in chromatin mobility. Our data suggest that changes in chromatin dynamics underlie the transitions in cellular plasticity and that higher chromatin mobility is at the nuclear foundations of totipotency. PMID:24831699

  19. On the physical and chemical dynamics of chromatin

    NASA Astrophysics Data System (ADS)

    Apratim, Manjul

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

  20. A chromatin remodelling complex that loads cohesin onto human chromosomes

    NASA Astrophysics Data System (ADS)

    Hakimi, Mohamed-Ali; Bochar, Daniel A.; Schmiesing, John A.; Dong, Yuanshu; Barak, Orr G.; Speicher, David W.; Yokomori, Kyoko; Shiekhattar, Ramin

    2002-08-01

    Nucleosomal DNA is arranged in a higher-order structure that presents a barrier to most cellular processes involving protein DNA interactions. The cellular machinery involved in sister chromatid cohesion, the cohesin complex, also requires access to the nucleosomal DNA to perform its function in chromosome segregation. The machineries that provide this accessibility are termed chromatin remodelling factors. Here, we report the isolation of a human ISWI (SNF2h)-containing chromatin remodelling complex that encompasses components of the cohesin and NuRD complexes. We show that the hRAD21 subunit of the cohesin complex directly interacts with the ATPase subunit SNF2h. Mapping of hRAD21, SNF2h and Mi2 binding sites by chromatin immunoprecipitation experiments reveals the specific association of these three proteins with human DNA elements containing Alu sequences. We find a correlation between modification of histone tails and association of the SNF2h/cohesin complex with chromatin. Moreover, we show that the association of the cohesin complex with chromatin can be regulated by the state of DNA methylation. Finally, we present evidence pointing to a role for the ATPase activity of SNF2h in the loading of hRAD21 on chromatin.

  1. CCSI: a database providing chromatin–chromatin spatial interaction information

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  3. A map of open chromatin in human pancreatic islets.

    PubMed

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

    2010-03-01

    Tissue-specific transcriptional regulation is central to human disease. To identify regulatory DNA active in human pancreatic islets, we profiled chromatin by formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq). We identified approximately 80,000 open chromatin sites. Comparison of FAIRE-seq data from islets to that from five non-islet cell lines revealed approximately 3,300 physically linked clusters of islet-selective open chromatin sites, which typically encompassed single genes that have islet-specific expression. We mapped sequence variants to open chromatin sites and found that rs7903146, a TCF7L2 intronic variant strongly associated with type 2 diabetes, is located in islet-selective open chromatin. We found that human islet samples heterozygous for rs7903146 showed allelic imbalance in islet FAIRE signals and that the variant alters enhancer activity, indicating that genetic variation at this locus acts in cis with local chromatin and regulatory changes. These findings illuminate the tissue-specific organization of cis-regulatory elements and show that FAIRE-seq can guide the identification of regulatory variants underlying disease susceptibility. PMID:20118932

  4. A Bayesian mixture model for chromatin interaction data.

    PubMed

    Niu, Liang; Lin, Shili

    2015-02-01

    Chromatin interactions mediated by a particular protein are of interest for studying gene regulation, especially the regulation of genes that are associated with, or known to be causative of, a disease. A recent molecular technique, Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET), that uses chromatin immunoprecipitation (ChIP) and high throughput paired-end sequencing, is able to detect such chromatin interactions genomewide. However, ChIA-PET may generate noise (i.e., pairings of DNA fragments by random chance) in addition to true signal (i.e., pairings of DNA fragments by interactions). In this paper, we propose MC_DIST based on a mixture modeling framework to identify true chromatin interactions from ChIA-PET count data (counts of DNA fragment pairs). The model is cast into a Bayesian framework to take into account the dependency among the data and the available information on protein binding sites and gene promoters to reduce false positives. A simulation study showed that MC_DIST outperforms the previously proposed hypergeometric model in terms of both power and type I error rate. A real data study showed that MC_DIST may identify potential chromatin interactions between protein binding sites and gene promoters that may be missed by the hypergeometric model. An R package implementing the MC_DIST model is available at http://www.stat.osu.edu/~statgen/SOFTWARE/MDM. PMID:25485614

  5. 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

  6. 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

  7. Evaluation of human sperm chromatin status after selection using a modified Diff-Quik stain indicates embryo quality and pregnancy outcomes following in vitro fertilization.

    PubMed

    Tavares, R S; Silva, A F; Lourenço, B; Almeida-Santos, T; Sousa, A P; Ramalho-Santos, J

    2013-11-01

    Sperm chromatin/DNA damage can be measured by a variety of assays. However, it has been reported that these tests may lose prognostic value in Assisted Reproductive Technology (ART) cycles when assessed in post-prepared samples, possibly due to the normalizing effect promoted by sperm preparation procedures. We have recently implemented a modified version of the Diff-Quik staining assay that allows for the evaluation of human sperm chromatin status in native samples, together with standard sperm morphology assessment. However, the value of this parameter in terms of predicting in vitro fertilization (IVF) and Intracytoplasmic sperm injection (ICSI) outcomes after sperm selection is unknown. In this study, data from 138 couples undergoing in vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI) treatments showed that sperm chromatin integrity was significantly improved after density gradient centrifugation and swim up (p < 0.001), but no correlations were found with fertilization or embryo development rates (p > 0.05). However, sperm samples presenting lower percentages of damaged chromatin were associated with better quality (Grade I) embryos in both ART procedures (p < 0.05) and clinical pregnancy among IVF couples (p < 0.05). Furthermore, regression analysis confirmed the clinical value of Diff-Quik staining in predicting IVF (but not ICSI) clinical pregnancy (OR: 0.927, 95% CI: 0.871-0.985, p = 0.015), and a threshold value of 34.25% for this parameter was established. The proportion of IVF couples achieving a clinical pregnancy was reduced 1.9-fold when the percentage of abnormal dark staining was ≥34.25% (p = 0.05). In conclusion, the Diff-Quik staining assay provides useful information regarding ART success, particularly in IVF cycles, where some degree of 'natural' sperm selection may occur; but not in ICSI, where sperm selection is operator dependent. This quick and low-cost assay is suggested as an alternative method to detect sperm chromatin status in minimal clinical settings, when no other well-established and robust assays (e.g. Sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling) are available. PMID:24124136

  8. 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.

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

    PubMed Central

    Comet, Itys; Schuettengruber, Bernd; Sexton, Tom; Cavalli, Giacomo

    2011-01-01

    Regulation of gene expression involves long-distance communication between regulatory elements and target promoters, but how this is achieved remains unknown. Insulator elements have been proposed to modulate the communication between regulatory elements and promoters due to their ability to insulate genes from regulatory elements or to take part in long-distance interactions. Using a high-resolution chromatin conformation capture (H3C) method, we show that the Drosophila gypsy insulator behaves as a conformational chromatin border that is able to prohibit contacts between a Polycomb response element (PRE) and a distal promoter. On the other hand, two spaced gypsy elements form a chromatin loop that is able to bring an upstream PRE in contact with a downstream gene to mediate its repression. Chromatin immunoprecipitation (ChIP) profiles of the Polycomb protein and its associated H3K27me3 histone mark reflect this insulator-dependent chromatin conformation, suggesting that Polycomb action at a distance can be organized by local chromatin topology. PMID:21262819

  10. Evaluation of genetic damage in open-cast coal mine workers using the buccal micronucleus cytome assay.

    PubMed

    Rohr, Paula; da Silva, Juliana; da Silva, Fernanda R; Sarmento, Merielen; Porto, Carem; Debastiani, Rafaela; Dos Santos, Carla E I; Dias, Johnny F; Kvitko, Kátia

    2013-01-01

    Coal is the largest fossil fuel source used for the generation of energy. However, coal extraction and its use constitute important pollution factors; thus, risk characterization and estimation are extremely important for the safety of coal workers and the environment. Candiota is located to the southeast of the state of Rio Grande do Sul and has the largest coal reserves in Brazil, and the largest thermal power complex in the state. In the open-cast mines, the coal miners are constantly exposed to coal dust. The human buccal micronucleus cytome (BMCyt) assay has been used widely to investigate biomarkers for DNA damage, cell death, and basal cell frequency in buccal cells. The aim of this study was to assess whether prolonged exposure to coal dust could lead to an increase in genomic instability, cell death, and frequency of basal cells using the BMCyt assay. In the analysis of epithelial cells, the exposed group (n = 41) presented with a significantly higher frequency of basal cells, micronuclei in basal and differentiated cells, and binucleated cells compared to the non-exposed group (n = 29). The exposed group showed a significantly lower frequency of condensed chromatin cells than the non-exposed group. However, we found no correlation between DNA damage and metal concentration in the blood of mine workers. DNA damage observed in the mine workers may be a consequence of oxidative damage resulting from exposure to coal residue mixtures. In addition, our findings confirm that the BMCyt assay can be used to identify occupational risk. PMID:23055270

  11. 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.

  12. Targeting a SWI/SNF-related chromatin remodeling complex to the ?-globin promoter in erythroid cells

    PubMed Central

    Lee, Chang-Hun; Murphy, Michael R.; Lee, Jong-Soo; Chung, Jay H.

    1999-01-01

    Chromatin remodeling complexes such as the SWI/SNF complex make DNA accessible to transcription factors by disrupting nucleosomes. However, it is not known how such complexes are targeted to the promoter. For example, a SWI/SNF1-like chromatin remodeling complex erythroid Krüppel-like factor (EKLF) coactivator-remodeling complex 1 (E-RC1) disrupts the nucleosomes over the human ?-globin promoter in an EKLF-dependent manner. However, it is not known whether E-RC1 is targeted specifically to the ?-globin promoter or whether E-RC1 is randomly targeted, but its activity is evident only at the ?-globin promoter. Because E-RC1 cannot remodel chromatin over the ?-globin promoter without EKLF in vitro, it has been proposed that SWI/SNF1-like complexes such as E-RC1 are targeted specifically to the promoter by selectively interacting with promoter-associated transcription factors such as EKLF. In this report, we test this hypothesis in the cellular context by using the ProteIN POsition Identification with Nuclease Tail (PIN*POINT) assay. We find that the Brahma-related gene (BRG) 1 and BRG1-associated factor (BAF) 170 subunits of E-RC1 are both recruited near the transcription initiation site of the ?-globin promoter. On transiently transfected templates, both the locus control region and the EKLF-binding site are important for their recruitment to the ?-globin promoter in mouse erythroleukemia cells. When the ?-globin promoter was linked to the cytomegalovirus enhancer, the E-RC1 complex was not recruited, suggesting that recruitment of the E-RC1 complex is not a general property of enhancers. PMID:10535918

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

    PubMed Central

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

    2015-01-01

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

  14. Exploration of mechanisms in nutriepigenomics: Identification of chromatin-modifying compounds from Olea Europaea.

    PubMed

    Bonvino, Natalie P; Ray, Nancy B; Luu, Vi T; Liang, Julia; Hung, Andrew; Karagiannis, Tom C

    2015-01-01

    Chemical modification of histones represents an important epigenetic mechanism critical for DNA metabolism including, transcription, replication and repair. A well-known example is maintenance of histone acetylation status by the opposing actions of histone acetyltransferase and histone deacetylase enzymes which add and remove acetyl groups on lysine residues on histone tails, respectively. Similarly, histone methyltransferase and histone demethylase enzymes are responsible for adding and removing methyl groups on histone tails, respectively. Further, there is accumulated evidence indicating a histone code where combinations of different chemical modifications on histone tails act in concert to regulate DNA metabolic events. Although numerous compounds have been developed to specifically alter the function of chromatin modifying enzymes (for example, histone deacetylase inhibitors are relatively well-investigated), we are only at the early stages of understanding the epigenetic effects of dietary compounds. Here we used in silico molecular modeling approaches combined with known experimental affinities for controls, to identify potential chromatin modifying compounds derived from Olea Europaea. Our findings indicate that various compounds derived from Olea Europaea have the ability to bind to the active site of different chromatin modifying enzymes, with an affinity analogous or higher than that for a known positive control. Further, we initiated the process of validating targets using in vitro binding and enzyme activity inhibition assays and provide initial findings of potential epigenetic effects in a clinical context. Overall, our findings can be considered as the first instalment of a comprehensive endeavour to catalogue and detail the epigenetic effects of compounds derived from Olea Europaea. PMID:26665212

  15. Ha-ras Oncogene Effect on DNA Content and Chromatin Supraorganization in benzo[a]pyrene-Transformed Human Breast Epithelial Cells

    PubMed Central

    Mello, Maria Luiza S.; de Campos Vidal, Benedicto; Russo, Jose

    1999-01-01

    When transfected to benzo[a]pyrene (BP)?transformed MCF?10F human breast epithelial cells (BP1 cell line) the c?Ha?ras oncogene has proven to enhance the neoplastic changes initiated by exposure to BP, giving rise to an aggressive tumorigenic cell line, BP1?Tras. We have previously demonstrated by image analysis that BP affects the DNA content and the chromatin supraorganization of MCF?10F cells. Here Feulgen?stained BP1?Tras cells were studied by image analysis in order to evaluate possible additional changes in DNA content and chromatin texture induced by insertion of the ras oncogene. A high variability in DNA content also including polyploidy or near?polyploidy, and an increase in the packing states of the chromatin which became still condensed in BP1 cells were found in BP1?Tras cells. The results differed from those reported for the BP1?E1 cell line which is also an aggressive tumorigenic cell line, but was attained through progressive passages of BP?transformed cells. It was demonstrated that different patterns of changes in DNA content and chromatin organization may be involved in equally aggressive tumorigenic BP?transformed cell lines originated from the same cell line by different mechanisms. PMID:10746437

  16. 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 into characteristics of X-ray emission laser beams from solidstate cathode medium of high-current glow discharge / A. B. Karabut. Charged particles from Ti and Pd foils / L. Kowalski ... [et al.]. Cr-39 track detectors in cold fusion experiments: review and perspectives / A. S. Roussetski. Energetic particle shower in the vapor from electrolysis / R. A. Oriani and J. C. Fisher. Nuclear reactions produced in an operating electrolysis cell / R. A. Oriani and J. C. Fisher. Evidence of microscopic ball lightning in cold fusion experiments / E. H. Lewis. Neutron emission from D[symbol] gas in magnetic fields under low temperature / T. Mizuno ... [et al.]. Energetic charged particle emission from hydrogen-loaded Pd and Ti cathodes and its enhancement by He-4 implantation / A. G. Lipson ... [et al.]. H-D permeation. Observation of nuclear transmutation reactions induced by D[symbol] gas permeation through Pd complexes / Y. Iwamura ... [et al.]. Deuterium (hydrogen) flux permeating through palladium and condensed matter nuclear science / Q. M. Wei ... [et al.]. Triggering. Precursors and the fusion reactions in polarized Pd/D-D[symbol]O system: effect of an external electric field / S. Szpak, P. A. Mosier-Boss, and F. E. Gordon. Calorimetric and neutron diagnostics of liquids during laser irradiation / Yu. N. Bazhutov ... [et al.]. Anomalous neutron capture and plastic deformation of Cu and Pd cathodes during electrolysis in a weak thermalized neutron field: evidence of nuclei-lattice exchange / A. G. Lipson and G. H. Miley. H-D loading. An overview of experimental studies on H/Pd over-loading with thin Pd wires and different electrolytic solutions / A. Spallone ... [et al.] -- 3. Transmutations. Photon and particle emission, heat production, and surface transformation in Ni-H system / E. Campari ... [et al.]. Surface analysis of hydrogen-loaded nickel alloys / E. Campari ... [et al.]. Low-energy nuclear reactions and the leptonic monopole / G. Lochak and L. Urutskoev. Results of analysis of Ti foil after glow discharge with deuterium / I. B. Savvatimova and D. V. Gavritenkov. Enhancement mechanisms of low-energy nuclear reactions / F. A. Gareev, I. E. Zhidkova, and Y. L. Ratis. Co-deposition of palladium with hydrogen isotopes / J. Dash and A. Ambadkar. Variation of the concentration of isotopes copper and zinc in human plasmas of patients affected by cancer / A. Triassi. Transmutation of metal at low energy in a confined plasma in water / D. Cirillo and V. Iorio. The conditions and realization of self-similar Coulomb collapse of condensed target and low-energy laboratory nucleosynthesis / S. V. Adamenko and V. I. Vysotskii. The spatial structure of water and the problem of controlled low-energy nuclear reactions in water matrix / V. I. Vysotskii and A. A. Kornilova. Experiments on controlled decontamination of water mixture of longlived active isotopes in biological cells / V. I. Vysotskii. Assessment of the biological effects of "strange" radiation / E. A. Pryakhin ... [et al.]. Possible nuclear transmutation of nitrogen in the earth's atmosphere / M. Fukuhara. Evidences on the occurrence of LENR-type processes in alchemical transmutations / J. Pérez-Pariente. History of the discovery of transmutation at Texas A&M University / J. O.-M. Bockris -- 4. Theory. Quantum electrodynamics. Concerning the modeling of systems in terms of quantum electro dynamics: the special case of "cold fusion" / M. Abyaneh ... [et al.]. Screening. Theoretical model of the probability of fusion between deuterons within deformed lattices with microcracks at room temperature / F. Fulvio. Resonant tunnelling. Effective interaction potential in the deuterium plasma and multiple resonance scattering / T. Toimela. Multiple scattering theory and condensed matter nuclear science - "super-absorption" in a crystal latice / X. Z. Li ... [et al.]. Ion band states. Framework for understanding LENR processes, using conventional condensed matter physics / S. R. Chubb. I. Bloch ions / T. A. Chubb. II. Inhibited diffusion driven surface transmutations / T. A. Chubb. III. Bloch nuclides, Iwamura transmutations, and Oriani showers / T. A. Chubb. Bose-Einstein condensate. Theoretical study of nuclear reactions induced by Bose-Einstein condensation in Pd / K.-I. Tsuchiya and H. Okumura. Proposal for new experimental tests of the Bose-Einstein condensation mechanism for low-energy nuclear reaction and transmutation processes in deuterium loaded micro- and nano-scale cavities / Y. E. Kim ... [et al.]. Mixtures of charged bosons confined in harmonic traps and Bose-Einstein condensation mechanism for low-energy nuclear reactions and transmutation processes in condensed matters / Y. E. Kim and A. L. Zubarev. Alternative interpretation of low-energy nuclear reaction processes with deuterated metals based on the Bose-Einstein condensation mechanism / Y. E. Kim and T. O. Passell. Multi-body fusion. [symbol]He/[symbol]He production ratios by tetrahedral symmetric condensation / A. Takahashi. Phonon coupling. Phonon-exchange models: some new results / P. L. Hagelstein. Neutron clusters. Cold fusion phenomenon and solid state nuclear physics / H. Kozima. Neutrinos, magnetic monopoles. Neutrino-driven nuclear reactions of cold fusion and transmutation / V. Filimonov. Light monopoles theory: an overview of their effects in physics, chemistry, biology, and nuclear science (weak interactions) / G. Lochak. Electrons clusters and magnetic monopoles / M. Rambaut. Others. Effects of atomic electrons on nuclear stability and radioactive decay / D. V. Filippov, L. I. Urutskoev, and A. A. Rukhadze. Search for erzion nuclear catalysis chains from cosmic ray erzions stopping in organic scintillator / Yu. N. Bazhutov and E. V. Pletnikov. Low-energy nuclear reactions resulting as picometer interactions with similarity to K-shell electron capture / H. Hora ... [et al.] -- 5. Other topics. On the possible magnetic mechanism of shortening the runaway of RBMK-1000 reactor at Chernobyl Nuclear Power Plant / D. V. Filippov ... [et al.]. Cold fusion in the context of a scientific revolution in physics: history and economic ramifications / E. Lewis. The nucleovoltaic cell / D. D. Moon. Introducing the book "Cold Fusion and the Future" / J. Rothwell. Recent cold fusion claims: are they valid? / L. Kowalski. History of attempts to publish a paper / L. Kowalski.

  17. Gravitational Condensate Stars

    NASA Astrophysics Data System (ADS)

    Mazur, P.; Mottola, E.

    The issue of the final state of the gravitational collapse will be addressed. Ishall present physical arguments to the effect that the remnant of the gravitationalcollapse of super-massive stars is a cold and dark super-dense object which isthermodynamically and dynamically stable: a Gravitational Condensate Star orQuasi Black Hole (QBH). A QBH is characterized by a huge, but not an infinite,surface redshift. This surface redshift depends universally on the total mass of aQBH and the proper thickness of a thin shell of an exotic matter described bythe Zel'dovich equation of state p = c2 . The velocity of sound in a thin shell isequal to the velocity of light. Hence, this thin shell replaces the event horizon of amathematical black hole ( = 0). Inside a thin shell the zero entropy gravitationalcondensate characterized by the cosmological equation of state p = -c2 resides.A QBH is described by a new static and spherically symmetric solution of Ein-stein's equations supplemented with the proper boundary conditions based on mi-crophysics considerations. The new solution has no singularities and no eventhorizons. Its entropy is maximized under small fluctuations and is given by thestandard hydrodynamic entropy of the thin shell which is proportional to the to-tal mass instead of the Bekenstein-Hawking entropy which is proportional to thesquare of the total mass. This resolves the paradox of an excessively high en-tropy of black holes as compared to their progenitors. The formation of such acold gravitational condensate stellar remnant very likely would require a violentcollapse process with an explosive output of energy. Some observational conse-quences of the formation of gravitational condensate stars will be described.

  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. Confinement contains condensates

    NASA Astrophysics Data System (ADS)

    Brodsky, Stanley J.; Roberts, Craig D.; Shrock, Robert; Tandy, Peter C.

    2012-06-01

    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 space-time, 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.

  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. 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 in multipotent stem cells. PMID:24413398

  2. 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. PMID:26391293

  3. 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. PMID:26391293

  4. Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity.

    PubMed

    Kim, Tae-Hee; Li, Fugen; Ferreiro-Neira, Isabel; Ho, Li-Lun; Luyten, Annouck; Nalapareddy, Kodandaramireddy; Long, Henry; Verzi, Michael; Shivdasani, Ramesh A

    2014-02-27

    Cells differentiate when transcription factors bind accessible cis-regulatory elements to establish specific gene expression programs. In differentiating embryonic stem cells, chromatin at lineage-restricted genes becomes sequentially accessible, probably by means of 'pioneer' transcription factor activity, but tissues may use other strategies in vivo. Lateral inhibition is a pervasive process in which one cell forces a different identity on its neighbours, 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 signalling mediates lateral inhibition to assign progenitor cells into absorptive or secretory lineages. Transcript profiles in isolated LGR5(+) intestinal stem cells 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 DNase I hypersensitivity--signifying accessible, permissive chromatin-at most of the same cis-elements. Enhancers acting uniquely in progenitors were well demarcated in LGR5(+) intestinal stem cells, 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 transcription factor, controls lateral inhibition through delta-like notch ligand genes and also drives the expression of numerous secretory lineage genes. Depletion of ATOH1 from specified secretory cells converted them into functional enterocytes, indicating prolonged responsiveness of marked enhancers to the presence or absence of a key transcription factor. Thus, lateral inhibition and intestinal crypt lineage plasticity involve interaction of a lineage-restricted transcription factor with broadly permissive chromatin established in multipotent stem cells. PMID:24413398

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

    PubMed Central

    Collepardo-Guevara, Rosana; Schlick, Tamar

    2014-01-01

    Deciphering the factors that control chromatin fiber structure is key to understanding fundamental chromosomal processes. Although details remain unknown, it is becoming clear that chromatin is polymorphic depending on internal and external factors. In particular, different lengths of the linker DNAs joining successive nucleosomes (measured in nucleosome-repeat lengths or NRLs) that characterize different cell types and cell cycle stages produce different structures. NRL is also nonuniform within single fibers, but how this diversity affects chromatin fiber structure is not clear. Here we perform Monte Carlo simulations of a coarse-grained oligonucleosome model to help interpret fiber structure subject to intrafiber NRL variations, as relevant to proliferating cells of interphase chromatin, fibers subject to remodeling factors, and regulatory DNA sequences. We find that intrafiber NRL variations have a profound impact on chromatin structure, with a wide range of different architectures emerging (highly bent narrow forms, canonical and irregular zigzag fibers, and polymorphic conformations), depending on the NRLs mixed. This stabilization of a wide range of fiber forms might allow NRL variations to regulate both fiber compaction and selective DNA exposure. The polymorphic forms spanning canonical to sharply bent structures, like hairpins and loops, arise from large NRL variations and are surprisingly more compact than uniform NRL structures. They are distinguished by tail-mediated far-nucleosome interactions, in addition to the near-nucleosome interactions of canonical 30-nm fibers. Polymorphism is consistent with chromatin’s diverse biological functions and heterogeneous constituents. Intrafiber NRL variations, in particular, may contribute to fiber bending and looping and thus to distant communication in associated regulatory processes. PMID:24847063

  6. Chromatin remodeling in the aging genome of Drosophila

    PubMed Central

    Wood, Jason G.; Hillenmeyer, Sara; Lawrence, Charles; Chang, Chengyi; Hosier, Suzanne; Lightfoot, Will; Mukherjee, Eric; Jiang, Nan; Schorl, Christoph; Brodsky, Alexander S.; Neretti, Nicola; Helfand, Stephen L.

    2010-01-01

    Summary Chromatin structure affects the accessibility of DNA to transcription, repair and replication. Changes in chromatin structure occur during development, but less is known about changes during aging. We examined the state of chromatin structure and its effect on gene expression during aging in Drosophila at the whole genome and cellular level using whole genome tiling microarrays of activation and repressive chromatin marks, whole genome transcriptional microarrays and single cell immunohistochemistry. We found dramatic reorganization of chromosomal regions with age. Mapping of H3K9me3 and HP1 signals to fly chromosomes reveals in young flies the expected high enrichment in the pericentric regions, the 4th chromosome and islands of facultative heterochromatin dispersed throughout the genome. With age there is a striking reduction in this enrichment resulting in a nearly equivalent level of H3K9me3 and HP1 in the pericentric regions, the 4th chromosome, facultative heterochromatin and euchromatin. These extensive changes in repressive chromatin marks are associated with alterations in age-related gene expression. Large-scale changes in repressive marks with age are further substantiated by single cell immunohistochemistry that show changes in nuclear distribution of H3K9me3 and HP1 marks with age. Such epigenetic changes are expected to directly or indirectly impinge upon important cellular functions such as gene expression, DNA repair and DNA replication. The combination of genome-wide approaches such as whole genome chromatin immunoprecipitation and transcriptional studies in conjunction with single cell immunohistochemistry as shown here provide a first step toward defining how changes in chromatin may contribute to the process of aging in metazoans. PMID:20961390

  7. SPERM CHROMATIN STRUCTURE ASSAY PARAMETERS AS PREDICTORS OF FAILED PREGNANCY FOLLOWING ASSISTED REPRODUCTIVE TECHNIQUES. (R827019)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  8. EVALUATION OF SPERM CHROMATIN STRUCTURE ASSAY (SCSA REGISTERED TRADEMARK) IN HUMAN SPERM AFTER SIMULATED OVERNIGHT SHIPMENT

    EPA Science Inventory

    Home semen collection kits allow men to collect a sample at their convenience and send it via overnight mail to the laboratory. Benefits of this approach include facilitated sample collection from different geographic locations, minimized variability through analysis by a central...

  9. 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.

  10. eRNAs Promote Transcription by Establishing Chromatin Accessibility at Defined Genomic Loci

    PubMed Central

    Mousavi, Kambiz; Zare, Hossein; Dell’Orso, Stefania; Grontved, Lars; Gutierrez-Cruz, Gustavo; Derfoul, Assia; Hager, Gordon L.; Sartorelli, Vittorio

    2013-01-01

    SUMMARY Transcription factors and DNA regulatory binding motifs are fundamental components of the gene regulatory network. Here, by using genome-wide binding profiling, we show extensive occupancy of transcription factors of myogenesis (MyoD and Myogenin) at extragenic enhancer regions coinciding with RNA synthesis (i.e. eRNA). In particular, multiple regions were transcribed to eRNA within regulatory region of MYOD1, including previously characterized Distal Regulatory Regions (DRR) and Core Enhancer (CE). While CERNA enhanced RNA polymerase II (PolII) occupancy and transcription at MYOD1, DRRRNA acted to activate the downstream myogenic genes. The deployment of transcriptional machinery to appropriate loci is contingent on chromatin accessibility, a rate-limiting step preceding PolII assembly. By nuclease sensitivity assay, it appear that eRNAs regulate genomic access of the transcriptional complex to defined regulatory regions. In conclusion, our data suggest that eRNAs contribute to establishing a cell-type-specific transcriptional circuitry by directing chromatin-remodeling events. PMID:23993744

  11. SIRT7 Is Activated by DNA and Deacetylates Histone H3 in the Chromatin Context.

    PubMed

    Tong, Zhen; Wang, Yi; Zhang, Xiaoyu; Kim, David D; Sadhukhan, Sushabhan; Hao, Quan; Lin, Hening

    2016-03-18

    Mammalian sirtuins (SIRT1-7) are members of a highly conserved family of nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylases that regulate many biological processes including metabolism, genome stability, and transcription. Among the seven human sirtuins, SIRT7 is the least understood, to a large extent due to the lack of enzymatic activity in vitro. Here, we reported that SIRT7 can be activated by DNA to hydrolyze the acetyl group from lysine residues in vitro on histone peptides and histones in the chromatin context. Both N- and C- termini of SIRT7 are important for the DNA-activated deacetylase activity. The regulatory mechanism of SIRT7 is different from that of SIRT6, which also showed increased activity on chromatin substrates, but the deacetylase activity of SIRT6 on a peptide substrate cannot be activated by DNA. This finding provides an improved enzymatic activity assay of SIRT7 that will promote the development of SIRT7 modulators. Further investigation into the activation mechanism of SIRT7 by DNA could provide new insights into its biological function and help the development of sirtuin activators. PMID:26907567

  12. Developmentally stable chromatin structure in the human. beta. -globin gene cluster

    SciTech Connect

    Forrester, W.C.; Thompson, C.; Elder, J.T.; Groudine, M.

    1986-03-01

    The DNase I-hypersensitive sites in the human embryonic ..beta..-globin gene region have been mapped in erythroid-enriched fractions of disaggregated fetal livers, in adult nucleated red blood cells, and in fetal brain tissue. The analysis of a region extending 11 kilobases (kb) 5' of the epsilion-globin gene reveals many minor nuclease-hypersensitive sites and one major site located 6.1 kb upstream of the epsilon-globin gene. All of these hypersensitive sites are erythroid-specific, and the major site is stable throughout erythroid development. As assayed by nuclear runoff transcription, little or no epsilon-globin gene expression is detectable in fetal or adult erythroid cells. Thus, the presence of the major hypersensitive site 5' of the epsilon-globin gene in both fetal and adult erythroid cells demonstrates that this site is not specifically correlated with transcription of the gene or with a particular stage of development. Rather, this site may reflect an early event in erythroid differentiation. In addition, DNase I has been used to probe the overall sensitivity of epsilon-globin chromatin in fetal erythroid cells. The findings indicate that the epsilon-globin gene as well as the other genes in the ..beta..-globin cluster reside within the chomatin domain that is more DNase I-sensitive than bulk chromatin.

  13. Chromatin redistribution of the DEK oncoprotein represses hTERT transcription in leukemias.

    PubMed

    Karam, Maroun; Thenoz, Morgan; Capraro, Valérie; Robin, Jean-Philippe; Pinatel, Christiane; Lancon, Agnès; Galia, Perrine; Sibon, David; Thomas, Xavier; Ducastelle-Lepretre, Sophie; Nicolini, Franck; El-Hamri, Mohamed; Chelghoun, Youcef; Wattel, Eric; Mortreux, Franck

    2014-01-01

    Although numerous factors have been found to modulate hTERT transcription, the mechanism of its repression in certain leukemias remains unknown. We show here that DEK represses hTERT transcription through its enrichment on the hTERT promoter in cells from chronic and acute myeloid leukemias, chronic lymphocytic leukemia, but not acute lymphocytic leukemias where hTERT is overexpressed. We isolated DEK from the hTERT promoter incubated with nuclear extracts derived from fresh acute myelogenous leukemia (AML) cells and from cells expressing Tax, an hTERT repressor encoded by the human T cell leukemia virus type 1. In addition to the recruitment of DEK, the displacement of two potent known hTERT transactivators from the hTERT promoter characterized both AML cells and Tax-expressing cells. Reporter and chromatin immunoprecipitation assays permitted to map the region that supports the repressive effect of DEK on hTERT transcription, which was proportionate to the level of DEK-promoter association but not with the level of DEK expression. Besides hTERT repression, this context of chromatin redistribution of DEK was found to govern about 40% of overall transcriptional modifications, including those of cancer-prone genes. In conclusion, DEK emerges as an hTERT repressor shared by various leukemia subtypes and seems involved in the deregulation of numerous genes associated with leukemogenesis. PMID:24563617

  14. Compaction of chromatin by diverse Polycomb group proteins requires localized regions of high charge

    PubMed Central

    Grau, Daniel J.; Chapman, Brad A.; Garlick, Joe D.; Borowsky, Mark; Francis, Nicole J.; Kingston, Robert E.

    2011-01-01

    Polycomb group (PcG) proteins are required for the epigenetic maintenance of developmental genes in a silent state. Proteins in the Polycomb-repressive complex 1 (PRC1) class of the PcG are conserved from flies to humans and inhibit transcription. One hypothesis for PRC1 mechanism is that it compacts chromatin, based in part on electron microscopy experiments demonstrating that Drosophila PRC1 compacts nucleosomal arrays. We show that this function is conserved between Drosophila and mouse PRC1 complexes and requires a region with an overrepresentation of basic amino acids. While the active region is found in the Posterior Sex Combs (PSC) subunit in Drosophila, it is unexpectedly found in a different PRC1 subunit, a Polycomb homolog called M33, in mice. We provide experimental support for the general importance of a charged region by predicting the compacting capability of PcG proteins from species other than Drosophila and mice and by testing several of these proteins using solution assays and microscopy. We infer that the ability of PcG proteins to compact chromatin in vitro can be predicted by the presence of domains of high positive charge and that PRC1 components from a variety of species conserve this highly charged region. This supports the hypothesis that compaction is a key aspect of PcG function. PMID:22012622

  15. Cosmic curvature and condensation

    NASA Technical Reports Server (NTRS)

    Harwit, Martin

    1992-01-01

    It is shown that the universe may consist of a patchwork of domains with different Riemann curvature constants k = 0, +/-1. Features of a phase transition in which flat space breaks up in a transition 2k0 - k(-) + k(+) with initial scale factors R(-) = R(+) are postulated and explored. It is shown that such a transition is energetically permitted, has the equivalent of a Curie temperature, and can lead in a natural way to the formation of voids and galaxies. It is predicted that, if the ambient universe on average is well fitted by a purely k(-) space, with only occasional domains of k(+) containing galaxies, a density parameter of (A(z sub c + 1)) super -1 should be expected, where z sub c represents the redshift of the earliest objects to have condensed, and A takes on values ranging from about 5 to 3. Present observations of quasars would suggest a density of about 0.03 or 0.05, respectively, but it could be lower if earlier condensation took place.

  16. Mesons condensate and Fermi momentum

    NASA Astrophysics Data System (ADS)

    Chandra, A.; Bhattacharya, A.; Chakrabarti, B.

    2013-01-01

    Quark-antiquark condensation has been investigated in the framework of the NJL model. Using the Fermi momentum of the particle as cut-off parameter the gap energy and coherence length for meson condensates have been studied. The Fermi momentum for the < {qbar q} rangle condensate has been extracted considering the coherence length ? ˜ 1 fm. The results are compared with existing data. Some interesting observations are made.

  17. Pion condensation in holographic QCD

    SciTech Connect

    Albrecht, Dylan; Erlich, Joshua

    2010-11-01

    We study pion condensation at zero temperature in a hard-wall holographic model of hadrons with isospin chemical potential. We find that the transition from the hadronic phase to the pion condensate phase is first order except in a certain limit of model parameters. Our analysis suggests that immediately across the phase boundary the condensate acts as a stiff medium approaching the Zel'dovich limit of equal energy density and pressure.

  18. Linear patterning of mesenchymal condensations is modulated by geometric constraints

    PubMed Central

    Klumpers, Darinka D.; Mao, Angelo S.; Smit, Theo H.; Mooney, David J.

    2014-01-01

    The development of the vertebral column starts with the formation of a linear array of mesenchymal condensations, forming the blueprint for the eventual alternating pattern of bone and cartilage. Despite growing insight into the molecular mechanisms of morphogenesis, the impact of the physical aspects of the environment is not well understood. We hypothesized that geometric boundary conditions may play a pivotal role in the linear patterning of condensations, as neighbouring tissues provide physical constraints to the cell population. To study the process of condensation and the patterning thereof under tightly controlled geometric constraints, we developed a novel in vitro model that combines micropatterning with the established micromass assay. The spacing and alignment of condensations changed with the width of the cell adhesive patterns, a phenomenon that could not be explained by cell availability alone. Moreover, the extent of chondrogenic commitment was increased on substrates with tighter geometric constraints. When the in vivo pattern of condensations was investigated in the developing vertebral column of chicken embryos, the measurements closely fit into the quantitative relation between geometric constraints and inter-condensation distance found in vitro. Together, these findings suggest a potential role of geometric constraints in skeletal patterning in a cellular process of self-organization. PMID:24718453

  19. Remodeling of chromatin under low intensity diffuse ultrasound

    PubMed Central

    Noriega, Sandra; Budhiraja, Gaurav; Subramanian, Anuradha

    2012-01-01

    A variety of mechanotransduction pathways mediate the response of fibroblasts or chondrocytes to ultrasound stimulation. In addition, regulatory pathways that co-ordinate stimulus-specific cellular responses are likely to exist. In this study, analysis was confined to the hypothesis that ultrasound stimulation (US) influences the chromatin structure, and that these changes may reflect a regulatory pathway that connects nuclear architecture, chromatin structure and gene expression. Murine fibroblasts seeded on tissue culture plates were stimulated with US (5.0 MHz (14 kPa), 51-s per application) and the thermal denaturation profiles of nuclei isolated from fibroblasts were assessed by dynamic scanning calorimetry (DSC). When compared to the thermal profiles obtained from the nuclei of non-stimulated cells, the nuclei obtained from stimulated cells showed a change in peak profiles and peak areas, which is indicative of chromatin remodeling. Independently, US was also observed to impact the histone (H1):chromatin association as measured indirectly by DAPI staining. Based on our work, it appears plausible that US can produce a remodeling of chromatin, thus triggering signal cascade and other intracellular mechanisms. PMID:22575092

  20. 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

  1. Looking at plant cell cycle from the chromatin window.

    PubMed

    Desvoyes, Bénédicte; Fernández-Marcos, María; Sequeira-Mendes, Joana; Otero, Sofía; Vergara, Zaida; Gutierrez, Crisanto

    2014-01-01

    The cell cycle is defined by a series of complex events, finely coordinated through hormonal, developmental and environmental signals, which occur in a unidirectional manner and end up in producing two daughter cells. Accumulating evidence reveals that chromatin is not a static entity throughout the cell cycle. In fact, there are many changes that include nucleosome remodeling, histone modifications, deposition and exchange, among others. Interestingly, it is possible to correlate the occurrence of several of these chromatin-related events with specific processes necessary for cell cycle progression, e.g., licensing of DNA replication origins, the E2F-dependent transcriptional wave in G1, the activation of replication origins in S-phase, the G2-specific transcription of genes required for mitosis or the chromatin packaging occurring in mitosis. Therefore, an emerging view is that chromatin dynamics must be considered as an intrinsic part of cell cycle regulation. In this article, we review the main features of several key chromatin events that occur at defined times throughout the cell cycle and discuss whether they are actually controlling the transit through specific cell cycle stages. PMID:25120553

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  3. 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

  4. Nucleosomal chromatin in the mature sperm of Drosophila melanogaster

    PubMed Central

    Elnfati, Abdul Hakim; Iles, David; Miller, David

    2015-01-01

    During spermiogenesis in mammals and many other vertebrate classes, histone-containing nucleosomes are replaced by protamine toroids, which can repackage chromatin at a 10 to 20-fold higher density than in a typical somatic nucleus. However, recent evidence suggests that sperm of many species, including human and mouse retain a small compartment of nucleosomal chromatin, particularly near genes important for embryogenesis. As in mammals, spermiogenesis in the fruit fly, Drosophila melanogaster has also been shown to undergo a programmed substitution of nucleosomes with protamine-like proteins. Using chromatin immunoprecipitation (ChIP) and whole-genome tiling array hybridization (ChIP-chip), supported by immunocytochemical evidence, we show that in a manner analogous to nucleosomal chromatin retention in mammalian spermatozoa, distinct domains packaged by the canonical histones H2A, H2B, H3 and H4 are present in the fly sperm nucleus. We also find evidence for the retention of nucleosomes with specific histone H3 trimethylation marks characteristic of chromatin repression (H3K9me3, H3K27me3) and active transcription (H3K36me3). Raw and processed data from the experiments are available at GEO, accession GSE52165. PMID:26981400

  5. Estrogen represses gene expression through reconfiguring chromatin structures

    PubMed Central

    Osmanbeyoglu, Hatice Ulku; Lu, Kevin N.; Oesterreich, Steffi; Day, Roger S.; Benos, Panayiotis V.; Coronnello, Claudia; Lu, Xinghua

    2013-01-01

    Estrogen regulates over a thousand genes, with an equal number of them being induced or repressed. The distinct mechanisms underlying these dual transcriptional effects remain largely unknown. We derived comprehensive views of the transcription machineries assembled at estrogen-responsive genes through integrating multiple types of genomic data. In the absence of estrogen, the majority of genes formed higher-order chromatin structures, including DNA loops tethered to protein complexes involving RNA polymerase II (Pol II), estrogen receptor alpha (ER?) and ER?-pioneer factors. Genes to be ‘repressed’ by estrogen showed active transcription at promoters and throughout the gene bodies; genes to be ‘induced’ exhibited active transcription initiation at promoters, but with transcription paused in gene bodies. In the presence of estrogen, the majority of estrogen-induced genes retained the original higher-order chromatin structures, whereas most estrogen-repressed genes underwent a chromatin reconfiguration. For estrogen-induced genes, estrogen enhances transcription elongation, potentially through recruitment of co-activators or release of co-repressors with unique roles in elongation. For estrogen-repressed genes, estrogen treatment leads to chromatin structure reconfiguration, thereby disrupting the originally transcription-efficient chromatin structures. Our in silico studies have shown that estrogen regulates gene expression, at least in part, through modifying previously assembled higher-order complexes, rather than by facilitating de novo assembly of machineries. PMID:23821662

  6. Micron-scale coherence in interphase chromatin dynamics

    PubMed Central

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

    2013-01-01

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

  7. 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

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

    PubMed Central

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

    2012-01-01

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

  9. Recovering ensembles of chromatin conformations from contact probabilities

    PubMed Central

    Meluzzi, Dario; Arya, Gaurav

    2013-01-01

    The 3D higher order organization of chromatin within the nucleus of eukaryotic cells has so far remained elusive. A wealth of relevant information, however, is increasingly becoming available from chromosome conformation capture (3C) and related experimental techniques, which measure the probabilities of contact between large numbers of genomic sites in fixed cells. Such contact probabilities (CPs) can in principle be used to deduce the 3D spatial organization of chromatin. Here, we propose a computational method to recover an ensemble of chromatin conformations consistent with a set of given CPs. Compared with existing alternatives, this method does not require conversion of CPs to mean spatial distances. Instead, we estimate CPs by simulating a physically realistic, bead-chain polymer model of the 30-nm chromatin fiber. We then use an approach from adaptive filter theory to iteratively adjust the parameters of this polymer model until the estimated CPs match the given CPs. We have validated this method against reference data sets obtained from simulations of test systems with up to 45 beads and 4 loops. With additional testing against experiments and with further algorithmic refinements, our approach could become a valuable tool for researchers examining the higher order organization of chromatin. PMID:23143266

  10. Topological constraints strongly affect chromatin reconstitution in silico

    PubMed Central

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

    2015-01-01

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

  11. Chromosome bands, their chromatin flavors, and their functional features.

    PubMed Central

    Holmquist, G P

    1992-01-01

    To show that the input pattern of chromosomal mutations is highly organized relative to the band patterns along human chromosomes, a new term, "metaphase chromatin flavor," is introduced. Five different flavors of euchromatic metaphase bands are cytologically identified along a human ideogram. These are G-bands and, based upon combinations of extreme Alu richness and GC richness, four different R-band flavors. The two flavors with extremely GC-rich components, traditionally called "T-bands," represent only 15% of all bands. However, they contain 65% of mapped genes, 19 of 25 mapped oncogenes, most cancer-associated rearrangements, evolutionary rearrangements, meiotic chiasmata, and X-ray-induced breaks. Flavors with extremely Alu-rich flavors are also involved in melphalan-induced rearrangements, pachytene stretching, and mitotic chiasmata. Frequencies of CpG islands, CCGCCC boxes, retroposon families, and genes are characteristic to each chromatin flavor and will facilitate alignment of genome sequences onto ideograms of chromatin flavor. The influence of chromatin flavor on the evolution of a gene's sequence is so strong that one can infer the flavor of the band in which a gene resides from the sequence of the gene itself. Correlation coefficients for many pairs of mapped genetic variables, while globally high, are quite low within bands of one flavor, implicating a concerted mode of evolution for bands of one chromatin flavor. Images p24-a Figure 2 Figure 5 PMID:1609794

  12. Analysis of epigenetic alterations to chromatin during development

    PubMed Central

    Minard, Meghan E.; Jain, Abhinav K.; Barton, Michelle Craig

    2009-01-01

    Each cell within a multicellular organism has distinguishable characteristics established by its unique patterns of gene expression. This individual identity is determined by the expression of genes in a time and place-dependent manner. It is becoming increasingly clear that chromatin plays a fundamental role in the control of gene transcription in multicellular organisms. Understanding the regulation of chromatin, and how the distinct identity of a cell is passed to daughter cells during development, is paramount. Techniques with which to study chromatin have advanced rapidly over the past decade. Development of high throughput techniques and their proper applications has provided us essential tools to understand the regulation of epigenetic phenomena and its effect on gene expression. Understanding the changes that occur in chromatin during the course of development will not only contribute to our knowledge of normal gene expression, but will also add to our knowledge of how gene expression goes awry during disease. This review opens with an introduction to some of the key premises of epigenetic regulation of gene expression. A discussion of experimental techniques with which one can study epigenetic alterations to chromatin during development follows, emphasizing recent breakthroughs in this area. We then present examples of epigenetic mechanisms exploited in the control of developmental cell fate and regulation of tissue-specific gene expression. Finally, we discuss some of the frontiers and challenges in this area of research. PMID:19603511

  13. In vitro dynamics of chromatin organization and migration.

    PubMed

    Doisy, A; Paillasson, S; Tracqui, P; Germain, F; Leitner, F; Robert-Nicoud, M; Ronot, X

    1996-12-01

    The organization of eukaryotic chromatin is not static but changes as a function of cell status during processes such as proliferation, differentiation, and migration. DNA quantification has not been used extensively to investigate chromatin dynamics in combination with cellular migration. In this context, an optimized DNA-specific, nonperturbant method has been developed for studying chromatin organization, using the fluorescent vital bisbenzimidazole probe Hoechst 33342: this property has been described by Hamori et al. (1980). Computer-assisted image analysis was used to follow migratory activity and chromatin organization of L929 fibroblasts during in vitro wound healing. Cell movements were analyzed using an optical flow technique, which consists in the calculation of the velocity field of cells and nuclear movements in the frame. This system allows the correlation of cell migration and position in the cell cycle. It makes it possible to study chromatin dynamics using a quantitative analysis of nuclear differentiation reorganization (nuclear texture) and to correlate this with migration characteristics. The present system would be of interest for studying cell-extracellular matrix interactions using differing substrates, and also the migratory response to chemotactic factors. Such a model is a prerequisite for gaining better understanding of drug action. PMID:9034635

  14. 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

  15. Nucleosomal chromatin in the mature sperm of Drosophila melanogaster.

    PubMed

    Elnfati, Abdul Hakim; Iles, David; Miller, David

    2016-03-01

    During spermiogenesis in mammals and many other vertebrate classes, histone-containing nucleosomes are replaced by protamine toroids, which can repackage chromatin at a 10 to 20-fold higher density than in a typical somatic nucleus. However, recent evidence suggests that sperm of many species, including human and mouse retain a small compartment of nucleosomal chromatin, particularly near genes important for embryogenesis. As in mammals, spermiogenesis in the fruit fly, Drosophila melanogaster has also been shown to undergo a programmed substitution of nucleosomes with protamine-like proteins. Using chromatin immunoprecipitation (ChIP) and whole-genome tiling array hybridization (ChIP-chip), supported by immunocytochemical evidence, we show that in a manner analogous to nucleosomal chromatin retention in mammalian spermatozoa, distinct domains packaged by the canonical histones H2A, H2B, H3 and H4 are present in the fly sperm nucleus. We also find evidence for the retention of nucleosomes with specific histone H3 trimethylation marks characteristic of chromatin repression (H3K9me3, H3K27me3) and active transcription (H3K36me3). Raw and processed data from the experiments are available at GEO, accession GSE52165. PMID:26981400

  16. H4K20 methylation regulates quiescence and chromatin compaction

    PubMed Central

    Evertts, Adam G.; Manning, Amity L.; Wang, Xin; Dyson, Nicholas J.; Garcia, Benjamin A.; Coller, Hilary A.

    2013-01-01

    The transition between proliferation and quiescence is frequently associated with changes in gene expression, extent of chromatin compaction, and histone modifications, but whether changes in chromatin state actually regulate cell cycle exit with quiescence is unclear. We find that primary human fibroblasts induced into quiescence exhibit tighter chromatin compaction. Mass spectrometry analysis of histone modifications reveals that H4K20me2 and H4K20me3 increase in quiescence and other histone modifications are present at similar levels in proliferating and quiescent cells. Analysis of cells in S, G2/M, and G1 phases shows that H4K20me1 increases after S phase and is converted to H4K20me2 and H4K20me3 in quiescence. Knockdown of the enzyme that creates H4K20me3 results in an increased fraction of cells in S phase, a defect in exiting the cell cycle, and decreased chromatin compaction. Overexpression of Suv4-20h1, the enzyme that creates H4K20me2 from H4K20me1, results in G2 arrest, consistent with a role for H4K20me1 in mitosis. The results suggest that the same lysine on H4K20 may, in its different methylation states, facilitate mitotic functions in M phase and promote chromatin compaction and cell cycle exit in quiescent cells. PMID:23924899

  17. Rules of Engagement for Base Excision Repair in Chromatin

    PubMed Central

    Odell, Ian D.; Wallace, Susan S.; Pederson, David S.

    2012-01-01

    Most of the DNA in eukaryotes is packaged in tandemly arrayed nucleosomes that, together with numerous DNA- and nucleosome-associated enzymes and regulatory factors, make up chromatin. Chromatin modifying and remodeling agents help regulate access to selected DNA segments in chromatin, thereby facilitating transcription and DNA replication and repair. Studies of nucleotide excision repair (NER), single strand break repair (SSBR), and the homology-directed (HDR) and non-homologous end-joining (NHEJ) double strand break repair pathways have led to an ‘access-repair-restore’ paradigm, in which chromatin in the vicinity of damaged DNA is disrupted, thereby enabling efficient repair and the subsequent repackaging of DNA into nucleosomes. When damage is extensive, these repair processes are accompanied by cell cycle checkpoint activation, which provides cells with sufficient time to either complete the repair or initiate apoptosis. It is not clear, however, if base excision repair (BER) of the ~20,000 or more oxidative DNA damages that occur daily in each nucleated human cell can be viewed through this same lens. Until recently, we did not know if BER requires or is accompanied by nucleosome disruption, and it is not yet clear that anything short of overwhelming oxidative damage (resulting in the shunting of DNA substrates into other repair pathways) results in checkpoint activation. This review highlights studies of how oxidatively damaged DNA in nucleosomes is discovered and repaired, and offers a working model of events associated with BER in chromatin that we hope will have heuristic value. PMID:22718094

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

    NASA Astrophysics Data System (ADS)

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

    1995-03-01

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