Sample records for chromatin condensation assay

  1. Sperm chromatin structure assay (SCSA®).

    PubMed

    Evenson, Donald P

    2013-01-01

    The SCSA(®) is the pioneering assay for the detection of damaged sperm DNA and altered proteins in sperm nuclei via flow cytometry of acridine orange (AO) stained sperm. The SCSA(®) is considered to be the most precise and repeatable test providing very unique, dual parameter data (red vs. green fluorescence) on a 1,024 × 1,024 channel scale, not only on DNA fragmentation but also on abnormal sperm characterized by lack of normal exchange of histones to protamines. Raw semen/sperm aliquots or purified sperm can be flash frozen, placed in a box with dry ice and shipped by overnight courier to an experienced SCSA(®) lab. The samples are individually thawed, prepared, and analyzed in ?10 min. Of significance, data on 5,000 individual sperm are recorded on a 1,024 × 1,024 dot plot of green (native DNA) and red (broken DNA) fluorescence. Repeat measurements have virtually identical dot plot patterns demonstrating that the low pH treatment that opens up the DNA strands at the sites of breaks and staining by acridine orange (AO) are highly precise and repeatable (CVs of 1-3%) and the same between fresh and frozen samples. SCSAsoft(®) software transforms the X-Y data to total DNA stainability versus red/red + green fluoresence (DFI) providing a more accurate determination of % DFI as well as the more sensitive value of standard deviation of DFI (SD DFI) as demonstrated by animal fertility and dose-response toxicology studies. The current established clinical threshold is 25% DFI for placing a man into a statistical probability of the following: (a) longer time to natural pregnancy, (b) low odds of IUI pregnancy, (c) more miscarriages, or (d) no pregnancy. Changes in lifestyle as well as medical intervention can lower the %DFI to increase the probability of natural pregnancy. Couples of men with >25% DFI are counseled to try ICSI and when in the >50% range may consider TESE/ICSI. The SCSA(®) simultaneously determines the % of sperm with high DNA stainability (%HDS) related to retained nuclear histones consistent with immature sperm; high HDS values are predictive of pregnancy failure.The SCSA(®) is considered to be the most technician friendly, time- and cost-efficient, precise and repeatable DNA fragmentation assay, with the most data and the only fragmentation assay with an accepted clinical threshold for placing a man at risk for infertility. SCSA(®) data are more predictive of male factor infertility than classical semen analyses. PMID:22992911

  2. 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

  3. DNA methyltransferase 3b preferentially associates with condensed chromatin

    PubMed Central

    Kashiwagi, Katsunobu; Nimura, Keisuke; Ura, Kiyoe; Kaneda, Yasufumi

    2011-01-01

    In mammals, DNA methylation is catalyzed by DNA methyltransferases (DNMTs) encoded by Dnmt1, Dnmt3a and Dnmt3b. Since, the mechanisms of regulation of Dnmts are still largely unknown, the physical interaction between Dnmt3b and chromatin was investigated in vivo and in vitro. In embryonic stem cell nuclei, Dnmt3b preferentially associated with histone H1-containing heterochromatin without any significant enrichment of silent-specific histone methylation. Recombinant Dnmt3b preferentially associated with nucleosomal DNA rather than naked DNA. Incorporation of histone H1 into nucleosomal arrays promoted the association of Dnmt3b with chromatin, whereas histone acetylation reduced Dnmt3b binding in vitro. In addition, Dnmt3b associated with histone deacetylase SirT1 in the nuclease resistant chromatin. These findings suggest that Dnmt3b is preferentially recruited into hypoacetylated and condensed chromatin. We propose that Dnmt3b is a ‘reader’ of higher-order chromatin structure leading to gene silencing through DNA methylation. PMID:20923784

  4. 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 ?-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

  5. Chromatin Position Effects Assayed by Thousands of Reporters

    E-print Network

    Zhang, Jianzhi

    elements, compartmentalization of the genome into various types of chromatin domains, and spatial). This phenome- non has been exploited extensively to deduce causal relation- ships in the interplay among DNA sequence, chromatin context, and gene activity. For example, detailed analysis of position effects in yeast

  6. Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure

    PubMed Central

    Kost, Nils; Kaiser, Sophie; Ostwal, Yogesh; Riedel, Dietmar; Stützer, Alexandra; Nikolov, Miroslav; Rathke, Christina; Renkawitz-Pohl, Renate; Fischle, Wolfgang

    2015-01-01

    Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure. PMID:25735749

  7. The sperm chromatin structure assay as a diagnostic tool in the human fertility clinic

    Microsoft Academic Search

    Gry Brandt Boe-Hansen; Jens Fedder; Annette Kjær Ersbøll; Preben Christensen

    2006-01-01

    BACKGROUND: Sperm DNA integrity has been shown to be necessary for achieving and sustaining embryo devel- opment. The objective was to evaluate the sperm chromatin structure assay (SCSA) as a diagnostic tool in clinical practice for intrauterine insemination (IUI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatments. METHODS: A total of 385 semen samples from 234 couples were

  8. 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.

  9. 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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Gotoh, Eisuke

    2015-01-01

    Chromosome analysis is a fundamental technique which is used in wide areas of cytogenetic study including karyotyping species, hereditary diseases diagnosis, or chromosome biology study. Chromosomes are usually prepared from mitotic cells arrested by colcemid block protocol. However, obtaining mitotic chromosomes is often hampered under several circumstances. As a result, cytogenetic analysis will be sometimes difficult or even impossible in such cases. Premature chromosome condensation (PCC) (see Note 1) is an alternative method that has proved to be a unique and useful way in chromosome analysis. Former, PCC has been achieved following cell fusion method (cell-fusion PCC) mediated either by fusogenic viruses (e.g., Sendai virus) or cell fusion chemicals (e.g., polyethylene glycol), but the cell fusion PCC has several drawbacks. The novel drug-induced PCC using protein phosphatase inhibitors was introduced about 20 years ago. This method is much simpler and easier even than the conventional mitotic chromosome preparation protocol use with colcemid block and furthermore obtained PCC index (equivalent to mitotic index for metaphase chromosome) is usually much higher than colcemid block method. Moreover, this method allows the interphase chromatin to be condensed to visualize like mitotic chromosomes. Therefore drug-induced PCC has opened the way for chromosome analysis not only in metaphase chromosomes but also in interphase chromatin. The drug-induced PCC has thus proven the usefulness in cytogenetics and other cell biology fields. For this second edition version, updated modifications/changes are supplemented in Subheadings 2, 3, and 4, and a new section describing the application of PCC in chromosome science fields is added with citation of updated references. PMID:25827875

  12. Immunohistochemical distribution of hyperacetylated histone H4 in testis of paddlefish Polyodon spathula : ultrastructural correlation with chromatin condensation

    Microsoft Academic Search

    Otilia Zarnescu

    2007-01-01

    The acetylation of core histones has been correlated with the deposition of free histones onto newly replicated DNA, transcriptional\\u000a activity and the displacement of histones by protamines during spermiogenesis. The aim of the present study was to investigate\\u000a the immunohistochemical distribution of hyperacetylated H4 during spermatogenesis in Polyodon spathula and to correlate these findings with the pattern of chromatin condensation

  13. Immunohistochemical distribution of hyperacetylated histone H4 in testis of paddlefish Polyodon spathula: ultrastructural correlation with chromatin condensation.

    PubMed

    Zarnescu, Otilia

    2007-05-01

    The acetylation of core histones has been correlated with the deposition of free histones onto newly replicated DNA, transcriptional activity and the displacement of histones by protamines during spermiogenesis. The aim of the present study was to investigate the immunohistochemical distribution of hyperacetylated H4 during spermatogenesis in Polyodon spathula and to correlate these findings with the pattern of chromatin condensation in spermatids. In immature testis, the Sertoli cells showed more intense immunoreactivity for highly acetylated H4 than that of most primary spermatogonia. The testis of paddlefish at the beginning of spermatogenesis possessed early secondary spermatogonia and late secondary spermatogonia/preleptotene primary spermatocyte with intense nuclear immunoreactivity for highly acetylated H4. In seminiferous tubules in which secondary spermatogonia nuclei were intensely immunostained, Sertoli cell nuclei were unstained. Testes in late spermatogenesis contained tubules in which the immunohistochemical reaction for highly acetylated H4 was positive in the nuclei of preleptotene primary spermatocytes and secondary spermatocytes and in spermatids at the beginning of the elongation process. No immunostaining was found in round spermatids and spermatozoa. During the resting stage, immunostaining was confined to the nuclei of spermatogonia and the cells from the interstitial tissue. Transmission electron microscopy revealed that early spermatids had a round nucleus with filaments of fine chromatin that were dispersed or condensed in clumps. In later stages of maturation, the spermatids had slightly oval nuclei and homogeneous granular chromatin. The chromatin of advanced spermatids was organized into thick fibres. At the end of spermiogenesis, spermatozoan nuclei consisted of homogeneous highly compacted chromatin. PMID:17252243

  14. Transcriptional characteristics of in vitro assembled chromatin assayed by microinjection into Xenopus laevis oocytes.

    PubMed

    Steinbeisser, H; Hofmann, A; Oudet, P; Trendelenburg, M F

    1989-06-01

    Plasmid DNA was in vitro assembled into chromatin using an S-150 extract of Xenopus laevis oocytes. By varying the assembly temperature and DNA concentration it is possible to generate fully or partially assembled molecules. The fate of the in vitro preassembled molecules injected into X. laevis oocyte nuclei and their transcriptional activity were studied. Completely reconstituted molecules underwent a rearrangement of their chromatin structure after injection and showed reduced transcriptional activity compared to protein-free DNA or partially reconstituted chromatin. PMID:2737294

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

    PubMed

    Hu, Yan; Kireev, Igor; Plutz, Matt; Ashourian, Nazanin; Belmont, Andrew S

    2009-04-01

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

  16. 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

  17. Nuclear growth and chromatin relaxation-condensation cycle in hepatocytes during the proliferative activation of rat liver.

    PubMed

    Serratosa, J; Domingo, J; Enrich, C; Bachs, O

    1988-01-01

    In order to quantify the changes in nucleolar and nuclear volumes and in chromatin condensation produced during proliferative activation we have carried out morphometric studies on hepatocyte nuclei during rat liver regeneration using electron microscopy. To minimize the artefactual effects produced by fixation on subcellular structures we have fixed the livers by perfusion with glutaraldehyde. The mean values for the nucleolar and nuclear volumes were progressively increased until 28 h after 66% partial hepatectomy. The maximum values raised for the nuclei and nucleoli at this time were 3 and 4.28 times, respectively, those of controls. Later, nuclear and nucleolar volumes progressively declined. Two waves of diminution in nuclear electron-dense material were produced after hepatectomy. The first occurred between 0 and 12 h, with minimum values 1.34 times lower than those from control animals at 8 h. The second occurred between 12 and 28 h, with minimum values 2.56 times lower than those from control rats at 24 h. These two waves in chromatin relaxation correlate very well with the transcriptional changes described by other authors during the pre-replicative, replicative and mitotic phases of liver regeneration. PMID:2898834

  18. Frozen-thawed rhinoceros sperm exhibit DNA damage shortly after thawing when assessed by the sperm chromatin dispersion assay.

    PubMed

    Portas, T; Johnston, S D; Hermes, R; Arroyo, F; López-Fernadez, C; Bryant, B; Hildebrandt, T B; Göritz, F; Gosalvez, J

    2009-09-15

    This study reports on the successful validation (via in situ nick translation and neutral comet assay) of the equine Sperm-Halomax kit as an appropriate methodology for the assessment of sperm DNA fragmentation in three species of rhinoceros. Rhinoceros sperm nuclei with fragmented DNA (validated using in situ nick translation) were evident as large halos with dispersed DNA fragments, whereas those with nonfragmented DNA displayed small halos of nondispersed DNA within the microgel. There was a high correlation (r) of 0.974 (R(2) value=0.949; P<0.01; n=16) between the respective assessments of the Sperm Chromatin Dispersion test (SCDt) and the neutral comet assay. Application of the SCDt to determine the DNA fragmentation dynamics of rhinoceros (n=6) sperm frozen in liquid nitrogen vapor and incubated postthaw at 37 degrees C for up to 48 h to mimic in vitro conditions in the female reproductive tract, revealed an increase (P=0.001) in DNA damage, as soon as 4h after the start of incubation. Linear regression equations were calculated for all six rhinoceroses over the first 6h of incubation and revealed individual animal variation. Freshly collected and incubated (37 degrees C) rhinoceros (n=3) sperm had no increase in the basal level of DNA fragmentation for up to 48 h, indicating that the cryopreservation of rhinoceros sperm in liquid nitrogen vapor, as used in this study, appeared to result in freeze-thaw DNA damage. PMID:19560805

  19. 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...

  20. 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

  1. The Influence of Condensed Tannin Structure on Rate of Microbial Mineralization and Reactivity to Chemical Assays

    Microsoft Academic Search

    Charlotte E. Norris; Caroline M. Preston; Karen E. Hogg; Brian D. Titus

    2011-01-01

    We examined how tannin structure influences reactivity in tannin assays and carbon and nitrogen mineralization. Condensed\\u000a tannins from the foliage of ten tree and shrub species and from pecan shells (Carya illinoensis) had different proportions of: (a) epicatechin (cis) and catechin (trans) isomers, (b) procyanidin (PC) and prodelphinidin (PD) monomers, and (c) different chain lengths. The response of each tannin

  2. 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...

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

    SciTech Connect

    Evenson, Donald P. [HCLD, Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007 (United States) and SCSA Diagnostics, 807 32nd Avenue, Brookings, SD 57007 (United States)]. E-mail: scsa@brookings.net; Wixon, Regina [SCSA Diagnostics, 807 32nd Avenue, Brookings, SD 57007 (United States)

    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.

  4. Chromatin Remodeling

    NSDL National Science Digital Library

    Neal F. Lue (Weill Medical College of Cornell University; Department of Microbiology and Immunology REV)

    2005-07-26

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

  5. 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

  6. Ectopically tethered CP190 induces large-scale chromatin decondensation

    PubMed Central

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

    2014-01-01

    Insulator mediated alteration in higher-order chromatin and/or nucleosome organization is an important aspect of epigenetic gene regulation. Recent studies have suggested a key role for CP190 in such processes. In this study, we analysed the effects of ectopically tethered insulator factors on chromatin structure and found that CP190 induces large-scale decondensation when targeted to a condensed lacO array in mammalian and Drosophila cells. In contrast, dCTCF alone, is unable to cause such a decondensation, however, when CP190 is present, dCTCF recruits it to the lacO array and mediates chromatin unfolding. The CP190 induced opening of chromatin may not be correlated with transcriptional activation, as binding of CP190 does not enhance luciferase activity in reporter assays. We propose that CP190 may mediate histone modification and chromatin remodelling activity to induce an open chromatin state by its direct recruitment or targeting by a DNA binding factor such as dCTCF. PMID:24472778

  7. Phase Transition in Reconstituted Chromatin

    E-print Network

    Tonau Nakai; Kohji Hizume; Shige. H. Yoshimura; Kunio Takeyasu; Kenichi Yoshikawa

    2004-09-10

    By observing reconstituted chromatin by fluorescence microscopy (FM) and atomic force microscopy (AFM), we found that the density of nucleosomes exhibits a bimodal profile, i.e., there is a large transition between the dense and dispersed states in reconstituted chromatin. Based on an analysis of the spatial distribution of nucleosome cores, we deduced an effective thermodynamic potential as a function of the nucleosome-nucleosome distance. This enabled us to interpret the folding transition of chromatin in terms of a first-order phase transition. This mechanism for the condensation of chromatin is discussed in terms of its biological significance.

  8. 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...

  9. Condensation

    NSDL National Science Digital Library

    2012-06-26

    In this activity, learners explore the process of condensation. After seeing water vapor condense, learners will help design a test to see if cooling water vapor has an effect on the rate of condensation.

  10. Two Isoforms of Drosophila TRF2 Are Involved in Embryonic Development, Premeiotic Chromatin Condensation, and Proper Differentiation of Germ Cells of Both Sexes

    PubMed Central

    Kopytova, Daria V.; Krasnov, Aleksey N.; Kopantceva, Marina R.; Nabirochkina, Elena N.; Nikolenko, Julia V.; Maksimenko, Oksana; Kurshakova, Maria M.; Lebedeva, Lubov A.; Yerokhin, Maksim M.; Simonova, Olga B.; Korochkin, Leonid I.; Tora, Laszlo; Georgiev, Pavel G.; Georgieva, Sofia G.

    2006-01-01

    The Drosophila TATA box-binding protein (TBP)-related factor 2 (TRF2 or TLF) was shown to control a subset of genes different from that controlled by TBP. Here, we have investigated the structure and functions of the trf2 gene. We demonstrate that it encodes two protein isoforms: the previously described 75-kDa TRF2 and a newly identified 175-kDa version in which the same sequence is preceded by a long N-terminal domain with coiled-coil motifs. Chromatography of Drosophila embryo extracts revealed that the long TRF2 is part of a multiprotein complex also containing ISWI. Both TRF2 forms are detected at the same sites on polytene chromosomes and have the same expression patterns, suggesting that they fulfill similar functions. A study of the manifestations of the trf2 mutation suggests an essential role of TRF2 during embryonic Drosophila development. The trf2 gene is strongly expressed in germ line cells of adult flies. High levels of TRF2 are found in nuclei of primary spermatocytes and trophocytes with intense transcription. In ovaries, TRF2 is present both in actively transcribing nurse cells and in the transcriptionally inactive oocyte nuclei. Moreover, TRF2 is essential for premeiotic chromatin condensation and proper differentiation of germ cells of both sexes. PMID:17015475

  11. 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

  12. 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

  13. Cytochemical evaluation of sperm chromatin and DNA integrity in couples with unexplained recurrent spontaneous abortions.

    PubMed

    Talebi, A R; Vahidi, S; Aflatoonian, A; Ghasemi, N; Ghasemzadeh, J; Firoozabadi, R D; Moein, M R

    2012-05-01

    The aim of this study was to examine the possible relationship between sperm DNA integrity and chromatin packaging evaluated by cytochemical assays, traditional sperm parameters and recurrent spontaneous abortion (RSA) of unknown origin. In this cohort study, 40 couples with a history of RSA and 40 couples with proven fertility were considered as case and control groups respectively. The semen samples of all husbands were analysed for sperm parameters and also sperm chromatin and DNA integrity assessed using cytochemical tests including aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), acridine orange (AOT) and nuclear chromatin stability assay. Among different sperm parameters, only slow motility was significantly different between the two groups. In sperm chromatin evaluations, there were significant differences between the two groups in all of the tests. In addition, the majority of semen samples in RSA patients exhibited upper percentages of abnormal spermatozoa than the cut-off values regarding different cytochemical assays. Our study showed that in the cases of RSA, slow motility had a significant reduction in comparison with controls and also spermatozoa of men from RSA group had less chromatin condensation and poorer DNA integrity than spermatozoa that obtained from fertile men with no history of RSA. PMID:21806662

  14. The dynamics of chromatin condensation: redistribution of topoisomerase II in the 87A7 heat shock locus during induction and recovery.

    PubMed Central

    Udvardy, A; Schedl, P

    1993-01-01

    We have examined the in vivo sites of action for topoisomerases II in the 87A7 heat shock locus as a function of gene activity. When the hsp70 genes are induced, there is a dramatic redistribution of topoisomerase II in the locus which parallels many of the observed alterations in chromatin structure. In addition to changes in the topoisomerase II distribution within the locus, we find topoisomerase II localized around the putative domain boundaries scs and scs'. During recovery, when the chromatin fiber of the locus recondenses, the major sites of action for topoisomerase II appear to be located within the two hsp70 genes and in the intergenic spacer separating the two genes. Images PMID:8246970

  15. Chromatin Compaction Protects Genomic DNA from Radiation Damage

    PubMed Central

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

    2013-01-01

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

  16. 1 Modeling Studies of Chromatin Fiber Structure as a 2 Function of DNA Linker Length

    E-print Network

    Schlick, Tamar

    1 Modeling Studies of Chromatin Fiber Structure as a 2 Function of DNA Linker Length 3 Ognjen chromatin; 15 mesoscale modeling; 16 linker histone; 17 fiber condensation 18 Chromatin fibers encountered in various species and tissues are character- 19 ized by different nucleosome repeat lengths (NRLs

  17. 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

  18. 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

  19. Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear ( Ursus arctos) spermatozoa

    Microsoft Academic Search

    M. Álvarez; V. García-Macías; F. Martínez-Pastor; F. Martínez; S. Borragán; M. Mata; J. Garde; L. Anel; P. De Paz

    2008-01-01

    The Cantabrian brown bear (Ursus arctos) is a highly endangered species in Spain and basic studies are necessary in order to bank its germplasm. Sperm heads are mainly made up of chromatin, thus their shape depends partly on chromatin structure. Thawed semen from 10 bears was used to analyze chromatin status by sperm chromatin structure assay (SCSA) and head morphometry

  20. Mutagenicity of the fractionated organic emissions from diesel, cigarette smoke condensate, coke oven, and roofing tar in the Ames assay.

    PubMed

    Austin, A C; Claxton, L D; Lewtas, J

    1985-01-01

    Mobile and stationary combustion sources emit particle-bound organics that, after extraction, are mutagenic to Salmonella typhimurium. In this study, the organic emissions from diesel, cigarette smoke condensate (CSC), coke oven, and roofing tar were fractionated and compared for mutagenicity in the Ames assay. This study demonstrated major differences in the distribution of mutagenicity among the four emission sources. Within each source, the relative mutagenicity of each fraction was significantly different in the presence and absence of an exogenous metabolic activation. In the diesel sample, over 90% of the mutagenic activity is located in the aromatic and polar neutral (PN) fractions; nitrated polynuclear aromatic hydrocarbons (NO2-PNAs) can account for a significant portion of this activity. Most of the mutagenicity of the coke oven main sample was found in the organic base (BASE) and PN fractions, which contained aromatic amines and nitrogen heterocycles. The CSC sample also had a high percentage of the mutagenic activity in the BASE fraction. Chemical analysis, however, indicates that the components in the CSC differed from those of the coke oven main sample. The roofing tar sample, which was not mutagenic in the absence of metabolic activation, contained several components that were very mutagenic after fractionation. This may be due to the separation of toxic components from the mutagenic components. The roofing tar emissions contained aromatic and polar mutagenic constituents. Although the specific mutagens in these different sources are not identical, they all cause frameshift mutations and appear to be compounds that could be classified as polycyclic organic matter. PMID:2414094

  1. RuvB-like ATPases function in chromatin decondensation at the end of mitosis.

    PubMed

    Magalska, Adriana; Schellhaus, Anna Katharina; Moreno-Andrés, Daniel; Zanini, Fabio; Schooley, Allana; Sachdev, Ruchika; Schwarz, Heinz; Madlung, Johannes; Antonin, Wolfram

    2014-11-10

    Chromatin undergoes extensive structural changes during the cell cycle. Upon mitotic entry, metazoan chromatin undergoes tremendous condensation, creating mitotic chromosomes with 50-fold greater compaction relative to interphase chromosomes. At the end of mitosis, chromosomes reestablish functional interphase chromatin competent for replication and transcription through a decondensation process that is cytologically well described. However, the underlying molecular events and factors remain unidentified. We describe a cell-free system that recapitulates chromatin decondensation based on purified mitotic chromatin and Xenopus egg extracts. Using biochemical fractionation, we identify RuvB-like ATPases as chromatin decondensation factors and demonstrate that their ATPase activity is essential for decondensation. Our results show that decompaction of metaphase chromosomes is not merely an inactivation of known chromatin condensation factors but rather an active process requiring specific molecular machinery. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its coordination with concomitant processes. PMID:25443297

  2. 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

  3. 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

  4. Chromatin proteins: key responders to stress.

    PubMed

    Smith, Karen T; Workman, Jerry L

    2012-01-01

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

  5. Bromodomain protein Brd4 associated with acetylated chromatin is important for maintenance of higher-order chromatin structure.

    PubMed

    Wang, Ranran; Li, Qing; Helfer, Christine M; Jiao, Jing; You, Jianxin

    2012-03-30

    Chromatin structure organization is crucial for regulating many fundamental cellular processes. However, the molecular mechanism that regulates the assembly of higher-order chromatin structure remains poorly understood. In this study, we demonstrate that Brd4 (bromodomain-containing protein 4) protein participates in the maintenance of the higher-order chromatin structure. Brd4, a member of the BET family of proteins, has been shown to play important roles in cellular growth control, cell cycle progression, and cancer development. We apply in situ single cell chromatin imaging and micrococcal nuclease (MNase) assay to show that Brd4 depletion leads to a large scale chromatin unfolding. A dominant-negative inhibitor encoding the double bromodomains (BDI/II) of Brd4 can competitively dissociate endogenous Brd4 from chromatin to trigger severely fragmented chromatin morphology. Mechanistic studies using Brd4 truncation mutants reveal that the Brd4 C-terminal domain is crucial for maintaining normal chromatin structure. Using bimolecular fluorescence complementation technology, we demonstrate that Brd4 molecules interact intermolecularly on chromatin and that replacing Brd4 molecules by BDI/II causes abnormal nucleosome aggregation and chromatin fragmentation. These studies establish a novel structural role of Brd4 in supporting the higher chromatin architecture. PMID:22334664

  6. Chromatin remodelling during development

    PubMed Central

    Ho, Lena; Crabtree, Gerald R.

    2011-01-01

    New methods for the genome-wide analysis of chromatin are providing insight into its roles in development and their underlying mechanisms. Current studies indicate that chromatin is dynamic, with its structure and its histone modifications undergoing global changes during transitions in development and in response to extracellular cues. In addition to DNA methylation and histone modification, ATP-dependent enzymes that remodel chromatin are important controllers of chromatin structure and assembly, and are major contributors to the dynamic nature of chromatin. Evidence is emerging that these chromatin-remodelling enzymes have instructive and programmatic roles during development. Particularly intriguing are the findings that specialized assemblies of ATP-dependent remodellers are essential for establishing and maintaining pluripotent and multipotent states in cells. PMID:20110991

  7. Targeting Chromatin Readers

    PubMed Central

    James, LI; Frye, SV

    2015-01-01

    Modulation of gene expression through epigenetic signaling has recently emerged as a novel approach in treating human disease. Specifically, chromatin reader proteins, which mediate protein–protein interactions via binding to modified lysine residues, are gaining traction as potential therapeutic targets. Herein, we review recent efforts to understand and modulate the activity of chromatin reader proteins with small-molecule ligands. PMID:23403847

  8. 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

  9. Gearing up chromatin

    PubMed Central

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

    2014-01-01

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

  10. The Silent Information Regulator 3 Protein, SIR3p, Binds to Chromatin Fibers and Assembles a Hypercondensed Chromatin Architecture in the Presence of Salt

    Microsoft Academic Search

    Steven J. McBryant; Christine Krause; Christopher L. Woodcock; Jeffrey C. Hansen

    2008-01-01

    The telomeres and mating-type loci of budding yeast adopt a condensed, heterochromatin-like state through recruitment of the silent information regulator (SIR) proteins SIR2p, SIR3p, and SIR4p. In this study we characterize the chromatin binding determinants of recombinant SIR3p and identify how SIR3p mediates chromatin fiber condensation in vitro. Purified full-length SIR3p was incubated with naked DNA, nucleosome core particles, or

  11. Chromatin elongation factors.

    PubMed

    Svejstrup, Jesper Q

    2002-04-01

    As RNA polymerase II leaves a gene promoter to transcribe the coding region, it faces a major obstacle - nucleosomes tightly wrapped into chromatin. Mechanisms to deal with this obstacle clearly exist in cells, as transcription through chromatin is very efficient in vivo, whereas nucleosomal templates pose a considerable problem for polymerase progression in reconstituted in vitro systems. Advances in our understanding of transcriptional elongation through chromatin have been made possible recently by the identification of several accessory factors that assist polymerase in the process. Insights into the function of these factors have been gained by a combination of yeast genetics and biochemical studies in mammalian systems. PMID:11893488

  12. EBV Latency Types Adopt Alternative Chromatin Conformations

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2012-01-01

    Current methods to study chromatin configuration are not well suited for high throughput drug screening since they require large cell numbers and multiple experimental steps that include centrifugation for isolation of nuclei or DNA. Here we show that site specific chromatin analysis can be achieved in one step by simply performing direct chromatin PCR (DC-PCR) on cells. The basic underlying observation was that standard hypotonic PCR buffers prevent global cellular chromatin solubilization during thermal cycling while more loosely organized chromatin can be amplified. Despite repeated heating to >90 °C, 41 of 61 tested 5' sequences of silenced genes (CDKN2A, PU.1, IRF4, FOSB, CD34) were not amplifiable while 47 could be amplified from expressing cells. Two gene regions (IRF4, FOSB) even required pre-heating of cells in isotonic media to allow this differentiation; otherwise none of 19 assayed sequences yielded PCR products. Cells with baseline expression or epigenetic reactivation gave similar DC-PCR results. Silencing during differentiation of CD34 positive cord blood cells closed respective chromatin while treatment of myeloma cells with an IRF4 transcriptional inhibitor opened a site to DC-PCR that was occupied by RNA polymerase II and NF?B as determined by ChIP. Translation into real-time PCR can not be achieved with commercial real-time PCR buffers which potently open chromatin, but even with simple ethidium bromide addition to standard PCR mastermix we were able to identify hits in small molecules screens that suppressed IRF4 expression or reactivated CDKN2A in myeloma cells using densitometry or visual inspection of PCR plates under UV light. While need in drug development inspired this work, application to genome-wide analysis appears feasible using phi29 for selective amplification of open cellular chromatin followed by library construction from supernatants since such supernatants yielded similar results as gene specific DC-PCR. PMID:22984542

  14. 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

  15. 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

  16. HMGN dynamics and chromatin function.

    PubMed

    Catez, Frédéric; Lim, Jae-Hwan; Hock, Robert; Postnikov, Yuri V; Bustin, Michael

    2003-06-01

    Recent studies indicate that most nuclear proteins, including histone H1 and HMG are highly mobile and their interaction with chromatin is transient. These findings suggest that the structure of chromatin is dynamic and the protein composition at any particular chromatin site is not fixed. Here we discuss how the dynamic behavior of the nucleosome binding HMGN proteins affects the structure and function of chromatin. The high intranuclear mobility of HMGN insures adequate supply of protein throughout the nucleus and serves to target these proteins to their binding sites. Transient interactions of the proteins with nucleosomes destabilize the higher order chromatin, enhance the access to nucleosomal DNA, and impart flexibility to the chromatin fiber. While roaming the nucleus, the HMGN proteins encounter binding partners and form metastable multiprotein complexes, which modulate their chromatin interactions. Studies with HMGN proteins underscore the important role of protein dynamics in chromatin function. PMID:12897844

  17. Chromatin dynamics during DNA repair revealed by pair correlation analysis of molecular flow in the nucleus.

    PubMed

    Hinde, Elizabeth; Kong, Xiangduo; Yokomori, Kyoko; Gratton, Enrico

    2014-07-01

    Chromatin dynamics modulate DNA repair factor accessibility throughout the DNA damage response. The spatiotemporal scale upon which these dynamics occur render them invisible to live cell imaging. Here we present a believed novel assay to monitor the in vivo structural rearrangements of chromatin during DNA repair. By pair correlation analysis of EGFP molecular flow into chromatin before and after damage, this assay measures millisecond variations in chromatin compaction with submicron resolution. Combined with laser microirradiation we employ this assay to monitor the real-time accessibility of DNA at the damage site. We find from comparison of EGFP molecular flow with a molecule that has an affinity toward double-strand breaks (Ku-EGFP) that DNA damage induces a transient decrease in chromatin compaction at the damage site and an increase in compaction to adjacent regions, which together facilitate DNA repair factor recruitment to the lesion with high spatiotemporal control. PMID:24988341

  18. The control of sex chromatin

    Microsoft Academic Search

    O. J. Miller; Dorothy Warburton

    1968-01-01

    Sex chromatin counts for more than 50 individuals with three or more X chromosomes in their complement were analyzed. The observed distribution of cells with 0, 1, 2,...n sex chromatin bodies in polysomic-X individuals was compared with the binomial distribution expected on the assumption of a constant probability that an X chromosome will form a visible sex chromatin body. The

  19. Low 17beta-estradiol levels in CNR1 knock-out mice affect spermatid chromatin remodeling by interfering with chromatin reorganization.

    PubMed

    Cacciola, Giovanna; Chioccarelli, Teresa; Altucci, Lucia; Ledent, Catherine; Mason, J Ian; Fasano, Silvia; Pierantoni, Riccardo; Cobellis, Gilda

    2013-06-01

    The type 1-cannabinoid receptor, CNR1, regulates differentiation of spermatids. Indeed, we have recently reported that the genetic inactivation of Cnr1 in mice influenced chromatin remodeling of spermatids, by reducing histone displacement and then sperm chromatin quality indices (chromatin condensation and DNA integrity). Herein, we have studied, at both central and testicular levels, the molecular signals potentially involved in histone displacement. In particular, investigation of the neuroendocrine axis involved in estrogen production demonstrated down-regulation of the axis supporting FSH/estrogen secretion in Cnr1-knockout male mice. Conversely, Cnr1-knockout male mice treated with 17beta-estradiol showed a weak increase of pituitary Fsh-beta subunit mRNA levels and a rescue of sperm chromatin quality indices demonstrating that estrogens, possibly in combination with FSH secretion, play an important role in regulating chromatin remodeling of spermatids. PMID:23677985

  20. BAF chromatin remodeling complex

    PubMed Central

    Tuoc, Tran Cong; Narayanan, Ramanathan; Stoykova, Anastassia

    2013-01-01

    The multi-subunit chromatin remodeling BAF complex controls different developmental processes. Using cortex-specific conditional knockout and overexpression mouse models, we have recently reported that BAF170, a subunit of the vertebrate BAF chromatin remodeling complex, interacts with transcription factor (TF) Pax6 to control cortical size and volume. The mechanistic basis includes suppression of the expression of Pax6 target genes, which are required for genesis of cortical intermediate progenitors (IPs) and specification of late neuronal subtype identity. In addition, we showed that a dynamic competition between BAF170 and BAF155 subunits within the BAF complex during progression of neurogenesis is a primary event in modulating the size of the mammalian cortex. Here, we present additional insights into the interaction between the BAF complex and TF Pax6 in the genesis of IPs of the developing cortex. Furthermore, we show that such competition between BAF170 and BAF155 is involved as well in the determination of the size of the embryonic body. Our results add new insights into a cell-intrinsic mechanism, mediated by the chromatin remodeling BAF complex that controls vertebrate body shape and size. PMID:23974113

  1. Cell cycle-dependent binding of HMGN proteins to chromatin.

    PubMed

    Cherukuri, Srujana; Hock, Robert; Ueda, Tetsuya; Catez, Frédéric; Rochman, Mark; Bustin, Michael

    2008-05-01

    Throughout the cell cycle, the histones remain associated with DNA, but the repertoire of proteins associated with the chromatin fiber continuously changes. The chromatin interaction of HMGNs, a family of nucleosome binding proteins that modulates the structure and activity of chromatin, during the cell cycle is controversial. Immunofluorescence studies demonstrated that HMGNs are not associated with chromatin, whereas live cell imaging indicated that they are present in mitotic chromosomes. To resolve this controversy, we examined the organization of wild-type and mutated HMGN1 and HMGN2 proteins in the cell nucleus by using immunofluorescence studies, live cell imaging, gel mobility shift assays, and bimolecular fluorescence complementation (BiFC). We find that during interphase, HMGNs bind specifically to nucleosomes and form homodimeric complexes that yield distinct BiFC signals. In metaphase, the nucleosomal binding domain of the protein is inactivated, and the proteins associate with chromatin with low affinity as monomers, and they do not form specific complexes. Our studies demonstrate that the mode of binding of HMGNs to chromatin is cell cycle dependent. PMID:18287527

  2. Ultrastructural organization of yeast chromatin

    PubMed Central

    1982-01-01

    The ultrastructural organization of yeast chromatin was examined in Miller spread preparations of samples prepared from spheroplasts or isolated nuclei of Saccharomyces cerevisiae. Micrographs from preparations dispersed in 1 mM Tris (pH 7.2) illustrate that the basic chromatin fiber in yeast exists in two ultrastructurally distinct conformations. The majority (up to 95%) of the chromatin displays a beaded nucleosomal organization, although adjacent nucleosomes are separated by internucleosomal linkers of variable lengths. Ribonucleoprotein (RNP) fibrils are only occasionally associated with chromatin displaying the conformation. The remaining 5-10% of the chromatin appears to be devoid of discrete nucleosomes and has a smooth contour with a fiber diameter of 30-40 A. Transcriptional units, including putative ribosomal precursor RNA genes, defined by the presence of nascent RNP fibrils are restricted to chromatin displaying this smooth morphology. Chromatin released from nuclei in the presence of 5 mM Mg++ displays higher-order chromatin fibers, 200-300 A in diameter, these fibers appear to be arranged in a manner than reflects the two forms of the basic chromatin fiber. PMID:7040415

  3. Stretching and twisting chromatin

    E-print Network

    Dobrovolskaia, Irina V.; Dobrovolskaia, Irina V.

    2012-01-01

    fully uncoiled [1]. Metaphase chromosome Condensed scaffold-30-nm fiber, and metaphase chromosome. Adapted from [23].metaphase as the multiple classic four-arm structures, the tightly packed linear chromosomes

  4. Measuring chromatin interaction dynamics on the second time scale at single-copy genes.

    PubMed

    Poorey, Kunal; Viswanathan, Ramya; Carver, Melissa N; Karpova, Tatiana S; Cirimotich, Shana M; McNally, James G; Bekiranov, Stefan; Auble, David T

    2013-10-18

    The chromatin immunoprecipitation (ChIP) assay is widely used to capture interactions between chromatin and regulatory proteins, but it is unknown how stable most native interactions are. Although live-cell imaging suggests short-lived interactions at tandem gene arrays, current methods cannot measure rapid binding dynamics at single-copy genes. We show, by using a modified ChIP assay with subsecond temporal resolution, that the time dependence of formaldehyde cross-linking can be used to extract in vivo on and off rates for site-specific chromatin interactions varying over a ~100-fold dynamic range. By using the method, we show that a regulatory process can shift weakly bound TATA-binding protein to stable promoter interactions, thereby facilitating transcription complex formation. This assay provides an approach for systematic, quantitative analyses of chromatin binding dynamics in vivo. PMID:24091704

  5. Chromatin modification mapping in nanochannels

    PubMed Central

    Lim, Shuang Fang; Karpusenko, Alena; Blumers, Ansel L.; Streng, Diana E.; Riehn, Robert

    2013-01-01

    We report the simultaneous mapping of multiple histone tail modifications on chromatin that has been confined to nanofluidic channels. In these channels, chromatin is elongated, and histone modification can be detected using fluorescently tagged monoclonal antibodies. Using reconstituted chromatin with three distinct histone sources and two histone tail modification probes (H3K4me3 and H3K9ac), we were able to distinguish chromatin from the different sources. Determined ratios of the two modifications were consistent with the bulk composition of histone mixtures. We determined that the major difficulty in transitioning the mapping method to site-specific profiling within single genomic molecules is the interference of naturally aggregating, off-the shelf antibodies with the internal structure of chromatin. PMID:24396539

  6. Deciphering how the chromatin factor RCC1 recognizes the nucleosome: the importance of individuals in the scientific discovery process

    PubMed Central

    Tan, Song

    2012-01-01

    The nucleosome repeating unit of chromatin is the target of chromatin enzymes and factors which regulate gene activity in a eukaryotic cell. How the nucleosome is recognized by chromatin enzymes and factors is poorly understood even though such interaction is fundamental to gene regulation and chromatin biology. My laboratory recently determined the structural basis for how the RCC1 (regulator of chromosome condensation) chromatin factor binds to the nucleosome, including the first atomic crystal structure of a chromatin protein complexed with the nucleosome core particle. I describe here how we developed and investigated structural models for RCC1 binding to the nucleosome using biochemical methods, and how we crystallized the 300 kDa complex of RCC1 with the nucleosome core particle. This article highlights the contributions made by key laboratory members and explains our thinking and rationale during the discovery process. PMID:22435811

  7. 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.

  8. Chromatin immunoprecipitation in fission yeast.

    PubMed

    Volpe, Thomas A; Demaio, Jessica

    2011-01-01

    A tremendous amount of information regarding the nature and regulation of heterochromatin has emerged in the past 10 years. This rapid progress is largely due to the development of techniques such as chromatin immunoprecipitation or "ChIP," which allow analysis of chromatin structure. Further technological advances such as microarray analysis and, more recently, deep sequencing technologies, have made ChIP an even more powerful tool. ChIP allows the investigator to identify protein interactions and/or the presence of various chromatin modifications at specific genomic loci. PMID:21528444

  9. Impaired sperm chromatin integrity in obese mice.

    PubMed

    Duale, N; Steffensen, I-L; Andersen, J; Brevik, A; Brunborg, G; Lindeman, B

    2014-03-01

    An increased global prevalence of obesity coincides with an apparent decline in male sperm quality and a possible association between these pathologies has been suggested. In this study, we examined the effects of obesity on sperm chromatin integrity using two mouse models of obesity. In one group of mice, obesity was induced by a high-fat diet (HFD) (diet-induced obesity; DIO model), whereas in the other group, leptin deficiency was used to study the effects of obesity independently of the influence of dietary factors. Sperm chromatin integrity is recognized as an important measure of male infertility, and was analysed by the sperm chromatin structure assay. We found increased sperm DNA fragmentation in both groups of obese mice compared to lean mice, whereas the percentage of immature spermatozoa was not increased by obesity. The DIO model reflects the human condition more closely than the leptin-deficient model and was therefore selected for examination of the transcriptional response of a selection of marker genes in the testis by quantitative real-time PCR. The analysis of transcript levels of the selected testicular marker genes showed moderate, but significant, up-regulation of the Cyp2e1, Cyp19a1, Tnf and Pparg genes in DIO mice compared to lean mice. In conclusion, a clear positive correlation between body mass index and sperm DNA fragmentation was found in two mouse models of obesity. However, the variability in sperm DNA fragmentation within the two groups of obese animals was high. The observed changes in the transcript level of the marker genes suggest that there may be a local response in testicular cells to the HFD regimen with a potential impact on intratesticular signalling and spermatogenesis. PMID:24459046

  10. 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

  11. 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

  12. Magnetic Tweezers Instrumentation: We have used magnetic tweezers to study chromatin assembly and disassembly and RNA

    E-print Network

    Leuba, Sanford

    Magnetic Tweezers Instrumentation: We have used magnetic tweezers to study chromatin assembly and disassembly and RNA transcription. Magnetic tweezers surface magnetic bead F DNA external magnets F =kBT l/> l F x surface Instrumental set-up video camera beam condenser hollow bearing with magnet 90x oil

  13. Structural and functional genome analysis using extended chromatin

    SciTech Connect

    Heaf, T.; Ward, D.C. [Yale Univ., New Haven, CT (United States)

    1994-09-01

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

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

    PubMed Central

    2013-01-01

    Background The activity of transposable elements can be regulated by different means. DNA CpG methylation is known to decrease or inhibit transpositional activity of diverse transposons. However, very surprisingly, it was previously shown that CpG methylation of the Sleeping Beauty (SB) transposon significantly enhanced transposition in mouse embryonic stem cells. Results In order to investigate the unexpected response of SB transposition to CpG methylation, related transposons from the Tc1/mariner superfamily, that is, Tc1, Himar1, Hsmar1, Frog Prince (FP) and Minos were tested to see how transposition was affected by CpG methylation. A significant increase of >20-fold in transposition of SB, FP and Minos was seen, whereas Tc1, Himar1 and Hsmar1 showed no difference in transposition upon CpG-methylation. The terminal inverted repeats (TIRs) of the SB, FP and Minos elements share a common structure, in which each TIR contains two functionally important binding sites for the transposase (termed the IR/DR structure). The group of IR/DR elements showed increased excision after CpG methylation compared to untreated transposon donor plasmids. We found that de novo CpG methylation is not required for transposition. A mutated FP donor plasmid with depleted CpG sites in both TIRs was as efficient in transposition as the wild-type transposon, indicating that CpG sites inside the TIRs are not responsible for altered binding of factors potentially modulating transposition. By using an in vivo one-hybrid DNA-binding assay in cultured human cells we found that CpG methylation had no appreciable effect on the affinity of SB transposase to its binding sites. However, chromatin immunoprecipitation indicated that CpG-methylated transposon donor plasmids are associated with a condensed chromatin structure characterized by trimethylated histone H3K9. Finally, DNA compaction by protamine was found to enhance SB transposition. Conclusions We have shown that DNA CpG methylation upregulates transposition of IR/DR elements in the Tc1/mariner superfamily. CpG methylation provokes the formation of a tight chromatin structure at the transposon DNA, likely aiding the formation of a catalytically active complex by facilitating synapsis of sites bound by the transposase. PMID:23676100

  15. Epigenetics. Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing.

    PubMed

    Cusanovich, Darren A; Daza, Riza; Adey, Andrew; Pliner, Hannah A; Christiansen, Lena; Gunderson, Kevin L; Steemers, Frank J; Trapnell, Cole; Shendure, Jay

    2015-05-22

    Technical advances have enabled the collection of genome and transcriptome data sets with single-cell resolution. However, single-cell characterization of the epigenome has remained challenging. Furthermore, because cells must be physically separated before biochemical processing, conventional single-cell preparatory methods scale linearly. We applied combinatorial cellular indexing to measure chromatin accessibility in thousands of single cells per assay, circumventing the need for compartmentalization of individual cells. We report chromatin accessibility profiles from more than 15,000 single cells and use these data to cluster cells on the basis of chromatin accessibility landscapes. We identify modules of coordinately regulated chromatin accessibility at the level of single cells both between and within cell types, with a scalable method that may accelerate progress toward a human cell atlas. PMID:25953818

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

    PubMed Central

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

    1974-01-01

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

  17. The influence of microwave radiation on the state of chromatin in human cells

    E-print Network

    Y. G. Shckorbatov; V. N. Pasiuga; V. A. Grabina; N. N. Kolchigin; D. O. Batrakov; V. V. Kalashnikov; D. D. Ivanchenko; V. N. Bykov

    2008-09-03

    Isolated human buccal epithelium cell were irradiated by microwaves at frequency f=35 GHz and surface power density E=30 mcW/cm2. The state of chromatin in human cells was determined by methodsof light and electron microscopy. The state of cell membranes was evaluated by the method of vital indigo carmine staining. The microwave-induced condensation of chromatin in human cells was revealed. Left side circulary polarized waves induced less effect than linearly polarized radiation. The linearly polarized electromagnetic waves induced cell membrane damage revealed by the increase of cell stainability. The data obtained are discussed in connection with the mechanisms of biologica effect of electromagnetic waves.

  18. Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3?-OH Single-strand DNA Breaks*

    PubMed Central

    Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Sánchez-Osuna, María; Casanelles, Elisenda; García-Belinchón, Mercè; Comella, Joan X.; Yuste, Victor J.

    2013-01-01

    Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD?/? cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3?-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3?-OH ends in single-strand rather than double-strand DNA nicks/breaks. PMID:23430749

  19. Chromatin Structure in Telomere Dynamics

    PubMed Central

    Galati, Alessandra; Micheli, Emanuela; Cacchione, Stefano

    2013-01-01

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

  20. Automated microfluidic chromatin immunoprecipitation from 2,000 cells Angela R. Wu,a

    E-print Network

    Quake, Stephen R.

    such as certain cancer and stem cells. We have designed a microfluidic device to perform sensitive ChIP analysis to the medical and biological fields. Chromatin immunoprecipitation (ChIP) is a powerful tech- nique. Second, the assays are lengthy (2­7 days) and cumbersome to perform. Consequently, the quality

  1. SP1 is required for basal activation and chromatin accessibility of CD151 promoter in liver cancer cells

    Microsoft Academic Search

    Jiayi Wang; Xiangfan Liu; Peihua Ni; Zhidong Gu; Qishi Fan

    2010-01-01

    CD151 plays an important role in liver cancer metastasis. The mechanism on how CD151 is expressed remains unclear. Here we have identified SP1 is a protein functioning in constitutive activation of CD151. Applying a PCR-based chromatin accessibility assay, an open chromatin conformation was discovered localized around the transcription start site of the CD151 gene. Deletion constructs of the 5? flanking

  2. Chromatin architecture reorganization during stem cell differentiation.

    PubMed

    Dixon, Jesse R; Jung, Inkyung; Selvaraj, Siddarth; Shen, Yin; Antosiewicz-Bourget, Jessica E; Lee, Ah Young; Ye, Zhen; Kim, Audrey; Rajagopal, Nisha; Xie, Wei; Diao, Yarui; Liang, Jing; Zhao, Huimin; Lobanenkov, Victor V; Ecker, Joseph R; Thomson, James A; Ren, Bing

    2015-02-19

    Higher-order chromatin structure is emerging as an important regulator of gene expression. Although dynamic chromatin structures have been identified in the genome, the full scope of chromatin dynamics during mammalian development and lineage specification remains to be determined. By mapping genome-wide chromatin interactions in human embryonic stem (ES) cells and four human ES-cell-derived lineages, we uncover extensive chromatin reorganization during lineage specification. We observe that although self-associating chromatin domains are stable during differentiation, chromatin interactions both within and between domains change in a striking manner, altering 36% of active and inactive chromosomal compartments throughout the genome. By integrating chromatin interaction maps with haplotype-resolved epigenome and transcriptome data sets, we find widespread allelic bias in gene expression correlated with allele-biased chromatin states of linked promoters and distal enhancers. Our results therefore provide a global view of chromatin dynamics and a resource for studying long-range control of gene expression in distinct human cell lineages. PMID:25693564

  3. Chromatin signatures of the Drosophila replication program.

    PubMed

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

    2011-02-01

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

  4. Buccal micronucleus cytome assay: results of an intra- and inter-laboratory scoring comparison.

    PubMed

    Bolognesi, Claudia; Roggieri, Paola; Ropolo, Monica; Thomas, Philip; Hor, Maryam; Fenech, Michael; Nersesyan, Armen; Knasmueller, Siegfried

    2015-07-01

    The buccal micronucleus cytome (BMCyt) assay is a minimally invasive approach for measuring DNA damage, cell proliferation, cell differentiation and cell death in exfoliated buccal cells. The main limitation for its use is the lack of knowledge about inter- and intra-laboratory variability in scoring micronuclei and other end points included in the cytome approach. In order to identify the main sources of variability across the BMCyt biomarkers, a scoring exercise was carried out between three experienced laboratories using the same set of slides and an identical set of detailed scoring criteria and associated images for the different end points. Single batches of slides were prepared from pooled samples of four groups of subjects characterised by different frequencies of cell types and micronuclei, namely Down syndrome patients, head and neck cancer patients undergoing radiotherapy and two age- and gender-matched control groups. A good agreement among the laboratories in the identification of normal differentiated cells and of micronuclei was obtained. A 3-fold and 20-fold increase in the frequency of micronucleated cells and micronuclei in differentiated cells of Down syndrome patients and in cancer patients, respectively, compared to matched controls, was a consistent result in the three laboratories. The scores of other cell types and nuclear anomalies, such as basal, binucleated, condensed chromatin and karyorrhectic cells showed significant disagreement between and within laboratories indicating that their evaluation using the current visual scoring protocol does not yield robust results for these parameters. The guidelines for BMCyt assay application could be improved by combining the anomalies associated with cell death (condensed chromatin and karyorrhectic cells) in a single category and by defining more stringent criteria in classifying basal cell, binucleated cells and buds. PMID:25795005

  5. Characterization of a lipophilic plasmid DNA condensate formed with a cationic peptide fatty acid conjugate.

    PubMed

    Do, Trinh T; Tang, Vicky J; Aguilera, Joe A; Perry, Christopher C; Milligan, Jamie R

    2011-05-01

    In the presence of cationic ligands, DNA molecules can become aggregated into larger particles in a process known as condensation. DNA condensates are of interest as models for the dense packing found in naturally occurring structures such as phage heads and chromatin. They have found extensive application in DNA transfection and also provide convenient models with which to study DNA damage by the direct effect of ionizing radiation. Further, conjugates of cationic peptides with fatty acids may represent a class of attractive ligands for these areas because of their simple synthesis. When plasmid pUC18 is used as the DNA target and N-caproyl-penta-arginine amide (Cap-R(5)-NH(2)) is used as the ligand, the physical properties of the resulting mixtures were characterized using static and dynamic light scattering, sedimentation, dye exclusion, circular dichroism, nanoparticle tracking, and atomic force microscopy. Their chemical properties were assayed using solvent extraction and protection against hydroxyl radical attack and nuclease digestion. Titration of the plasmid with the Cap-R(5)-NH(2) ligand produced sharply defined changes in both chemical and physical properties, which was associated with the formation of condensed DNA particles in the 100-2000 nm size range. The caproyl group at the ligand's N-terminus produced a large increase in the partitioning of the resulting condensate from water into chloroform and in its binding to the neutral detergent Pluronic F-127. Both the physical and chemical data were all consistent with condensation of the plasmid by the ligand where the presence in the ligand of the caproyl group conferred an extensive lipophilic character upon the condensate. PMID:21410151

  6. Chromatin Landscape Dictates HSF Binding to Target DNA Elements

    PubMed Central

    Guertin, Michael J.; Lis, John T.

    2010-01-01

    Sequence-specific transcription factors (TFs) are critical for specifying patterns and levels of gene expression, but target DNA elements are not sufficient to specify TF binding in vivo. In eukaryotes, the binding of a TF is in competition with a constellation of other proteins, including histones, which package DNA into nucleosomes. We used the ChIP-seq assay to examine the genome-wide distribution of Drosophila Heat Shock Factor (HSF), a TF whose binding activity is mediated by heat shock-induced trimerization. HSF binds to 464 sites after heat shock, the vast majority of which contain HSF Sequence-binding Elements (HSEs). HSF-bound sequence motifs represent only a small fraction of the total HSEs present in the genome. ModENCODE ChIP-chip datasets, generated during non-heat shock conditions, were used to show that inducibly bound HSE motifs are associated with histone acetylation, H3K4 trimethylation, RNA Polymerase II, and coactivators, compared to HSE motifs that remain HSF-free. Furthermore, directly changing the chromatin landscape, from an inactive to an active state, permits inducible HSF binding. There is a strong correlation of bound HSEs to active chromatin marks present prior to induced HSF binding, indicating that an HSE's residence in “active” chromatin is a primary determinant of whether HSF can bind following heat shock. PMID:20844575

  7. Modifications of chromatin structure and gene expression following induced alterations of cellular shape.

    PubMed

    Vergani, Laura; Grattarola, Myriam; Nicolini, Claudio

    2004-08-01

    In higher eukaryotes cellular shape is a dynamic element which can be altered by external and internal factors (i.e. surface interactions, temperature, ionic strength). Our question was: might modifications of cell shape reflect on nuclear morphology and architecture and hence on chromatin function, in order to represent a mechanism of cell regulation? We altered the shape of cultured fibroblasts by coating the growth substratum with synthetic polymers, which alternatively increased and decreased the adhesiveness. By means of Fluorescence microscopy we analysed the modifications of cell and nucleus architecture induced by the different substrata. Then we used differential scanning calorimetry to investigate if a remodelling of chromatin structure was associated with the induced morphological changes. Finally, we evaluated if the observed modifications of chromatin condensation affect the transcriptional profile. At this stage of the work we focused on just four genes (c-myc, c-fos, c-jun and collagen) and we analysed their expression by dot blot hybridization and RT-PCR. The results confirm that mechanical factors external to the cell, such as the physico-chemical features of the substratum, are able to modulate gene transcription through a remodelling of chromatin structure. Therefore the work supports our starting hypothesis of a regulatory pathway connecting in sequence cellular morphomety/nuclear architecture/chromatin structure/gene expression. PMID:15147724

  8. The chromatin remodelling complex FACT associates with actively transcribed regions of the Arabidopsis genome.

    PubMed

    Duroux, Meg; Houben, Andreas; R?zicka, Kamil; Friml, Jirí; Grasser, Klaus D

    2004-12-01

    The packaging of the genomic DNA into chromatin in the cell nucleus requires machineries that facilitate DNA-dependent processes such as transcription in the presence of repressive chromatin structures. Using co-immunoprecipitation we have identified in Arabidopsis thaliana cells the FAcilitates Chromatin Transcription (FACT) complex, consisting of the 120-kDa Spt16 and the 71-kDa SSRP1 proteins. Indirect immunofluorescence analyses revealed that both FACT subunits co-localize to nuclei of the majority of cell types in embryos, shoots and roots, whereas FACT is not present in terminally differentiated cells such as mature trichoblasts or cells of the root cap. In the nucleus, Spt16 and SSRP1 are found in the cytologically defined euchromatin of interphase cells independent of the status of DNA replication, but the proteins are not associated with heterochromatic chromocentres and condensed mitotic chromosomes. FACT can be detected by chromatin immunoprecipitation over the entire transcribed region (5'-UTR, coding sequence, 3'-UTR) of actively transcribed genes, whereas it does not occur at transcriptionally inactive heterochromatic regions and intergenic regions. FACT localizes to inducible genes only after induction of transcription, and the association of the complex with the genes correlates with the level of transcription. Collectively, these results indicate that FACT assists transcription elongation through plant chromatin. PMID:15546350

  9. Chromatin Remodelers: From Function to Dysfunction.

    PubMed

    Längst, Gernot; Manelyte, Laura

    2015-01-01

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

  10. 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

  11. Higher Order Chromatin Degradation: Implications for Neurodegeneration

    Microsoft Academic Search

    Gregory W. Konat

    2002-01-01

    Higher order chromatin degradation (HOCD) is a hallmark of programmed cell death. HOCD is mediated by enzymatic digestion of the DNA backbone at matrix attachment regions, and ultimately results in the excision of chromatin loops and their oligomers from chromosomes. We have recently demonstrated that hydrogen peroxide (H2O2), the major mediator of oxidative stress, rapidly induces HOCD. This demonstration allowed

  12. Mapping chromatin conformation Peter J Shaw

    E-print Network

    Shaw, Peter

    Mapping chromatin conformation Peter J Shaw Address: Department of Cell and Developmental Biology and can be found at: http://f1000.com/reports/biology/content/2/18 Abstract Chromatin conformation capture using high-throughput DNA sequencing to generate global interaction maps for the entire genome

  13. Regulation of chromatin by histone modifications

    Microsoft Academic Search

    Andrew J Bannister; Tony Kouzarides

    2011-01-01

    Chromatin is not an inert structure, but rather an instructive DNA scaffold that can respond to external cues to regulate the many uses of DNA. A principle component of chromatin that plays a key role in this regulation is the modification of histones. There is an ever-growing list of these modifications and the complexity of their action is only just

  14. Chromatin patterns associated with lung adenocarcinoma progression

    PubMed Central

    Druliner, Brooke R.; Fincher, Justin A.; Sexton, Brittany S.; Vera, Daniel L.; Roche, Michael; Lyle, Stephen; Dennis, Jonathan H.

    2013-01-01

    The development and progression of lung adenocarcinoma, one of the most common cancers, is driven by the interplay of genetic and epigenetic changes and the role of chromatin structure in malignant transformation remains poorly understood. We used systematic nucleosome distribution and chromatin accessibility microarray mapping platforms to analyze the genome-wide chromatin structure from normal tissues and from primary lung adenocarcinoma of different grades and stages. We identified chromatin-based patterns across different patients with lung adenocarcinoma of different cancer grade and stage. Low-grade cancers had nucleosome distributions very different compared with the corresponding normal tissue but had nearly identical chromatin accessibility. Conversely, nucleosome distributions of high-grade cancers showed few differences. Substantial disruptions in chromosomal accessibility were seen in a patient with a high-grade and high-stage tumor. These data imply that chromatin structure changes during the progression of lung adenocarcinoma. We have therefore developed a model in which low-grade lung adenocarcinomas are linked to changes in nucleosome distributions, whereas higher-grade tumors are linked to large-scale chromosomal changes. These results provide a foundation for the development of a comprehensive framework linking the general and locus-specific roles of chromatin structure to lung cancer progression. We propose that this strategy has the potential to identify a new class of chromatin-based diagnostic, prognostic and therapeutic markers in cancer progression. PMID:23598721

  15. 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

  16. Understanding the Words of Chromatin Regulation

    PubMed Central

    Wu, Jiang I.; Lessard, Julie; Crabtree, Gerald R.

    2009-01-01

    Recent studies indicate that chromatin regulatory complexes produce biological specificity in the way that letters produce meanings by combinations into words. Combinatorial assembly of chromatin regulatory complexes may be critical for maximizing the information content provided by arrays of histone modifications. PMID:19167321

  17. Overlapping Chromatin Remodeling Systems Collaborate Genome-wide at Dynamic Chromatin Transitions

    PubMed Central

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

    2013-01-01

    ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4, and Snf2h. The localization patterns of all three proteins significantly overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome-wide, and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Many regions require the activity of more than one remodeler to regulate accessibility. These findings provide a dynamic view of chromatin organization, and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes. PMID:24317492

  18. 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

  19. 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

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

    SciTech Connect

    Namdar, Mandana [Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS (United Kingdom); Kearsey, Stephen E. [Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS (United Kingdom)]. 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.

  1. Biphasic chromatin binding of histone chaperone FACT during eukaryotic chromatin DNA replication

    PubMed Central

    Kundu, Lena R.; Seki, Masayuki; Watanabe, Nanae; Murofushi, Hiromu; Furukohri, Asako; Waga, Shou; Score, Alan J.; Blow, J. Julian; Horikoshi, Masami; Enomoto, Takemi; Tada, Shusuke

    2012-01-01

    The facilitates chromatin transcription (FACT) complex affects nuclear DNA transactions in a chromatin context. Though the involvement of FACT in eukaryotic DNA replication has been revealed, a clear understanding of its biochemical behavior during DNA replication still remains elusive. Here, we analyzed the chromatin-binding dynamics of FACT using Xenopus egg extract cell-free system. We found that FACT has at least two distinct chromatin-binding phases: (1) a rapid chromatin-binding phase at the onset of DNA replication that did not involve origin licensing and (2) a second phase of chromatin binding that initiated after origin licensing. Intriguingly, early-binding FACT dissociated from chromatin when DNA replication was blocked by the addition of Cdc6 in the licensed state before origin ring. Cdc6-induced removal of FACT was blocked by the inhibition of origin licensing with geminin, but not by suppressing the activity of DNA polymerases, CDK, or Cdc7. Furthermore, chromatin transfer experiments revealed that impairing the later binding of FACT severely compromises DNA replication activity. Taken together, we propose that even though FACT has rapid chromatin-binding activity, the binding pattern of FACT on chromatin changes after origin licensing, which may contribute to the establishment of its functional link to the DNA replication machinery. PMID:21232560

  2. Biphasic chromatin binding of histone chaperone FACT during eukaryotic chromatin DNA replication.

    PubMed

    Kundu, Lena R; Seki, Masayuki; Watanabe, Nanae; Murofushi, Hiromu; Furukohri, Asako; Waga, Shou; Score, Alan J; Blow, J Julian; Horikoshi, Masami; Enomoto, Takemi; Tada, Shusuke

    2011-06-01

    The facilitates chromatin transcription (FACT) complex affects nuclear DNA transactions in a chromatin context. Though the involvement of FACT in eukaryotic DNA replication has been revealed, a clear understanding of its biochemical behavior during DNA replication still remains elusive. Here, we analyzed the chromatin-binding dynamics of FACT using Xenopus egg extract cell-free system. We found that FACT has at least two distinct chromatin-binding phases: (1) a rapid chromatin-binding phase at the onset of DNA replication that did not involve origin licensing and (2) a second phase of chromatin binding that initiated after origin licensing. Intriguingly, early-binding FACT dissociated from chromatin when DNA replication was blocked by the addition of Cdc6 in the licensed state before origin firing. Cdc6-induced removal of FACT was blocked by the inhibition of origin licensing with geminin, but not by suppressing the activity of DNA polymerases, CDK, or Cdc7. Furthermore, chromatin transfer experiments revealed that impairing the later binding of FACT severely compromises DNA replication activity. Taken together, we propose that even though FACT has rapid chromatin-binding activity, the binding pattern of FACT on chromatin changes after origin licensing, which may contribute to the establishment of its functional link to the DNA replication machinery. PMID:21232560

  3. 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

  4. Chromatin remodelling initiation during human spermiogenesis

    PubMed Central

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

    2012-01-01

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

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

    SciTech Connect

    Pina-Guzman, B. [Seccion Externa de Toxicologia, CINVESTAV-IPN, Ave. IPN 2508, Col. Zacatenco, Mexico City 07360 (Mexico); Solis-Heredia, M.J. [Seccion Externa de Toxicologia, CINVESTAV-IPN, Ave. IPN 2508, Col. Zacatenco, Mexico City 07360 (Mexico); Quintanilla-Vega, B. [Seccion Externa de Toxicologia, CINVESTAV-IPN, Ave. IPN 2508, Col. Zacatenco, Mexico City 07360 (Mexico)]. 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.

  6. UV laser radiosensitivity of normal and tumoral chromatin

    NASA Astrophysics Data System (ADS)

    Radu, Liliana; Mihailescu, Ion N.; Preoteasa, V.; Radulescu, Irina; Gostian, Doina; Hening, Alexandru; Radu, S.

    1998-07-01

    A normal and a tumoral chromatin radiosensitivity to an UV laser radiation was determined. The characteristics of these chromatin samples were established by the analysis of the absorption and emission spectra of chromatin complexes with a specific DNA ligand-ethidium bromide, by Scatchard representations of ligand binding to chromatin and by the Forster energy transfer efficiency determination between two fluorescent ligands coupled at chromatin: dansyl chloride and acridine orange. The effects of excimer laser beam with (lambda) equals 248 nm on chromatin structure were analyzed by the above methods and also by the establishment of the intrinsic chromatin fluorescence and of the excited state lifetimes of a specific DNA ligand.

  7. 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

  8. 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

  9. 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.

  10. Identifying chromatin interactions at high spatial resolution

    E-print Network

    Reeder, Christopher Campbell

    2014-01-01

    This thesis presents two computational approaches for identifying chromatin interactions at high spatial resolution from ChIA-PET data. We introduce SPROUT which is a hierarchical probabilistic model that discovers high ...

  11. 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

  12. The Current State of Chromatin Immunoprecipitation

    Microsoft Academic Search

    Philippe Collas

    2010-01-01

    The biological significance of interactions of nuclear proteins with DNA in the context of gene expression, cell differentiation,\\u000a or disease has immensely been enhanced by the advent of chromatin immunoprecipitation (ChIP). ChIP is a technique whereby\\u000a a protein of interest is selectively immunoprecipitated from a chromatin preparation to determine the DNA sequences associated\\u000a with it. ChIP has been widely used

  13. Postnatal Effects of Sperm Chromatin Damage

    Microsoft Academic Search

    Miriam Pérez-Crespo; Raúl Fernández-González; Miguel Ángel Ramírez; Eva Pericuesta; Alexandra Calle; Alfonso Gutiérrez-Adán

    \\u000a The use of spermatozoa with fragmented DNA has been linked to ­developmental and postnatal effects in animal models. Environmental\\u000a and toxic factors such as radiation, heat stress, air pollution, chemotherapeutic agents, etc. are known to have detrimental\\u000a effects on sperm chromatin. Sperm chromatin damage has also been observed following sperm manipulation techniques (freeze–thawing\\u000a without cryoprotectants, freeze-drying, preincubation under different conditions,

  14. The influence of chromatin structure on initial DNA damage and radiosensitivity in CHO-K1 and xrs1 cells at low doses of irradiation (1-10 Gy)

    Microsoft Academic Search

    W. P. Roos; A. Binder; L. Böhm

    2002-01-01

    Mitotic compaction of chromatin was generated by treatment of cells with nocodazole. Alternatively, chromatin structure was altered by incubating cells in 500 mM NaCl. The irradiation response in the dose range of 1-10 Gy was measured by colony assay and by a modified fluorometric analysis of DNA unwinding (FADU) assay which measures the amount of undamaged DNA by EtBr fluorescence.

  15. DNA integrity in sexed bull sperm assessed by neutral Comet assay and sperm chromatin structure assay

    Microsoft Academic Search

    Gry B. Boe-Hansen; Ian D. Morris; Annette K. Ersbøll; Torben Greve; Preben Christensen

    2005-01-01

    During the production of sex-sorted spermatozoa from bull semen, the cells are exposed to a number of potential hazards including: dilution, centrifugation, incubation, exposure to DNA stains and laser light. These factors may affect the survival capacity and fertilization potential of the sperm. The objective of this study was to determine whether sex-sorted bull spermatozoa have more DNA damage than

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

    PubMed

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

    2015-07-21

    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

  17. A macrohistone variant links dynamic chromatin compaction to BRCA1-dependent genome maintenance.

    PubMed

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

    2014-08-21

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

  18. Cohesin codes – interpreting chromatin architecture and the many facets of cohesin function

    PubMed Central

    Rudra, Soumya; Skibbens, Robert V.

    2013-01-01

    Summary Sister chromatid tethering is maintained by cohesin complexes that minimally contain Smc1, Smc3, Mcd1 and Scc3. During S-phase, chromatin-associated cohesins are modified by the Eco1/Ctf7 family of acetyltransferases. Eco1 proteins function during S phase in the context of replicated sister chromatids to convert chromatin-bound cohesins to a tethering-competent state, but also during G2 and M phases in response to double-stranded breaks to promote error-free DNA repair. Cohesins regulate transcription and are essential for ribosome biogenesis and complete chromosome condensation. Little is known, however, regarding the mechanisms through which cohesin functions are directed. Recent findings reveal that Eco1-mediated acetylation of different lysine residues in Smc3 during S phase promote either cohesion or condensation. Phosphorylation and SUMOylation additionally impact cohesin functions. Here, we posit the existence of a cohesin code, analogous to the histone code introduced over a decade ago, and speculate that there is a symphony of post-translational modifications that direct cohesins to function across a myriad of cellular processes. We also discuss evidence that outdate the notion that cohesion defects are singularly responsible for cohesion-mutant-cell inviability. We conclude by proposing that cohesion establishment is linked to chromatin formation. PMID:23516328

  19. Opsonophagocytic assay.

    PubMed

    Dwyer, Markryan; Gadjeva, Mihaela

    2014-01-01

    The opsonophagocytic killing (OPK) assay is used as a correlate for protection to measure the functional capacities of vaccine-candidate-raised antibodies. This in vitro assay aids selecting promising vaccines by demonstrating whether the vaccine-induced antibodies drive efficient complement deposition and subsequent opsonophagocytic killing. Here, we describe two protocols for an OPK assay using either human-derived PMNs or cultured HL-60 cells. PMID:24218277

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

    PubMed

    Polo, Sophie E

    2015-02-13

    DNA damage signaling and repair machineries operate in a nuclear environment where DNA is wrapped around histone proteins and packaged into chromatin. Understanding how chromatin structure is restored together with the DNA sequence during DNA damage repair has been a topic of intense research. Indeed, chromatin integrity is central to cell functions and identity. However, chromatin shows remarkable plasticity in response to DNA damage. This review presents our current knowledge of chromatin dynamics in the mammalian cell nucleus in response to DNA double strand breaks and UV lesions. I provide an overview of the key players involved in regulating histone dynamics in damaged chromatin regions, focusing on histone chaperones and their concerted action with histone modifiers, chromatin remodelers and repair factors. I also discuss how these dynamics contribute to reshaping chromatin and, by altering the chromatin landscape, may affect the maintenance of epigenetic information. PMID:24887097

  1. Chromatin remodeling factors Isw2 and Ino80 regulate checkpoint activity and chromatin structure in S phase.

    PubMed

    Lee, Laura; Rodriguez, Jairo; Tsukiyama, Toshio

    2015-04-01

    When cells undergo replication stress, proper checkpoint activation and deactivation are critical for genomic stability and cell survival and therefore must be highly regulated. Although mechanisms of checkpoint activation are well studied, mechanisms of checkpoint deactivation are far less understood. Previously, we reported that chromatin remodeling factors Isw2 and Ino80 attenuate the S-phase checkpoint activity in Saccharomyces cerevisiae, especially during recovery from hydroxyurea. In this study, we found that Isw2 and Ino80 have a more pronounced role in attenuating checkpoint activity during late S phase in the presence of methyl methanesulfonate (MMS). We therefore screened for checkpoint factors required for Isw2 and Ino80 checkpoint attenuation in the presence of MMS. Here we demonstrate that Isw2 and Ino80 antagonize checkpoint activators and attenuate checkpoint activity in S phase in MMS either through a currently unknown pathway or through RPA. Unexpectedly, we found that Isw2 and Ino80 increase chromatin accessibility around replicating regions in the presence of MMS through a novel mechanism. Furthermore, through growth assays, we provide additional evidence that Isw2 and Ino80 partially counteract checkpoint activators specifically in the presence of MMS. Based on these results, we propose that Isw2 and Ino80 attenuate S-phase checkpoint activity through a novel mechanism. PMID:25701287

  2. Purification of specific chromatin loci for proteomic analysis.

    PubMed

    Byrum, Stephanie D; Taverna, Sean D; Tackett, Alan J

    2015-01-01

    Purification of small, native chromatin regions for proteomic identification of specifically bound proteins and histone posttranslational modifications is a powerful approach for studying mechanisms of chromosome metabolism. Here we detail a Chromatin Affinity Purification with Mass Spectrometry (ChAP-MS) approach for affinity purification of 1 kb regions of chromatin for targeted proteomic analysis. This approach utilizes quantitative, high resolution mass spectrometry to categorize proteins and histone posttranslational modifications co-enriched with the given chromatin region as either "specific" to the targeted chromatin or "nonspecific" contamination. In this way, the ChAP-MS approach can help define and redefine mechanisms of chromatin-templated activities. PMID:25311124

  3. Higher-order structure of nucleosome oligomers from short-repeat chromatin.

    PubMed Central

    Pearson, E C; Butler, P J; Thomas, J O

    1983-01-01

    Sedimentation measurements and electron microscopy at a series of ionic strengths suggest that chromatin from neurons of the cerebral cortex is able to form condensed structures in vitro that are probably several turns of a solenoid with about six nucleosomes per turn. Since neuronal chromatin has a short nucleosomal repeat (approximately 165 bp) allowing virtually no linker DNA between nucleosomes, and yet forms apparently 'normal' elements of solenoid, the packing of nucleosomes in the solenoid must be highly constrained. This permits only a limited number of possible models, and enables tentative suggestions to be made about the location of the linker DNA in the typical solenoid. Images Fig. 1. Fig. 4. Fig. 5. PMID:10872332

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

    PubMed Central

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

    2011-01-01

    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. While RCC1 has been shown to bind directly with the nucleosome, the molecular details of this interaction were not known. We have determined the crystal structure of the RCC1-nucleosome core particle complex at 2.9 Å resolution, providing the first 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 our nucleosomes forms a 145 bp and not the expected canonical 147 bp nucleosome core particle. PMID:20739938

  5. Models incorporating chromatin modification data identify functionally important p53 binding sites

    PubMed Central

    Lim, Ji-Hyun; Iggo, Richard D.; Barker, Daniel

    2013-01-01

    Genome-wide prediction of transcription factor binding sites is notoriously difficult. We have developed and applied a logistic regression approach for prediction of binding sites for the p53 transcription factor that incorporates sequence information and chromatin modification data. We tested this by comparison of predicted sites with known binding sites defined by chromatin immunoprecipitation (ChIP), by the location of predictions relative to genes, by the function of nearby genes and by analysis of gene expression data after p53 activation. We compared the predictions made by our novel model with predictions based only on matches to a sequence position weight matrix (PWM). In whole genome assays, the fraction of known sites identified by the two models was similar, suggesting that there was little to be gained from including chromatin modification data. In contrast, there were highly significant and biologically relevant differences between the two models in the location of the predicted binding sites relative to genes, in the function of nearby genes and in the responsiveness of nearby genes to p53 activation. We propose that these contradictory results can be explained by PWM and ChIP data reflecting primarily biophysical properties of protein–DNA interactions, whereas chromatin modification data capture biologically important functional information. PMID:23599002

  6. Genetic Architecture of Transcription and Chromatin Regulation

    PubMed Central

    Kim, Kwoneel; Bang, Hyoeun; Lee, Kibaick

    2015-01-01

    DNA microarray and next-generation sequencing provide data that can be used for the genetic analysis of multiple quantitative traits such as gene expression levels, transcription factor binding profiles, and epigenetic signatures. In particular, chromatin opening is tightly coupled with gene transcription. To understand how these two processes are genetically regulated and associated with each other, we examined the changes of chromatin accessibility and gene expression in response to genetic variation by means of quantitative trait loci mapping. Regulatory patterns commonly observed in yeast and human across different technical platforms and experimental designs suggest a higher genetic complexity of transcription regulation in contrast to a more robust genetic architecture of chromatin regulation. PMID:26175661

  7. 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

  8. 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

  9. 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

  10. 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

  11. A Chromatin-Mediated Reversible Drug-Tolerant State

    E-print Network

    Oregon, University of

    viability via engagement of IGF-1 receptor signaling and an altered chromatin state that requires by treatment with IGF-1 receptor inhibitors or chromatin-modifying agents, potentially yielding a therapeutic

  12. The Chromatin Fingerprint of Gene Enhancer Elements*

    PubMed Central

    Zentner, Gabriel E.; Scacheri, Peter C.

    2012-01-01

    Different cell types within a single organism are generally distinguished by strikingly different patterns of gene expression, which are dynamic throughout development and adult life. Distal enhancer elements are key drivers of spatiotemporal specificity in gene regulation. Often located tens of kilobases from their target promoters and functioning in an orientation-independent manner, the identification of bona fide enhancers has proved a formidable challenge. With the development of ChIP-seq, global cataloging of putative enhancers has become feasible. Here, we review the current understanding of the chromatin landscape at enhancers and how these chromatin features enable robust identification of tissue-specific enhancers. PMID:22952241

  13. 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.

  14. The Roles of Chromatin Remodelling Factors in Replication

    Microsoft Academic Search

    Ana Neves-Costa; Patrick Varga-Weisz

    Dynamic changes of chromatin structure control DNA-dependent events, including DNA replication.\\u000a Along with DNA, chromatin organization must be replicated to maintain genetic and epigenetic information\\u000a through cell generations. Chromatin remodelling is important for several steps in replication: determination\\u000a and activation of origins of replication, replication machinery progression, chromatin assembly and\\u000a DNA repair. Histone chaperones such as the FACT complex assist

  15. 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.

  16. 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.

  17. Mapping accessible chromatin regions using Sono-Seq

    E-print Network

    Gerstein, Mark

    Mapping accessible chromatin regions using Sono-Seq Raymond K. Auerbacha,1 , Ghia Euskirchenb,1, CT 06520; and dDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School arises from local changes in chromatin conformation. Previous methods for mapping chromatin accessibility

  18. GENE EXPRESSION & METABOLISM Chromatin and Transcription in Yeast

    E-print Network

    Winston, Fred

    for H2A.Z in transcription 360 Chromatin-Remodeling Factors 361 Identification of the Swi/Snf and RSC complexes 361 Swi/Snf complexes have chromatin-remodeling activity 362 Regulation of transcription by Swi/Snf 362 RSC plays broad roles in gene expression and chromatin structure 363 Bromodomains in Swi/Snf

  19. Chromatin regulation: how complex does it get?

    PubMed

    Meier, Karin; Brehm, Alexander

    2014-11-01

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

  20. PERSPECTIVES Chromatin Conformation of Yeast Centromeres

    E-print Network

    PERSPECTIVES Chromatin Conformation of Yeast Centromeres KERRY S. BLOOM, ENRIQUE AMAYA, JOHN CARBON The centromere region of Saccharomyces cerevisiae chromosome III has been replaced by various DNA fragments from the centromere regions of yeast chromosomes III and Xl. A 289-base pair centromere (CEN3) sequence can stabilize

  1. Chemical biology: Chromatin chemistry goes cellular

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  2. 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

  3. Hajjoul/Mathon et al. Chromatin dynamics in living yeasts High throughput chromatin motion tracking in living yeast reveals the

    E-print Network

    Boyer, Edmond

    Hajjoul/Mathon et al. Chromatin dynamics in living yeasts - 1 - High throughput chromatin motion tracking in living yeast reveals the flexibility of the fiber throughout the genome Houssam Hajjoul1.1101/gr.157008.113 #12;Hajjoul/Mathon et al. Chromatin dynamics in living yeasts - 2 - ABSTRACT (211 words

  4. Nucleas action on chromatin: evidence for discrete, repeated nucleoprotein units along chromatin fibrils.

    PubMed

    Oosterhof, D K; Hozier, J C; Rill, R L

    1975-02-01

    The time course of the fragmentation of calf thymus chromatin by DNase II (deoxyribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.6) has been examined by sedimentation of chromatin digests through linear (5-20%) sucrose gradients. The action of nuclease is decidedly nonrandom, ultimately producing roughly equal amounts of acid-soluble oligonucleotides and 11S nucleoprotein particles. The 11S particles contain double-stranded DNA that is approximately 400 A or 120 base-pairs long, as measured by electron microscopic examination of deproteinized samples, and is maintained in a compact conformation within the intact particles. In addition, 15S nucleoprotein particles containing predominantly 800-A lengths of DNA have been isolated from less extensively digested chromatin. Evidence is presented which indicates that the 11S particles are fundamental structural units that are arranged in tandem along certain regions of chromatin fibrils. Preliminary experiments with different nucleases and with chromatin from different mammalian species indicate that these results are a natural consequence of the arrangement of DNA and proteins in mammalian chromatin and are not peculiar to the system described in detail. PMID:1054845

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

    PubMed

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

    2014-04-01

    The base excision repair (BER) pathway is a conserved DNA repair system required to maintain genomic integrity and prevent mutagenesis in all eukaryotic cells. Nevertheless, how BER operates in vivo (i.e. in the context of chromatin) is poorly understood. We have investigated the role of an essential ATP-dependent chromatin remodelling (ACR) complex RSC (Remodels the Structure of Chromatin) in BER of intact yeast cells. We show that depletion of STH1, the ATPase subunit of RSC, causes enhanced sensitivity to the DNA alkylating agent methyl methanesulfonate (MMS) and results in a substantial inhibition of BER, at the GAL1 locus and in the genome overall. Consistent with this observation, the DNA in chromatin is less accessible to micrococcal nuclease digestion in the absence of RSC. Quantitative PCR results indicate that repair deficiency in STH1 depleted cells is not due to changes in the expression of BER genes. Collectively, our data indicates the RSC complex promotes efficient BER in chromatin. These results provide, for the first time, a link between ATP-dependent chromatin remodelling and BER in living cells. PMID:24674626

  6. Inferential modeling of 3D chromatin structure.

    PubMed

    Wang, Siyu; Xu, Jinbo; Zeng, Jianyang

    2015-04-30

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

  7. 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

  8. 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 ?m 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

  9. Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain.

    PubMed Central

    Kalos, M; Fournier, R E

    1995-01-01

    The human apolipoprotein B (apoB) gene resides within a 47.5-kb chromatin domain that is flanked by sequences that bind to the nuclear matrix. These matrix attachment regions (MARs) are boundaries between nuclease-sensitive and -resistant chromatin. As domain boundaries are thought to function as insulator elements, shielding sequences between them from effects of neighboring chromatin, this raised the possibility that the apoB MARs have functions that could be assayed by transfection. To test this possibility, we examined effects of the apoB MARs on transgene expression in transiently and stably transfected rat and human hepatoma cells. The apoB MARs had no effects on expression of transiently transfected reporters, but they altered expression of stably integrated transgenes in dramatic and reproducible ways. Single integrated copies of transgenes that contained the apoB promoter and second intron enhancer, which are sufficient for high-level expression in transient assays, were expressed at low and variable levels in stable transfectant clones. In contrast, transgenes containing the apoB 5' and 3' MARs were expressed at levels nearly 200-fold higher than levels of the minimal reporters in stable transfectants, and expression was position independent. Transgenes that contained the apoB MARs and an additional 3.3 kb of apoB 5' flanking sequence were also expressed in an elevated, position-independent manner. Surprisingly, tandem transgene arrays in multicopy transfectants were transcriptionally inactive. These observations suggest that the apoB MARs function as insulator elements, shielding transgene expression from effects of neighboring chromatin domains. PMID:7799927

  10. 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

  11. Alleviation of histone H1-mediated transcriptional repression and chromatin compaction by the acidic activation region in chromosomal protein HMG-14.

    PubMed Central

    Ding, H F; Bustin, M; Hansen, U

    1997-01-01

    Histone H1 promotes the generation of a condensed, transcriptionally inactive, higher-order chromatin structure. Consequently, histone H1 activity must be antagonized in order to convert chromatin to a transcriptionally competent, more extended structure. Using simian virus 40 minichromosomes as a model system, we now demonstrate that the nonhistone chromosomal protein HMG-14, which is known to preferentially associate with active chromatin, completely alleviates histone H1-mediated inhibition of transcription by RNA polymerase II. HMG-14 also partially disrupts histone H1-dependent compaction of chromatin. Both the transcriptional enhancement and chromatin-unfolding activities of HMG-14 are mediated through its acidic, C-terminal region. Strikingly, transcriptional and structural activities of HMG-14 are maintained upon replacement of the C-terminal fragment by acidic regions from either GAL4 or HMG-2. These data support the model that the acidic C terminus of HMG-14 is involved in unfolding higher-order chromatin structure to facilitate transcriptional activation of mammalian genes. PMID:9315642

  12. A defined structure of the 30 nm chromatin fibre which accommodates different nucleosomal repeat lengths.

    PubMed Central

    Butler, P J

    1984-01-01

    Earlier work on the condensation of chromatins of different repeat lengths into the 30 nm fibre has been surveyed and it is shown that the external geometry of the fibre must be the same for all the chromatins. This can only be fitted by a helical coiling of nucleosomes into a solenoid with the linker DNA disposed internally. On this basis, various models were calculated and compared with published electric dichroism data. The only good fit is found with a 'reverse-loop' model, where the linker DNA forms a complete turn into the hole of the solenoid, of opposite hand to the nucleosomal DNA superhelix. This gives a topological linking number of one per nucleosome and would resolve the 'linking number paradox' if the DNA screw is the same in chromatin as in solution. The feasibility of a reverse-loop for short linkers (down to 15 base pairs) was investigated by model building and kinks of approximately 120 degrees into both DNA grooves are described, which will allow such packing. There will, however, be a 'forbidden' range for the linker DNA length, between approximately 1 and 14 bp, corresponding to nucleosomal repeats of 163 and 176 bp. PMID:6510410

  13. Prp22 and Spliceosome Components Regulate Chromatin Dynamics in Germ-Line Polyploid Cells

    PubMed Central

    Klusza, Stephen; Novak, Amanda; Figueroa, Shirelle; Palmer, William; Deng, Wu-Min

    2013-01-01

    During Drosophila oogenesis, the endopolyploid nuclei of germ-line nurse cells undergo a dramatic shift in morphology as oogenesis progresses; the easily-visible chromosomes are initially polytenic during the early stages of oogenesis before they transiently condense into a distinct ‘5-blob’ configuration, with subsequent dispersal into a diffuse state. Mutations in many genes, with diverse cellular functions, can affect the ability of nurse cells to fully decondense their chromatin, resulting in a ‘5-blob arrest’ phenotype that is maintained throughout the later stages of oogenesis. However, the mechanisms and significance of nurse-cell (NC) chromatin dispersal remain poorly understood. Here, we report that a screen for modifiers of the 5-blob phenotype in the germ line isolated the spliceosomal gene peanuts, the Drosophila Prp22. We demonstrate that reduction of spliceosomal activity through loss of peanuts promotes decondensation defects in NC nuclei during mid-oogenesis. We also show that the Prp38 spliceosomal protein accumulates in the nucleoplasm of nurse cells with impaired peanuts function, suggesting that spliceosomal recycling is impaired. Finally, we reveal that loss of additional spliceosomal proteins impairs the full decondensation of NC chromatin during later stages of oogenesis, suggesting that individual spliceosomal subcomplexes modulate expression of the distinct subset of genes that are required for correct morphology in endopolyploid nurse cells. PMID:24244416

  14. Nuclear condensation and free radical scavenging: a dual mechanism of bisbenzimidazoles to modulate radiation damage to DNA

    Microsoft Academic Search

    Urmila Tawar; Sandhya Bansal; Shiteshu Shrimal; Manish Singh; Vibha Tandon

    2007-01-01

    The complexing of histones with DNA and the resulting condensation of chromatin protects mammalian cell, from radiation-induced\\u000a strand breakage. In the present study, benzimidazoles DMA and TBZ showed marked radioprotection through drug-induced compaction\\u000a of chromatin and direct quenching of free radicals generated by radiation. The mammalian cells were incubated with 100 ?M\\u000a concentration of DMA and TBZ and irradiated at 5 Gy;

  15. Differences of supranucleosomal organization in different kinds of chromatin: cell type-specific globular subunits containing different numbers of nucleosomes

    PubMed Central

    1984-01-01

    Fractions of homogeneously-sized supranucleosomal particles can be obtained in high yield and purity from various types of cells by brief micrococcal nuclease digestion (10 or 20 s) of condensed chromatin in 100 mM NaCl followed by sucrose gradient centrifugation and agarose gel electrophoresis. These chromatin particles, which contain only DNA and histones, differed according to cell type. Sea urchin spermatozoa (Paracentrotus lividus) gave rise to heavy particles (ca. 260 S) with a mean diameter (48 nm). These resembled the unit chromatin fibrils fixed in situ, which contain an average of 48 nucleosomes, as determined both by electron microscopy after unraveling in low salt buffer and gel electrophoresis. In contrast, higher order particles from chicken erythrocyte chromatin were smaller (105 S; 36-nm diam) and contained approximately 20 nucleosomes. The smallest type of supranucleosomal particle was obtained from chicken and rat liver (39 S; 32-nm diam; eight nucleosomes). Oligomeric chains of such granular particles could be recognized in regions of higher sucrose density, indicating that distinct supranucleosomal particles of globular shape are not an artifact of exposure to low salt concentrations but can be obtained at near-physiological ionic strength. The demonstration of different particle sizes in chromatin from different types of nuclei is contrary to the view that such granular particles are produced by artificial breakdown into "detached turns" from a uniform and general solenoid structure of approximately six nucleosomes per turn. Our observations indicate that the higher order packing of the nucleosomal chain can differ greatly in different types of nuclei and the supranucleosomal organization of chromatin differs between cell types and is related to the specific state of cell differentiation. PMID:6736129

  16. Chromatin remodeling — a novel strategy to control excessive alcohol drinking

    PubMed Central

    Warnault, V; Darcq, E; Levine, A; Barak, S; Ron, D

    2013-01-01

    Harmful excessive use of alcohol has a severe impact on society and it remains one of the major causes of morbidity and mortality in the population. However, mechanisms that underlie excessive alcohol consumption are still poorly understood, and thus available medications for alcohol use disorders are limited. Here, we report that changing the level of chromatin condensation by affecting DNA methylation or histone acetylation limits excessive alcohol drinking and seeking behaviors in rodents. Specifically, we show that decreasing DNA methylation by inhibiting the activity of DNA methyltransferase (DNMT) with systemic administration of the FDA-approved drug, 5-azacitidine (5-AzaC) prevents excessive alcohol use in mice. Similarly, we find that increasing histone acetylation via systemic treatment with several histone deacetylase (HDAC) inhibitors reduces mice binge-like alcohol drinking. We further report that systemic administration of the FDA-approved HDAC inhibitor, SAHA, inhibits the motivation of rats to seek alcohol. Importantly, the actions of both DNMT and HDAC inhibitors are specific for alcohol, as no changes in saccharin or sucrose intake were observed. In line with these behavioral findings, we demonstrate that excessive alcohol drinking increases DNMT1 levels and reduces histone H4 acetylation in the nucleus accumbens (NAc) of rodents. Together, our findings illustrate that DNA methylation and histone acetylation control the level of excessive alcohol drinking and seeking behaviors in preclinical rodent models. Our study therefore highlights the possibility that DNMT and HDAC inhibitors can be used to treat harmful alcohol abuse. PMID:23423140

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

    PubMed Central

    1992-01-01

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

  18. Total globozoospermia associated with increased frequency of immature spermatozoa with chromatin defects and aneuploidy: a case report.

    PubMed

    Vozdova, M; Rybar, R; Kloudova, S; Prinosilova, P; Texl, P; Rubes, J

    2014-10-01

    Globozoospermia, characterised by the presence of round spermatozoa lacking acrosomes in an ejaculate, is a known cause of male infertility. Semen analysis, including sperm chromatin structure assay, toluidine blue, chromomycin A3 and aniline blue staining and fluorescence in situ hybridisation, was performed in an infertile globozoospermic patient to establish to which extent these genetic factors contributed to his infertility. No spermatozoa capable of hyaluronan (HA) binding were detected in the HA binding assay. Increased rates of immature spermatozoa with defective replacement of histones by protamines, DNA breaks and disturbed chromatin integrity and sperm aneuploid for the sex chromosomes were observed. Intracytoplasmic sperm injection (ICSI) was used in three in vitro fertilisation (IVF) cycles, and enough morphologically well-developing embryos were obtained in each cycle. However, no pregnancy was achieved. The infertility of our couple, resistant to IVF/ICSI treatment, was most probably caused by a combination of male and female factors. PMID:24007278

  19. Chromatin structure modification in an excimer laser field

    NASA Astrophysics Data System (ADS)

    Radu, Liliana; Mihailescu, I.; Gazdaru, Doina

    2002-04-01

    Chromatin is the complex of deoxyribonucleic acid (DNA) with proteins that exists in eukaryotic cell nuclei. Chromatin was extracted from livers of Wistar rats and subjected to a 248-nm excimer laser radiation, in doses of 0.5 3 MJ/m2. An UV excimer laser Iofan 1701, with 40-mJ dose/pulse and frequency of 30 Hz was used. The radiolysis of chromatin was analyzed by (1) 1H-NMR spectroscopy, (2) steady-state fluorescence, (3) time-resolved fluorescence, and (4) fluorescence resonance energy transfer (FRET) methods. The laser action on chromatin determines bigger values of the transverse relaxation time (T2), which indicates less bound water in the chromatin structure, therefore a more injured one. The chromatin intrinsic fluorescence decreases on laser action, proving the destruction of the chromatin protein structure. By the time-resolved fluorescence we established that the relative contribution of the excited state lifetime of bound ethidium bromide to chromatin DNA diminishes with the laser dose. This denotes single- and double-strand breaks produced in DNA structure. By the FRET method, the energy transfer efficiency and the distance between dansyl chloride and acridine orange coupled at chromatin were determined. The distance increases with laser action. The determination of the chromatin structure modification in an excimer laser field can be of real interest in medical applications.

  20. Measuring Chromatin Structure in Budding Yeast.

    PubMed

    Belton, Jon-Matthew; Dekker, Job

    2015-01-01

    Chromosome conformation capture (3C) has revolutionized the ways in which the conformation of chromatin and its relationship to other molecular functions can be studied. 3C-based techniques are used to determine the spatial arrangement of chromosomes in organisms ranging from bacteria to humans. In particular, they can be applied to the study of chromosome folding and organization in model organisms with small genomes and for which powerful genetic tools exist, such as budding yeast. Studies in yeast allow the mechanisms that establish or maintain chromatin structure to be analyzed at very high resolution with relatively low cost, and further our understanding of these fundamental processes in higher eukaryotes as well. Here we provide an overview of chromatin structure and introduce methods for performing 3C, with a focus on studies in budding yeast. Variations of the basic 3C approach (e.g., 3C-PCR, 5C, and Hi-C) can be used according to the scope and goals of a given experiment. PMID:26134912

  1. Modulation of chromatin organization by RH-3, a preparation of Hippophae rhamnoides, a possible role in radioprotection.

    PubMed

    Kumar, I Prem; Namita, Samanta; Goel, H C

    2002-09-01

    The present study was aimed to understand the mode of action of alcoholic extract of whole berries of Hippophae rhamnoides (RH-3) which has already been reported to render more than 80 % protection against radiation induced mortality in mice. Direct and indirect antioxidant action (free radical scavenging and metal chelating potential) were assayed using 2-deoxy ribose degradation and 2,2'-bipiridyl assays. Effect of RH-3 on radiation and chemical oxidant mediated DNA damage was evaluated using single cell gel electrophoresis (Comet assay) and alkaline halo assay. Ability of RH-3 to bind with calf thymus DNA was assayed through change in melting temperature (Tm) while toxicity was assayed in thymocytes by trypan blue exclusion. RH-3 inhibited 2-deoxy ribose degradation in a dose dependent manner (IC 50 approximately 500 microg/ml). 2,2'-bipiridyl assay revealed the inability of RH-3 to chelate Fe2+ ions. RH-3 inhibited radiation and tertiary butyl hydroperoxide induced DNA strand breaks in a dose dependent manner and at concentrations of 100 and 120 microg/ml the length of comet tail was considerably reduced and became almost similar to that of untreated control. RH-3 at a concentration of 120 pg/ml or more induced a strong compaction of chromatin as was evident from lack of tail and appearance of intensely stained circular bodies. This made the nuclei resistant even to a radiation dose of 1,000 Gy. The compaction of chromatin was not reversed even by relaxation buffer indicating that salt concentration had no role in RH-3 induced chromatin compaction. Alkaline halo assay also corroborated the results of comet assay. Lower DNA-RH-3 concentrations (1:0.5 and 1:1) induced a shift of Tm towards left by 2 and 5 degrees C respectively; however higher concentrations (1:8 and 1:16) shifted the Tm towards right increasing it by 10 and 21 degrees C correspondingly. RH-3, evinced only a mild free radical scavenging activity at concentrations used in the present study, therefore its ability to protect DNA could mainly be attributed to direct modulation of chromatin organization. Further work to unravel these facts would be necessary. PMID:12349895

  2. Histone Acetylation and Chromatin Remodeling Are Required for UV-B–Dependent Transcriptional Activation of Regulated Genes in Maize[W

    PubMed Central

    Casati, Paula; Campi, Mabel; Chu, Feixia; Suzuki, Nagi; Maltby, David; Guan, Shenheng; Burlingame, Alma L.; Walbot, Virginia

    2008-01-01

    The nuclear proteomes of maize (Zea mays) lines that differ in UV-B tolerance were compared by two-dimensional gel electrophoresis after UV light treatment. Differential accumulation of chromatin proteins, particularly histones, constituted the largest class identified by mass spectrometry. UV-B–tolerant landraces and the B73 inbred line show twice as many protein changes as the UV-B–sensitive b, pl W23 inbred line and transgenic maize expressing RNA interference constructs directed against chromatin factors. Mass spectrometic analysis of posttranslational modifications on histone proteins demonstrates that UV-B–tolerant lines exhibit greater acetylation on N-terminal tails of histones H3 and H4 after irradiation. These acetylated histones are enriched in the promoter and transcribed regions of the two UV-B–upregulated genes examined; radiation-sensitive lines lack this enrichment. DNase I and micrococcal nuclease hypersensitivity assays indicate that chromatin adopts looser structures around the selected genes in the UV-B–tolerant samples. Chromatin immunoprecipitation experiments identified additional chromatin factor changes associated with the nfc102 test gene after UV-B treatment in radiation-tolerant lines. Chromatin remodeling is thus shown to be a key process in acclimation to UV-B, and lines deficient in this process are more sensitive to UV-B. PMID:18398050

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

    PubMed Central

    2010-01-01

    Background Regulator of chromosome condensation 1 (RCC1) is the guanine nucleotide exchange factor for Ran GTPase. Localised generation of Ran-GTP by RCC1 on chromatin is critical for nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Both the N-terminal tail of RCC1 and its association with Ran are important for its interaction with chromatin in cells. In vitro, the association of Ran with RCC1 induces a conformational change in the N-terminal tail that promotes its interaction with DNA. Results We have investigated the mechanism of the dynamic interaction of the ? isoform of human RCC1 (RCC1?) with chromatin in live cells using fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) fusions. We show that the N-terminal tail stabilises the interaction of RCC1? with chromatin and this function can be partially replaced by another lysine-rich nuclear localisation signal. Removal of the tail prevents the interaction of RCC1? with chromatin from being stabilised by RanT24N, a mutant that binds stably to RCC1?. The interaction of RCC1? with chromatin is destabilised by mutation of lysine 4 (K4Q), which abolishes ?-N-terminal methylation, and this interaction is no longer stabilised by RanT24N. However, ?-N-terminal methylation of RCC1? is not regulated by the binding of RanT24N. Conversely, the association of Ran with precipitated RCC1? does not require the N-terminal tail of RCC1? or its methylation. The mobility of RCC1? on chromatin is increased by mutation of aspartate 182 (D182A), which inhibits guanine-nucleotide exchange activity, but RCC1?D182A can still bind nucleotide-free Ran and its interaction with chromatin is stabilised by RanT24N. Conclusions These results show that the stabilisation of the dynamic interaction of RCC1? with chromatin by Ran in live cells requires the N-terminal tail of RCC1?. ?-N-methylation is not regulated by formation of the binary complex with Ran, but it promotes chromatin binding through the tail. This work supports a model in which the association of RCC1? with chromatin is promoted by a conformational change in the ?-N-terminal methylated tail that is induced allosterically in the binary complex with Ran. PMID:20565941

  4. Chromatin response to DNA double-strand break damage.

    PubMed

    Bao, Yunhe

    2011-06-01

    Manipulation of chromatin, in which genomic DNA is packaged, is a fundamental requirement for all DNA-based metabolic processes in eukayotic cells. Histone variant incorporation, histone post-translational modifications, and ATP-dependent chromatin remodeling are three major strategies for chromatin manipulation, and are relatively well characterized in transcriptional regulation. Emerging lines of evidence indicate that histone variants (H2AX and H2A.Z), histone post-translational modifications (acetylation, phosphorylation, methylation and ubiquitination) and chromatin-remodeling complexes (INO80, SWR1, SWI/SNF, RSC and NuRD) are important and direct players in the DNA double-strand break (DSB) response as well. New studies also reveal that incorporation of histone variants into nucleosomes, histone modifications and ATP-dependent chromatin remodeling are specifically and intimately connected during the DSB damage response. This article summarizes the recent advances in our understanding of the relationship between chromatin modifications and the DSB damage response. PMID:22122340

  5. 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. (Institut fuer Strahlenschutz, Neuherberg (Germany, F.R.))

    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.

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

    SciTech Connect

    Kuo, Wei-Hung W. [Department of Dermatology and Skin Science, Jack Bell Research Centre, Vancouver Coastal Health Research Institute, University of British Columbia, 2660 Oak Street, Vancouver, BC V6H 3Z6 (Canada); Wang Yemin [Department of Dermatology and Skin Science, Jack Bell Research Centre, Vancouver Coastal Health Research Institute, University of British Columbia, 2660 Oak Street, Vancouver, BC V6H 3Z6 (Canada); Wong, Ronald P.C. [Department of Dermatology and Skin Science, Jack Bell Research Centre, Vancouver Coastal Health Research Institute, University of British Columbia, 2660 Oak Street, Vancouver, BC V6H 3Z6 (Canada); Campos, Eric I. [Department of Dermatology and Skin Science, Jack Bell Research Centre, Vancouver Coastal Health Research Institute, University of British Columbia, 2660 Oak Street, Vancouver, BC V6H 3Z6 (Canada); Li Gang [Department of Dermatology and Skin Science, Jack Bell Research Centre, Vancouver Coastal Health Research Institute, University of British Columbia, 2660 Oak Street, Vancouver, BC V6H 3Z6 (Canada)]. 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.

  7. Developmental patterns of chromatin structure and DNA methylation responsible for epigenetic expression of a maize regulatory gene.

    PubMed Central

    Hoekenga, O A; Muszynski, M G; Cone, K C

    2000-01-01

    Epigenetic regulatory mechanisms heritably alter patterns of gene expression without changes in DNA sequence. Epigenetic states are often correlated with developmentally imposed alterations in genomic DNA methylation and local chromatin structure. Pl-Blotched is a stable epigenetic allele of the maize anthocyanin regulatory gene, purple plant1(pl). Pl-Blotched plants display a variegated pattern of pigmentation that contrasts sharply with the uniformly dark purple pigmentation of plants carrying the dominant Pl-Rhoades allele. Previously, we showed that the lower level of pigmentation in Pl-Blotched is correlated with lower pl mRNA levels and increased DNA methylation at some sites. To explore how DNA methylation, chromatin structure, and developmental stage might contribute to the expression of Pl-Blotched, we used methylation-sensitive restriction enzymes and DNaseI sensitivity assays to compare the methylation status and chromatin structure of Pl-Blotched and Pl-Rhoades at different stages in development. Both alleles exhibit developmentally sensitive changes in methylation. In Pl-Blotched, methylation of two diagnostic HpaII/MspI sites increases progressively, coincident with the juvenile-to-adult transition in growth. In seedlings, the chromatin encompassing the coding region of the gene is less sensitive to DNaseI digestion in Pl-Blotched than in Pl-Rhoades. Developmental maturation from seedling to adult is accompanied by expansion of this closed chromatin domain to include the promoter and downstream flanking sequences. We provide evidence to show that chromatin structure, rather than DNA methylation, is the primary epigenetic determinant for the phenotypic differences between Pl-Blotched and Pl-Rhoades. PMID:10924483

  8. Physical properties of chemically acetylated rat liver chromatin.

    PubMed Central

    Wallace, R B; Sargent, T D; Murphy, R F; Bonner, J

    1977-01-01

    The physical properties of rat liver chromatin and nucleosomes acetylated with acetic anhydride were examined in order to clarify the mechanism by which chemical acetylation of histones increases template activity in vitro [Marushige, K. (1976) Proc. Natl. Acad. Sci. USA 73, 3937-3941]. Acetylation was found to have dramatic effects on the magnesium solubility, nuclease sensitivity, thermal denaturation, and sedimentation of chromatin and nucleosomes. The significance of the results to models of gene activation and chromatin replication is considered. Images PMID:269387

  9. Chromatin structure of Xenopus rDNA transcription termination sites

    Microsoft Academic Search

    Michael F. Trendelenburg

    1982-01-01

    The ultrastructure of Xenopus laevis (X.l.) 40S pre-rRNA transcription termination sites was investigated by electron microscopy. Amplified nucleolar chromatin was rapidly dispersed and processed for chromatin spread preparations. This type of preparation revealed that many rDNA termination sites appeared as 50 nm long segments of chromatin axis covered by a complex of three closely spaced RNA polymerase particles. Particle (Pt1)

  10. Depletion of the Chromatin Looping Proteins CTCF and Cohesin Causes Chromatin Compaction: Insight into Chromatin Folding by Polymer Modelling

    PubMed Central

    Tark-Dame, Mariliis; Jerabek, Hansjoerg; Manders, Erik M. M.; Heermann, Dieter W.; van Driel, Roel

    2014-01-01

    Folding of the chromosomal fibre in interphase nuclei is an important element in the regulation of gene expression. For instance, physical contacts between promoters and enhancers are a key element in cell-type–specific transcription. We know remarkably little about the principles that control chromosome folding. Here we explore the view that intrachromosomal interactions, forming a complex pattern of loops, are a key element in chromosome folding. CTCF and cohesin are two abundant looping proteins of interphase chromosomes of higher eukaryotes. To investigate the role of looping in large-scale (supra Mb) folding of human chromosomes, we knocked down the gene that codes for CTCF and the one coding for Rad21, an essential subunit of cohesin. We measured the effect on chromosome folding using systematic 3D fluorescent in situ hybridization (FISH). Results show that chromatin becomes more compact after reducing the concentration of these two looping proteins. The molecular basis for this counter-intuitive behaviour is explored by polymer modelling usingy the Dynamic Loop model (Bohn M, Heermann DW (2010) Diffusion-driven looping provides a consistent framework for chromatin organization. PLoS ONE 5: e12218.). We show that compaction can be explained by selectively decreasing the number of short-range loops, leaving long-range looping unchanged. In support of this model prediction it has recently been shown by others that CTCF and cohesin indeed are responsible primarily for short-range looping. Our results suggest that the local and the overall changes in of chromosome structure are controlled by a delicate balance between short-range and long-range loops, allowing easy switching between, for instance, open and more compact chromatin states. PMID:25299688

  11. 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

  12. 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

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

    PubMed

    Fukagawa, Tatsuo; Earnshaw, William C

    2014-09-01

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

  14. Regulation of Chromatin Structure in the Cardiovascular System

    PubMed Central

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

    2013-01-01

    It has been appreciated for some time that cardiovascular disease involves large-scale transcriptional changes in various cell types. What has become increasingly clear only in the last few years, however, is the role of chromatin remodeling in cardiovascular phenotypes in normal physiology as well as in development and disease. This review summarizes the state of the chromatin field in terms of distinct mechanisms to regulate chromatin structure in vivo, identifying when these modes of regulation have been demonstrated in cardiovascular tissues. We describe areas in which a better understanding of chromatin structure is leading to new insights into the fundamental biology of cardiovascular disease. PMID:23575346

  15. Multiple Modes of Interaction between the Methylated DNA Binding Protein MeCP2 and Chromatin?

    PubMed Central

    Nikitina, Tatiana; Shi, Xi; Ghosh, Rajarshi P.; Horowitz-Scherer, Rachel A.; Hansen, Jeffrey C.; Woodcock, Christopher L.

    2007-01-01

    Mutations of the methylated DNA binding protein MeCP2, a multifunctional protein that is thought to transmit epigenetic information encoded as methylated CpG dinucleotides to the transcriptional machinery, give rise to the debilitating neurodevelopmental disease Rett syndrome (RTT). In this in vitro study, the methylation-dependent and -independent interactions of wild-type and mutant human MeCP2 with defined DNA and chromatin substrates were investigated. A combination of electrophoretic mobility shift assays and visualization by electron microscopy made it possible to understand the different conformational changes underlying the gel shifts. MeCP2 is shown to have, in addition to its well-established methylated DNA binding domain, a methylation-independent DNA binding site (or sites) in the first 294 residues, while the C-terminal portion of MeCP2 (residues 295 to 486) contains one or more essential chromatin interaction regions. All of the RTT-inducing mutants tested were quantitatively bound to chromatin under our conditions, but those that tend to be associated with the more severe RTT symptoms failed to induce the extensive compaction observed with wild-type MeCP2. Two modes of MeCP2-driven compaction were observed, one promoting nucleosome clustering and the other forming DNA-MeCP2-DNA complexes. MeCP2 binding to DNA and chromatin involves a number of different molecular interactions, some of which result in compaction and oligomerization. The multifunctional roles of MeCP2 may be reflected in these different interactions. PMID:17101771

  16. 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

  17. Facilitated diffusion of proteins on chromatin

    E-print Network

    O. Benichou; C. Chevalier; B. Meyer; R. Voituriez

    2011-01-26

    We present a theoretical model of facilitated diffusion of proteins in the cell nucleus. This model, which takes into account the successive binding/unbinding events of proteins to DNA, relies on a fractal description of the chromatin which has been recently evidenced experimentally. Facilitated diffusion is shown quantitatively to be favorable for a fast localization of a target locus by a transcription factor, and even to enable the minimization of the search time by tuning the affinity of the transcription factor with DNA. This study shows the robustness of the facilitated diffusion mechanism, invoked so far only for linear conformations of DNA.

  18. 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.

    PubMed

    Khalifa, Tarek; Lymberopoulos, Aristotelis

    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. PMID:23288451

  19. 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

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

    Microsoft Academic Search

    N. G. Kornet

    2008-01-01

    Stem cells replenish the cells present in an organism throughout its lifetime and sustain growth. They have unique characteristics: the capability to self-renew and the potential to differentiate into several cell types. Recently, it has become clear that chromatin factors support these unique features in mammalian stem cells (Chapter 1). The role of chromatin factors in plant stem cell control

  1. Nucleosome conformational flexibility and implications for chromatin dynamics

    Microsoft Academic Search

    Taras Shevchenko; M. Curie

    2004-01-01

    The active role of chromatin in the regulation of gene activity seems to imply a conformational flexibility of the basic chromatin structural unit, the nucleosome. This review is devoted to our recent results pertaining to this subject, using an original approach based on the topology of single particles reconstituted on DNA minicircles, combined with their theoretical simulation. Three types of

  2. Genome organisation and chromatin structure in Escherichia coli

    Microsoft Academic Search

    David Ussery; Thomas Schou Larsen; K. Trevor Wilkes; Carsten Friis; Peder Worning; Anders Krogh; Søren Brunak

    2001-01-01

    We have analysed the complete sequence of the Escherichia coli K12 isolate MG1655 genome for chromatin-associated protein binding sites, and compared the predicted location of predicted sites with experimental expression data from ‘DNA chip’ experiments. Of the dozen proteins associated with chromatin in E. coli, only three have been shown to have significant binding preferences: integration host factor (IHF) has

  3. Proteomic Interrogation of Human Chromatin Mariana P. Torrente1

    E-print Network

    Shorter, James

    Proteomic Interrogation of Human Chromatin Mariana P. Torrente1 , Barry M. Zee2 , Nicolas L. Young2 and other chromatin-related processes. Citation: Torrente MP, Zee BM, Young NL, Baliban RC, LeRoy G, et al; Accepted August 16, 2011; Published September 14, 2011 Copyright: ß 2011 Torrente et al. This is an open

  4. Dynamic reprogramming of chromatin accessibility during Drosophila embryo development

    PubMed Central

    2011-01-01

    Background The development of complex organisms is believed to involve progressive restrictions in cellular fate. Understanding the scope and features of chromatin dynamics during embryogenesis, and identifying regulatory elements important for directing developmental processes remain key goals of developmental biology. Results We used in vivo DNaseI sensitivity to map the locations of regulatory elements, and explore the changing chromatin landscape during the first 11 hours of Drosophila embryonic development. We identified thousands of conserved, developmentally dynamic, distal DNaseI hypersensitive sites associated with spatial and temporal expression patterning of linked genes and with large regions of chromatin plasticity. We observed a nearly uniform balance between developmentally up- and down-regulated DNaseI hypersensitive sites. Analysis of promoter chromatin architecture revealed a novel role for classical core promoter sequence elements in directing temporally regulated chromatin remodeling. Another unexpected feature of the chromatin landscape was the presence of localized accessibility over many protein-coding regions, subsets of which were developmentally regulated or associated with the transcription of genes with prominent maternal RNA contributions in the blastoderm. Conclusions Our results provide a global view of the rich and dynamic chromatin landscape of early animal development, as well as novel insights into the organization of developmentally regulated chromatin features. PMID:21569360

  5. Tau promotes neurodegeneration through global chromatin relaxation

    PubMed Central

    Frost, Bess; Hemberg, Martin; Lewis, Jada; Feany, Mel B.

    2014-01-01

    The microtubule-associated protein tau is involved in a number of neurodegenerative disorders, including Alzheimer’s disease (AD). Previous studies link oxidative stress and subsequent DNA damage to neuronal death in AD and related tauopathies. Since DNA damage can significantly alter chromatin structure, we examined epigenetic changes in tau-induced neurodegeneration. We have found widespread loss of heterochromatin in tau transgenic Drosophila and mice, and in human AD. Importantly, genetic rescue of tau-induced heterochromatin loss substantially reduced neurodegeneration in Drosophila. We identified oxidative stress and subsequent DNA damage as a mechanistic link between transgenic tau expression and heterochromatin relaxation, and found that heterochromatin loss permits aberrant gene expression in tauopathies. Furthermore, large-scale analyses from human AD brains revealed a widespread transcriptional increase in genes that are heterochromatically silenced in controls. Our results establish heterochromatin loss as a toxic effector of tau-induced neurodegeneration, and identify chromatin structure as a potential therapeutic target in AD. PMID:24464041

  6. Mapping chromatin interactions with 5C technology

    PubMed Central

    Ferraiuolo, Maria A.; Sanyal, Amartya; Naumova, Natalia; Dekker, Job; Dostie, Josée

    2013-01-01

    In eukaryotes, genome organization can be observed on many levels and at different scales. This organization is important not only to reduce chromosome length but also for the proper execution of various biological processes. High-resolution mapping of spatial chromatin structure was made possible by the development of the chromosome conformation capture (3C) technique. 3C uses chemical cross-linking followed by proximity-based ligation of fragmented DNA to capture frequently interacting chromatin segments in cell populations. Several 3C-related methods capable of higher chromosome conformation mapping throughput were reported afterwards. These techniques include the 3C-carbon copy (5C) approach, which offers the advantage of being highly quantitative and reproducible. We provide here a reference protocol for the production of 5C libraries analyzed by next-generation sequencing or onto microarrays. A procedure used to verify that 3C library templates bear the high quality required to produce superior 5C libraries is also described. We believe that this comprehensive detailed protocol will help guide researchers in probing spatial genome organization and its role in various biological processes. PMID:23137922

  7. Chromatin Dynamics during Nucleotide Excision Repair: Histones on the Move.

    PubMed

    Adam, Salomé; Polo, Sophie E

    2012-01-01

    It has been a long-standing question how DNA damage repair proceeds in a nuclear environment where DNA is packaged into chromatin. Several decades of analysis combining in vitro and in vivo studies in various model organisms ranging from yeast to human have markedly increased our understanding of the mechanisms underlying chromatin disorganization upon damage detection and re-assembly after repair. Here, we review the methods that have been developed over the years to delineate chromatin alterations in response to DNA damage by focusing on the well-characterized Nucleotide Excision Repair (NER) pathway. We also highlight how these methods have provided key mechanistic insight into histone dynamics coupled to repair in mammals, raising new issues about the maintenance of chromatin integrity. In particular, we discuss how NER factors and central players in chromatin dynamics such as histone modifiers, nucleosome remodeling factors, and histone chaperones function to mobilize histones during repair. PMID:23109890

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

    PubMed Central

    Majumder, Parijat; Dasgupta, Dipak

    2011-01-01

    Background Distamycin A is a prototype minor groove binder, which binds to B-form DNA, preferentially at A/T rich sites. Extensive work in the past few decades has characterized the binding at the level of double stranded DNA. However, effect of the same on physiological DNA, i.e. DNA complexed in chromatin, has not been well studied. Here we elucidate from a structural perspective, the interaction of distamycin with soluble chromatin, isolated from Sprague-Dawley rat. Methodology/Principal Findings Chromatin is a hierarchical assemblage of DNA and protein. Therefore, in order to characterize the interaction of the same with distamycin, we have classified the system into various levels, according to the requirements of the method adopted, and the information to be obtained. Isothermal titration calorimetry has been employed to characterize the binding at the levels of chromatin, chromatosome and chromosomal DNA. Thermodynamic parameters obtained thereof, identify enthalpy as the driving force for the association, with comparable binding affinity and free energy for chromatin and chromosomal DNA. Reaction enthalpies at different temperatures were utilized to evaluate the change in specific heat capacity (?Cp), which, in turn, indicated a possible binding associated structural change. Ligand induced structural alterations have been monitored by two complementary methods - dynamic light scattering, and transmission electron microscopy. They indicate compaction of chromatin. Using transmission electron microscopy, we have visualized the effect of distamycin upon chromatin architecture at di- and trinucleosome levels. Our results elucidate the simultaneous involvement of linker bending and internucleosomal angle contraction in compaction process induced by distamycin. Conclusions/Significance We summarize here, for the first time, the thermodynamic parameters for the interaction of distamycin with soluble chromatin, and elucidate its effect on chromatin architecture. The study provides insight into a ligand induced compaction phenomenon, and suggests new mechanisms of chromatin architectural alteration. PMID:22046291

  9. Enhanced Condensation Heat Transfer

    NASA Astrophysics Data System (ADS)

    Rose, John Winston

    The paper gives some personal observations on various aspects of enhanced condensation heat transfer. The topics discussed are external condensation (horizontal low-finned tubes and wire-wrapped tubes), internal condensation (microfin tubes and microchannels) and Marangoni condensation of binary mixtures.

  10. Quantitative Analysis of Chromosome Condensation in Fission Yeast

    PubMed Central

    Petrova, Boryana; Dehler, Sascha; Kruitwagen, Tom; Hériché, Jean-Karim; Miura, Kota

    2013-01-01

    Chromosomes undergo extensive conformational rearrangements in preparation for their segregation during cell divisions. Insights into the molecular mechanisms behind this still poorly understood condensation process require the development of new approaches to quantitatively assess chromosome formation in vivo. In this study, we present a live-cell microscopy-based chromosome condensation assay in the fission yeast Schizosaccharomyces pombe. By automatically tracking the three-dimensional distance changes between fluorescently marked chromosome loci at high temporal and spatial resolution, we analyze chromosome condensation during mitosis and meiosis and deduct defined parameters to describe condensation dynamics. We demonstrate that this method can determine the contributions of condensin, topoisomerase II, and Aurora kinase to mitotic chromosome condensation. We furthermore show that the assay can identify proteins required for mitotic chromosome formation de novo by isolating mutants in condensin, DNA polymerase ?, and F-box DNA helicase I that are specifically defective in pro-/metaphase condensation. Thus, the chromosome condensation assay provides a direct and sensitive system for the discovery and characterization of components of the chromosome condensation machinery in a genetically tractable eukaryote. PMID:23263988

  11. In vivo dissection of the chromosome condensation machinery

    PubMed Central

    Lavoie, Brigitte D.; Hogan, Eileen; Koshland, Douglas

    2002-01-01

    The machinery mediating chromosome condensation is poorly understood. To begin to dissect the in vivo function(s) of individual components, we monitored mitotic chromosome structure in mutants of condensin, cohesin, histone H3, and topoisomerase II (topo II). In budding yeast, both condensation establishment and maintenance require all of the condensin subunits, but not topo II activity or phospho-histone H3. Structural maintenance of chromosome (SMC) protein 2, as well as each of the three non-SMC proteins (Ycg1p, Ycs4p, and Brn1p), was required for chromatin binding of the condensin complex in vivo. Using reversible condensin alleles, we show that chromosome condensation does not involve an irreversible modification of condensin or chromosomes. Finally, we provide the first evidence of a mechanistic link between condensin and cohesin function. A model discussing the functional interplay between cohesin and condensin is presented. PMID:11864994

  12. SATB1-mediated functional packaging of chromatin into loops.

    PubMed

    Kohwi-Shigematsu, Terumi; Kohwi, Yoshinori; Takahashi, Keiko; Richards, Hunter W; Ayers, Stephen D; Han, Hye-Jung; Cai, Shutao

    2012-11-01

    Mammalian genomes are organized into multiple layers of higher-order chromatin structure, and in this organization chromatin looping is a striking and crucial feature that brings together distal genomic loci into close spatial proximity. Such three-dimensional organization of chromatin has been suggested to be functionally important in gene regulation. Many important questions need to be addressed, such as what types of nuclear proteins are responsible for folding chromatin into loops, whether there are any genomic marks that serve as the core sites of chromatin folding events, how distal genomic sites are brought together, and what are the biological consequences for interactions between distal genomic loci. In order to address these fundamental questions, it is essential to devise and employ methods that can capture higher-order structures formed by specific nuclear proteins at high resolution. In this article, in order to describe methods of analyzing protein-mediated chromatin interactions, we will use as an example a global genome-organizer protein, SATB1, which mediates chromatin looping. PMID:22782115

  13. Innovation in gene regulation: the case of chromatin computation.

    PubMed

    Prohaska, Sonja J; Stadler, Peter F; Krakauer, David C

    2010-07-01

    Chromatin regulation is understood to be one of the fundamental modes of gene regulation in eukaryotic cells. We argue that the basic proteins that determine the chromatin architecture constitute an evolutionary ancient layer of transcriptional regulation common to all three domains of life. We explore phylogenetically, sources of innovation in chromatin regulation, focusing on protein domains related to chromatin structure and function, demonstrating a step-wise increase of complexity in chromatin regulation. Building upon the highly conserved use of variants of chromosomal architectural proteins to distinguish chromosomal states, Eukarya secondarily acquired mechanisms for "writing" chemical modifications onto chromatin that constitute persistent signals. The acquisition of reader domains enabled decoding of these complex, signal combinations and a decoupling of the signal from immediate biochemical effects. We show how the coupling of reading and writing, which is most prevalent in crown-group Eukarya, could have converted chromatin into a powerful computational device capable of storing and processing more information than pure cis-regulatory networks. PMID:20303358

  14. PRC2-independent chromatin compaction and transcriptional repression in cancer.

    PubMed

    Vallot, C; Hérault, A; Boyle, S; Bickmore, W A; Radvanyi, F

    2015-02-01

    The silencing of large chromosomal regions by epigenetic mechanisms has been reported to occur frequently in cancer. Epigenetic marks, such as histone methylation and acetylation, are altered at these loci. However, the mechanisms of formation of such aberrant gene clusters remain largely unknown. Here, we show that, in cancer cells, the epigenetic remodeling of chromatin into hypoacetylated domains covered with histone H3K27 trimethylation is paralleled by changes in higher-order chromatin structures. Using fluorescence in situ hybridization, we demonstrate that regional epigenetic silencing corresponds to the establishment of compact chromatin domains. We show that gene repression is tightly correlated to the state of chromatin compaction and not to the levels of H3K27me3-its removal through the knockdown of EZH2 does not induce significant gene expression nor chromatin decompaction. Moreover, transcription can occur with intact high-H3K27me3 levels; treatment with histone deacetylase inhibitors can relieve chromatin compaction and gene repression, without altering H3K27me3 levels. Our findings imply that compaction and subsequent repression of large chromatin domains are not direct consequences of PRC2 deregulation in cancer cells. By challenging the role of EZH2 in aberrant gene silencing in cancer, these findings have therapeutical implications, notably for the choice of epigenetic drugs for tumors with multiple regional epigenetic alterations. PMID:24469045

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

    PubMed Central

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

    2015-01-01

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

  16. PARP1 enhances inflammatory cytokine expression by alteration of promoter chromatin structure in microglia

    PubMed Central

    Martínez-Zamudio, Ricardo Iván; Ha, Hyo Chol

    2014-01-01

    Background Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated enzyme that participates in processes such as transcription and DNA repair through the regulation of chromatin structure. Accumulating evidence suggests an important role for PARP1 enzymatic activity in promoting CNS inflammation by facilitating the expression of inflammatory cytokines in glial cells. However, the molecular mechanisms by which PARP1 enzymatic activity mediates this process are not well understood. In this report we sought to determine the molecular mechanisms by which PARP1 enzymatic activity facilitates the expression of Il1? and TNF in LPS-stimulated BV2 cells. Methods PARP1 enzymatic activity and histone ADP-ribosylation were measured in LPS-stimulated BV2 cells by radioactive labelling with 32P-NAD+. To assess the effect of histone ADP-ribosylation on nucleosome structure, in vitro nucleosome remodeling, nuclease accessibility and binding assays were performed. These studies were complemented by chromatin immunoprecipitation assays in resting and LPS-stimulated BV2 cells in order to determine the occupancy of PARP1, nucleosomes and the RelA subunit of NF-?B, as well as ADP-ribosylation, at the Il1? and Tnf promoters. Finally, we determined the effect of pharmacological inhibition of PARP1 enzymatic activity on the LPS stimulation-dependent induction of Il1? and Tnf mRNA. Results Our results indicate that LPS stimulation induces PARP1 enzymatic activity and histone ADP-ribosylation in the chromatin compartment of BV2 cells. In vitro studies show that nucleosome-bound PARP1 disrupts nucleosome structure histone ADP-ribosylation, increasing the accessibility of nucleosomal DNA. Consistent with this PARP1 is constitutively associated with at the Il1? and Tnf promoters in resting BV2 cells. Upon stimulation with LPS, ADP-ribosylation is observed at these promoters, and this is correlated with increased recruitment of the transcription factor NF-?B, resulting in robust transcription of these inflammatory cytokines. Accordingly, pharmacological inhibition of PARP1 enzymatic activity reduces NF-?B recruitment, and Il1? and Tnf expression in LPS-stimulated microglia. Conclusions Collectively, our data suggest that PARP1 facilitates inflammatory cytokine expression in microglia by increasing the accessibility of promoter DNA via histone ADP-riboyslation. PMID:25161822

  17. 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

  18. In vivo dissection of the chromosome condensation machinery: reversibility of condensation distinguishes contributions of condensin and cohesin.

    PubMed

    Lavoie, Brigitte D; Hogan, Eileen; Koshland, Douglas

    2002-03-01

    The machinery mediating chromosome condensation is poorly understood. To begin to dissect the in vivo function(s) of individual components, we monitored mitotic chromosome structure in mutants of condensin, cohesin, histone H3, and topoisomerase II (topo II). In budding yeast, both condensation establishment and maintenance require all of the condensin subunits, but not topo II activity or phospho-histone H3. Structural maintenance of chromosome (SMC) protein 2, as well as each of the three non-SMC proteins (Ycg1p, Ycs4p, and Brn1p), was required for chromatin binding of the condensin complex in vivo. Using reversible condensin alleles, we show that chromosome condensation does not involve an irreversible modification of condensin or chromosomes. Finally, we provide the first evidence of a mechanistic link between condensin and cohesin function. A model discussing the functional interplay between cohesin and condensin is presented. PMID:11864994

  19. 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

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

    PubMed

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

    2015-02-18

    The dynamic organization of chromatin plays an essential role in the regulation of gene expression and in other fundamental cellular processes. The underlying physical basis of these activities lies in the sequential positioning, chemical composition, and intermolecular interactions of the nucleosomes-the familiar assemblies of ?150 DNA base pairs and eight histone proteins-found on chromatin fibers. Here we introduce a mesoscale model of short nucleosomal arrays and a computational framework that make it possible to incorporate detailed structural features of DNA and histones in simulations of short chromatin constructs. We explore the effects of nucleosome positioning and the presence or absence of cationic N-terminal histone tails on the 'local' inter-nucleosomal interactions and the global deformations of the simulated chains. The correspondence between the predicted and observed effects of nucleosome composition and numbers on the long-range communication between the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We also extract effective nucleosome-nucleosome potentials from the simulations and implement the potentials in a larger-scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable effect of nucleosome spacing on chromatin flexibility, with small changes in DNA linker length significantly altering the interactions of nucleosomes and the dimensions of the fiber as a whole. In addition, we find that these changes in nucleosome positioning influence the statistical properties of long chromatin constructs. That is, simulated chromatin fibers with the same number of nucleosomes exhibit polymeric behaviors ranging from Gaussian to worm-like, depending upon nucleosome spacing. These findings suggest that the physical and mechanical properties of chromatin can span a wide range of behaviors, depending on nucleosome positioning, and that care must be taken in the choice of models used to interpret the experimental properties of long chromatin fibers. PMID:25564155

  1. Centromeric heterochromatin assembly in fission yeast--balancing transcription, RNA interference and chromatin modification.

    PubMed

    Alper, Benjamin J; Lowe, Brandon R; Partridge, Janet F

    2012-07-01

    Distinct regions of the eukaryotic genome are packaged into different types of chromatin, with euchromatin representing gene rich, transcriptionally active regions and heterochromatin more condensed and gene poor. The assembly and maintenance of heterochromatin is important for many aspects of genome control, including silencing of gene transcription, suppression of recombination, and to ensure proper chromosome segregation. The precise mechanisms underlying heterochromatin establishment and maintenance are still unclear, but much progress has been made towards understanding this process during the last few years, particularly from studies performed in fission yeast. In this review, we hope to provide a conceptual model of centromeric heterochromatin in fission yeast that integrates our current understanding of the competing forces of transcription, replication, and RNA decay that influence its assembly and propagation. PMID:22733402

  2. Detection of DNA primary damage by premature chromosome condensation in human peripheral blood lymphocytes treated with methyl methanesulfonate

    Microsoft Academic Search

    C. Lorenti Garcia; M. Carloni; N. Palma de la Pena

    2001-01-01

    chromatin conformation changes in the conversion of DNA atmosphere at 80% humidity. Phytohaemagglutinin (PHA) (Murex) was added lesions into chromosome aberrations. The efficiency of chro- to complete medium at a concentration of 2% to stimulate cell growth. matin condensation and decondensation soon after irradiation Drug treatments greatly affect transformation of DNA lesions into chromosome MMS (Sigma Chemical Co.) was dissolved

  3. Chromatin is a Complex of whose Structure is Only Visible During Mitosis in Eukaryotic Cells

    E-print Network

    Cutler, Chris

    Chromatin Chromatin is a Complex of whose Structure is Only Visible During Mitosis in Eukaryotic Cells Chromatin is Comprised of Tightly which are Uncoiled at the End of Mitosis and Disappear During Chromatin During Mitosis is the Result of Contraction of in Length of the Chromosomal DNA The Organization

  4. p53 and the PWWP domain containing effector proteins in chromatin damage repair

    PubMed Central

    Hu, Jing; Wang, Yanming

    2013-01-01

    In eukaryotic cells, DNA damage repair occurs on a template DNA that is organized with histones to form nucleosomes and chromatin structures. As such, chromatin plays an important role in DNA damage repair. In this review, we will use “chromatin damage repair” as a framework and highlight recent progress in understanding the role of chromatin, chromatin modifiers, chromatin binding effectors (e.g., the PWWP domain proteins), and the p53 tumor suppressor. We view chromatin as an active participant during DNA damage repair. PMID:25264544

  5. Atomic force microscope imaging of chromatin assembled in Xenopus laevis egg extract.

    PubMed

    Fu, Hongxia; Freedman, Benjamin S; Lim, Chwee Teck; Heald, Rebecca; Yan, Jie

    2011-06-01

    Gaps persist in our understanding of chromatin lower- and higher-order structures. Xenopus egg extracts provide a way to study essential chromatin components which are difficult to manipulate in living cells, but nanoscale imaging of chromatin assembled in extracts poses a challenge. We describe a method for preparing chromatin assembled in extracts for atomic force microscopy (AFM) utilizing restriction enzyme digestion followed by transferring to a mica surface. Using this method, we find that buffer dilution of the chromatin assembly extract or incubation of chromatin in solutions of low ionic strength results in loosely compacted chromatin fibers that are prone to unraveling into naked DNA. We also describe a method for direct AFM imaging of chromatin which does not utilize restriction enzymes and reveals higher-order fibers of varying widths. Due to the capability of controlling chromatin assembly conditions, we believe these methods have broad potential for studying physiologically relevant chromatin structures. PMID:21369955

  6. Conservation and divergence in higher order chromatin structure 

    E-print Network

    Chambers, Emily Victoria

    2013-11-29

    Aspects of higher order chromatin structure such as replication timing, lamina association and Hi-C inter-locus interactions have been recently studied in several human and mouse cell types and it has been suggested that ...

  7. Shaping the landscape: mechanistic consequences of ubiquitin modification of chromatin.

    PubMed

    Braun, Sigurd; Madhani, Hiten D

    2012-07-01

    The organization of eukaryotic chromosomes into transcriptionally active euchromatin and repressed heterochromatin requires mechanisms that establish, maintain and distinguish these canonical chromatin domains. Post-translational modifications are fundamental in these processes. Monoubiquitylation of histones was discovered more than three decades ago, but its precise function has been enigmatic until recently. It is now appreciated that the spectrum of chromatin ubiquitylation is not restricted to monoubiquitylation of histones, but includes degradatory ubiquitylation of histones, histone-modifying enzymes and non-histone chromatin factors. These occur in a spatially and temporally controlled manner. In this review, we summarize our understanding of these mechanisms with a particular emphasis on how ubiquitylation shapes the physical landscape of chromatin. PMID:22688965

  8. Shaping the landscape: mechanistic consequences of ubiquitin modification of chromatin

    PubMed Central

    Braun, Sigurd; Madhani, Hiten D

    2012-01-01

    The organization of eukaryotic chromosomes into transcriptionally active euchromatin and repressed heterochromatin requires mechanisms that establish, maintain and distinguish these canonical chromatin domains. Post-translational modifications are fundamental in these processes. Monoubiquitylation of histones was discovered more than three decades ago, but its precise function has been enigmatic until recently. It is now appreciated that the spectrum of chromatin ubiquitylation is not restricted to monoubiquitylation of histones, but includes degradatory ubiquitylation of histones, histone-modifying enzymes and non-histone chromatin factors. These occur in a spatially and temporally controlled manner. In this review, we summarize our understanding of these mechanisms with a particular emphasis on how ubiquitylation shapes the physical landscape of chromatin. PMID:22688965

  9. 33rd Annual International Asilomar Chromatin and Chromosomes Conference

    E-print Network

    Eirin Lopez, Jose Maria

    of histone variants in bivalve molluscs and their relevance in the development of chromatin-based tests.Z variants, actively expressed (transcribed and translated) in a bivalve mollusc, the mussel Mytilus

  10. Superstructure and CD spectrum as probes of chromatin integrity.

    PubMed Central

    de Murcia, G; Das, G C; Erard, M; Daune, M

    1978-01-01

    Two types of chromatin were extracted from the same stock of rat liver nuclei by a short exposure to micrococcal nuclease and by shearing respectively. These two materials which are identical in their protein/DNA content and by the presence of the five histones, were compared by means of circular dichroism and electron microscopy. Under the electron microscope and in absence of any divalent cation a superstructure of the unfixed chromatin fiber can be viewed only with native material but is no more present in sheared one. The increase of CD signal at 280 nm (from 2000 to about 4000 cm2 deg.dmole-1) in the case of sheared chromatin is not related to the loss of superstructure but to the structural changes of DNA inside the nucleosomal core which are always produced by shearing. These two correlated observations offer new sensitive probes of the integrity of any native or reconstituted chromatin. Images PMID:634797

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

    PubMed

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

    2015-07-01

    Chromatin remodeling ATPases and their associated complexes can alter the accessibility of the genome in the context of chromatin by using energy derived from the hydrolysis of ATP to change the positioning, occupancy and composition of nucleosomes. In animals and plants, these remodelers have been implicated in diverse processes ranging from stem cell maintenance and differentiation to developmental phase transitions and stress responses. Detailed investigation of their roles in individual processes has suggested a higher level of selectivity of chromatin remodeling ATPase activity than previously anticipated, and diverse mechanisms have been uncovered that can contribute to the selectivity. This review summarizes recent advances in understanding the roles and activities of chromatin remodeling ATPases in plants. PMID:25977075

  12. Facts about FACT and transcript elongation through chromatin.

    PubMed

    Belotserkovskaya, Rimma; Reinberg, Danny

    2004-04-01

    The regulation of transcription elongation within the context of chromatin is a topic of great interest. Even though chromatin presents a barrier to transcription by the PolII machinery in vitro, this process is rather efficient in vivo. Importantly, the chromatin structure of the actively transcribed genes is altered as part of this process. A large number of factors implicated in the control of transcript elongation have been identified through genetics, biochemistry and targeted proteomics approaches. However the precise roles and mechanisms of action of these factors remain obscure. A significant advance came about this past year with the elucidation of the roles of FACT and Spt6 in transcription elongation. These factors facilitate PolII passage through chromatin by destabilizing the nucleosome structure as well as reassemble nucleosomes traversed by PolII. PMID:15196460

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

    PubMed

    Rios, Ester C S; Szczesny, Bartosz; Soriano, Francisco G; Olah, Gabor; Szabo, Csaba

    2015-06-01

    Hydrogen sulfide (H2S) is an endogenous gaseous biological mediator, which regulates, among others, the oxidative balance of cells under normal physiological conditions, as well as in various diseases. Several previous studies have reported that H2S attenuates inflammatory mediator production. In this study, we investigated the role of H2S in chromatin modulation in an in vitro model of lipopolysaccharide (LPS)-induced inflammation and evaluated its effects on inflammatory cytokine production. Tamm-Horsfall protein 1 (THP-1) differentiated macrophages were pre-treated with sodium hydrosulfide (NaHS) (an H2S donor) at 0.01, 0.1, 0.5 or 1 mM for 30 min. To stimulate cytokine production, the cells were challenged with bacterial LPS (1 µg/ml) for 1, 4, 8 or 24 h. Histone H3 acetylation was analyzed by chromatin immunoprecipitation (ChIP), cytokine production was measured by ELISA and histone deacetylase (HDAC) activity was analyzed using a standard biochemical assay. H2S inhibited the production of interleukin-6 (IL-6) and tumor necrosis factor-? (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

  15. Chromatin dynamics at the Saccharomyces cerevisiae PHO5 promoter 

    E-print Network

    Jessen, Walter Joseph

    2006-04-12

    Neighboring UASp1 Persists at Extended Times of Activation??????????????... 112 4-5 Chromatin Remodeling of the Wild-Type PHO5 Promoter Spreads from UASp1??????????????????????... 115 4-6 Activation Localizes SWI/SNF Preferentially at the UAS... propagation of chromatin remodeling from enhancer-bound activators, and (ii) that interaction of SWI/SNF with the promoter localizes to the enhancer region. SIGNIFICANCE Early studies of the mechanisms by which activator proteins stimulate transcription...

  16. 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

  17. Sequence-specific packaging of DNA in human sperm chromatin

    Microsoft Academic Search

    J. M. Gatewood; G. R. Cook; R. Balhorn; E. M. Bradbury; C. W. Schmid

    1987-01-01

    The DNA in human sperm chromatin is packaged into nucleoprotamine (approx.85%) and nucleohistone (approx.15%). Whether these two chromatin fractions are sequence-specific subsets of the spermatozoon genome is the question addressed in this report. Sequence-specific packaging would suggest distinct structural and functional roles for nucleohistone and nucleoprotamine in late spermatogenesis or early development or both. After removal of histones with 0.65

  18. Chromatin dynamics at the Saccharomyces cerevisiae PHO5 promoter

    E-print Network

    Jessen, Walter Joseph

    2006-04-12

    Neighboring UASp1 Persists at Extended Times of Activation??????????????... 112 4-5 Chromatin Remodeling of the Wild-Type PHO5 Promoter Spreads from UASp1??????????????????????... 115 4-6 Activation Localizes SWI/SNF Preferentially at the UAS... propagation of chromatin remodeling from enhancer-bound activators, and (ii) that interaction of SWI/SNF with the promoter localizes to the enhancer region. SIGNIFICANCE Early studies of the mechanisms by which activator proteins stimulate transcription...

  19. Environmental-stress-induced Chromatin Regulation and its Heritability.

    PubMed

    Fang, Lei; Wuptra, Kenly; Chen, Danqi; Li, Hongjie; Huang, Shau-Ku; Jin, Chunyuan; Yokoyama, Kazunari K

    2014-01-15

    Chromatin is subject to proofreading and repair mechanisms during the process of DNA replication, as well as repair to maintain genetic and epigenetic information and genome stability. The dynamic structure of chromatin modulates various nuclear processes, including transcription and replication, by altering the accessibility of the DNA to regulatory factors. Structural changes in chromatin are affected by the chemical modification of histone proteins and DNA, remodeling of nucleosomes, incorporation of variant histones, noncoding RNAs, and nonhistone DNA-binding proteins. Phenotypic diversity and fidelity can be balanced by controlling stochastic switching of chromatin structure and dynamics in response to the environmental disruptors and endogenous stresses. The dynamic chromatin remodeling can, therefore, serve as a sensor, through which environmental and/or metabolic agents can alter gene expression, leading to global cellular changes involving multiple interactive networks. Furthermore its recent evidence also suggests that the epigenetic changes are heritable during the development. This review will discuss the environmental sensing system for chromatin regulation and genetic and epigenetic controls from developmental perspectives. PMID:25045581

  20. Lessons from senescence: chromatin maintenance in non-proliferating cells

    PubMed Central

    Rai, Taranjit Singh; Adams, Peter D.

    2013-01-01

    Cellular senescence is an irreversible proliferation arrest, thought to contribute to tumour suppression, proper wound healing and, perhaps, tissue and organismal aging. Two classical tumor suppressors, p53 and pRB, control cell cycle arrest associated with senescence. Profound molecular changes occur in cells undergoing senescence. At the level of chromatin, for example, senescence associated heterochromatic foci (SAHF) form in some cell types. Chromatin is inherently dynamic and likely needs to be actively maintained to achieve a stable cell phenotype. In proliferating cells chromatin is maintained in conjunction with DNA replication, but how non-proliferating cells maintain chromatin structure is poorly understood. Some histone variants, such as H3.3 and macroH2A increase as cells undergo senescence, suggesting histone variants and their associated chaperones could be important in chromatin structure maintenance in senescent cells. Here, we discuss options available for senescent cells to maintain chromatin structure and the relative contribution of histone variants and chaperones in this process. PMID:21839870

  1. 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.

  2. Integrative annotation of chromatin elements from ENCODE data

    PubMed Central

    Hoffman, Michael M.; Ernst, Jason; Wilder, Steven P.; Kundaje, Anshul; Harris, Robert S.; Libbrecht, Max; Giardine, Belinda; Ellenbogen, Paul M.; Bilmes, Jeffrey A.; Birney, Ewan; Hardison, Ross C.; Dunham, Ian; Kellis, Manolis; Noble, William Stafford

    2013-01-01

    The ENCODE Project has generated a wealth of experimental information mapping diverse chromatin properties in several human cell lines. Although each such data track is independently informative toward the annotation of regulatory elements, their interrelations contain much richer information for the systematic annotation of regulatory elements. To uncover these interrelations and to generate an interpretable summary of the massive datasets of the ENCODE Project, we apply unsupervised learning methodologies, converting dozens of chromatin datasets into discrete annotation maps of regulatory regions and other chromatin elements across the human genome. These methods rediscover and summarize diverse aspects of chromatin architecture, elucidate the interplay between chromatin activity and RNA transcription, and reveal that a large proportion of the genome lies in a quiescent state, even across multiple cell types. The resulting annotation of non-coding regulatory elements correlate strongly with mammalian evolutionary constraint, and provide an unbiased approach for evaluating metrics of evolutionary constraint in human. Lastly, we use the regulatory annotations to revisit previously uncharacterized disease-associated loci, resulting in focused, testable hypotheses through the lens of the chromatin landscape. PMID:23221638

  3. Integrative epigenomic mapping defines four main chromatin states in Arabidopsis

    PubMed Central

    Roudier, François; Ahmed, Ikhlak; Bérard, Caroline; Sarazin, Alexis; Mary-Huard, Tristan; Cortijo, Sandra; Bouyer, Daniel; Caillieux, Erwann; Duvernois-Berthet, Evelyne; Al-Shikhley, Liza; Giraut, Laurène; Després, Barbara; Drevensek, Stéphanie; Barneche, Frédy; Dèrozier, Sandra; Brunaud, Véronique; Aubourg, Sébastien; Schnittger, Arp; Bowler, Chris; Martin-Magniette, Marie-Laure; Robin, Stéphane; Caboche, Michel; Colot, Vincent

    2011-01-01

    Post-translational modification of histones and DNA methylation are important components of chromatin-level control of genome activity in eukaryotes. However, principles governing the combinatorial association of chromatin marks along the genome remain poorly understood. Here, we have generated epigenomic maps for eight histone modifications (H3K4me2 and 3, H3K27me1 and 2, H3K36me3, H3K56ac, H4K20me1 and H2Bub) in the model plant Arabidopsis and we have combined these maps with others, produced under identical conditions, for H3K9me2, H3K9me3, H3K27me3 and DNA methylation. Integrative analysis indicates that these 12 chromatin marks, which collectively cover ?90% of the genome, are present at any given position in a very limited number of combinations. Moreover, we show that the distribution of the 12 marks along the genomic sequence defines four main chromatin states, which preferentially index active genes, repressed genes, silent repeat elements and intergenic regions. Given the compact nature of the Arabidopsis genome, these four indexing states typically translate into short chromatin domains interspersed with each other. This first combinatorial view of the Arabidopsis epigenome points to simple principles of organization as in metazoans and provides a framework for further studies of chromatin-based regulatory mechanisms in plants. PMID:21487388

  4. Integrative epigenomic mapping defines four main chromatin states in Arabidopsis.

    PubMed

    Roudier, François; Ahmed, Ikhlak; Bérard, Caroline; Sarazin, Alexis; Mary-Huard, Tristan; Cortijo, Sandra; Bouyer, Daniel; Caillieux, Erwann; Duvernois-Berthet, Evelyne; Al-Shikhley, Liza; Giraut, Laurène; Després, Barbara; Drevensek, Stéphanie; Barneche, Frédy; Dèrozier, Sandra; Brunaud, Véronique; Aubourg, Sébastien; Schnittger, Arp; Bowler, Chris; Martin-Magniette, Marie-Laure; Robin, Stéphane; Caboche, Michel; Colot, Vincent

    2011-05-18

    Post-translational modification of histones and DNA methylation are important components of chromatin-level control of genome activity in eukaryotes. However, principles governing the combinatorial association of chromatin marks along the genome remain poorly understood. Here, we have generated epigenomic maps for eight histone modifications (H3K4me2 and 3, H3K27me1 and 2, H3K36me3, H3K56ac, H4K20me1 and H2Bub) in the model plant Arabidopsis and we have combined these maps with others, produced under identical conditions, for H3K9me2, H3K9me3, H3K27me3 and DNA methylation. Integrative analysis indicates that these 12 chromatin marks, which collectively cover ?90% of the genome, are present at any given position in a very limited number of combinations. Moreover, we show that the distribution of the 12 marks along the genomic sequence defines four main chromatin states, which preferentially index active genes, repressed genes, silent repeat elements and intergenic regions. Given the compact nature of the Arabidopsis genome, these four indexing states typically translate into short chromatin domains interspersed with each other. This first combinatorial view of the Arabidopsis epigenome points to simple principles of organization as in metazoans and provides a framework for further studies of chromatin-based regulatory mechanisms in plants. PMID:21487388

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

    PubMed

    Birch, Joanna L; Tan, Bertrand C-M; Panov, Kostya I; Panova, Tatiana B; Andersen, Jens S; Owen-Hughes, Tom A; Russell, Jackie; Lee, Sheng-Chung; Zomerdijk, Joost C B M

    2009-04-01

    Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle. The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III and facilitates their transcription in cells. Our findings indicate that, beyond the established role in Pol II transcription, FACT has physiological functions in chromatin transcription by all three nuclear RNA Pols. Our data also imply that local chromatin dynamics influence transcription of the active rRNA genes by Pol I and of Pol III-transcribed genes. PMID:19214185

  6. Economical Condensing Turbines? 

    E-print Network

    Dean, J. E.

    1997-01-01

    Steam turbines have long been used at utilities and in industry to generate power. There are three basic types of steam turbines: condensing, letdown and extraction/condensing. • Letdown turbines reduce the pressure of the incoming steam to one...

  7. Condensed Matter Physics

    Microsoft Academic Search

    Michael P. Marder

    2000-01-01

    A modern, unified treatment of condensed matter physics This new work presents for the first time in decades a sweeping review of the whole field of condensed matter physics. It consolidates new and classic topics from disparate sources, teaching \\

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

    PubMed Central

    Zhou, Qi; Bachtrog, Doris

    2015-01-01

    Sex chromosomes evolve distinctive types of chromatin from a pair of ancestral autosomes that are usually euchromatic. In Drosophila, the dosage-compensated X becomes enriched for hyperactive chromatin in males (mediated by H4K16ac), while the Y chromosome acquires silencing heterochromatin (enriched for H3K9me2/3). Drosophila autosomes are typically mostly euchromatic but the small dot chromosome has evolved a heterochromatin-like milieu (enriched for H3K9me2/3) that permits the normal expression of dot-linked genes, but which is different from typical pericentric heterochromatin. In Drosophila busckii, the dot chromosomes have fused to the ancestral sex chromosomes, creating a pair of ‘neo-sex’ chromosomes. Here we collect genomic, transcriptomic and epigenomic data from D. busckii, to investigate the evolutionary trajectory of sex chromosomes from a largely heterochromatic ancestor. We show that the neo-sex chromosomes formed <1 million years ago, but nearly 60% of neo-Y linked genes have already become non-functional. Expression levels are generally lower for the neo-Y alleles relative to their neo-X homologs, and the silencing heterochromatin mark H3K9me2, but not H3K9me3, is significantly enriched on silenced neo-Y genes. Despite rampant neo-Y degeneration, we find that the neo-X is deficient for the canonical histone modification mark of dosage compensation (H4K16ac), relative to autosomes or the compensated ancestral X chromosome, possibly reflecting constraints imposed on evolving hyperactive chromatin in an originally heterochromatic environment. Yet, neo-X genes are transcriptionally more active in males, relative to females, suggesting the evolution of incipient dosage compensation on the neo-X. Our data show that Y degeneration proceeds quickly after sex chromosomes become established through genomic and epigenetic changes, and are consistent with the idea that the evolution of sex-linked chromatin is influenced by its ancestral configuration. PMID:26114585

  9. Protocol: Chromatin immunoprecipitation (ChIP) methodology to investigate histone modifications in two model diatom species

    PubMed Central

    2012-01-01

    In this report we describe a chromatin immunoprecipitation (ChIP) protocol for two fully sequenced model diatom species Phaeodactylum tricornutum and Thalassiosira pseudonana. This protocol allows the extraction of satisfactory amounts of chromatin and gives reproducible results. We coupled the ChIP assay with real time quantitative PCR. Our results reveal that the two major histone marks H3K4me2 and H3K9me2 exist in P. tricornutum and T. pseudonana. As in other eukaryotes, H3K4me2 marks active genes whereas H3K9me2 marks transcriptionally inactive transposable elements. Unexpectedly however, T. pseudonana housekeeping genes also show a relative enrichment of H3K9me2. We also discuss optimization of the procedure, including growth conditions, cross linking and sonication. Validation of the protocol provides a set of genes and transposable elements that can be used as controls for studies using ChIP in each diatom species. This protocol can be easily adapted to other diatoms and eukaryotic phytoplankton species for genetic and biochemical studies. PMID:23217141

  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. Assessing sites of NF-?B DNA binding using chromatin immunoprecipitation.

    PubMed

    Colleran, Amy; Collins, Patricia E; Carmody, Ruaidhrí J

    2015-01-01

    The NF-?B transcription factor is in fact a family of related proteins which dimerize to form at least 12 distinct complexes which regulate the expression of hundred of genes of importance to a range of physiological and pathological processes. The binding of NF-?B to the regulatory regions and promoters of target genes is influenced by a number of factors including the sequence of DNA-binding sites, the posttranslational modification of NF-?B, and the interaction of cofactors and co-regulators of transcription. In addition, the binding of NF-?B to promoters is highly dynamic and the recruitment of specific subunits to specific binding sites may occur with distinct kinetics. Moreover, genome-wide analysis of NF-?B chromatin binding indicates that the majority of DNA-binding events are not associated with changes in transcriptional activity. Thus, the analysis of NF-?B recruitment and activity at specific binding sites is of critical importance in understanding the regulation of transcription. In this chapter we describe a chromatin immunoprecipitation assay to investigate the in situ binding of NF-?B to specific sites in the genome. PMID:25736743

  12. Centromeric chromatin and its dynamics in plants.

    PubMed

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

    2015-07-01

    Centromeres are chromatin structures that are required for proper separation of chromosomes during mitosis and meiosis. The centromere is composed of centromeric DNA, often enriched in satellite repeats, and kinetochore complex proteins. To date, over 100 kinetochore components have been identified in various eukaryotes. Kinetochore assembly begins with incorporation of centromeric histone H3 variant CENH3 into centromeric nucleosomes. Protein components of the kinetochore are either present at centromeres throughout the cell cycle or localize to centromeres transiently, prior to attachment of microtubules to each kinetochore in prometaphase of mitotic cells. This is the case for the spindle assembly checkpoint (SAC) proteins in animal cells. The SAC complex ensures equal separation of chromosomes between daughter nuclei by preventing anaphase onset before metaphase is complete, i.e. the sister kinetochores of all chromosomes are attached to spindle fibers from opposite poles. In this review, we focus on the organization of centromeric DNA and the kinetochore assembly in plants. We summarize recent advances regarding loading of CENH3 into the centromere, and the subcellular localization and protein-protein interactions of Arabidopsis thaliana proteins involved in kinetochore assembly and function. We describe the transcriptional activity of corresponding genes based on in silico analysis of their promoters and cell cycle-dependent expression. Additionally, barley homologs of all selected A. thaliana proteins have been identified in silico, and their sequences and domain structures are presented. PMID:25976696

  13. Condensation in Microchannels

    Microsoft Academic Search

    Yongping Chen; Mingheng Shi; Ping Cheng; G. P. Peterson

    2008-01-01

    Condensation in microchannels has applications in a wide variety of advanced microthermal devices. Presented here is a review of both experimental and theoretical analyses of condensation in these microchannels, with special attention given to the effects of channel diameter and surface conditions on the flow regimes of condensing flows occurring in these channels. This review suggests that surface tension, rather

  14. Global Nature of Dynamic Protein-Chromatin Interactions In Vivo: Three-Dimensional Genome Scanning and Dynamic Interaction Networks of Chromatin Proteins

    Microsoft Academic Search

    Robert D. Phair; Paola Scaffidi; Cem Elbi; Jaromíra Vecerova; Anup Dey; Keiko Ozato; David T. Brown; Gordon Hager; Michael Bustin; Tom Misteli

    2004-01-01

    Genome structure and gene expression depend on a multitude of chromatin-binding proteins. The binding properties of these proteins to native chromatin in intact cells are largely unknown. Here, we describe an approach based on combined in vivo photobleaching microscopy and kinetic modeling to analyze globally the dynamics of binding of chromatin-associated proteins in living cells. We have quantitatively determined basic

  15. The NuRD Chromatin-Remodeling Enzyme CHD4 Promotes Embryonic Vascular Integrity by Transcriptionally Regulating Extracellular Matrix Proteolysis

    PubMed Central

    Ingram, Kyle G.; Curtis, Carol D.; Silasi-Mansat, Robert; Lupu, Florea; Griffin, Courtney T.

    2013-01-01

    The extracellular matrix (ECM) supports vascular integrity during embryonic development. Proteolytic degradation of ECM components is required for angiogenesis, but excessive ECM proteolysis causes blood vessel fragility and hemorrhage. Little is understood about how ECM proteolysis is transcriptionally regulated during embryonic vascular development. We now show that the NuRD ATP-dependent chromatin-remodeling complex promotes vascular integrity by preventing excessive ECM proteolysis in vivo. Mice lacking endothelial CHD4—a catalytic subunit of NuRD complexes—died at midgestation from vascular rupture. ECM components surrounding rupture-prone vessels in Chd4 mutants were significantly downregulated prior to embryonic lethality. Using qPCR arrays, we found two critical mediators of ECM stability misregulated in mutant endothelial cells: the urokinase-type plasminogen activator receptor (uPAR or Plaur) was upregulated, and thrombospondin-1 (Thbs1) was downregulated. Chromatin immunoprecipitation assays showed that CHD4-containing NuRD complexes directly bound the promoters of these genes in endothelial cells. uPAR and THBS1 respectively promote and inhibit activation of the potent ECM protease plasmin, and we detected increased plasmin activity around rupture-prone vessels in Chd4 mutants. We rescued ECM components and vascular rupture in Chd4 mutants by genetically reducing urokinase (uPA or Plau), which cooperates with uPAR to activate plasmin. Our findings provide a novel mechanism by which a chromatin-remodeling enzyme regulates ECM stability to maintain vascular integrity during embryonic development. PMID:24348274

  16. Dynamic chromatin boundaries delineate a latency control region of Epstein-Barr virus.

    PubMed

    Chau, Charles M; Lieberman, Paul M

    2004-11-01

    The oncogenic potential of latent Epstein-Barr virus (EBV) can be regulated by epigenetic factors controlling LMP1 and EBNA2 gene transcription. The EBV latency control region (LCR) constitutes approximately 12 kb of viral sequence spanning the divergent promoters of LMP1 and EBNA2 and encompasses the EBV latent replication origin OriP and RNA polymerase III-transcribed EBV-encoded RNA genes. We have used the chromatin immunoprecipitation assay to examine the chromatin architecture of the LCR in different types of EBV latency programs. We have found that histone H3 K4 methylation (H3mK4) was enriched throughout a large domain that extended from internal repeat 1 (IR1) to the terminal repeat in type III latency where EBNA2 and LMP1 genes are expressed. In type I latency where EBNA2 and LMP1 genes are transcriptionally silent, the H3mK4 domain contracts and does not enter the EBNA2 or LMP1 promoters. In contrast, histone H3 K9 methylation (H3mK9), associated with silent heterochromatin, was enriched in the EBNA2 and LMP1 upstream control regions in type I but not type III cells. MTA [5'-deoxy-5'(methylthio)adenosine], a pharmacological inhibitor of protein methylation, globally reduced histone H3mK4 and inhibited EBNA2 transcription in type III cells. 5'-Azacytidine, an inhibitor of DNA methylation that derepresses EBNA2 transcription in type I latency, caused H3mK4 expansion and a corresponding loss of H3mK9 at IR1. The chromatin boundary protein and transcription repressor CCCTC-binding factor was enriched at the EBNA2 transcription control region in type I but not type III cells. We also present evidence that OriP binding factors EBNA1 and ORC2 can interact with sequences outside of OriP including a region within IR1 that may influence EBNA2 transcription status. These results indicate that types I and III latency programs have distinct histone methylation patterns in the LCR and suggest that chromatin architecture coordinates gene expression of LMP1 and EBNA2. PMID:15507618

  17. Partial inhibition of histone deacetylase in active chromatin by HMG 14 and HMG 17.

    PubMed Central

    Reeves, R; Candido, E P

    1980-01-01

    Digestion of isolated Friend erythroleukemic cell nuclei with DNase I under conditions which selectively destroy the DNA of transcriptionally "active" genes releases into the supernatant fraction proteins of the non-histone "High Mobility Group" (HMGs). Two of these, HMG-14 and HMG-17(identified by solubility in trichloroacetic acid, electrophoretic mobility on SDS-polyacrylamide gels and by amino acid composition) will partially inhibit the endogenous mouse cell histone deacetylase enzymes when added to in vitro assay mixtures. Other closely related proteins do not share this inhibitory ability and thus the reaction with the enzymes appears to be specific. Since these two HMG proteins appear to be preferentially associated with the "active" fraction of chromatin, these findings have important implications for possible models of eukaryotic gene regulatory mechanisms. Images PMID:6448990

  18. Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq

    PubMed Central

    Adli, Mazhar; Bernstein, Bradley E.

    2015-01-01

    Chromatin immunoprecipitation (ChIP) combined with high-throughput sequencing (ChIP-seq) has become the gold standard for whole-genome mapping of protein-DNA interactions. However, conventional ChIP protocols necessitate the use of large numbers of cells, and library preparation steps associated with current high-throughput sequencing platforms require substantial amounts of DNA; both of these factors preclude the application of ChIP-seq technology to many biologically important but rare cell types. Here we describe a nano-ChIP-seq protocol that combines a high-sensitivity small-scale ChIP assay and a tailored procedure for generating high-throughput sequencing libraries from scarce amounts of ChIP DNA. In terms of the numbers of cells required, the method provides two to three orders of magnitude of improvement over the conventional ChIP-seq method and the entire procedure can be completed within 4 d. PMID:21959244

  19. 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

  20. 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...

  1. BioVyon Protein A, an alternative solid-phase affinity matrix for chromatin immunoprecipitation.

    PubMed

    Chernukhin, Igor; Kang, Sung Yun; Brown, Sam; Gretton, Svetlana; Mendez-Catala, Claudia Fabiola; Cowieson, Dave; Klenova, Elena

    2011-05-15

    Chromatin immunoprecipitation (ChIP) is an important technique in the study of DNA/protein interactions. The ChIP procedure, however, has limitations in that it is lengthy, can be inconsistent, and is prone to nonspecific binding of DNA and proteins to the bead-based solid-phase matrices that are often used for the immunoprecipitation step. In this investigation, we examined the utility of a new matrix for ChIP assays, BioVyon Protein A, a solid support based on porous polyethylene. In ChIP experiments carried out using two antibodies and seven DNA loci, the performance of BioVyon Protein A was significantly better, with a greater percentage of DNA pull-down in all of the assays tested compared with bead-based matrices, Protein A Sepharose, and Dynabeads Protein A. Furthermore, the rigid porous disc format within a column made the BioVyon matrix much easier to use with fewer steps and less equipment requirements, resulting in a significant reduction in the time taken to process the ChIP samples. In summary, BioVyon Protein A provides a column-based assay method for ChIP and other immunoprecipitation-based procedures; the rigid porous structure of BioVyon enables a fast and robust protocol with higher ChIP enrichment ratios. PMID:21284925

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

    PubMed Central

    Tong, Kevin; Skibbens, Robert V.

    2015-01-01

    Cohesins are required both for the tethering together of sister chromatids (termed cohesion) and subsequent condensation into discrete structures—processes fundamental for faithful chromosome segregation into daughter cells. Differentiating between cohesin roles in cohesion and condensation would provide an important advance in studying chromatin metabolism. Pds5 is a cohesin-associated factor that is essential for both cohesion maintenance and condensation. Recent studies revealed that ELG1 deletion suppresses the temperature sensitivity of pds5 mutant cells. However, the mechanisms through which Elg1 may regulate cohesion and condensation remain unknown. Here, we report that ELG1 deletion from pds5-1 mutant cells results in a significant rescue of cohesion, but not condensation, defects. Based on evidence that Elg1 unloads the DNA replication clamp PCNA from DNA, we tested whether PCNA overexpression would similarly rescue pds5-1 mutant cell cohesion defects. The results indeed reveal that elevated levels of PCNA rescue pds5-1 temperature sensitivity and cohesion defects, but do not rescue pds5-1 mutant cell condensation defects. In contrast, RAD61 deletion rescues the condensation defect, but importantly, neither the temperature sensitivity nor cohesion defects exhibited by pds5-1 mutant cells. In combination, these findings reveal that cohesion and condensation are separable pathways and regulated in nonredundant mechanisms. These results are discussed in terms of a new model through which cohesion and condensation are spatially regulated. PMID:25986377

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

    PubMed

    Tong, Kevin; Skibbens, Robert V

    2015-06-01

    Cohesins are required both for the tethering together of sister chromatids (termed cohesion) and subsequent condensation into discrete structures-processes fundamental for faithful chromosome segregation into daughter cells. Differentiating between cohesin roles in cohesion and condensation would provide an important advance in studying chromatin metabolism. Pds5 is a cohesin-associated factor that is essential for both cohesion maintenance and condensation. Recent studies revealed that ELG1 deletion suppresses the temperature sensitivity of pds5 mutant cells. However, the mechanisms through which Elg1 may regulate cohesion and condensation remain unknown. Here, we report that ELG1 deletion from pds5-1 mutant cells results in a significant rescue of cohesion, but not condensation, defects. Based on evidence that Elg1 unloads the DNA replication clamp PCNA from DNA, we tested whether PCNA overexpression would similarly rescue pds5-1 mutant cell cohesion defects. The results indeed reveal that elevated levels of PCNA rescue pds5-1 temperature sensitivity and cohesion defects, but do not rescue pds5-1 mutant cell condensation defects. In contrast, RAD61 deletion rescues the condensation defect, but importantly, neither the temperature sensitivity nor cohesion defects exhibited by pds5-1 mutant cells. In combination, these findings reveal that cohesion and condensation are separable pathways and regulated in nonredundant mechanisms. These results are discussed in terms of a new model through which cohesion and condensation are spatially regulated. PMID:25986377

  4. Molecular characteristics and chromatin texture features in acute promyelocytic leukemia

    PubMed Central

    2012-01-01

    Background Acute promyelocytic leukemia is a cytogenetically well defined entity. Nevertheless, some features observed at diagnosis are related to a worse outcome of the patients. Methods In a prospective study, we analyzed peripheral (PB) leukocyte count, immunophenotype, methylation status of CDKN2B, CDKN2A and TP73; FLT3 and NPM1 mutations besides nuclear chromatin texture characteristics of the leukemic cells. We also examined the relation of these features with patient’s outcome. Results Among 19 cases, 4 had a microgranular morphology, 7 presented PB leukocytes >10x109/l, 2 had FLT3-ITD and 3 had FLT3-TKD (all three presenting a methylated CDKN2B). NPM1 mutation was not observed. PB leukocyte count showed an inverse relation with standard deviation of gray levels, contrast, cluster prominence, and chromatin fractal dimension (FD). Cases with FLT3-ITD presented a microgranular morphology, PB leukocytosis and expression of HLA-DR, CD34 and CD11b. Concerning nuclear chromatin texture variables, these cases had a lower entropy, contrast, cluster prominence and FD, but higher local homogeneity, and R245, in keeping with more homogeneously distributed chromatin. In the univariate Cox analysis, a higher leukocyte count, FLT3-ITD mutation, microgranular morphology, methylation of CDKN2B, besides a higher local homogeneity of nuclear chromatin, a lower chromatin entropy and FD were associated to a worse outcome. All these features lost significance when the cases were stratified for FLT3-ITD mutation. Methylation status of CDNK2A and TP73 showed no relation to patient’s survival. Conclusion in APL, patients with FLT3-ITD mutation show different clinical characteristics and have blasts with a more homogeneous chromatin texture. Texture analysis demonstrated that FLTD-ITD was accompanied not only by different cytoplasmic features, but also by a change in chromatin structure in routine cytologic preparations. Yet we were not able to detect chromatin changes by nuclear texture analysis of patients with the FTLD-TKD or methylation of specific genes. PMID:22742960

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

    PubMed

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

    2015-01-01

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

  6. Dissecting the chromatin interactome of microRNA genes

    PubMed Central

    Chen, Dijun; Fu, Liang-Yu; Zhang, Zhao; Li, Guoliang; Zhang, Hang; Jiang, Li; Harrison, Andrew P.; Shanahan, Hugh P.; Klukas, Christian; Zhang, Hong-Yu; Ruan, Yijun; Chen, Ling-Ling; Chen, Ming

    2014-01-01

    Our knowledge of the role of higher-order chromatin structures in transcription of microRNA genes (MIRs) is evolving rapidly. Here we investigate the effect of 3D architecture of chromatin on the transcriptional regulation of MIRs. We demonstrate that MIRs have transcriptional features that are similar to protein-coding genes. RNA polymerase II–associated ChIA-PET data reveal that many groups of MIRs and protein-coding genes are organized into functionally compartmentalized chromatin communities and undergo coordinated expression when their genomic loci are spatially colocated. We observe that MIRs display widespread communication in those transcriptionally active communities. Moreover, miRNA–target interactions are significantly enriched among communities with functional homogeneity while depleted from the same community from which they originated, suggesting MIRs coordinating function-related pathways at posttranscriptional level. Further investigation demonstrates the existence of spatial MIR–MIR chromatin interacting networks. We show that groups of spatially coordinated MIRs are frequently from the same family and involved in the same disease category. The spatial interaction network possesses both common and cell-specific subnetwork modules that result from the spatial organization of chromatin within different cell types. Together, our study unveils an entirely unexplored layer of MIR regulation throughout the human genome that links the spatial coordination of MIRs to their co-expression and function. PMID:24357409

  7. 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

  8. 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

  9. Micron-scale coherence in interphase chromatin dynamics.

    PubMed

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

    2013-09-24

    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

  10. 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

  11. 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

  12. Chromatin remodeling by the small RNA machinery in mammalian cells

    PubMed Central

    Li, Long-Cheng

    2014-01-01

    Chromatin states, quite different from changes in DNA sequence, can impact fundamental cellular processes such as determination of cell identity and development of disease. However, how chromatin states are established and regulated remain to be fully elucidated. In several lower eukaryotes, the small RNA machinery comprised of small RNA and its partners, the Argonaute proteins, is known to play important roles in the establishment of heterochromatin and silencing of repetitive sequences. In mammalian cells, however, the nuclear function of the small RNA machinery is largely unknown. Emerging evidence suggests that components of the small RNA pathway interact with chromatin to regulate nuclear events, including gene transcription and alternative splicing. In addition, these endogenous mechanisms are being exploited to target specific genomic loci for manipulation of gene expression and splicing events. In this review, I summarize current understanding of chromatin remodeling by small RNAs in mammalian cells and highlight recent efforts to map genome-wide interactions between RNAi-related factors and chromatin. PMID:24149777

  13. The Open Chromatin Landscape of Kaposi's Sarcoma-Associated Herpesvirus

    PubMed Central

    Hilton, Isaac B.; Simon, Jeremy M.; Lieb, Jason D.; Davis, Ian J.; Damania, Blossom

    2013-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus which establishes latent infection in endothelial and B cells, as well as in primary effusion lymphoma (PEL). During latency, the viral genome exists as a circular DNA minichromosome (episome) and is packaged into chromatin analogous to human chromosomes. Only a small subset of promoters, those which drive latent RNAs, are active in latent episomes. In general, nucleosome depletion (“open chromatin”) is a hallmark of eukaryotic regulatory elements such as promoters and transcriptional enhancers or insulators. We applied formaldehyde-assisted isolation of regulatory elements (FAIRE) followed by next-generation sequencing to identify regulatory elements in the KSHV genome and integrated these data with previously identified locations of histone modifications, RNA polymerase II occupancy, and CTCF binding sites. We found that (i) regions of open chromatin were not restricted to the transcriptionally defined latent loci; (ii) open chromatin was adjacent to regions harboring activating histone modifications, even at transcriptionally inactive loci; and (iii) CTCF binding sites fell within regions of open chromatin with few exceptions, including the constitutive LANA promoter and the vIL6 promoter. FAIRE-identified nucleosome depletion was similar among B and endothelial cell lineages, suggesting a common viral genome architecture in all forms of latency. PMID:23986576

  14. Role of chromatin in water stress responses in plants

    PubMed Central

    Han, Soon-Ki; Wagner, Doris

    2014-01-01

    As sessile organisms, plants are exposed to environmental stresses throughout their life. They have developed survival strategies such as developmental and morphological adaptations, as well as physiological responses, to protect themselves from adverse environments. In addition, stress sensing triggers large-scale transcriptional reprogramming directed at minimizing the deleterious effect of water stress on plant cells. Here, we review recent findings that reveal a role of chromatin in water stress responses. In addition, we discuss data in support of the idea that chromatin remodelling and modifying enzymes may be direct targets of stress signalling pathways. Modulation of chromatin regulator activity by these signaling pathways may be critical in minimizing potential trade-offs between growth and stress responses. Alterations in the chromatin organization and/or in the activity of chromatin remodelling and modifying enzymes may furthermore contribute to stress memory. Mechanistic insight into these phenomena derived from studies in model plant systems should allow future engineering of broadly drought-tolerant crop plants that do not incur unnecessary losses in yield or growth. PMID:24302754

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

    PubMed

    Singh, Purva; Schwarzbauer, Jean E

    2014-10-15

    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

  16. The Chromatin “Landscape” of a Murine Adult ?-Globin Gene Is Unaffected by Deletion of Either the Gene Promoter or a Downstream Enhancer

    PubMed Central

    Cadiz-Rivera, Brenda; Fromm, George; de Vries, Christina; Fields, Jennifer; McGrath, Kathleen E.; Fiering, Steven; Bulger, Michael

    2014-01-01

    In mammals, the complex tissue- and developmental-specific expression of genes within the ?-globin cluster is known to be subject to control by the gene promoters, by a locus control region (LCR) located upstream of the cluster, and by sequence elements located across the intergenic regions. Despite extensive investigation, however, the complement of sequences that is required for normal regulation of chromatin structure and gene expression within the cluster is not fully defined. To further elucidate regulation of the adult ?-globin genes, we investigate the effects of two deletions engineered within the endogenous murine ?-globin locus. First, we find that deletion of the ?2-globin gene promoter, while eliminating ?2-globin gene expression, results in no additional effects on chromatin structure or gene expression within the cluster. Notably, our observations are not consistent with competition among the ?-globin genes for LCR activity. Second, we characterize a novel enhancer located 3? of the ?2-globin gene, but find that deletion of this sequence has no effect whatsoever on gene expression or chromatin structure. This observation highlights the difficulty in assigning function to enhancer sequences identified by the chromatin “landscape” or even by functional assays. PMID:24817273

  17. The chromatin "landscape" of a murine adult ?-globin gene is unaffected by deletion of either the gene promoter or a downstream enhancer.

    PubMed

    Cadiz-Rivera, Brenda; Fromm, George; de Vries, Christina; Fields, Jennifer; McGrath, Kathleen E; Fiering, Steven; Bulger, Michael

    2014-01-01

    In mammals, the complex tissue- and developmental-specific expression of genes within the ?-globin cluster is known to be subject to control by the gene promoters, by a locus control region (LCR) located upstream of the cluster, and by sequence elements located across the intergenic regions. Despite extensive investigation, however, the complement of sequences that is required for normal regulation of chromatin structure and gene expression within the cluster is not fully defined. To further elucidate regulation of the adult ?-globin genes, we investigate the effects of two deletions engineered within the endogenous murine ?-globin locus. First, we find that deletion of the ?2-globin gene promoter, while eliminating ?2-globin gene expression, results in no additional effects on chromatin structure or gene expression within the cluster. Notably, our observations are not consistent with competition among the ?-globin genes for LCR activity. Second, we characterize a novel enhancer located 3' of the ?2-globin gene, but find that deletion of this sequence has no effect whatsoever on gene expression or chromatin structure. This observation highlights the difficulty in assigning function to enhancer sequences identified by the chromatin "landscape" or even by functional assays. PMID:24817273

  18. A Generic Tool for Transcription Factor Target Gene Discovery in Arabidopsis Cell Suspension Cultures Based on Tandem Chromatin Affinity Purification1[W][OPEN

    PubMed Central

    Verkest, Aurine; Abeel, Thomas; Heyndrickx, Ken S.; Van Leene, Jelle; Lanz, Christa; Van De Slijke, Eveline; De Winne, Nancy; Eeckhout, Dominique; Persiau, Geert; Van Breusegem, Frank; Inzé, Dirk; Vandepoele, Klaas; De Jaeger, Geert

    2014-01-01

    Genome-wide identification of transcription factor (TF) binding sites is pivotal to our understanding of gene expression regulation. Although much progress has been made in the determination of potential binding regions of proteins by chromatin immunoprecipitation, this method has some inherent limitations regarding DNA enrichment efficiency and antibody necessity. Here, we report an alternative strategy for assaying in vivo TF-DNA binding in Arabidopsis (Arabidopsis thaliana) cells by tandem chromatin affinity purification (TChAP). Evaluation of TChAP using the E2Fa TF and comparison with traditional chromatin immunoprecipitation and single chromatin affinity purification illustrates the suitability of TChAP and provides a resource for exploring the E2Fa transcriptional network. Integration with transcriptome, cis-regulatory element, functional enrichment, and coexpression network analyses demonstrates the quality of the E2Fa TChAP sequencing data and validates the identification of new direct E2Fa targets. TChAP enhances both TF target mapping throughput, by circumventing issues related to antibody availability, and output, by improving DNA enrichment efficiency. PMID:24453163

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

    PubMed Central

    Calo, Eliezer; Wysocka, Joanna

    2013-01-01

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

  20. Chromatin organization at the nuclear pore favours HIV replication

    PubMed Central

    Lelek, Mickaël; Casartelli, Nicoletta; Pellin, Danilo; Rizzi, Ermanno; Souque, Philippe; Severgnini, Marco; Di Serio, Clelia; Fricke, Thomas; Diaz-Griffero, Felipe; Zimmer, Christophe; Charneau, Pierre; Di Nunzio, Francesca

    2015-01-01

    The molecular mechanisms that allow HIV to integrate into particular sites of the host genome are poorly understood. Here we tested if the nuclear pore complex (NPC) facilitates the targeting of HIV integration by acting on chromatin topology. We show that the integrity of the nuclear side of the NPC, which is mainly composed of Tpr, is not required for HIV nuclear import, but that Nup153 is essential. Depletion of Tpr markedly reduces HIV infectivity, but not the level of integration. HIV integration sites in Tpr-depleted cells are less associated with marks of active genes, consistent with the state of chromatin proximal to the NPC, as analysed by super-resolution microscopy. LEDGF/p75, which promotes viral integration into active genes, stabilizes Tpr at the nuclear periphery and vice versa. Our data support a model in which HIV nuclear import and integration are concerted steps, and where Tpr maintains a chromatin environment favourable for HIV replication. PMID:25744187

  1. Structural plasticity of single chromatin fibers revealed by torsional manipulation

    E-print Network

    Aurelien Bancaud; Natalia Conde e Silva; Maria Barbi; Gaudeline Wagner; Jean-Francois Allemand; Julien Mozziconacci; Christophe Lavelle; Vincent Croquette; Jean-Marc Victor; Ariel Prunell; Jean-Louis Viovy

    2007-07-13

    Magnetic tweezers are used to study the mechanical response under torsion of single nucleosome arrays reconstituted on tandem repeats of 5S positioning sequences. Regular arrays are extremely resilient and can reversibly accommodate a large amount of supercoiling without much change in length. This behavior is quantitatively described by a molecular model of the chromatin 3-D architecture. In this model, we assume the existence of a dynamic equilibrium between three conformations of the nucleosome, which are determined by the crossing status of the entry/exit DNAs (positive, null or negative). Torsional strain, in displacing that equilibrium, extensively reorganizes the fiber architecture. The model explains a number of long-standing topological questions regarding DNA in chromatin, and may provide the ground to better understand the dynamic binding of most chromatin-associated proteins.

  2. Higher order structure in a short repeat length chromatin

    PubMed Central

    1984-01-01

    Polynucleosomes from calf brain cortical neurone nuclei have an average repeat length of less than 168 base pairs. The ability of this material to adopt higher order structure has been assessed by various physical techniques. Although containing on average less DNA per nucleosome than is required to form a chromatosome, this short repeat length chromatin folded in an H1 dependent manner to a structure with properties similar to those observed for longer repeat length chromatins such as that of chicken erythrocyte (McGhee, J.D., D.C. Rau, E. Charney, and G. Felsenfeld, 1980, Cell, 22:87-96). These observations are discussed in the context of H1 location in the higher order chromatin fiber. PMID:6715407

  3. Heterogeneity of chromatin fragments produced by micrococcal nuclease action.

    PubMed

    Rill, R L; Oosterhof, D K; Hozier, J C; Nelson, D A

    1975-09-01

    Digestion of calf thymus chromatin with micrococcal nuclease produces a mixture of apparently well defined nucleoprotein fragments which have been partially resolved by sedimentation on linear (5-20%) sucrose gradients. Sedimentation patterns reveal a predominant peak at the 11S position, three slower components, which have not previously been reported, at the 3.4S, 5.3S and 8.6S positions, and three faster components at the 17S, 22S and 26S positions. DNA isolated from the 3S to 12S region of gradients has been resolved on polyacrylamide gels into nine to ten discrete components ranging from 47 to 156 base pairs in length. A nearly identical pattern of small DNA products was obtained from chromatin digested in intact nuclei. These data suggest that chromatin contains either several types of subunits or predominently a single type of subunit which can be asymmetrically cleaved at any one of four or more sites. PMID:1178527

  4. Chromatin and epigenetic regulation of pre-mRNA processing

    PubMed Central

    Brown, Seth J.; Stoilov, Peter; Xing, Yi

    2012-01-01

    New data are revealing a complex landscape of gene regulation shaped by chromatin states that extend into the bodies of transcribed genes and associate with distinct RNA elements such as exons, introns and polyadenylation sites. Exons are characterized by increased levels of nucleosome positioning, DNA methylation and certain histone modifications. As pre-mRNA splicing occurs co-transcriptionally, changes in the transcription elongation rate or epigenetic marks can influence exon splicing. These new discoveries broaden our understanding of the epigenetic code and ascribe a novel role for chromatin in controlling pre-mRNA processing. In this review, we summarize the recently discovered interplay between the modulation of chromatin states and pre-mRNA processing with the particular focus on how these processes communicate with one another to control gene expression. PMID:22936691

  5. Chromatin organization at the nuclear pore favours HIV replication.

    PubMed

    Lelek, Mickaël; Casartelli, Nicoletta; Pellin, Danilo; Rizzi, Ermanno; Souque, Philippe; Severgnini, Marco; Di Serio, Clelia; Fricke, Thomas; Diaz-Griffero, Felipe; Zimmer, Christophe; Charneau, Pierre; Di Nunzio, Francesca

    2015-01-01

    The molecular mechanisms that allow HIV to integrate into particular sites of the host genome are poorly understood. Here we tested if the nuclear pore complex (NPC) facilitates the targeting of HIV integration by acting on chromatin topology. We show that the integrity of the nuclear side of the NPC, which is mainly composed of Tpr, is not required for HIV nuclear import, but that Nup153 is essential. Depletion of Tpr markedly reduces HIV infectivity, but not the level of integration. HIV integration sites in Tpr-depleted cells are less associated with marks of active genes, consistent with the state of chromatin proximal to the NPC, as analysed by super-resolution microscopy. LEDGF/p75, which promotes viral integration into active genes, stabilizes Tpr at the nuclear periphery and vice versa. Our data support a model in which HIV nuclear import and integration are concerted steps, and where Tpr maintains a chromatin environment favourable for HIV replication. PMID:25744187

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

    PubMed

    Collepardo-Guevara, Rosana; Schlick, Tamar

    2014-06-01

    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

  7. Statistical physics of nucleosome positioning and chromatin structure

    NASA Astrophysics Data System (ADS)

    Morozov, Alexandre

    2012-02-01

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

  8. 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

  9. Demonstration of extensive chromatin cleavage in transplanted Morris hepatoma 7777 tissue: apoptosis or necrosis?

    PubMed Central

    Fukuda, K.; Kojiro, M.; Chiu, J. F.

    1993-01-01

    Cell death may occur by either of two mechanisms: necrosis or apoptosis (programmed cell death). In this paper, we demonstrate extensive chromatin cleavage into oligonucleosome-length fragments (DNA ladder) in transplanted Morris hepatoma 7777 tissue, which is suggestive of the stimulation of an endogenous endonuclease activity previously found to be involved in the process of apoptosis. The existence of many apoptotic cells, which are morphologically characterized by condensed cytoplasm and basophilic nuclear fragments, were also seen in this tissue. In vivo and in vitro experiments were designed to further differentiate the morphological and biochemical features of necrosis and apoptosis in liver and hepatoma cells. Liver tissue undergoing ischemic necrosis showed a distinct DNA ladder pattern without demonstrating the morphology of apoptosis, indicating that chromatin cleavage into oligonucleosomal-length fragments is not confined to apoptotic cell death, at least in liver cells. In in vitro-cultured McA-RH7777 cells, however, DNA ladder pattern was detected only in cells showing characteristic morphology of apoptosis. From these two criteria (i.e., characteristic morphology and DNA ladder), it was strongly suggested that the apoptotic process is highly activated in the transplanted 7777 tissue. Based on the results obtained from in vitro experiments, it was suggested that tumor apoptosis may represent a residual attempt at autoregulation within the expanding tumor population and/or may result from mild cellular injuries such as hypoxia, nutrient deficiency, or other unknown noxious factor(s). We also showed evidence that apoptosis is inducible in hepatoma cells in vitro by a wide range of mild injuries or stimuli. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8384410

  10. Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by

    E-print Network

    Langowski, Jörg

    . Gasser*¶ *Department of Molecular Biology and National Center of Competence in Research Frontiers of Biophysics of Macromolecules, German Cancer Research Center (Deutsche Krebsforschungszentrum), B040, Im compact chromatin structure for transcriptionally competent chromatin in living yeast cells suggests

  11. Combinatorial Patterning of Chromatin Regulators Uncovered by Genome-wide Location Analysis in Human Cells

    E-print Network

    Ram, Oren

    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 ...

  12. Systematic Dissection of Roles for Chromatin Regulators in a Yeast Stress Response

    E-print Network

    Pfeffner, Jenna

    Packaging of eukaryotic genomes into chromatin has wide-ranging effects on gene transcription. Curiously, it is commonly observed that deletion of a global chromatin regulator affects expression of only a limited subset ...

  13. Chromatin Structure and the Inheritance of Epigenetic Information

    PubMed Central

    Margueron, Raphaël; Reinberg, Danny

    2013-01-01

    Although it is widely accepted that the regulation of the chromatin landscape is pivotal to conveying epigenetic phenomena, it is still unclear how a defined chromatin domain is reproduced following replication and transmitted from one generation to another. Here we review multiple mechanisms that contribute to the inheritance of epigenetic information with emphasis on the recycling of old histones following replication, the requirement for a positive feedback loop, long-range gene interactions, and the complex network of trans-acting factors. PMID:20300089

  14. Chromatin modification by the RNA Polymerase II elongation complex.

    PubMed

    Tanny, Jason C

    2014-01-01

    Transcription elongation by RNA polymerase II (RNAP II) involves the coordinated action of numerous regulatory factors. Among these are chromatin-modifying enzymes, which generate a stereotypic and conserved pattern of histone modifications along transcribed genes. This pattern implies a precise coordination between regulators of histone modification and the RNAP II elongation complex. Here I review the pathways and molecular events that regulate co-transcriptional histone modifications. Insight into these events will illuminate the assembly of functional RNAP II elongation complexes and how the chromatin landscape influences their composition and function. PMID:25494544

  15. Chromatin modifications, epigenetics, and how protozoan parasites regulate their lives

    PubMed Central

    Croken, Matthew M.; Nardelli, Sheila C.; Kim, Kami

    2012-01-01

    Chromatin structure plays a vital role in epigenetic regulation of protozoan parasite gene expression. Epigenetic gene regulation impacts parasite virulence, differentiation and cell cycle control. Recent work in many laboratories has elucidated the functions of histone modifying proteins that regulate parasite gene expression by chemical modification of constituent nucleosomes. A major focus of investigation has been characterizing post-translational modifications (PTM) of histones and identifying the enzymes that are responsible. Despite conserved features and specificity common to all eukaryotes, parasite enzymes involved in chromatin modification have unique functions that regulate unique aspects of parasite biology. PMID:22480826

  16. CYTOGENETIC ABNORMALITY IN MAN—Wider Implications of Theories of Sex Chromatin Origin

    PubMed Central

    Miles, Charles P.

    1962-01-01

    Female nuclei may be identified by means of sex chromatin. In general the number of sex chromatin bodies is one less than the number of X chromosomes. An exception to this rule is a case of sex chromatin-positive XO Turner's syndrome. This case suggests the possibility of sex chromatin-positive XY males, and it may be evidence for chromosomal differentiation. PMID:14473851

  17. Dynamic binding of histone H1 to chromatin in living cells

    Microsoft Academic Search

    Tom Misteli; Akash Gunjan; Robert Hock; Michael Bustin; David T. Brown

    2000-01-01

    The linker histone H1 is believed to be involved in chromatin organization by stabilizing higher-order chromatin structure. Histone H1 is generally viewed as a repressor of transcription as it prevents the access of transcription factors and chromatin remodelling complexes to DNA. Determining the binding properties of histone H1 to chromatin in vivo is central to understanding how it exerts these

  18. Determination of the RNA-content in samples of isolated chromatin by circular dichroism measurements

    Microsoft Academic Search

    Holger Notbohm

    1988-01-01

    A method is presented to determine the RNA content in isolated chromatin. An increased elipticity at 260nm–290nm in the circular dichroism spectrum of chromatin is due to RNA released together with chromatin from the cell nuclei. Mixtures of chicken erythrocyte chromatin and pure 5s-RNA were used to reconstruct such spectra. From the obtained data an RNA content higher than 3–5%

  19. The role of chromatin proteins in DNA damage recognition and repair Mini-review

    Microsoft Academic Search

    Piotr Widlak; Monika Pietrowska; Joanna Lanuszewska

    2006-01-01

    The structure of chromatin is the major factor determining the rate and efficiency of DNA repair. Chromatin remodeling events\\u000a such as rearrangement of nucleosomes and higher order chromatin structures are indispensable features of repair processes.\\u000a During the last decade numerous chromatin proteins have been identified that preferentially bind to different types of DNA\\u000a damage. The HMGB proteins, which preferentially interact

  20. The Emerging Roles of ATP-Dependent Chromatin Remodeling Enzymes in Nucleotide Excision Repair

    PubMed Central

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

    2012-01-01

    DNA repair in eukaryotic cells takes place in the context of chromatin, where DNA, including damaged DNA, is tightly packed into nucleosomes and higher order chromatin structures. Chromatin intrinsically restricts accessibility of DNA repair proteins to the damaged DNA and impacts upon the overall rate of DNA repair. Chromatin is highly responsive to DNA damage and undergoes specific remodeling to facilitate DNA repair. How damaged DNA is accessed, repaired and restored to the original chromatin state, and how chromatin remodeling coordinates these processes in vivo, remains largely unknown. ATP-dependent chromatin remodelers (ACRs) are the master regulators of chromatin structure and dynamics. Conserved from yeast to humans, ACRs utilize the energy of ATP to reorganize packing of chromatin and control DNA accessibility by sliding, ejecting or restructuring nucleosomes. Several studies have demonstrated that ATP-dependent remodeling activity of ACRs plays important roles in coordination of spatio-temporal steps of different DNA repair pathways in chromatin. This review focuses on the role of ACRs in regulation of various aspects of nucleotide excision repair (NER) in the context of chromatin. We discuss current understanding of ATP-dependent chromatin remodeling by various subfamilies of remodelers and regulation of the NER pathway in vivo. PMID:23109894

  1. Human centromeric chromatin is a dynamic chromosomal domain that can spread

    E-print Network

    Sullivan, Beth A.

    Human centromeric chromatin is a dynamic chromosomal domain that can spread over noncentromeric DNA for review September 12, 2005) Human centromeres are specialized chromatin domains containing the centromeric dimethy- lated at lysine 4, distinguishing centromeric chromatin (CEN chro- matin) from flanking

  2. Effects of aluminum and other cations on the structure of brain and liver chromatin

    SciTech Connect

    Walker, P.R.; LeBlanc, J.; Sikorska, M. (National Research Council of Canada, Ottawa, Ontario)

    1989-05-02

    The reactivity of aluminum and several other divalent and trivalent metallic cations toward chromatin from rat brain and liver has been investigated. Two criteria are used to determine the relative reactivity of these cations toward chromatin. The first involves the ability of the ions to compact the chromatin fibers to the point where chromatin precipitates. The second criterion measures the ability of cations to interfere with the accessibility of exogenous structural probes (nucleases) to chromatin. Of the divalent cations tested, nickel, cobalt, zinc, cadmium, and mercury were the most reactive toward chromatin, on the basis of their ability to induce precipitation of chromatin in the micromolar concentration range. The divalent cations magnesium, calcium, copper, strontium, and barium were much less effective, although all cations precipitate chromatin if their concentration is increased. Of the trivalent cations tested, aluminum indium, and gallium were very effective precipitants, whereas iron and scandium were without effect at the concentrations tested. Of all the cations tested, aluminum was the most reactive. Aluminum's ability to alter the structure of chromatin was investigated further by testing its ability to interfere with nuclease accessibility. This test confirmed that aluminum does induce considerable changes in chromatin structure at micromolar concentrations. Furthermore, chromatin from cortical areas of the brain was much more sensitive to aluminum than chromatin from liver. These results are discussed in light of the known toxicity of these cations, with particular emphasis on the possible role of aluminum in Alzheimer's disease.

  3. The Adenomatous Polyposis Coli Protein Contributes to Normal Compaction of Mitotic Chromatin

    Microsoft Academic Search

    Dina Dikovskaya; Guennadi Khoudoli; Ian P. Newton; Gaganmeet S. Chadha; Daniel Klotz; Ashwat Visvanathan; Angus Lamond; Jason R. Swedlow; Inke S. Näthke

    2012-01-01

    The tumour suppressor Adenomatous Polyposis Coli (APC) is required for proper mitosis; however, the exact role of APC in mitosis is not understood. Using demembranated sperm chromatin exposed to meiotic Xenopus egg extract and HeLa cells expressing fluorescently labelled histones, we established that APC contributes to chromatin compaction. Sperm chromatin in APC-depleted Xenopus egg extract frequently formed tight round or

  4. The DNA damage response in chromatin: a focus on histone variants and heterochromatin proteins

    E-print Network

    1 The DNA damage response in chromatin: a focus on histone variants and heterochromatin in distinct levels of chromatin organization to integrate them as real players in the DNA damage response (DDR and contribute to the fine-tuning of damage signaling, DNA and chromatin repair. To take into account

  5. The Arabidopsis SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA Targets Directly to PINs and Is Required for Root Stem Cell Niche Maintenance.

    PubMed

    Yang, Songguang; Li, Chenlong; Zhao, Linmao; Gao, Sujuan; Lu, Jingxia; Zhao, Minglei; Chen, Chia-Yang; Liu, Xuncheng; Luo, Ming; Cui, Yuhai; Yang, Chengwei; Wu, Keqiang

    2015-06-01

    BRAHMA (BRM), a SWI/SNF chromatin remodeling ATPase, is essential for the transcriptional reprogramming associated with development and cell differentiation in Arabidopsis thaliana. In this study, we show that loss-of-function mutations in BRM led to defective maintenance of the root stem cell niche, decreased meristematic activity, and stunted root growth. Mutations of BRM affected auxin distribution by reducing local expression of several PIN-FORMED (PIN) genes in the stem cells and impaired the expression of the stem cell transcription factor genes PLETHORA (PLT1) and PLT2. Chromatin immunoprecipitation assays showed that BRM could directly target to the chromatin of PIN1, PIN2, PIN3, PIN4, and PIN7. In addition, genetic interaction assays indicate that PLTs acted downstream of BRM, and overexpression of PLT2 partially rescued the stem cell niche defect of brm mutants. Taken together, these results support the idea that BRM acts in the PLT pathway to maintain the root stem cell niche by altering the expression of PINs. PMID:25991732

  6. Soft Condensed Matter Biopolymers

    E-print Network

    Schüler, Axel

    Keywords Soft Condensed Matter Biopolymers Cell Elasticity Neuronal Networks Biomimetic properties of membranes and bi- opolymers. Current interests are fo- cused on the plasma membrane condensed matter. In the living cell, this matter is often far from equilib- rium and also behaving in a non

  7. Topoisomerase II: a fitted mechanism for the chromatin landscape

    PubMed Central

    Roca, Joaquim

    2009-01-01

    The mechanism by which type-2A topoisomerases transport one DNA duplex through a transient double-strand break produced in another exhibits fascinating traits. One of them is the fine coupling between inter-domainal movements and ATP usage; another is their preference to transport DNA in particular directions. These capabilities have been inferred from in vitro studies but we ignore their significance inside the cell, where DNA configurations markedly differ from those of DNA in free solution. The eukaryotic type-2A enzyme, topoisomerase II, is the second most abundant chromatin protein after histones and its biological roles include the decatenation of newly replicated DNA and the relaxation of polymerase-driven supercoils. Yet, topoisomerase II is also implicated in other cellular processes such as chromatin folding and gene expression, in which the topological transformations catalysed by the enzyme are uncertain. Here, some capabilities of topoisomerase II that might be relevant to infer the enzyme performance in the context of chromatin architecture are discussed. Some aspects addressed are the importance of the DNA rejoining step to ensure genome stability, the regulation of the enzyme activity and of its putative structural role, and the selectively of DNA transport in the chromatin milieu. PMID:19059997

  8. Vascular smooth muscle cell phenotypic plasticity: focus on chromatin remodelling

    PubMed Central

    Spin, Joshua M.; Maegdefessel, Lars; Tsao, Philip S.

    2012-01-01

    Differentiated vascular smooth muscle cells (SMCs) retain the capacity to modify their phenotype in response to inflammation or injury. This phenotypic switching is a crucial component of vascular disease, and is partly dependent on epigenetic regulation. An appreciation has been building in the literature for the essential role chromatin remodelling plays both in SMC lineage determination and in influencing changes in SMC behaviour and state. This process includes numerous chromatin regulatory elements and pathways such as histone acetyltransferases, deacetylases, and methyltransferases and other factors that act at SMC-specific marker sites to silence or permit access to the cellular transcriptional machinery and on other key regulatory elements such as myocardin and Kruppel-like factor 4 (KLF4). Various stimuli known to alter the SMC phenotype, such as transforming growth factor beta (TGF-?), platelet-derived growth factor (PDGF), oxidized phospholipids, and retinoic acid, appear to act in part through effects upon SMC chromatin structure. In recent years, specific covalent histone modifications that appear to establish SMC determinacy have been identified, while others alter the differentiation state. In this article, we review the mechanisms of chromatin remodelling as it applies to the SMC phenotype. PMID:22362814

  9. Nuclear body movement is determined by chromatin accessibility and dynamics

    E-print Network

    Rippe, Karsten

    Nuclear body movement is determined by chromatin accessibility and dynamics Sabine M. Go¨ risch) Promyelocytic leukemia (PML) and Cajal bodies are mobile sub- nuclear organelles, which are involved in understanding their biological functions is their mobility. The diffusion properties of PML and Cajal bodies

  10. Chromatin-bound RNA and the Neurobiology of Psychiatric Disease

    PubMed Central

    Tushir, Jogender Singh; Akbarian, Schahram

    2013-01-01

    A large, and still rapidly expanding literature on epigenetic regulation in the nervous system has provided fundamental insights into the dynamic regulation of DNA methylation and post-translational histone modifications in the context of neuronal plasticity in health and disease. Remarkably, however, very little is known about the potential role of chromatin-bound RNAs, including many long non-coding transcripts and various types of small RNAs. Here, we provide an overview on RNA-mediated regulation of chromatin structure and function, with focus on histone lysine methylation and psychiatric disease. Examples of recently discovered chromatin-bound long non-coding RNAs important for neuronal health and function include the Brain-derived Neurotrophic Factor antisense transcript (Bdnf-AS) which regulates expression of the corresponding sense transcript, and LOC389023 which is associated with human-specific histone methylation signatures at the chromosome 2q14.1 neurodevelopmental risk locus by regulating expression of DPP10, an auxillary subunit for voltage-gated K(+) channels. We predict that the exploration of chromatin-bound RNA will significantly advance our current knowledge base in neuroepigenetics and biological psychiatry. PMID:23831425

  11. RESEARCH ARTICLES Embryo and Endosperm Inherit Distinct Chromatin and

    E-print Network

    Paris-Sud XI, Université de

    RESEARCH ARTICLES Embryo and Endosperm Inherit Distinct Chromatin and Transcriptional States from that RNA POLYMERASE II is less active in the embryo than in the endosperm. This dimorphic pattern cell versus the central cell. Thus, distinct epigenetic and transcriptional patterns in the embryo

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

    PubMed Central

    Beneke, Sascha

    2012-01-01

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

  13. Chromatin in pluripotent embryonic stem cells and differentiation

    Microsoft Academic Search

    Eran Meshorer; Tom Misteli

    2006-01-01

    Embryonic stem (ES) cells are unique in that they are pluripotent and have the ability to self-renew. The molecular mechanisms that underlie these two fundamental properties are largely unknown. We discuss how unique properties of chromatin in ES cells contribute to the maintenance of pluripotency and the determination of differentiation properties.

  14. Chromatin multiprotein complexes involved in the maintenance of transcription patterns

    Microsoft Academic Search

    Valerio Orlando; Renato Paro

    1995-01-01

    In Drosophila, the maintenance of active and inactive patterns of gene expression during development involves the activity of two genetically complex systems. Molecular analysis of the components, apparently acting in large multiprotein complexes, has allowed a substantial advancement in our understanding of the role of chromatin higher order structures in gene regulation and nuclear organization. The Polycomb-group factors induce heterochromatin-like

  15. Chromatin fiber structure: morphology, molecular determinants, structural transitions.

    PubMed Central

    Zlatanova, J; Leuba, S H; van Holde, K

    1998-01-01

    Despite more than 20 years of research, the structure of the chromatin fiber and its molecular determinants remain enigmatic. Recent developments in high-resolution microscopic techniques, as well as the application of mathematical modeling to chromatin fiber structure, have allowed the acquisition of some new insights into the structure and its determinants. Here we present some of the newest data on the structure of the chromatin fiber in both its extended and compacted states, and bring together this new knowledge with older data in an attempt to provide a unified view of how chromatin components interact with each other to form its various conformations. The structural transitions that are believed to take place during transcriptional activation and its cessation are also discussed. It becomes obvious that despite some progress in our understanding of the fiber structure and its dynamics, huge gaps continue to exist. Bridging these gaps will require further improvements in already available techniques and the introduction of completely new approaches. PMID:9591681

  16. Influence of CpG islands on chromatin structure 

    E-print Network

    Wachter, Elisabeth

    2014-06-28

    CpG islands (CGIs) are short GC rich sequences with a high frequency of CpGs that are associated with the active chromatin mark H3K4me3. Most occur at gene promoters and are often free of cytosine methylation. Recent ...

  17. Coregulator-dependent facilitation of chromatin occupancy by GATA-1

    E-print Network

    Bresnick, Emery H.

    Coregulator-dependent facilitation of chromatin occupancy by GATA-1 Saumen Pal*, Alan B. Cantor-protein interactions, which regulate tran- scription. However, the mechanism by which the Friend of GATA (FOG) coregulator mediates GATA factor-dependent transcription is unknown. We showed previously that GATA-1 replaces

  18. Nucleosome Geometry and Internucleosomal Interactions Control the Chromatin Fiber Conformation

    Microsoft Academic Search

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

    2008-01-01

    Based on model structures with atomic resolution, a coarse-grained model for the nucleosome geometry was implemented. The dependence of the chromatin fiber conformation on the spatial orientation of nucleosomes and the path and length of the linker DNA was systematically explored by Monte Carlo simulations. Two fiber types were analyzed in detail that represent nucleosome chains without and with linker

  19. From DNA Sequence to Chromatin Dynamics: Computational Analysis of Transcriptional

    E-print Network

    Friedman, Nir

    factors with no such extensive data? In my dissertation, I developed a novel structure-based approach with the experimental group of Erin O'Shea (HHMI/Harvard), I developed a novel analytical approach where the expressionFrom DNA Sequence to Chromatin Dynamics: Computational Analysis of Transcriptional Regulation

  20. New insights into chromatin function in transcriptional control

    Microsoft Academic Search

    ALAN P. WOLFFE

    Transcription requires the recognition of numerous DNA sequences by diverse transcription fac- tors, which together assemble large nucleoprotein com- plexes that tether RNA polymerase and facilitate the in- itiation of RNA synthesis. In vivo the assembly of these transcription complexes occurs in a nuclear environment where the template DNA is compacted more than 103-fold through the assembly of chromatin. Our

  1. The size of the thyroid hormone receptor in chromatin

    SciTech Connect

    Gruol, D.J. (The Salk Institute, San Diego, CA); Kempner, E.S.

    1982-01-01

    We have used radiation inactivation and target theory to determine the size of the functional unit for T/sub 3/ binding in rat liver chromatin. The process involves exposure of frozen chromatin samples to a beam of high energy electrons produced in a linear accelerator and subsequent measurement of the residual capacity to bind hormone. Our experiments were carried out using three forms of solubilized chromatin: 1) sonicated, containing the receptor in fragments which sedimented faster than 30 S; 2) digested by nuclease, containing the receptor in a form which sedimented at 5-6 S; 3) digested by nuclease and made 0.5 M in KCl, containing the receptor in a form which sedimented at 3.8 S. We have shown that in each sample preparation the receptor retained the ability to bind T/sub 3/ with the same capacity and affinity that had previously been measured with high molecular weith chromatin. Irradiation caused a reduction in the capacity to bind T/sub 3/ but did not change the affinity of the remaining receptors for the hormone. In each preparation, the radiation resulted in a simple exponential loss of binding capacity with dose, indicating that a single target size was detected. Within the variation of the measurements, the target size for each form of the receptor was the same, 59,000 daltons.

  2. Tripartite organization of centromeric chromatin in budding yeast

    E-print Network

    Henikoff, Steven

    Tripartite organization of centromeric chromatin in budding yeast Kristina Krassovskya,b , Jorja G by nucleosomes containing the CenH3 histone variant, whereas in budding yeast, a 120-bp centromere DNA element this is the case in the budding yeast, Saccharomyces cerevisiae, where each of the 16 centromeres consists of a 120

  3. Frequent involvement of chromatin remodeler alterations in gastric field cancerization.

    PubMed

    Takeshima, Hideyuki; Niwa, Tohru; Takahashi, Takamasa; Wakabayashi, Mika; Yamashita, Satoshi; Ando, Takayuki; Inagawa, Yuki; Taniguchi, Hirokazu; Katai, Hitoshi; Sugiyama, Toshiro; Kiyono, Tohru; Ushijima, Toshikazu

    2015-02-01

    A field for cancerization, or a field defect, is formed by the accumulation of genetic and epigenetic alterations in normal-appearing tissues, and is involved in various cancers, especially multiple cancers. Epigenetic alterations are frequently present in chronic inflammation-exposed tissues, but information on individual genes involved in the formation of a field defect is still fragmental. Here, using non-cancerous gastric tissues of cancer patients, we isolated 16 aberrantly methylated genes, and identified chromatin remodelers ACTL6B and SMARCA1 as novel genes frequently methylated in non-cancerous tissues. SMARCA1 was expressed at high levels in normal gastric tissues, but was frequently silenced by aberrant methylation in gastric cancer cells. Moreover, somatic mutations of additional chromatin remodelers, such as ARID1A, SMARCA2, and SMARCA4, were found in 30% of gastric cancers. Mutant allele frequency suggested that the majority of cancer cells harbored a mutation when present. Depletion of a chromatin remodeler, SMARCA1 or SMARCA2, in cancer cell lines promoted their growth. These results showed that epigenetic and genetic alterations of chromatin remodelers are induced at an early stage of carcinogenesis and are frequently involved in the formation of a field defect. PMID:25462860

  4. Relative contributions of chromatin and kinetochores to mitotic spindle assembly

    PubMed Central

    Lon?arek, Jadranka; Kaláb, Petr; Khodjakov, Alexey

    2009-01-01

    During mitosis and meiosis in animal cells, chromosomes actively participate in spindle assembly by generating a gradient of Ran guanosine triphosphate (RanGTP). A high concentration of RanGTP promotes microtubule nucleation and stabilization in the vicinity of chromatin. However, the relative contributions of chromosome arms and centromeres/kinetochores in this process are not known. In this study, we address this issue using cells undergoing mitosis with unreplicated genomes (MUG). During MUG, chromatin is rapidly separated from the forming spindle, and both centrosomal and noncentrosomal spindle assembly pathways are active. MUG chromatin is coated with RCC1 and establishes a RanGTP gradient. However, a robust spindle forms around kinetochores/centromeres outside of the gradient peak. When stable kinetochore microtubule attachment is prevented by Nuf2 depletion in both MUG and normal mitosis, chromatin attracts astral microtubules but cannot induce spindle assembly. These results support a model in which kinetochores play the dominant role in the chromosome-mediated pathway of mitotic spindle assembly. PMID:19805628

  5. ATP-dependent chromatin remodeling: genetics, genomics and mechanisms

    PubMed Central

    Hargreaves, Diana C; Crabtree, Gerald R

    2011-01-01

    Macromolecular assemblies that regulate chromatin structure using the energy of ATP hydrolysis have critical roles in development, cancer, and stem cell biology. The ATPases of this family are encoded by 27 human genes and are usually associated with several other proteins that are stable, non-exchangeable subunits. One fundamental mechanism used by these complexes is thought to be the movement or exchange of nucleosomes to regulate transcription. However, recent genetic studies indicate that chromatin remodelers may also be involved in regulating other aspects of chromatin structure during many cellular processes. The SWI/SNF family in particular appears to have undergone a substantial change in subunit composition and mechanism coincident with the evolutionary advent of multicellularity and the appearance of linking histones. The differential usage of this greater diversity of mammalian BAF subunits is essential for the development of specific cell fates, including the progression from pluripotency to multipotency to committed neurons. Recent human genetic screens have revealed that BRG1, ARID1A, BAF155, and hSNF5 are frequently mutated in tumors, indicating that BAF complexes also play a critical role in the initiation or progression of cancer. The mechanistic bases underlying the genetic requirements for BAF and other chromatin remodelers in development and cancer are relatively unexplored and will be a focus of this review. PMID:21358755

  6. Chromatin-regulating proteins as targets for cancer therapy

    PubMed Central

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

    2014-01-01

    Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium butyrate and valproic acid) have been shown to sensitize cancer cells to ionizing irradiation in preclinical models, and some of these molecules are being tested in clinical trials, either alone or in combination with radiotherapy. Meanwhile, recent large-scale genome analyses have identified frequent mutations in genes encoding chromatin-regulating proteins, especially in those encoding subunits of the SWI/SNF chromatin-remodeling complex, in various human cancers. These observations have driven researchers toward development of targeted therapies against cancers carrying these mutations. DOT1L inhibition in MLL-rearranged leukemia, EZH2 inhibition in EZH2-mutant or MLL-rearranged hematologic malignancies and SNF5-deficient tumors, BRD4 inhibition in various hematologic malignancies, and BRM inhibition in BRG1-deficient tumors have demonstrated promising anti-tumor effects in preclinical models, and these strategies are currently awaiting clinical application. Overall, the data collected so far suggest that targeting chromatin-regulating proteins is a promising strategy for tomorrow's cancer therapy, including radiotherapy and molecularly targeted chemotherapy. PMID:24522270

  7. Human lymphoid translocation fragile zones are hypomethylated and have accessible chromatin.

    PubMed

    Lu, Zhengfei; Lieber, Michael R; Tsai, Albert G; Pardo, Carolina E; Müschen, Markus; Kladde, Michael P; Hsieh, Chih-Lin

    2015-04-01

    Chromosomal translocations are a hallmark of hematopoietic malignancies. CG motifs within translocation fragile zones (typically 20 to 600 bp in size) are prone to chromosomal translocation in lymphomas. Here we demonstrate that the CG motifs in human translocation fragile zones are hypomethylated relative to the adjacent DNA. Using a methyltransferase footprinting assay on isolated nuclei (in vitro), we find that the chromatin at these fragile zones is accessible. We also examined in vivo accessibility using cellular expression of a prokaryotic methylase. Based on this assay, which measures accessibility over a much longer time interval than is possible with in vitro methods, these fragile zones were found to be more accessible than the adjacent DNA. Because DNA within the fragile zones can be methylated by both cellular and exogenous methyltransferases, the fragile zones are predominantly in a duplex DNA conformation. These observations permit more-refined models for why these zones are 100- to 1,000-fold more prone to undergo chromosomal translocation than the adjacent regions. PMID:25624348

  8. Low angle x-ray diffraction studies of chromatin structure in vivo and in isolated nuclei and metaphase chromosomes

    PubMed Central

    Langmore, JP; Paulson

    1983-01-01

    Diffraction of x-rays from living cells, isolated nuclei, and metaphase chromosomes gives rise to several major low angle reflections characteristic of a highly conserved pattern of nucleosome packing within the chromatin fibers. We answer three questions about the x-ray data: Which reflections are characteristic of chromosomes in vivo? How can these reflections be preserved in vitro? What chromosome structures give rise to the reflections? Our consistent observation of diffraction peaks at 11.0, 6.0, 3.8, 2.7 and 2.1 nm from a variety of living cells, isolated nuclei, and metaphase chromosomes establishes these periodicities as characteristic of eukaryotic chromosomes in vivo. In addition, a 30-40- nm peak is observed from all somatic cells that have substantial amounts of condensed chromatin, and a weak 18-nm reflection is observed from nucleated erythrocytes. These observations provide a standard for judging the structural integrity of isolated nuclei, chromosomes, and chromatin, and thus resolve long standing controversy about the “tru” nature of chromosome diffraction. All of the reflection seen in vivo can be preserved in vitro provided that the proper ionic conditions are maintained. Our results show clearly that the 30-40-nm maximum is a packing reflection. The packing we observe in vivo is directly correlated to the side-by-side arrangement of 20- 30-nm fibers observed in thin sections of fixed and dehydrated cells and isolated chromosomes. This confirms that such packing is present in living cells and is not merely an artifact of electron microscopy. As expected, the packing reflection is shifted to longer spacings when the fibers are spread apart by reducing the concentration of divalent cations in vitro. Because the 18-, 11.0-, 6.0-, 3.8-, 2.7-, and 2.1-nm reflections are not affected by the decondensation caused by removal of divalent cations, these periodicities must reflect the internal structure of the chromaticn fibers. PMID:6682117

  9. Low angle x-ray diffraction studies of chromatin structure in vivo and in isolated nuclei and metaphase chromosomes.

    PubMed

    Langmore, J P; Paulson, J R

    1983-04-01

    Diffraction of x-rays from living cells, isolated nuclei, and metaphase chromosomes gives rise to several major low angle reflections characteristic of a highly conserved pattern of nucleosome packing within the chromatin fibers. We answer three questions about the x-ray data: Which reflections are characteristic of chromosomes in vivo? How can these reflections be preserved in vitro? What chromosome structures give rise to the reflections? Our consistent observation of diffraction peaks at 11.0, 6.0, 3.8, 2.7 and 2.1 nm from a variety of living cells, isolated nuclei, and metaphase chromosomes establishes these periodicities as characteristic of eukaryotic chromosomes in vivo. In addition, a 30-40- nm peak is observed from all somatic cells that have substantial amounts of condensed chromatin, and a weak 18-nm reflection is observed from nucleated erythrocytes. These observations provide a standard for judging the structural integrity of isolated nuclei, chromosomes, and chromatin, and thus resolve long standing controversy about the "tru" nature of chromosome diffraction. All of the reflection seen in vivo can be preserved in vitro provided that the proper ionic conditions are maintained. Our results show clearly that the 30-40-nm maximum is a packing reflection. The packing we observe in vivo is directly correlated to the side-by-side arrangement of 20- 30-nm fibers observed in thin sections of fixed and dehydrated cells and isolated chromosomes. This confirms that such packing is present in living cells and is not merely an artifact of electron microscopy. As expected, the packing reflection is shifted to longer spacings when the fibers are spread apart by reducing the concentration of divalent cations in vitro. Because the 18-, 11.0-, 6.0-, 3.8-, 2.7-, and 2.1-nm reflections are not affected by the decondensation caused by removal of divalent cations, these periodicities must reflect the internal structure of the chromaticn fibers. PMID:6682117

  10. A proteomics approach for the identification of nucleophosmin and heterogeneous nuclear ribonucleoprotein C1/C2 as chromatin-binding proteins in response to DNA double-strand breaks

    PubMed Central

    Lee, Seung Yun; Park, Ji-Hye; Kim, Sungsu; Park, Eun-Jung; Yun, Yungdae; Kwon, Jongbum

    2005-01-01

    Double-strand breaks (DSBs) of chromosomal DNA trigger the cellular response that activates the pathways for DNA repair and cell-cycle checkpoints, and sometimes the pathways leading to cell death if the damage is too severe to be tolerated. Evidence indicates that, upon generation of DNA DSBs, many nuclear proteins that are involved in DNA repair and checkpoints are recruited to chromatin around the DNA lesions. In the present study we used a proteomics approach to identify DNA-damage-induced chromatin-binding proteins in a systematic way. Two-dimensional gel analysis for protein extracts of chromatin from DNA-damage-induced and control HeLa cells identified four proteins as the candidates for DNA-damage-induced chromatin-binding proteins. MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS analysis identified these proteins to be NPM (nucleophosmin), hnRNP (heterogeneous nuclear ribonucleoprotein) C1, hnRNP C2 and 37-kDa laminin-receptor precursor, and the identity of these proteins was further confirmed by immunoblot analysis with specific antibodies. We then demonstrated with chromatin-binding assays that NPM and hnRNP C1/C2, the abundant nuclear proteins with pleiotropic functions, indeed bind to chromatin in a DNA-damage-dependent manner, implicating these proteins in DNA repair and/or damage response. Immunofluorescence experiments showed that NPM, normally present in the nucleoli, is mobilized into the nucleoplasm after DNA damage, and that neither NPM nor hnRNP C1/C2 is actively recruited to the sites of DNA breaks. These results suggest that NPM and hnRNP C1/C2 may function at the levels of the global context of chromatin, rather than by specifically targeting the broken DNA. PMID:15737070

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  12. Distribution of histone variants in the sea urchin chromatin fractions obtained by selective micrococcal nuclease digestion.

    PubMed

    Jasinskiene, N E; Jasinskas, A L; Gineitis, A A

    1985-10-01

    Chromatin fractions differing in their transcriptional activity were isolated by selective micrococcal nuclease digestion of nuclei from sea urchin embryos (Strongylocentrotus droebachiensis) at the gastrula and pluteus stage. The electrophoretic analysis of the chromatin proteins at the gastrula stage showed that a soluble, transcriptionally active fraction of chromatin was enriched with early variants of histones H1 and H2A. The early and late variants of histone H2A at the pluteus stage were distributed randomly between chromatin fractions. However, the content of both variants of histone H1 was essentially decreased in the soluble transcriptionally active fraction of chromatin. PMID:4069105

  13. Epigenetic modifications, chromatin distribution and TP53 transcription in a model of breast cancer progression.

    PubMed

    Santos, Gilson C; da Silva, Ana P A; Feldman, Lucas; Ventura, Grasiella M; Vassetzky, Yegor; de Moura Gallo, Claudia V

    2015-04-01

    In the present paper we aimed to characterize epigenetic aspects and analyze TP53 transcription in the 21?T series, composed of breast cell lines: non-cancerous H16N2; Atypical Ductal Hyperplasia 21PT; Ductal Carcinoma in situ 21NT and Invasive Metastatic Carcinoma 21MT1. We detected a global genomic hypomethylation in 21NT and 21MT1. The histone modification markers analysis showed an important global decrease of the active chromatin mark H4Ac in 21MT1 relative to the other cell lines while the repressive mark H3K9Me3 were not significantly altered. The mRNA levels of DNA methylation and histone modification key enzymes are consistent with the observed genomic hypomethylation and histone hypoacetylation. The expression of DNMT3A/B increased at the initial stages of oncogenesis and the expression of DNMT1 and HAT1 decreased at the advanced stages of breast cancer. Using a confocal immunofluorescent assay, we observed that H4Ac was mostly located at the periphery and the repressive mark H3K9Me3, at the center of 21NT and 21MT1 cells nuclei. TP53 P1 promoter was found to be in an open chromatin state, with a relatively high enrichment of H4Ac and similar TP53 transcription levels in all 21?T cell lines. In conclusion, we observed epigenetic alterations (global genome hypomethylation, global hypoacetylation and accumulation of pericentric heterochromatin) in metastatic breast cancer cells of the 21?T series. These alterations may act at later stages of breast cancer progression and may not affect TP53 transcription at the P1 promoter. PMID:25358520

  14. Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens).

    PubMed

    Jenkins, J A; Draugelis-Dale, R O; Pinkney, A E; Iwanowicz, L R; Blazer, V S

    2015-03-15

    Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin-fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa. PMID:25559842

  15. Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens)

    USGS Publications Warehouse

    Jenkins, Jill A.; Draugelis-Dale, Rassa O.; Pinkney, Alfred E.; Iwanowicz, Luke R.; Blazer, Vicki S.

    2015-01-01

    Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin–fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  17. The chromatin remodelers RSC and ISW1 display functional and chromatin-based promoter antagonism.

    PubMed

    Parnell, Timothy J; Schlichter, Alisha; Wilson, Boris G; Cairns, Bradley R

    2015-01-01

    ISWI family chromatin remodelers typically organize nucleosome arrays, while SWI/SNF family remodelers (RSC) typically disorganize and eject nucleosomes, implying an antagonism that is largely unexplored in vivo. Here, we describe two independent genetic screens for rsc suppressors that yielded mutations in the promoter-focused ISW1a complex or mutations in the 'basic patch' of histone H4 (an epitope that regulates ISWI activity), strongly supporting RSC-ISW1a antagonism in vivo. RSC and ISW1a largely co-localize, and genomic nucleosome studies using rsc isw1 mutant combinations revealed opposing functions: promoters classified with a nucleosome-deficient region (NDR) gain nucleosome occupancy in rsc mutants, but this gain is attenuated in rsc isw1 double mutants. Furthermore, promoters lacking NDRs have the highest occupancy of both remodelers, consistent with regulation by nucleosome occupancy, and decreased transcription in rsc mutants. Taken together, we provide the first genetic and genomic evidence for RSC-ISW1a antagonism and reveal different mechanisms at two different promoter architectures. PMID:25821983

  18. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K. (Pleasanton, CA); Snyderman, Neal J. (Berkeley, CA); Rowland, Mark S. (Alamo, CA)

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  19. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K. (Pleasanton, CA); Snyderman, Neal J. (Berkeley, CA); Rowland, Mark S. (Alamo, CA)

    2012-05-15

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  20. Histone H2B ubiquitylation disrupts local and higher order chromatin compaction

    PubMed Central

    Fierz, Beat; Chatterjee, Champak; McGinty, Robert K.; Bar-Dagan, Maya; Raleigh, Daniel P.; Muir, Tom W.

    2010-01-01

    Regulation of chromatin structure involves histone post-translational modifications which can modulate intrinsic properties of the chromatin fiber to change the chromatin state. We used chemically defined nucleosome arrays to demonstrate that H2B ubiquitylation (uH2B), a modification associated with transcription, interferes with chromatin compaction and leads to an open and biochemically accessible fiber conformation. Importantly, these effects were specific for ubiquitin, as compaction of chromatin modified with a similar ubiquitin-sized protein, Hub1, was only weakly affected. Applying a fluorescence based method we found that uH2B acts through a mechanism distinct from H4 tail acetylation (acH4), a modification known to disrupt chromatin folding. Finally, incorporation of both uH2B and acH4 in nucleosomes resulted in synergistic inhibition of higher order chromatin structure formation, possibly a result of their distinct mode of action. PMID:21196936

  1. Chromatin analyses of Zymoseptoria tritici: Methods for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq).

    PubMed

    Soyer, Jessica L; Möller, Mareike; Schotanus, Klaas; Connolly, Lanelle R; Galazka, Jonathan M; Freitag, Michael; Stukenbrock, Eva H

    2015-06-01

    The presence or absence of specific transcription factors, chromatin remodeling machineries, chromatin modification enzymes, post-translational histone modifications and histone variants all play crucial roles in the regulation of pathogenicity genes. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) provides an important tool to study genome-wide protein-DNA interactions to help understand gene regulation in the context of native chromatin. ChIP-seq is a convenient in vivo technique to identify, map and characterize occupancy of specific DNA fragments with proteins against which specific antibodies exist or which can be epitope-tagged in vivo. We optimized existing ChIP protocols for use in the wheat pathogen Zymoseptoria tritici and closely related sister species. Here, we provide a detailed method, underscoring which aspects of the technique are organism-specific. Library preparation for Illumina sequencing is described, as this is currently the most widely used ChIP-seq method. One approach for the analysis and visualization of representative sequence is described; improved tools for these analyses are constantly being developed. Using ChIP-seq with antibodies against H3K4me2, which is considered a mark for euchromatin or H3K9me3 and H3K27me3, which are considered marks for heterochromatin, the overall distribution of euchromatin and heterochromatin in the genome of Z. tritici can be determined. Our ChIP-seq protocol was also successfully applied to Z. tritici strains with high levels of melanization or aberrant colony morphology, and to different species of the genus (Z. ardabiliae and Z. pseudotritici), suggesting that our technique is robust. The methods described here provide a powerful framework to study new aspects of chromatin biology and gene regulation in this prominent wheat pathogen. PMID:25857259

  2. 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

  3. CPF-associated phosphatase activity opposes condensin-mediated chromosome condensation.

    PubMed

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

    2014-06-01

    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 PP1(Dis2) 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

  4. 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.

  5. Mechanism of dropwise condensation

    E-print Network

    Umur, Aydin

    1963-01-01

    From a study of surface phenomena, information is obtained about conditions under which net condensation can occur. An experimental examination of the surface, using an optical method capable of detecting thin films of ...

  6. Rapid mercury assays

    SciTech Connect

    Szurdoki, S.; Kido, H.; Hammock, B.D. [Univ. of California, Davis, CA (United States)

    1996-10-01

    We have developed rapid assays with the potential of detecting mercury in environmental samples. our methods combine the simple ELISA-format with the selective, high affinity complexation of mercuric ions by sulfur-containing ligands. The first assay is based on a sandwich chelate formed by a protein-bound ligand immobilized on the wells of a microliter plate, mercuric ion of the analyzed sample, and another ligand conjugated to a reporter enzyme. The second assay involves competition between mercuric ions and an organomercury-conjugate to bind to a chelating conjugate. Several sulfur containing chelators (e.g., dithiocarbamates) and organomercurials linked to macromolecular carriers have been investigated in these assay formats. The assays detect mercuric ions in ppb/high ppt concentrations with high selectivity.

  7. CYTOKINE-INDUCED CHROMATIN MODIFICATIONS OF THE TYPE I COLLAGEN ALPHA 2 GENE DURING INTESTINAL ENDOTHELIAL-TO-MESENCHYMAL TRANSITION

    PubMed Central

    Sadler, Tammy; Scarpa, Melania; Rieder, Florian; West, Gail; Stylianou, Eleni

    2013-01-01

    Background Fibrosis of the intestine is currently an irreversible complication of Inflammatory Bowel Disease yet little is understood of the underlying pathogenesis and anti-fibrotic strategies remain elusive. To develop effective therapies, knowledge of the mechanism of transcription and excessive deposition of type I collagen - a hallmark of fibrosis, is needed. We have shown previously that endothelial-to-mesenchymal transition (EndoMT) contributes to the pool of intestinal fibrotic cells and that a cytokine cocktail (IL1-?, TNF-? and TGF-?) induces Collagen I alpha 2 (COL1A2) mRNA and protein. Methods Chromatin immunoprecipitation assays on pure cultures of human intestinal mucosal endothelial cells undergoing EndoMT were performed with antibodies to specific histone modifications and RNA polymerase II. RT-PCR was used to quantify the levels of Col1A2 and endothelial specific von Willebrand factor (vWF) mRNA. Results We show that cytokines induce selective chromatin modifications (histone 4 hyperacetylation and hypermethylation of histone 3) and phosphorylated RNA polymerase II at the COL1A2 promoter. Hypoacetylated and hypomethylated histone 3 was detected on the repressed vWF gene. Prolonged exposure to cytokines (16 days) retained hyperacetylation of select lysines in H4 on the COL1A2 promoter. Removal of cytokines after 16 days and continued culture for 10 days, showed persistent hyperacetylation at lysine 16 in histone H4. Conclusion This is the first study to show that COL1A2 gene expression is associated with cytokine-induced, temporally ordered and persistent chromatin modifications and suggests that these are important determinants of gene expression in EndoMT and intestinal fibrosis. PMID:23635716

  8. The epigenetic signature of CFTR expression is co-ordinated via chromatin acetylation through a complex intronic element.

    PubMed

    Paul, Thankam; Li, SiDe; Khurana, Sanjeev; Leleiko, Neal S; Walsh, Martin J

    2007-12-15

    The CFTR (cystic fibrosis transmembrane conductance regulator) gene is a tightly regulated and differentially expressed transcript in many mucosal epithelial cell types. It appears that DNA sequence variations alone do not explain CFTR-related gastrointestinal disease patterns and that epigenetic modifiers influence CFTR expression. Our aim was to characterize the native chromatin environment in cultured cells for intestinal CFTR expression by determining the relationship between histone acetylation and occupation of CFTR by multiple transcription factors, through a common regulatory element. We used HDAC (histone deacetylase) inhibition and ChIP (chromatin immunoprecipitation) analyses to define regions associated with acute acetylation of histone at the CFTR locus. We identified a region within the first intron associated with acute acetylation of histone H4 as an epigenetic signature corresponding to an intestine-specific enhancer element for CFTR. DHS (DNase I-hypersensitivity) assays and ChIP were used to specify control elements and occupation by regulatory factors. Quantitative ChIP procedures indicate that HNF1alpha (hepatic nuclear factor 1alpha) and Cdx2 (caudal homeobox protein 2) occupy and regulate through a novel intronic enhancer element of CFTR and that Tcf4 (T-cell factor 4) overlaps the same DNA element. RNAi (RNA interference) of Tcf4 and HNF1alpha decreased intestinal cell CFTR expression, identifying these as positive regulatory factors and CFTR as a target for Wnt signalling. We have linked the acetylation signature of nucleosomal histones to active intestinal CFTR expression and occupation by transcription factors HNF1alpha, Cdx2 and Tcf4 which converge to modify chromatin architecture. These studies suggest the therapeutic potential of histone modification strategies, such as inhibition of HDAC activity, to treat CFTR-associated disease by selectively enhancing CFTR expression. PMID:17848139

  9. CPTAC Assay Portal: a repository of targeted proteomic assays

    SciTech Connect

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Rodriguez, Henry; Abbateillo, Susan E.; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Paulovich, Amanda G.

    2014-06-27

    To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigators to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.

  10. Chromatin assembly in a yeast whole-cell?extract

    PubMed Central

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

    1997-01-01

    A simple in vitro system that supports chromatin assembly was developed for Saccharomyces cerevisiae. The assembly reaction is ATP-dependent, uses soluble histones and assembly factors, and generates physiologically spaced nucleosomes. We analyze the pathway of histone recruitment into nucleosomes, using this system in combination with genetic methods for the manipulation of yeast. This analysis supports the model of sequential recruitment of H3/H4 tetramers and H2A/H2B dimers into nucleosomes. Using a similar approach, we show that DNA ligase I can play an important role in template repair during assembly. These studies demonstrate the utility of this system for the combined biochemical and genetic analysis of chromatin assembly in yeast. PMID:9256430

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

    PubMed Central

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

    2014-01-01

    Summary The genetic architecture of autism spectrum disorder involves the interplay of common and rare variation and their impact on hundreds of genes. Using exome sequencing, analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, and a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic, transcriptional, and chromatin remodeling pathways. These include voltage-gated ion channels regulating propagation of action potentials, pacemaking, and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodelers, prominently histone post-translational modifications involving lysine methylation/demethylation. PMID:25363760

  12. Radiation-induced DNA damage and chromatin structure

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  13. Spectacle: fast chromatin state annotation using spectral learning.

    PubMed

    Song, Jimin; Chen, Kevin C

    2015-01-01

    Epigenomic data from ENCODE can be used to associate specific combinations of chromatin marks with regulatory elements in the human genome. Hidden Markov models and the expectation-maximization (EM) algorithm are often used to analyze epigenomic data. However, the EM algorithm can have overfitting problems in data sets where the chromatin states show high class-imbalance and it is often slow to converge. Here we use spectral learning instead of EM and find that our software Spectacle overcame these problems. Furthermore, Spectacle is able to find enhancer subtypes not found by ChromHMM but strongly enriched in GWAS SNPs. Spectacle is available at https://github.com/jiminsong/Spectacle. PMID:25786205

  14. Defining the replication program through the chromatin landscape

    PubMed Central

    Ding, Queying; MacAlpine, David M.

    2011-01-01

    DNA replication is an essential cell cycle event required for the accurate and timely duplication of the chromosomes. It is essential that the genome is replicated accurately and completely within the confines of S-phase. Failure to completely copy the genome has the potential to result in catastrophic genomic instability. Replication initiates in a coordinated manner from multiple locations, termed origins of replication, distributed across each of the chromosomes. The selection of these origins of replication is a dynamic process responding to both developmental and tissue specific signals. In this review, we explore the role of the local chromatin environment in regulating the DNA replication program at the level of origin selection and activation. Finally, there is increasing molecular evidence that the DNA replication program itself affects the chromatin landscape, suggesting that DNA replication is critical for both genetic and epigenetic inheritance. PMID:21417598

  15. Causes of death in X chromatin positive males (Klinefelter's syndrome).

    PubMed Central

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

    1985-01-01

    The causes of death in 466 X chromatin positive males (Klinefelter's syndrome) studied prospectively over the last 25 years have been analysed. We have previously reported the overall mortality to be increased by 50% and life expectancy reduced by about five years. A highly significant increase in mortality from cerebrovascular disease was observed in the sub group considered to be most representative of X chromatin positive males in general. In the age group up to 45 years this increase could be attributed to deaths from subarachnoid haemorrhage. An increase in mortality from respiratory diseases was observed in those ascertained in psychiatric hospitals. In the sample as a whole there were small but highly significant numbers of deaths from carcinoma of the breast and aortic valve disease. The deaths from carcinoma of the breast were comparable with those expected if female mortality rates were applied. PMID:4086964

  16. A dynamic ubiquitin equilibrium couples proteasomal activity to chromatin remodeling

    PubMed Central

    Dantuma, Nico P.; Groothuis, Tom A.M.; Salomons, Florian A.; Neefjes, Jacques

    2006-01-01

    Protein degradation, chromatin remodeling, and membrane trafficking are critically regulated by ubiquitylation. The presence of several coexisting ubiquitin-dependent processes, each of crucial importance to the cell, is remarkable. This brings up questions on how the usage of this versatile regulator is negotiated between the different cellular processes. During proteotoxic stress, the accumulation of ubiquitylated substrates coincides with the depletion of ubiquitylated histone H2A and chromatin remodeling. We show that this redistribution of ubiquitin during proteotoxic stress is a direct consequence of competition for the limited pool of free ubiquitin. Thus, the ubiquitin cycle couples various ubiquitin-dependent processes because of a rate-limiting pool of free ubiquitin. We propose that this ubiquitin equilibrium may allow cells to sense proteotoxic stress in a genome-wide fashion. PMID:16606690

  17. Chromatin Immunoprecipitation to Characterize the Epigenetic Profiles of Imprinted Domains

    PubMed Central

    Singh, Purnima; Szabó, Piroska E.

    2013-01-01

    Summary Imprinted genes are marked by parental allele specific DNA methylation and histone modifications which regulate their monoallelic expression. Chromatin immunoprecipitation (ChIP) is the technique of choice to characterize the histones associated with either the maternal or paternal chromosomes. To study allele-specific chromatin composition at imprinted regions, the method has to be efficient to work on limiting amount of starting material, and specific enough to recognize one of the parental alleles. We optimized the commonly used ChIP technique for efficient recovery of one parental allele from small number of cells. We provide examples to show that this ChIP protocol can specifically distinguish between parental alleles in mouse embryo fibroblasts carrying maternal and paternal duplication of mouse distal Chr7 and also in normal mouse embryo fibroblasts carrying single nucleotide polymorphism at imprinted regions. PMID:22907496

  18. Models of chromatin spatial organisation in the cell nucleus

    NASA Astrophysics Data System (ADS)

    Nicodemi, Mario

    2014-03-01

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

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

    PubMed Central

    Boivin, A; Dura, J M

    1998-01-01

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

  20. Condensate System Troubleshooting and Optimization 

    E-print Network

    Jenkins, B. V.

    1983-01-01

    , some of the carbon dioxide dissolves in the conden sate. This forms carbonic acid. Carbonic acid, being a weak acid, will cause four characteristic problems: The pH of the condensate will drop Dissolved iron content of the condensate... will increase Total dissolved solids content of the condensate will also increase A trough-like thinning of the bottom of the condensate pipe occurs. (Fig. 1) Figure 1 - Carbonic Acid Attack on Condensate Piping What is the impact? Iron is dissolved...

  1. The SAF-box domain of chromatin protein DEK

    Microsoft Academic Search

    Friederike Bohm; Ferdinand Kappes; Ingo Scholten; Nicole Richter; Hiroshi Matsuo; Rolf Knippers; Tanja Waldmann

    2005-01-01

    DEK is an abundant chromatin protein in metazoans reaching copy numbers of several millions\\/nucleus. Previous work has shown that human DEK, a protein of 375 amino acids, has two functional DNA-binding domains, of which one resides in a central part of the molecule and contains sequences corresponding to the scaffold attachment factor-box (SAF-box) domain as found in a growing number

  2. Coregulator-dependent facilitation of chromatin occupancy by GATA1

    Microsoft Academic Search

    Saumen Pal; Alan B. Cantor; Kirby D. Johnson; Tyler B. Moran; Meghan E. Boyer; Stuart H. Orkin; Emery H. Bresnick

    2004-01-01

    Coregulator recruitment by DNA-bound factors results in chromatin modification and protein-protein interactions, which regulate transcription. However, the mechanism by which the Friend of GATA (FOG) coregulator mediates GATA factor-dependent transcription is unknown. We showed previously that GATA-1 replaces GATA-2 at an upstream region of the GATA-2 locus, and that this GATA switch represses GATA-2. Genetic complementation analysis in FOG-1-null hematopoietic

  3. Conserved Organization of Centromeric Chromatin in Flies and Humans

    Microsoft Academic Search

    Michael D. Blower; Beth A. Sullivan; Gary H. Karpen

    2002-01-01

    Recent studies have highlighted the importance of centromere-specific histone H3-like (CENP-A) proteins in centromere function. We show that Drosophila CID and human CENP-A appear at metaphase as a three-dimensional structure that lacks histone H3. However, blocks of CID\\/CENP-A and H3 nucleosomes are linearly interspersed on extended chromatin fibers, and CID is close to H3 nucleosomes in polynucleosomal preparations. When CID

  4. MYC recruits the TIP60 histone acetyltransferase complex to chromatin

    Microsoft Academic Search

    Scott R. Frank; Tiziana Parisi; Stefan Taubert; Paula Fernandez; Miriam Fuchs; Ho-Man Chan; David M. Livingston; Bruno Amati

    2003-01-01

    The transcription factor MYC binds specific DNA sites in cellular chromatin and induces the acetylation of histones H3 and H4. However, the histone acetyltransferases (HATs) that are responsible for these modifications have not yet been identified. MYC associates with TRRAP, a subunit of distinct macromolecular complexes that contain the HATs GCN5\\/PCAF or TIP60. Although the association of MYC with GCN5

  5. Characterization of brain cell nuclei with decondensed chromatin.

    PubMed

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

    2015-07-01

    Although multipotent cell types have enlarged nuclei with decondensed chromatin, this property has not been exploited to enhance the characterization of neural progenitor cell (NPC) populations in the brain. We found that mouse brain cell nuclei that expressed exceptionally high levels of the pan neuronal marker NeuN/FOX3 (NeuN-High) had decondensed chromatin relative to most NeuN-Low or NeuN-Neg (negative) nuclei. Purified NeuN-High nuclei expressed significantly higher levels of transcripts encoding markers of neurogenesis, neuroplasticity, and learning and memory (ARC, BDNF, ERG1, HOMER1, NFL/NEF1, SYT1), subunits of chromatin modifying machinery (SIRT1, HDAC1, HDAC2, HDAC11, KAT2B, KAT3A, KAT3B, KAT5, DMNT1, DNMT3A, Gadd45a, Gadd45b) and markers of NPC and cell cycle activity (BRN2, FOXG1, KLF4, c-MYC, OCT4, PCNA, SHH, SOX2) relative to neuronal NeuN-Low or to mostly non-neuronal NeuN-Neg nuclei. NeuN-High nuclei expressed higher levels of HDAC1, 2, 4, and 5 proteins. The cortex, hippocampus, hypothalamus, thalamus, and nucleus accumbens contained high percentages of large decondensed NeuN-High nuclei, while the cerebellum, and pons contained very few. NeuN-High nuclei have the properties consistent with their being derived from extremely active neurons with elevated rates of chromatin modification and/or NPC-like cells with multilineage developmental potential. The further analysis of decondensed neural cell nuclei should provide novel insights into neurobiology and neurodegenerative disease. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 738-756, 2015. PMID:25369517

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

    PubMed

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

    1978-05-01

    Laser light scattering measurements have been made on a series of polynucleosomes containing from 50 to 150 nucleosomes. Radii of gyration have been determined as a function of polynucleosome length for different ionic strength solutions. The results suggest that at low ionic strength the chromatin adopts a loosely helical structure rather than a random coil. The helix becomes more regular on increasing the ionic strength, the dimension resembling those proposed by Finch and Klug for their solenoid model. PMID:662693

  7. Chromatin signature of embryonic pluripotency is established during genome activation

    PubMed Central

    Vastenhouw, Nadine L.; Zhang, Yong; Woods, Ian G.; Imam, Farhad; Regev, Aviv; Liu, X. Shirley; Rinn, John; Schier, Alexander F.

    2010-01-01

    After fertilization the embryonic genome is inactive until transcription is initiated during the maternal-zygotic transition1,2,3. This transition coincides with the formation of pluripotent cells, which in mammals can be used to generate embryonic stem cells. To study the changes in chromatin structure that accompany pluripotency and genome activation, we mapped the genomic locations of histone H3 molecules bearing Lysine trimethylation modifications before and after the maternal-zygotic transition in zebrafish. Trimethylation of Lysine 27, which is repressive, and trimethylation of Lysine 4, which is activating, were not detected before the transition. After genome activation, more than 80% of genes were marked by Lysine 4 trimethylation, including many inactive developmental regulatory genes that were also marked by Lysine 27 trimethylation. Sequential chromatin immunoprecipitation demonstrated that the same promoter regions had both trimethylation marks. Such bivalent chromatin domains also exist in embryonic stem cells and are thought to poise genes for activation while keeping them repressed4,5,6,7,8. In addition, we found many inactive genes that were uniquely marked by Lysine 4 trimethylation. Despite this activating modification, these monovalent genes were neither expressed nor stably bound by RNA polymerase II. Inspection of published datasets revealed similar monovalent domains in embryonic stem cells. Moreover, Lysine 4 trimethylation marks could form in the absence of both sequence-specific transcriptional activators and stable association of RNA pol II, as indicated by the analysis of an inducible transgene. These results suggest that bivalent and monovalent domains might poise embryonic genes for activation and that the chromatin profile associated with pluripotency is established during the maternal-zygotic transition. PMID:20336069

  8. Chromatin Architecture and the Generation of Antigen Receptor Diversity

    PubMed Central

    Jhunjhunwala, Suchit; van Zelm, Menno C.; Peak, Mandy; Murre, Cornelis

    2009-01-01

    The adaptive immune system generates a specific response to a vast spectrum of antigens. This remarkable property is achieved by lymphocytes that each express single and unique antigen receptors. During lymphocyte development, antigen receptor coding elements are assembled from widely dispersed gene segments. The assembly of antigen receptors is controlled at multiple levels, including epigenetic marking, nuclear location, and chromatin topology. Here we review recently uncovered mechanisms that underpin long-range genomic interactions and the generation of antigen receptor diversity. PMID:19665968

  9. Chromatin remodeling during Saccharomyces cerevisiae ADH2 gene activation.

    PubMed Central

    Verdone, L; Camilloni, G; Di Mauro, E; Caserta, M

    1996-01-01

    We have analyzed at both low and high resolution the distribution of nucleosomes over the Saccharomyces cerevisiae ADH2 promoter region in its chromosomal location, both under repressing (high-glucose) conditions and during derepression. Enzymatic treatments (micrococcal nuclease and restriction endonucleases) were used to probe the in vivo chromatin structure during ADH2 gene activation. Under glucose-repressed conditions, the ADH2 promoter was bound by a precise array of nucleosomes, the principal ones positioned at the RNA initiation sites (nucleosome +1), at the TATA box (nucleosome -1), and upstream of the ADR1-binding site (UAS1) (nucleosome -2). The UAS1 sequence and the adjacent UAS2 sequence constituted a nucleosome-free region. Nucleosomes -1 and +1 were destabilized soon after depletion of glucose and had become so before the appearance of ADH2 mRNA. When the transcription rate was high, nucleosomes -2 and +2 also underwent rearrangement. When spheroplasts were prepared from cells grown in minimal medium, detection of this chromatin remodeling required the addition of a small amount of glucose. Cells lacking the ADR1 protein did not display any of these chromatin modifications upon glucose depletion. Since the UAS1 sequence to which Adr1p binds is located immediately upstream of nucleosome -1, Adr1p is presumably required for destabilization of this nucleosome and for aiding the TATA-box accessibility to the transcription machinery. PMID:8628264

  10. Combinatorial epigenetic patterns as quantitative predictors of chromatin biology

    PubMed Central

    2014-01-01

    Background Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is the most widely used method for characterizing the epigenetic states of chromatin on a genomic scale. With the recent availability of large genome-wide data sets, often comprising several epigenetic marks, novel approaches are required to explore functionally relevant interactions between histone modifications. Computational discovery of "chromatin states" defined by such combinatorial interactions enabled descriptive annotations of genomes, but more quantitative approaches are needed to progress towards predictive models. Results We propose non-negative matrix factorization (NMF) as a new unsupervised method to discover combinatorial patterns of epigenetic marks that frequently co-occur in subsets of genomic regions. We show that this small set of combinatorial "codes" can be effectively displayed and interpreted. NMF codes enable dimensionality reduction and have desirable statistical properties for regression and classification tasks. We demonstrate the utility of codes in the quantitative prediction of Pol2-binding and the discrimination between Pol2-bound promoters and enhancers. Finally, we show that specific codes can be linked to molecular pathways and targets of pluripotency genes during differentiation. Conclusions We have introduced and evaluated a new computational approach to represent combinatorial patterns of epigenetic marks as quantitative variables suitable for predictive modeling and supervised machine learning. To foster widespread adoption of this method we make it available as an open-source software-package – epicode at https://github.com/mcieslik-mctp/epicode. PMID:24472558

  11. Chromatin structures: dissecting their mixed patterns in nuclease digests.

    PubMed Central

    Drinkwater, R D; Wilson, P J; Skinner, J D; Burgoyne, L A

    1987-01-01

    Four separate features could be distinguished in Fe-DNAase-1 digestions of human lymphoblast nuclei: a di-nucleosomal (2N) repeat, a mono-nucleosomal (1N) repeat, a component of "random" DNA, and triple splitting of major peaks. The random component is major, is unlikely to be completely artifactual, and is what would be expected from the face to face layering model of Subirana et. al., (1). The 2N pattern appeared to be associated with compact, metaphase-type chromatin, whereas the 1N pattern was associated with more exposed chromatin. These two modes are explained in terms of orderly back-to-back folding of zig-zag nucleofilaments, and face-to-face folding respectively. Hybridization studies indicated that the centromeric classes of repetitive DNA had the same digestion spectra as the major interspersed classes of repetitive DNA, and DNA enriched in transcriptionally active sequences. It is suggested that current coil models are all inadequate explanations of higher order chromatin packing. PMID:3671072

  12. Growth phase dependency of chromatin cleavage and degradation by bleomycin.

    PubMed Central

    Moore, C W; Jones, C S; Wall, L A

    1989-01-01

    Preferential cleavage of Saccharomyces cerevisiae chromosomes in internucleosomal (linker) regions and nonspecific degradation of chromatin by an anticancer antibiotic which degrades DNA were investigated and found to increase in consecutive stages of growth. Cleavage of DNA in internucleosomal regions and intensities and multiplicities of nucleosomal bands were dependent on drug concentration, growth phase of the cells, and length of incubation. Cellular DNA was least degraded during logarithmic phase. After cells progressed only one generation in logarithmic phase, low concentrations (6.7 x 10(-7) to 3.4 x 10(-6) M) of bleomycin produced approximately three to seven times more DNA breaks. Internucleosomal cleavage was highest, and the most extended oligonucleosomal series and extensive chromatin degradation were observed during stationary phase. It is concluded that the growth phase of cells is critical in determining amounts of the highly preferential cleavage in internucleosomal regions and overall breakage and degradation of DNA. Mononucleosomal bands were most intense, indicating the greatest accumulation of DNA of this size. Mean mononucleosomal lengths were 165.9 +/- 3.9 base pairs, in agreement with yeast mononucleosomal lengths. As high-molecular-weight chromatin was digested by bleomycin, oligonucleosomes and, eventually, mononucleosomes became digested. Therefore, it is also concluded that bleomycin degradation of oligonucleosomes and trimming of DNA linker regions proceed to degradation of the monosomes (core plus linker DNA). Images PMID:2479336

  13. Nucleolin: dual roles in rDNA chromatin transcription.

    PubMed

    Durut, Nathalie; Sáez-Vásquez, Julio

    2015-02-01

    Nucleolin is a major nucleolar protein conserved in all eukaryotic organisms. It is a multifunctional protein involved in different cellular aspects like chromatin organization and stability, DNA and RNA metabolism, assembly of ribonucleoprotein complexes, cytokinesis, cell proliferation and stress response. The multifunctionality of nucleolin is linked to its tripartite structure, post-translational modifications and its ability of shuttling from and to the nucleolus/nucleoplasm and cytoplasm. Nucleolin has been now studied for many years and its activities and properties have been described in a number of excellent reviews. Here, we overview the role of nucleolin in RNA polymerase I (RNAPI) transcription and describe recent results concerning its functional interaction with rDNA chromatin organization. For a long time, nucleolin has been associated with rRNA gene expression and pre-rRNA processing. However, the functional connection between nucleolin and active versus inactive rRNA genes is still not fully understood. Novel evidence indicates that the nucleolin protein might be required for controlling the transcriptional ON/OFF states of rDNA chromatin in both mammals and plants. PMID:25225127

  14. Chromatin signaling in muscle stem cells: interpreting the regenerative microenvironment

    PubMed Central

    Brancaccio, Arianna; Palacios, Daniela

    2015-01-01

    Muscle regeneration in the adult occurs in response to damage at expenses of a population of adult stem cells, the satellite cells. Upon injury, either physical or genetic, signals released within the satellite cell niche lead to the commitment, expansion and differentiation of the pool of muscle progenitors to repair damaged muscle. To achieve this goal satellite cells undergo a dramatic transcriptional reprogramming to coordinately activate and repress specific subset of genes. Although the epigenetics of muscle regeneration has been extensively discussed, less emphasis has been put on how extra-cellular cues are translated into the specific chromatin reorganization necessary for progression through the myogenic program. In this review we will focus on how satellite cells sense the regenerative microenvironment in physiological and pathological circumstances, paying particular attention to the mechanism through which the external stimuli are transduced to the nucleus to modulate chromatin structure and gene expression. We will discuss the pathways involved and how alterations in this chromatin signaling may contribute to satellite cells dysfunction during aging and disease. PMID:25904863

  15. OPERating ON chromatin, a colorful language where context matters

    PubMed Central

    Gardner, Kathryn E.; Allis, C. David; Strahl, Brian D.

    2011-01-01

    Histones, the fundamental packaging elements of eukaryotic DNA, are highly decorated with a diverse set of post-translational modifications (PTMs) that are recognized to govern the structure and function of chromatin. Ten years ago, we put forward the histone code hypothesis, which provided a model to explain how single and/or combinatorial PTMs on histones regulate the diverse activities associated with chromatin (e.g. gene transcription). At that time, there was a limited understanding of both the number of PTMs that occur on histones as well as the proteins that place, remove and interpret them. Since the conception of this hypothesis, the field has witnessed an unprecedented advance in our understanding of the enzymes that contribute to the establishment of histone PTMs, as well as the diverse effector proteins that bind them. While debate continues as to whether histone PTMs truly constitute a strict “code”, it is becoming clear that PTMs on histone proteins function in elaborate combinations to regulate the many activities associated with chromatin. In this special issue, we celebrate the 50th anniversary of the landmark publication of the lac operon with a review that provides a current view of the histone code hypothesis, the lessons we have learned over the last decade, and the technologies that will drive our understanding of histone PTMs forward in the future. PMID:21272588

  16. The accessible chromatin landscape of the human genome.

    PubMed

    Thurman, Robert E; Rynes, Eric; Humbert, Richard; Vierstra, Jeff; Maurano, Matthew T; Haugen, Eric; Sheffield, Nathan C; Stergachis, Andrew B; Wang, Hao; Vernot, Benjamin; Garg, Kavita; John, Sam; Sandstrom, Richard; Bates, Daniel; Boatman, Lisa; Canfield, Theresa K; Diegel, Morgan; Dunn, Douglas; Ebersol, Abigail K; Frum, Tristan; Giste, Erika; Johnson, Audra K; Johnson, Ericka M; Kutyavin, Tanya; Lajoie, Bryan; Lee, Bum-Kyu; Lee, Kristen; London, Darin; Lotakis, Dimitra; Neph, Shane; Neri, Fidencio; Nguyen, Eric D; Qu, Hongzhu; Reynolds, Alex P; Roach, Vaughn; Safi, Alexias; Sanchez, Minerva E; Sanyal, Amartya; Shafer, Anthony; Simon, Jeremy M; Song, Lingyun; Vong, Shinny; Weaver, Molly; Yan, Yongqi; Zhang, Zhancheng; Zhang, Zhuzhu; Lenhard, Boris; Tewari, Muneesh; Dorschner, Michael O; Hansen, R Scott; Navas, Patrick A; Stamatoyannopoulos, George; Iyer, Vishwanath R; Lieb, Jason D; Sunyaev, Shamil R; Akey, Joshua M; Sabo, Peter J; Kaul, Rajinder; Furey, Terrence S; Dekker, Job; Crawford, Gregory E; Stamatoyannopoulos, John A

    2012-09-01

    DNase?I hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ?2.9 million DHSs that encompass virtually all known experimentally validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. We connect ?580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is organized with dozens to hundreds of co-activated elements, and the transcellular DNase?I sensitivity pattern at a given region can predict cell-type-specific functional behaviours. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation. PMID:22955617

  17. Genomic response to Wnt signalling is highly context-dependent - Evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets

    SciTech Connect

    Railo, Antti [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland)] [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland); Pajunen, Antti [Department of Biochemistry, University of Oulu (Finland)] [Department of Biochemistry, University of Oulu (Finland); Itaeranta, Petri; Naillat, Florence; Vuoristo, Jussi; Kilpelaeinen, Pekka [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland)] [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland); Vainio, Seppo, E-mail: Seppo.Vainio@oulu.fi [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland)] [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland)

    2009-10-01

    Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-{beta}-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of {beta}-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays of gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.

  18. The rad9 gene of Coprinus cinereus encodes a proline-rich protein required for meiotic chromosome condensation and synapsis

    SciTech Connect

    Seitz, L.C.; Tang, Keliang; Cummings, W.J.; Zolan, M.E. [Indiana Univ., Bloomington, IN (United States)

    1996-04-01

    The rad9 gene of Coprinus cinereus is essential for the normal completion of meiosis. We examined surface-spread preparations of wild-type and rad9-1 nuclei from the meiotic stages of karyogamy through metaphase I, and we determined the primary sequence, structure, and meiotic expression of the rad9 gene. In wild-type C. cinereus, karyogamy is followed by condensation and alignment of homologous chromosomes. Condensation and axial core development largely precede synapsis, which often initiates at telomeres. A diffuse diplotene phase coincides with dissolution of the synaptonemal complex, and subsequently chromosomes further condense as the cells progress into metaphase I. In contrast, although karyogamy and nucleolar fusion are apparently normal in rad9-1 basidia, only short stretches of synaptonemal complex form. These correlate with stretches of condensed chromatin, mostly at apparent chromosome ends, and regions of presumptive triple synapsis are numerous. rad9-1 basidia enter the diffuse stages of early diplotene, and then 50% of these cells enter metaphase I by the criteria of nucleolar elimination and at least some chromatin condensation. rad9 gene expression is induced after gamma irradiation and during meiosis. The gene has 27 exons and encodes a predicted protein of 2157 amino acids, with a proline-rich amino terminus. 62 refs., 10 figs.

  19. Characterisation of a subpopulation of sperm with massive nuclear damage, as recognised with the sperm chromatin dispersion test.

    PubMed

    Gosálvez, J; Rodríguez-Predreira, M; Mosquera, A; López-Fernández, C; Esteves, S C; Agarwal, A; Fernández, J L

    2014-08-01

    Assessment of human sperm DNA fragmentation by the sperm chromatin dispersion (SCD) test is based on the detection of haloes of spreading DNA loops after sequential DNA denaturing and protamine removal. After the SCD test, sperm without DNA fragmentation show chromatin haloes emerging from the central nuclear core, while sperm containing fragmented DNA present small or no haloes. The nuclear degraded sperm are recognised as a differentiated category within the sperm with fragmented DNA, whose cores appear irregularly and/or faintly stained. This subpopulation is more prevalent in patients with varicocele. Protein staining with 2.7-dibrom-4-hydroxy-mercury-fluorescein demonstrated that degraded sperm intensely lose nuclear core proteins after the SCD processing. Moreover, degraded sperm are 65% more faintly labelled for DNA breaks after in situ nick translation (ISNT) on average, due to extensive DNA loss. A two-dimensional comet assay under sequential neutral and alkaline conditions demonstrated that degraded sperm contain both massive double- and single-strand DNA breaks. The degraded sperm appear as a subpopulation with stronger nuclear damage, affecting both DNA and protein fractions, possibly due to intense intratesticular oxidative stress, what could explain its higher proportion in patients with varicocele. PMID:23710631

  20. In vitro chromatin remodelling by chromatin accessibility complex (CHRAC) at the SV40 origin of DNA replication.

    PubMed Central

    Alexiadis, V; Varga-Weisz, P D; Bonte, E; Becker, P B; Gruss, C

    1998-01-01

    DNA replication is initiated by binding of initiation factors to the origin of replication. Nucleosomes are known to inhibit the access of the replication machinery to origin sequences. Recently, nucleosome remodelling factors have been identified that increase the accessibility of nucleosomal DNA to transcription regulators. To test whether the initiation of DNA replication from an origin covered by nucleosomes would also benefit from the action of nucleosome remodelling factors, we reconstituted SV40 DNA into chromatin in Drosophila embryo extracts. In the presence of T-antigen and ATP, a chromatin-associated cofactor allowed efficient replication from a nucleosomal origin in vitro. In search of the energy-dependent cofactor responsible we found that purified 'chromatin accessibility complex' (CHRAC) was able to alter the nucleosomal structure at the origin allowing the binding of T-antigen and efficient initiation of replication. These experiments provide evidence for the involvement of a nucleosome remodelling machine in structural changes at the SV40 origin of DNA replication in vitro. PMID:9628878

  1. Chromatin indexing in Arabidopsis: an epigenomic tale of tails and more.

    PubMed

    Roudier, François; Teixeira, Felipe Karam; Colot, Vincent

    2009-11-01

    Packaging DNA into chromatin is pivotal for the regulation of genome activity in eukaryotes. This chromatin-level control relies on a range of histone modifications and variants, chromatin-remodeling proteins and DNA methylation in plants and mammals. High-resolution maps have recently been obtained for several chromatin modifications in Arabidopsis, which provide a first glimpse at the organization of plant epigenomes. These maps suggest a pervasive involvement of transcriptional activity in indexing chromatin with reference to the underlying DNA sequence. However, to assess the contribution of chromatin dynamics to plant development and phenotypic plasticity, it will be necessary to shift from a static to a dynamic view of the Arabidopsis epigenome. PMID:19850370

  2. Assays without Borders

    Cancer.gov

    CPTAC researchers partner with international labs to demonstrate the ability of Targeted mass spectrometry–based assays to reproducibly quantify Human proteins across labs, countries and continents in a recently published journal article.

  3. Multi-omic Data Integration Links Deleted in Breast Cancer 1 (DBC1) Degradation to Chromatin Remodeling in Inflammatory Response*

    PubMed Central

    Nakayasu, Ernesto S.; Brown, Roslyn N.; Ansong, Charles; Sydor, Michael A.; Imtiaz, Sayed; Mihai, Cosmin; Sontag, Ryan; Hixson, Kim K.; Monroe, Matthew E.; Sobreira, Tiago J. P.; Orr, Galya; Petyuk, Vladislav A.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.

    2013-01-01

    This study investigated the dynamics of ubiquitinated proteins after the inflammatory stimulation of RAW 264.7 macrophage-like cells with bacterial lipopolysaccharide. Ubiquitination is a common protein post-translational modification that regulates many key cellular functions. We demonstrated that levels of global ubiquitination and K48 and K63 polyubiquitin chains change after lipopolysaccharide stimulation. Quantitative proteomic analysis identified 1199 ubiquitinated proteins, 78 of which exhibited significant changes in ubiquitination levels following stimulation. Integrating the ubiquitinome data with global proteomic and transcriptomic results allowed us to identify a subset of 88 proteins that were targeted for degradation after lipopolysaccharide stimulation. Using cellular assays and Western blot analyses, we biochemically validated DBC1 (a histone deacetylase inhibitor) as a degradation substrate that is targeted via an orchestrated mechanism utilizing caspases and the proteasome. The degradation of DBC1 releases histone deacetylase activity, linking lipopolysaccharide activation to chromatin remodeling in caspase- and proteasome-mediated signaling. PMID:23639857

  4. Large-Scale Chromatin Unfolding and Remodeling Induced by VP16 Acidic Activation Domain

    Microsoft Academic Search

    Tudorita Tumbar; Gail Sudlow; Andrew S. Belmont

    1999-01-01

    Analysis of the relationship between tran- scriptional activators and chromatin organization has focused largely on lower levels of chromatin structure. Here we describe striking remodeling of large-scale chromatin structure induced by a strong transcriptional activator. A VP16-lac repressor fusion protein targeted the VP16 acidic activation domain to chromosome re- gions containing lac operator repeats. Targeting was ac- companied by increased

  5. The Ino80 chromatin-remodeling enzyme regulates replisome function and stability

    Microsoft Academic Search

    Manolis Papamichos-Chronakis; Craig L Peterson

    2008-01-01

    Previous studies have demonstrated essential roles for ATP-dependent chromatin-remodeling and chromatin-modifying enzymes in gene transcription and DNA repair, but few studies have addressed how the replication machinery deals with chromatin. Here we show that the Ino80 remodeling enzyme is recruited to replication origins as cells enter S phase. Inducible degradation of Ino80 shows that it is required continuously for efficient

  6. The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins

    Microsoft Academic Search

    George Orphanides; Wei-Hua Wu; Michael Hampsey; Danny Reinberg

    1999-01-01

    The regulation of gene expression depends critically upon chromatin structure. Transcription of protein-coding genes can be reconstituted on naked DNA with only the general transcription factors and RNA polymerase II (ref. 2). This minimal system cannot transcribe DNA packaged into chromatin, indicating thataccessory factors may facilitate access to DNA. Two classes of accessory factor, ATP-dependent chromatin-remodelling enzymes and histone acetyltransferases,

  7. Probing the W chromosome of the codling moth, Cydia pomonella , with sequences from microdissected sex chromatin

    Microsoft Academic Search

    Iva Fuková; Walther Traut; Magda Vítková; Petr Nguyen; Svatava Kubí?ková; František Marec

    2007-01-01

    The W chromosome of the codling moth, Cydia pomonella, like that of most Lepidoptera species, is heterochromatic and forms a female-specific sex chromatin body in somatic cells.\\u000a We collected chromatin samples by laser microdissection from euchromatin and W-chromatin bodies. DNA from the samples was\\u000a amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) and used to prepare painting probes and\\u000a start

  8. Simple Simulations of DNA Condensation

    SciTech Connect

    STEVENS,MARK J.

    2000-07-12

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

  9. Chromosome condensation in the absence of the non-SMC subunits of MukBEF.

    PubMed

    Wang, Qinhong; Mordukhova, Elena A; Edwards, Andrea L; Rybenkov, Valentin V

    2006-06-01

    MukBEF is a bacterial SMC (structural maintenance of chromosome) complex required for chromosome partitioning in Escherichia coli. We report that overproduction of MukBEF results in marked chromosome condensation. This condensation is rapid and precedes the effects of overproduction on macromolecular synthesis. Condensed nucleoids are often mispositioned; however, cell viability is only mildly affected. The overproduction of MukB leads to a similar chromosome condensation, even in the absence of MukE and MukF. Thus, the non-SMC subunits of MukBEF play only an auxiliary role in chromosome condensation. MukBEF, however, was often a better condensin than MukB. Furthermore, the chromosome condensation by MukB did not rescue the temperature sensitivity of MukEF-deficient cells, nor did it suppress the high frequency of anucleate cell formation. We infer that the role of MukBEF in stabilizing chromatin architecture is more versatile than its role in controlling chromosome size. We further propose that MukBEF could be directly involved in chromosome segregation. PMID:16740950

  10. A chromatin binding site in the tail domain of nuclear lamins that interacts with core histones

    PubMed Central

    1995-01-01

    Interaction of chromatin with the nuclear envelope and lamina is thought to help determine higher order chromosome organization in the interphase nucleus. Previous studies have shown that nuclear lamins bind chromatin directly. Here we have localized a chromatin binding site to the carboxyl-terminal tail domains of both A- and B-type mammalian lamins, and have characterized the biochemical properties of this binding in detail. Recombinant glutathione-S-transferase fusion proteins containing the tail domains of mammalian lamins C, B1, and B2 were analyzed for their ability to associate with rat liver chromatin fragments immobilized on microtiter plate wells. We found that all three lamin tails specifically bind to chromatin with apparent KdS of 120-300 nM. By examining a series of deletion mutants, we have mapped the chromatin binding region of the lamin C tail to amino acids 396- 430, a segment immediately adjacent to the rod domain. Furthermore, by analysis of chromatin subfractions, we found that core histones constitute the principal chromatin binding component for the lamin C tail. Through cooperativity, this lamin-histone interaction could be involved in specifying the high avidity attachment of chromatin to the nuclear envelope in vivo. PMID:7559784

  11. Chromatin states reveal functional associations for globally defined transcription start sites in four human cell lines

    PubMed Central

    2014-01-01

    Background Deciphering the most common modes by which chromatin regulates transcription, and how this is related to cellular status and processes is an important task for improving our understanding of human cellular biology. The FANTOM5 and ENCODE projects represent two independent large scale efforts to map regulatory and transcriptional features to the human genome. Here we investigate chromatin features around a comprehensive set of transcription start sites in four cell lines by integrating data from these two projects. Results Transcription start sites can be distinguished by chromatin states defined by specific combinations of both chromatin mark enrichment and the profile shapes of these chromatin marks. The observed patterns can be associated with cellular functions and processes, and they also show association with expression level, location relative to nearby genes, and CpG content. In particular we find a substantial number of repressed inter- and intra-genic transcription start sites enriched for active chromatin marks and Pol II, and these sites are strongly associated with immediate-early response processes and cell signaling. Associations between start sites with similar chromatin patterns are validated by significant correlations in their global expression profiles. Conclusions The results confirm the link between chromatin state and cellular function for expressed transcripts, and also indicate that active chromatin states at repressed transcripts may poise transcripts for rapid activation during immune response. PMID:24669905

  12. Electric oscillation and coupling of chromatin regulate chromosome packaging and transcription in eukaryotic cells

    PubMed Central

    2012-01-01

    Transcription in eukaryotic cells is efficiently spatially and temporally regulated, but how this genome-wide regulation is achieved at the physical level remains unclear, given the limited transcriptional resources within the nucleus and the sporadic linear arrangements of genes within chromosomes. In this article, we provide a physical model for chromatin cluster formation, based on oscillation synchronization and clustering of different chromatin regions, enabling efficient systemic genome-wide regulation of transcription. We also propose that the electromagnetic field generated by oscillation of chromatin is the driving force for chromosome packing during M phase. We further explore the physical mechanisms for chromatin oscillation cluster (COC) formation, and long-distance chromatin kissing. The COC model, which connects the dots between chromatin epigenetic modification and higher-order nuclear organization, answers many important questions, such as how the CCCTC-binding factor CTCF contributes to higher-order chromatin organization, and the mechanism of sequential transcriptional activation of HOX clusters. In the COC model, long non-coding RNAs function as oscillation clustering adaptors to recruit chromatin modification factors to specific sub-nuclear regions, fine-tuning transcriptional events in the chromatin oscillation clusters. Introns of eukaryotic genes have evolved to promote the clustering of transcriptionally co-regulated genes in these sub-nuclear regions. PMID:22759343

  13. Chromatin states modify network motifs contributing to cell-specific functions.

    PubMed

    Zhao, Hongying; Liu, Tingting; Liu, Ling; Zhang, Guanxiong; Pang, Lin; Yu, Fulong; Fan, Huihui; Ping, Yanyan; Wang, Li; Xu, Chaohan; Xiao, Yun; Li, Xia

    2015-01-01

    Epigenetic modification can affect many important biological processes, such as cell proliferation and apoptosis. It can alter chromatin conformation and contribute to gene regulation. To investigate how chromatin states associated with network motifs, we assembled chromatin state-modified regulatory networks by combining 269 ChIP-seq data and chromatin states in four cell types. We found that many chromatin states were significantly associated with network motifs, especially for feedforward loops (FFLs). These distinct chromatin state compositions contribute to different expression levels and translational control of targets in FFLs. Strikingly, the chromatin state-modified FFLs were highly cell-specific and, to a large extent, determined cell-selective functions, such as the embryonic stem cell-specific bivalent modification-related FFL with an important role in poising developmentally important genes for expression. Besides, comparisons of chromatin state-modified FFLs between cancerous/stem and primary cell lines revealed specific type of chromatin state alterations that may act together with motif structural changes cooperatively contribute to cell-to-cell functional differences. Combination of these alterations could be helpful in prioritizing candidate genes. Together, this work highlights that a dynamic epigenetic dimension can help network motifs to control cell-specific functions. PMID:26169043

  14. Chromatin states modify network motifs contributing to cell-specific functions

    PubMed Central

    Zhao, Hongying; Liu, Tingting; Liu, Ling; Zhang, Guanxiong; Pang, Lin; Yu, Fulong; Fan, Huihui; Ping, Yanyan; Wang, Li; Xu, Chaohan; Xiao, Yun; Li, Xia

    2015-01-01

    Epigenetic modification can affect many important biological processes, such as cell proliferation and apoptosis. It can alter chromatin conformation and contribute to gene regulation. To investigate how chromatin states associated with network motifs, we assembled chromatin state-modified regulatory networks by combining 269 ChIP-seq data and chromatin states in four cell types. We found that many chromatin states were significantly associated with network motifs, especially for feedforward loops (FFLs). These distinct chromatin state compositions contribute to different expression levels and translational control of targets in FFLs. Strikingly, the chromatin state-modified FFLs were highly cell-specific and, to a large extent, determined cell-selective functions, such as the embryonic stem cell-specific bivalent modification-related FFL with an important role in poising developmentally important genes for expression. Besides, comparisons of chromatin state-modified FFLs between cancerous/stem and primary cell lines revealed specific type of chromatin state alterations that may act together with motif structural changes cooperatively contribute to cell-to-cell functional differences. Combination of these alterations could be helpful in prioritizing candidate genes. Together, this work highlights that a dynamic epigenetic dimension can help network motifs to control cell-specific functions. PMID:26169043

  15. 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

  16. Evaporation, Condensation, and Precipitation

    NSDL National Science Digital Library

    Miss Brown

    2009-10-21

    After completion of this project students should have an understanding of evaporation, condensation, and precipitation in the water cycle. Use the websites provided to answer the questions. Record your answers on the spreadsheet provided. Do you understand how the water cycle works? Begin by watching this short video about the water cycle.water cycle video Use the website to define condensation, precipitation, and evaporation?water cycle List the different types of precipitation from the site.types of precipitation Follow the directions to the experiment on this website to get a better understanding of how evaporation takes ...

  17. A comparison of alternative assays to measure DNA damage in stallion spermatozoa: TUNEL test versus 'Nicoletti assay'.

    PubMed

    Kakasi, Balázs; Nagy, Szabolcs; Pál, László; Czimber, Gyula E; Husvéth, Ferenc

    2015-03-01

    The aberrations of sperm DNA may cause various problems and have negative consequences on fertility. These influence embryonic development or might lead to early embryo loss. Sperm Chromatin Structure Assay (SCSA) is the flow cytometric method most often used for the detection of DNA lesions; however, some studies using that method reached confusing conclusions. The aim of this pilot study was to adjust and compare two alternative tests, namely the TUNEL test and the Nicoletti assay. The above-mentioned two flow cytometric methods capable of detecting the fragmented DNA of sperm were tested on 12 frozen-thawed stallion semen samples. The TUNEL test demonstrated much higher DNA fragmentation ratio than the Nicoletti assay (mean ± SD: 30.77 ± 13.03% vs. 1.93 ± 0.89%, respectively). A fluorescent microscopic check of the samples showed that TUNEL labelled the plasma membrane and the mitochondria in a nonspecific way, rather than detecting only the fragmented DNA, thus eventually resulting in a false positive sign. The Nicoletti assay is simpler, quicker and does not detect nonspecific binding; however, further analyses are required to determine its diagnostic value. PMID:25655419

  18. Interpreting clinical assays for histone deacetylase inhibitors

    PubMed Central

    Martinet, Nadine; Bertrand, Philippe

    2011-01-01

    As opposed to genetics, dealing with gene expressions by direct DNA sequence modifications, the term epigenetics applies to all the external influences that target the chromatin structure of cells with impact on gene expression unrelated to the sequence coding of DNA itself. In normal cells, epigenetics modulates gene expression through all development steps. When “imprinted” early by the environment, epigenetic changes influence the organism at an early stage and can be transmitted to the progeny. Together with DNA sequence alterations, DNA aberrant cytosine methylation and microRNA deregulation, epigenetic modifications participate in the malignant transformation of cells. Their reversible nature has led to the emergence of the promising field of epigenetic therapy. The efforts made to inhibit in particular the epigenetic enzyme family called histone deacetylases (HDACs) are described. HDAC inhibitors (HDACi) have been proposed as a viable clinical therapeutic approach for the treatment of leukemia and solid tumors, but also to a lesser degree for noncancerous diseases. Three epigenetic drugs are already arriving at the patient’s bedside, and more than 100 clinical assays for HDACi are registered on the National Cancer Institute website. They explore the eventual additive benefits of combined therapies. In the context of the pleiotropic effects of HDAC isoforms, more specific HDACi and more informative screening tests are being developed for the benefit of the patients. PMID:21625397

  19. Strategies in Optimizing Condensate Return

    E-print Network

    Bloom, D.

    Optimizing condensate return for reuse as boiler feedwater is often a viable means of reducing fuel costs and improving boiler system efficiency. As more condensate is returned, less makeup is required and savings on water and water treatment costs...

  20. Chromatin structure of transcriptionally competent and repressed genes.

    PubMed Central

    Kamakaka, R T; Thomas, J O

    1990-01-01

    We have compared transcriptionally competent and repressed genes with respect to their linker histone content and their ability to fold into higher-order structures. Histones were cross-linked covalently to DNA in chicken erythrocyte and oviduct nuclei by UV irradiation, and the DNA that was immunoprecipitated with anti-H1 and (for erythrocytes) anti-H5 antibodies was analysed for particular DNA sequences. None of the sequences investigated was free of H1 (H5). However, in mature erythrocytes the tissue-specific adult beta-globin gene (beta A) appears to be partially depleted of H5, and both the beta-globin gene and the H5 gene (also tissue-specific), as well as the 'housekeeping' beta-actin gene, appear to be partially depleted of H1 relative to inactive genes; in oviduct slight H1-depletion is detected on the ovalbumin gene relative to genes that are inactive in this tissue and the actin gene. Transcriptionally competent erythrocyte chromatin fragments, in contrast to inactive fragments, are unable to self-associate into 'pseudo-higher-order structures'. This is likely to be a consequence of the partial depletion of H5 and/or H1 in active chromatin, resulting in the breakdown of (probably cooperative) interactions between H5 and/or H1 molecules that otherwise mediate the assembly of pseudo-higher-order structures in vitro and a stable 30 nm chromatin filament in vivo. Images Fig. 2. Fig. 3. Fig. 6. Fig. 7. Fig. 8. PMID:2249661

  1. Chromatin proteins from normal, vegetalized, and animalized sea urchin embryos.

    PubMed

    Gineitis, A A; Stankeviciute, J V; Vorob'ev, V I

    1976-09-01

    The chemical composition of the chromatin, the fractional content of histones and nonhistone chromatin proteins (NHP), and the biosynthesis of these proteins in normal, vegetalized, and animalized embryos of the sea urchin Strongylocentrotus droebachiensis at the blastula, mesenchyme blastula, and gastrula stages have been studied. The amount of the NHP in the chromatin from normal and vegetalized embryos increases during early embryonic development while that in animalized embryos remains without change at the mesenchyme blastula stage and then decreases. During development the histone content in all three cases slightly decreases. Polyacrylamide gel electrophoresis reveals that both fractional composition of histones and their biosynthesis in normal, vegetalized, and animalized embryos display no differences. During development, however, some changes occur, so that the relative amount of histones F1 and F2a2 increases, F2b decreases, while F3 and F2a1 remains constant. Histone F1 at the blastula stage consists of two subfractions while at the gastrula stage it consists of three subfractions. The histone F2a1 consists of one and two, respectively. Histone F3 at all stages is made up of three subfractions; histone F2b is made up of two; and the histone F2a2 is electrophoretically homogeneous. Specific radioactivity of the arginine-rich histones F3 and F2a1 tends to increase during development, while that of moderately lysine-rich histones F2b and F2a2 does not change, and that of the lysine-rich histone F1 decreases. The NHP in normal, vegetalized, and animalized embryos at different developmental stages consist of 17 fractions that can be separated by isoelectrofocusing within the 4.5-8.8 pH range. Quantitative changes have been observed in the fractions focused at pH 4.5-6.1 during development and in normal and modified embryos at the gastrula stage. PMID:12194431

  2. Chromatin computation: epigenetic inheritance as a pattern reconstruction problem.

    PubMed

    Arnold, Christian; Stadler, Peter F; Prohaska, Sonja J

    2013-11-01

    Eukaryotic histones carry a diverse set of specific chemical modifications that accumulate over the life-time of a cell and have a crucial impact on the cell state in general and the transcriptional program in particular. Replication constitutes a dramatic disruption of the chromatin states that effectively amounts to partial erasure of stored information. To preserve its epigenetic state the cell reconstructs (at least part of) the histone modifications by means of processes that are still very poorly understood. A plausible hypothesis is that the different combinations of reader and writer domains in histone-modifying enzymes implement local rewriting rules that are capable of "recomputing" the desired parental modification patterns on the basis of the partial information contained in that half of the nucleosomes that predate replication. To test whether such a mechanism is theoretically feasible, we have developed a flexible stochastic simulation system (available at http://www.bioinf.uni-leipzig.de/Software/StoChDyn) for studying the dynamics of histone modification states. The implementation is based on Gillespie's approach, i.e., it models the master equation of a detailed chemical model. It is efficient enough to use an evolutionary algorithm to find patterns across multiple cell divisions with high accuracy. We found that it is easy to evolve a system of enzymes that can maintain a particular chromatin state roughly stable, even without explicit boundary elements separating differentially modified chromatin domains. However, the success of this task depends on several previously unanticipated factors, such as the length of the initial state, the specific pattern that should be maintained, the time between replications, and chemical parameters such as enzymatic binding and dissociation rates. All these factors also influence the accumulation of errors in the wake of cell divisions. PMID:23880640

  3. 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

  4. 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...

  5. Cloud Condensation Nuclei Sizes

    Microsoft Academic Search

    J. G. Hudson; S. Mishra

    2006-01-01

    The sizes of cloud condensation nuclei (CCN) can be determined by first passing an aerosol sample through a differential mobility analyzer (DMA) and then to a CCN counter or spectrometer (i.e., Hudson 1989), which provides a mean value of the critical supersaturation (Sc) of the particles. By sequentially dialing in several different DMA sizes a relationship between dry particle size

  6. Steam and Condensate Systems 

    E-print Network

    Yates, W.

    1979-01-01

    STEAM AND CONDENSATE SYSTEM Wesley Yates Yarway Corporation Houston, Texas In the late 60's and early 70's oil was plentiful and steam was relatively inexpen sive. The switch to low sulphur fuel oil and the oil embargo suddenly changed the pic...

  7. Asymmetric condensed dark matter

    E-print Network

    Anthony Aguirre; Alberto Diez-Tejedor

    2015-02-25

    We explore the viability of a boson dark matter candidate with an asymmetry between the number densities of particles and antiparticles. A simple thermal field theory analysis confirms that, under certain general conditions, this component would develop a Bose-Einstein condensate in the early universe that, for appropriate model parameters, could survive the ensuing cosmological evolution until now. The condensation of a dark matter component in equilibrium with the thermal plasma is a relativistic process, hence the amount of matter dictated by the charge asymmetry is complemented by a hot relic density frozen out at the time of decoupling. Contrary to the case of ordinary WIMPs, dark matter particles in a condensate can be very light, $10^{-22}\\,{\\rm eV} \\lesssim m \\lesssim 10^2\\,{\\rm eV}$; the lower limit arises from constraints on small-scale structure formation, while the upper bound ensures that the density from thermal relics is not too large. Big-Bang nucleosynthesis constrains the temperature of decoupling to the scale of the QCD phase transition or above. This requires large dark matter-to-photon ratios and very weak interactions with standard model particles. Finally, we argue that a given boson particle that was in thermal equilibrium in the early universe may be in a condensate, or in the form of thermal relics, but we cannot have a combination of both contributing significantly to the mass density today.

  8. Re-Condensation

    E-print Network

    Bhatia, P.; Kozman, T.

    2004-01-01

    When steam transfers its heat in a manufacturing process or heat exchanger, it may revert to a liquid phase called condensate. This paper presents a method to help certain manufacturing and petro-chemical companies to save energy costs by returning their...

  9. Re-Condensation 

    E-print Network

    Bhatia, P.; Kozman, T.

    2004-01-01

    When steam transfers its heat in a manufacturing process or heat exchanger, it may revert to a liquid phase called condensate. This paper presents a method to help certain manufacturing and petro-chemical companies to save energy costs by returning their...

  10. Soft condensed matter physics

    Microsoft Academic Search

    T. C. Lubensky

    1997-01-01

    Soft condensed matter physics is the study of materials, such as fluids, liquid crystals, polymers, colloids and emulsions, that are “soft” to the touch. This article will review some properties, such as the dominance of entropy, that are unique to soft materials and some properties such as the interplay between broken-symmetry, dynamic mode structure and topological defects that are common

  11. Condensed-Matter Physics.

    ERIC Educational Resources Information Center

    Hirsch, Jorge E.; Scalapino, Douglas J.

    1983-01-01

    Discusses ways computers are being used in condensed-matter physics by experimenters and theorists. Experimenters use them to control experiments and to gather and analyze data. Theorists use them for detailed predictions based on realistic models and for studies on systems not realizable in practice. (JN)

  12. Condensed matter physics journals

    Microsoft Academic Search

    R. Todorov

    1983-01-01

    On the basis of a citation\\/reference criterion, 20 core journals are selected in the field of condensed matter physics. Citation data and indicators from 1980Journal Citation Reports reveal their different characteristic features such as applied orientation, communication function and longevity. The manually obtained data for the core journals are written into a matrix in order to determine an appropriate ranking

  13. Condensed Matter Physics

    Microsoft Academic Search

    I Strzalkowski

    2000-01-01

    Condensed matter physics constitutes nowadays an enormous field of knowledge. To write a good textbook covering all main topics in that field in a suitable way and in a reasonable volume is very hard indeed. I believe Michael Marder has achieved this goal with great success. The text is arranged in six parts. Part I describes the atomic structure of

  14. Transcriptional regulation and chromatin remodeling mechanisms at PHO5 

    E-print Network

    Carvin, Christopher Dumas

    2005-08-29

    in nucleosome remodeling. The most widely studied complex is the Swi-Snf complex in yeast, named for its role in mating- type switching as well as sucrose fermentation (reviewed in Martens and Winston, 2003). Defects in the human Swi-Snf complex are linked... with permission from Archana Dhasarathy. 12 An earlier study showed that yeast genes could be classified into three distinct classes based on their requirement for the chromatin remodelers Swi- Snf and SAGA: 1) those genes which require both Swi-Snf...

  15. The structure of the chromatin core particle in solution.

    PubMed Central

    Pardon, J F; Worcester, D L; Wooley, J C; Cotter, R I; Lilley, D M; Richards, R M

    1977-01-01

    The shape and size of the nucleosomal core particle from chromatin has been examined by analysis of neutron and X-ray scattering data from dilute solutions. Calculations of scattering for many different models have been made and only one model was able to account for both the X-ray and neutron profiles. This model is an oblate structure with height about 50A and diameter 110A. The DNA is mainly confined to two annuli located at the top and bottom respectively of the core particle positioned on the outside of a compact protein core which has a height of about 40A and diameter about 73A. PMID:561952

  16. Cohesin: a critical chromatin organizer in mammalian gene regulation

    PubMed Central

    Chien, Richard; Zeng, Weihua; Ball, Alexander R.; Yokomori, Kyoko

    2014-01-01

    Cohesins are evolutionarily conserved essential multi-protein complexes important for higher-order chromatin organization. They play pivotal roles in the maintenance of genome integrity through mitotic chromosome regulation, DNA repair and replication, as well as gene regulation critical for proper development and cellular differentiation. In this review, we will discuss the multifaceted functions of mammalian cohesins and their apparent functional hierarchy in the cell, with particular focus on their actions in gene regulation and their relevance to human developmental disorders. PMID:21851156

  17. Transcriptional regulation and chromatin remodeling mechanisms at PHO5

    E-print Network

    Carvin, Christopher Dumas

    2005-08-29

    in nucleosome remodeling. The most widely studied complex is the Swi-Snf complex in yeast, named for its role in mating- type switching as well as sucrose fermentation (reviewed in Martens and Winston, 2003). Defects in the human Swi-Snf complex are linked... with permission from Archana Dhasarathy. 12 An earlier study showed that yeast genes could be classified into three distinct classes based on their requirement for the chromatin remodelers Swi- Snf and SAGA: 1) those genes which require both Swi-Snf...

  18. DEPARTMENT OF CONDENSED MATTER PHYSICS

    E-print Network

    Shyamasundar, R.K.

    DEPARTMENT OF CONDENSED MATTER PHYSICS AND MATERIALS SCIENCE Welcome to the Department of Condensed Matter Physics and Materials Science (CMPMS) at TIFR! Research in CMPMS asks questions about j a y a r a g h a v a n #12;Condensed Matter Physics & Materials Science Research areas A common

  19. Physics 232 Condensed Matter Physics

    E-print Network

    Young, A. Peter

    1 Physics 232 Condensed Matter Physics Instructor: Peter Young Office: 212 ISB Telephone: 459://apyoung.com/232 TOPICS This course on condensed matter physics will cover three areas: · magnetism, · optical physics such as Condensed Matter Physics by M. Mardar Solid State Physics by N. Ashroft and N. D. Mermin

  20. Against vaccine assay secrecy.

    PubMed

    Herder, Matthew; Hatchette, Todd F; Halperin, Scott A; Langley, Joanne M

    2015-01-01

    Increasing the transparency of the evidence base behind health interventions such as pharmaceuticals, biologics, and medical devices, has become a major point of critique, conflict, and policy focus in recent years. Yet the lack of publicly available information regarding the immunogenicity assays upon which many important, widely used vaccines are based has received no attention to date. In this paper we draw attention to this critical public health problem by reporting on our efforts to secure vaccine assay information in respect of 10 vaccines through Canada's access to information law. We argue, under Canadian law, that the public health interest in having access to the methods for these laboratory procedures should override claims by vaccine manufacturers and regulators that this information is proprietary; and, we call upon several actors to take steps to ensure greater transparency with respect to vaccine assays, including regulators, private firms, researchers, research institutions, research funders, and journal editors. PMID:25826194

  1. Rover waste assay system

    SciTech Connect

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  2. [Glutathione and glutathione assays].

    PubMed

    Rousar, Tomás; Cervinková, Zuzana; Muzáková, Vladimíra; Kucera, Otto; Lotková, Halka; Krivákovaá, Pavla

    2005-01-01

    Glutathione, the very important intracellular antioxidant, is present in intracelullar environment in milimolar concentrations. Glutathione is a tripeptide molecule, which plays an essential role in the antioxidant system, as well as in maintenance of the intracellular redox state. This thiol compound exists in two forms, the reduced (GSH) and the oxidized (GSSG), and the ratio of both forms is crucial for the characterization of the oxidative stress in cells. Number of analytical methods have been developed for the measurement of the glutathione. Especially, High Performance Liquid Chromatography methods (HPLC) are mostly used linked to different types of detection, including electrochemical, UV/VIS or fluorimetric detection. Another approach for glutathione assay is using the spectral methods, either fluorimetric or spectrophotometric assays. In enzymatic assay, glutathione reductase reduces GSSG with simultaneous oxidation of specific substrate, which is sequentially photometrically detected. The fluorimetric method is based on the detection of derivatized GSH molecule. PMID:16669486

  3. Functional Anatomy of Polycomb and Trithorax Chromatin Landscapes in Drosophila Embryos

    PubMed Central

    Leblanc, Benjamin; Portoso, Manuela; Jaschek, Rami; Tolhuis, Bas; van Lohuizen, Maarten; Tanay, Amos; Cavalli, Giacomo

    2009-01-01

    Polycomb group (PcG) and trithorax group (trxG) proteins are conserved chromatin factors that regulate key developmental genes throughout development. In Drosophila, PcG and trxG factors bind to regulatory DNA elements called PcG and trxG response elements (PREs and TREs). Several DNA binding proteins have been suggested to recruit PcG proteins to PREs, but the DNA sequences necessary and sufficient to define PREs are largely unknown. Here, we used chromatin immunoprecipitation (ChIP) on chip assays to map the chromosomal distribution of Drosophila PcG proteins, the N- and C-terminal fragments of the Trithorax (TRX) protein and four candidate DNA-binding factors for PcG recruitment. In addition, we mapped histone modifications associated with PcG-dependent silencing and TRX-mediated activation. PcG proteins colocalize in large regions that may be defined as polycomb domains and colocalize with recruiters to form several hundreds of putative PREs. Strikingly, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding, suggesting that recruiter proteins have a dual function in activation as well as silencing. One major discriminant between activation and silencing is the strong binding of Pleiohomeotic (PHO) to silenced regions, whereas its homolog Pleiohomeotic-like (PHOL) binds preferentially to active promoters. In addition, the C-terminal fragment of TRX (TRX-C) showed high affinity to PcG binding sites, whereas the N-terminal fragment (TRX-N) bound mainly to active promoter regions trimethylated on H3K4. Our results indicate that DNA binding proteins serve as platforms to assist PcG and trxG binding. Furthermore, several DNA sequence features discriminate between PcG- and TRX-N–bound regions, indicating that underlying DNA sequence contains critical information to drive PREs and TREs towards silencing or activation. PMID:19143474

  4. Sidestream condensate polishing for PWRs

    SciTech Connect

    Shor, S.W.W.; Yim, S.L.; Rios, J.; Liu, J.

    1986-06-01

    Condensate polishers are used in power plant condensate system to remove both particulate matter and ionized corrodents. Their conventional location is just downstream of the hotwell pumps (condensate pumps). Most polisher installations have enough flow capacity to polish 100% of the condensate. This inline configuration has some disadvantage, including a flow that varies with unit load and tends to disturb the polisher beds and reduce their effectiveness, and a potential for interrupting flow to the feedwater pumps. An alternate arrangement where water is extracted from either the condenser or the condensate system, polished and returned to the system, has been used in a few plants. Three different ways of doing this have been used: divide the condenser hotwell into two parts, one of which receives condensate from the tube bundles and the other of which is sheltered. Take unpolished condensate from the first part, purify it and return it to the other part from which the condensate pumps take suction; take unpolished condensate from one end of a divided header on the suction side of the hotwell pumps and after polishing it return it to the other end; and take unpolished condensate from a header on the discharge side of the condensate pumps, purify it and return it to the condensate system a short distance downstream. The three variants are analyzed in this report. It is concluded that the variant where the connections are on the discharge side of the condensate pumps is the most desirable for retrofitting, in all cases being far easier to retrofit than an inline polisher. In many cases it will be most desirable for new construction.

  5. A chromatin-modifying protein is a context-dependent modulator of cell survival in hematopoietic malignancies

    E-print Network

    Shingleton, Jennifer Ricks

    2014-01-01

    Proteins that modify chromatin architecture are important drivers of leukemia. Many genetic lesions in hematopoietic malignancies lead to altered function of one or more chromatin-modifying proteins, often resulting in ...

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

    NASA Astrophysics Data System (ADS)

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

    2007-09-01

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

  7. GenStructure/bionet.genome.gene-structure: Genome and Chromatin Structure and Function Newsgroup

    NSDL National Science Digital Library

    The purpose of this moderated newsgroup is to provide a proper forum for the discussion of issues pertaining to and involving genome and/or chromatin structure and function. Primarily it should enable those researchers who work in genome/chromatin structure or related fields to communicate ideas and information, as well as provide a chance for collaboration among national and international research groups.

  8. Chromatin higher-order structure studied by neutron scattering and scanning transmission electron microscopy

    Microsoft Academic Search

    S. E. Gerchman; V. Ramakrishnan

    1987-01-01

    Neutron scattering in solution and scanning transmission electron microscopy were simultaneously done on chicken erythrocyte chromatin at various salt and magnesium concentrations. We show that chromatin is organized into a higher-order structure even at low ionic strength and that the mass per unit length increases continuously as a function of salt concentration, reaching a limiting value of between six and

  9. Different Functional Modes of p300 in Activation of RNA Polymerase III Transcription from Chromatin Templates

    Microsoft Academic Search

    Claudia Mertens; Robert G. Roeder

    2008-01-01

    Transcriptional coactivators that regulate the activity of human RNA polymerase III (Pol III) in the context of chromatin have not been reported. Here, we describe a completely defined in vitro system for transcription of a human tRNA gene assembled into a chromatin template. Transcriptional activation and histone acetyla- tion in this system depend on recruitment of p300 by general initiation

  10. Features of endogenous cardiomyocyte chromatin revealed by super-resolution STED microscopy

    PubMed Central

    Mitchell-Jordan, Scherise; Chen, Haodong; Franklin, Sarah; Stefani, Enrico; Bentolila, Laurent A.; Vondriska, Thomas M.

    2013-01-01

    Despite the extensive knowledge of the functional unit of chromatin—the nucleosome—for which structural information exists at the atomic level, little is known about the endogenous structure of eukaryotic genomes. Chromosomal capture techniques and genome-wide chromatin immunoprecipitation and next generation sequencing have provided complementary insight into global features of chromatin structure, but these methods do not directly measure structural features of the genome in situ. This lack of insight is particularly troublesome in terminally differentiated cells which must reorganize their genomes for large scale gene expression changes in the absence of cell division. For example, cardiomyocytes, which are fully committed and reside in interphase, are capable of massive gene expression changes in response to physiological stimuli, but the global changes in chromatin structure that enable such transcriptional changes are unknown. The present study addressed this problem utilizing super-resolution stimulated emission depletion (STED) microscopy to directly measure chromatin features in mammalian cells. We demonstrate that immunolabeling of histone H3 coupled with STED imaging reveals chromatin domains on a scale of 40–70 nm, several folds better than the resolution of conventional confocal microscopy. An analytical workflow is established to detect changes in chromatin structure following acute stimuli and used to investigate rearrangements in cardiomyocyte genomes following agonists that induce cellular hypertrophy. This approach is readily adaptable to investigation of other nuclear features using a similar antibody-based labeling technique and enables direct measurements of chromatin domain changes in response to physiological stimuli. PMID:22846883

  11. Chromatin domain activation via GATA-1 utilization of a small subset of dispersed GATA motifs

    E-print Network

    Bresnick, Emery H.

    Chromatin domain activation via GATA-1 utilization of a small subset of dispersed GATA motifs occupancy of such motifs in chromatin are not understood. The transcription factor GATA-1 that regulates red blood cell development binds with high affinity to GATA motifs, and initial studies suggest

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

    PubMed

    Babaei, Sepideh; Mahfouz, Ahmed; Hulsman, Marc; Lelieveldt, Boudewijn P F; de Ridder, Jeroen; Reinders, Marcel

    2015-05-01

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

  13. Large-scale chromatin structural domains within mitotic and interphase chromosomes in vivo and in vitro

    Microsoft Academic Search

    Andrew S. Belmont; Michael B. Braunfeld; John W. Sedat; David A. Agard

    1989-01-01

    Higher-order chromatin structural domains approximately 130 nm in width are observed as prominent components of both Drosophila melanogaster and human mitotic chromosomes using buffer conditions which preserve chromosome morphology as determined by light microscopic comparison with chromosomes within living cells. Spatially discrete chromatin structural domains of similar size also exist as prominent components within interphase nuclei prepared under equivalent conditions.

  14. ZNF143 provides sequence specificity to secure chromatin interactions at gene promoters.

    PubMed

    Bailey, Swneke D; Zhang, Xiaoyang; Desai, Kinjal; Aid, Malika; Corradin, Olivia; Cowper-Sal Lari, Richard; Akhtar-Zaidi, Batool; Scacheri, Peter C; Haibe-Kains, Benjamin; Lupien, Mathieu

    2015-01-01

    Chromatin interactions connect distal regulatory elements to target gene promoters guiding stimulus- and lineage-specific transcription. Few factors securing chromatin interactions have so far been identified. Here, by integrating chromatin interaction maps with the large collection of transcription factor-binding profiles provided by the ENCODE project, we demonstrate that the zinc-finger protein ZNF143 preferentially occupies anchors of chromatin interactions connecting promoters with distal regulatory elements. It binds directly to promoters and associates with lineage-specific chromatin interactions and gene expression. Silencing ZNF143 or modulating its DNA-binding affinity using single-nucleotide polymorphisms (SNPs) as a surrogate of site-directed mutagenesis reveals the sequence dependency of chromatin interactions at gene promoters. We also find that chromatin interactions alone do not regulate gene expression. Together, our results identify ZNF143 as a novel chromatin-looping factor that contributes to the architectural foundation of the genome by providing sequence specificity at promoters connected with distal regulatory elements. PMID:25645053

  15. SWI2/SNF2 chromatin remodeling ATPases overcome polycomb repression and control floral organ identity

    E-print Network

    Plotkin, Joshua B.

    SWI2/SNF2 chromatin remodeling ATPases overcome polycomb repression and control floral organ that the SWI2/SNF2 chromatin-remodeling ATPases SPLAYED (SYD) and BRAHMA (BRM) are redundantly required for flower patterning and for the activation of AP3 and AG. The SWI2/SNF2 ATPases are recruited

  16. Recent advances in understanding chromatin remodeling by Swi/Snf complexes

    E-print Network

    Winston, Fred

    Recent advances in understanding chromatin remodeling by Swi/Snf complexes Joseph A Martens and Fred Winston Members of the Swi/Snf family of chromatin-remodeling complexes play critical roles aspects of Swi/Snf complexes, including the roles of specific subunits, the repression of transcription

  17. Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins

    E-print Network

    Blow, J. Julian

    Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated-free system DNA replication Chromatin a b s t r a c t The use of cell-free extracts prepared from eggs apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus

  18. Elongation by RNA polymerase II on chromatin templates requires topoisomerase activity

    Microsoft Academic Search

    Neelima Mondal; Ye Zhang; Zophonias Jonsson; Suman Kumar Dhar; Madhu Kannapiran; Jeffrey D. Parvin

    2003-01-01

    Transcription on chromatin by RNA polymerase II (pol II) is repressed as compared with transcription on histone-free DNA. In this study, we show that human topoisomerase I (topo I) and yeast topo- isomerase II (topo II), each of which relax both posi- tive and negative superhelical tension, reverse the transcriptional repression by chromatin. In the presence of bacterial topo I,

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

    SciTech Connect

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

    2010-05-01

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

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

    PubMed Central

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

    2015-01-01

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

  1. Roberts syndrome: New evidence supporting an altered metaphase chromatin structure

    SciTech Connect

    Shang, X.M.; Schultz, E.L.; Tonk, V. [Univ. of Texas Southwestern Medical Center, Dallas, TX (United States)

    1994-09-01

    Roberts syndrome is a rare autosomal recessive disease clinically manifested in the newborn by mental and growth retardation, tetraphocomelia, and a variety of craniofacial abnormalities. Cell lines derived from RS patients exhibit subtle mutagen hypersensitivity and cytogenetic abnormalities which include random chromosome loss and the splaying of heterochromatic chromosomal regions. The latter, typically detected on C-banded metaphases, has been used prenatally for the diagnosis of RS. To gain further insights into the RS defect, we have examined a number of parameters related to metaphase chromatin structure, with observations as follows. (1) The heterochromatic splaying associated with RS was found to be visible on G- as well as C-banded metaphases. (2) Quantitative evaluations using fluorescence image analysis revealed that RS metaphase chromosomes bind DAPI less efficiently than chromosomes from normal cells. (3) Denaturation of chromosomal DNA with either a C-banding procedure or 70% formamide at 70{degree}C each produced an aberrant hybridization pattern on RS chromosomes in FISH experiments employing biotinylated total human DNA as probe. (4) RS cells exhibited a >3-fold increase in sensitivity to VM-26, a potent inhibitor of topoisomerase II. Collectively, the aforementioned data support the notion that the primary defect in RS results in an altered metaphase chromatin structure.

  2. Sequence-specific packaging of DNA in human sperm chromatin

    SciTech Connect

    Gatewood, J.M.; Cook, G.R.; Balhorn, R.; Bradbury, E.M.; Schmid, C.W.

    1987-05-22

    The DNA in human sperm chromatin is packaged into nucleoprotamine (approx.85%) and nucleohistone (approx.15%). Whether these two chromatin fractions are sequence-specific subsets of the spermatozoon genome is the question addressed in this report. Sequence-specific packaging would suggest distinct structural and functional roles for nucleohistone and nucleoprotamine in late spermatogenesis or early development or both. After removal of histones with 0.65 M NaCl, exposed DNA was cleaved with Bam HI restriction endonuclease and separated by centrifugation from insoluble nucleoprotamine. The DNA sequence distribution of nucleohistone DNA in the supernatant and nucleoprotamine DNA in the pellet was compared by cloning size-selected single-copy sequences and by using the derived clones as probes of nucleohistone DNA and nucleoprotamine DNA. Two clones derived from nucleohistone DNA preferentially hybridized to nucleohistone DNA, and two clones derived from nucleoprotamine DNA preferentially hybridized to nucleoprotamine DNA, which demonstrated the existence of sequence-specific nucleohistone and nucleoprotamine components within the human spermatozoon.

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

    PubMed Central

    Kok, Kurtulus; Arnosti, David N.

    2015-01-01

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

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

    PubMed

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

    2014-11-13

    The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones. PMID:25363760

  5. Epigenetic Regulation of Chromatin States in Schizosaccharomyces pombe.

    PubMed

    Allshire, Robin C; Ekwall, Karl

    2015-01-01

    This article discusses the advances made in epigenetic research using the model organism fission yeast Schizosaccharomyces pombe. S. pombe has been used for epigenetic research since the discovery of position effect variegation (PEV). This is a phenomenon in which a transgene inserted within heterochromatin is variably expressed, but can be stably inherited in subsequent cell generations. PEV occurs at centromeres, telomeres, ribosomal DNA (rDNA) loci, and mating-type regions of S. pombe chromosomes. Heterochromatin assembly in these regions requires enzymes that modify histones and the RNA interference (RNAi) machinery. One of the key histone-modifying enzymes is the lysine methyltransferase Clr4, which methylates histone H3 on lysine 9 (H3K9), a classic hallmark of heterochromatin. The kinetochore is assembled on specialized chromatin in which histone H3 is replaced by the variant CENP-A. Studies in fission yeast have contributed to our understanding of the establishment and maintenance of CENP-A chromatin and the epigenetic activation and inactivation of centromeres. PMID:26134317

  6. The FACT chromatin modulator: genetic and structure/function relationships.

    PubMed

    Singer, Richard A; Johnston, Gerald C

    2004-08-01

    The chromatin configuration of DNA inhibits access by enzymes such as RNA polymerase II. This inhibition is alleviated by FACT, a conserved transcription elongation factor that has been found to reconfigure nucleosomes to allow transit along the DNA by RNA polymerase II, thus facilitating transcription. FACT also reorganizes nucleosomes after the passage of RNA polymerase II, as indicated by the effects of certain FACT mutations. The larger of the two subunits of FACT is Spt16/Cdc68, while the smaller is termed SSRP1 (vertebrates) or Pob3 (budding yeast). The HMG-box domain at the C terminus of SSRP1 is absent from Pob3; the function of this domain for yeast FACT is supplied by the small HMG-box protein Nhp6. In yeast, this "detachable" HMG domain is a general chromatin component, unlike FACT, which is found only in transcribed regions and associated with RNA polymerase II. The several domains of the larger FACT subunit are also likely to have different functions. Genetic studies suggest that FACT mediates nucleosome reorganization along several pathways, and reinforce the notion that protein unfolding and (or) refolding is involved in FACT activity for transcription. PMID:15284894

  7. Macroautophagic cargo sequestration assays.

    PubMed

    Seglen, Per O; Luhr, Morten; Mills, Ian G; Sætre, Frank; Szalai, Paula; Engedal, Nikolai

    2015-03-01

    Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy. PMID:25576638

  8. The tethering of chromatin to the nuclear envelope supports nuclear mechanics

    PubMed Central

    Schreiner, Sarah M.; Koo, Peter K.; Zhao, Yao; Mochrie, Simon G. J.; King, Megan C.

    2015-01-01

    The nuclear lamina is thought to be the primary mechanical defence of the nucleus. However, the lamina is integrated within a network of lipids, proteins and chromatin; the interdependence of this network poses a challenge to defining the individual mechanical contributions of these components. Here, we isolate the role of chromatin in nuclear mechanics by using a system lacking lamins. Using novel imaging analyses, we observe that untethering chromatin from the inner nuclear membrane results in highly deformable nuclei in vivo, particularly in response to cytoskeletal forces. Using optical tweezers, we find that isolated nuclei lacking inner nuclear membrane tethers are less stiff than wild-type nuclei and exhibit increased chromatin flow, particularly in frequency ranges that recapitulate the kinetics of cytoskeletal dynamics. We suggest that modulating chromatin flow can define both transient and long-lived changes in nuclear shape that are biologically important and may be altered in disease. PMID:26074052

  9. The tethering of chromatin to the nuclear envelope supports nuclear mechanics.

    PubMed

    Schreiner, Sarah M; Koo, Peter K; Zhao, Yao; Mochrie, Simon G J; King, Megan C

    2015-01-01

    The nuclear lamina is thought to be the primary mechanical defence of the nucleus. However, the lamina is integrated within a network of lipids, proteins and chromatin; the interdependence of this network poses a challenge to defining the individual mechanical contributions of these components. Here, we isolate the role of chromatin in nuclear mechanics by using a system lacking lamins. Using novel imaging analyses, we observe that untethering chromatin from the inner nuclear membrane results in highly deformable nuclei in vivo, particularly in response to cytoskeletal forces. Using optical tweezers, we find that isolated nuclei lacking inner nuclear membrane tethers are less stiff than wild-type nuclei and exhibit increased chromatin flow, particularly in frequency ranges that recapitulate the kinetics of cytoskeletal dynamics. We suggest that modulating chromatin flow can define both transient and long-lived changes in nuclear shape that are biologically important and may be altered in disease. PMID:26074052

  10. Enhanced chromatin dynamics by FACT promotes transcriptional restart after UV-induced DNA damage.

    PubMed

    Dinant, Christoffel; Ampatziadis-Michailidis, Giannis; Lans, Hannes; Tresini, Maria; Lagarou, Anna; Grosbart, Malgorzata; Theil, Arjan F; van Cappellen, Wiggert A; Kimura, Hiroshi; Bartek, Jiri; Fousteri, Maria; Houtsmuller, Adriaan B; Vermeulen, Wim; Marteijn, Jurgen A

    2013-08-22

    Chromatin remodeling is tightly linked to all DNA-transacting activities. To study chromatin remodeling during DNA repair, we established quantitative fluorescence imaging methods to measure the exchange of histones in chromatin in living cells. We show that particularly H2A and H2B are evicted and replaced at an accelerated pace at sites of UV-induced DNA damage. This accelerated exchange of H2A/H2B is facilitated by SPT16, one of the two subunits of the histone chaperone FACT (facilitates chromatin transcription) but largely independent of its partner SSRP1. Interestingly, SPT16 is targeted to sites of UV light-induced DNA damage-arrested transcription and is required for efficient restart of RNA synthesis upon damage removal. Together, our data uncover an important role for chromatin dynamics at the crossroads of transcription and the UV-induced DNA damage response. PMID:23973375

  11. Diverse lamin-dependent mechanisms interact to control chromatin dynamics. Focus on laminopathies.

    PubMed

    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

  12. Monomethylation of Lysine 20 on Histone H4 Facilitates Chromatin Maturation?

    PubMed Central

    Scharf, Annette N. D.; Meier, Karin; Seitz, Volker; Kremmer, Elisabeth; Brehm, Alexander; Imhof, Axel

    2009-01-01

    Histone modifications play an important role in shaping chromatin structure. Here, we describe the use of an in vitro chromatin assembly system from Drosophila embryo extracts to investigate the dynamic changes of histone modifications subsequent to histone deposition. In accordance with what has been observed in vivo, we find a deacetylation of the initially diacetylated isoform of histone H4, which is dependent on chromatin assembly. Immediately after deposition of the histones onto DNA, H4 is monomethylated at K20, which is required for an efficient deacetylation of the H4 molecule. H4K20 methylation-dependent dl(3)MBT association with chromatin and the identification of a dl(3)MBT-dRPD3 complex suggest that a deacetylase is specifically recruited to the monomethylated substrate through interaction with dl(3)MBT. Our data demonstrate that histone modifications are added and removed during chromatin assembly in a highly regulated manner. PMID:19001096

  13. Gravitational vacuum condensate stars

    PubMed Central

    Mazur, Pawel O.; Mottola, Emil

    2004-01-01

    A new final state of gravitational collapse is proposed. By extending the concept of Bose–Einstein condensation to gravitational systems, a cold, dark, compact object with an interior de Sitter condensate pv = -?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

  14. SWI/SNF-dependent long-range remodeling of yeast HIS3 chromatin

    PubMed Central

    Kim, Yeonjung; Clark, David J.

    2002-01-01

    Current models for the role of the SWI/SNF chromatin remodeling complex in gene regulation are focused on promoters, where the most obvious changes in chromatin structure occur. Here we present evidence that the SWI/SNF complex is involved in the remodeling of the chromatin structure of an entire gene in vivo. We compared the native chromatin structures of a small yeast plasmid containing the HIS3 gene purified from uninduced and induced cells. Relative to uninduced chromatin, induced chromatin displayed a large reduction in negative supercoiling, a large reduction in sedimentation rate, and increased accessibility to restriction enzymes with sites located both near and far from the HIS3 promoter. These observations indicate that the entire plasmid was remodeled as a result of induction. Loss of supercoiling required the presence of the SWI/SNF remodeling complex and the activator Gcn4p in vivo. The TATA boxes were not required, suggesting that remodeling was not the result of transcription. The induction-dependent loss of negative supercoiling was not apparent in cells, indicating that the supercoils were lost preferentially from induced chromatin during purification. Thus, induced HIS3 chromatin has a highly labile structure that is revealed as a result of purification. It is concluded that induction of HIS3 creates a domain of labile chromatin structure that extends far beyond the promoter to include the entire gene. We propose that the SWI/SNF complex is recruited to the HIS3 promoter by Gcn4p and then directs remodeling of a chromatin domain, with important implications for transcription. PMID:12432091

  15. Interplay between chromatin state, regulator binding, and regulatory motifs in six human cell types

    PubMed Central

    Ernst, Jason; Kellis, Manolis

    2013-01-01

    The regions bound by sequence-specific transcription factors can be highly variable across different cell types despite the static nature of the underlying genome sequence. This has been partly attributed to changes in chromatin accessibility, but a systematic picture has been hindered by the lack of large-scale data sets. Here, we use 456 binding experiments for 119 regulators and 84 chromatin maps generated by the ENCODE in six human cell types, and relate those to a global map of regulatory motif instances for these factors. We find specific and robust chromatin state preferences for each regulator beyond the previously reported open-chromatin association, suggesting a much richer chromatin landscape beyond simple accessibility. The preferentially bound chromatin states of regulators were enriched for sequence motifs of regulators relative to all states, suggesting that these preferences are at least partly encoded by the genomic sequence. Relative to all regions bound by a regulator, however, regulatory motifs were surprisingly depleted in the regulator's preferentially bound states, suggesting additional non-sequence-specific binding beyond the level predicted by the regulatory motifs. Such permissive binding was largely restricted to open-chromatin regions showing histone modification marks characteristic of active enhancer and promoter regions, whereas open-chromatin regions lacking such marks did not show permissive binding. Lastly, the vast majority of cobinding of regulator pairs is predicted by the chromatin state preferences of individual regulators. Overall, our results suggest a joint role of sequence motifs and specific chromatin states beyond mere accessibility in mediating regulator binding dynamics across different cell types. PMID:23595227

  16. Bose-Einstein Condensation

    SciTech Connect

    El-Sherbini, Th.M. [Physics Department, Faculty of Science, Cairo University, Giza (Egypt)

    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.

  17. Delineation of the Protein Module That Anchors HMGN Proteins to Nucleosomes in the Chromatin of Living Cells

    Microsoft Academic Search

    Tetsuya Ueda; Frederic Catez; Gabi Gerlitz; Michael Bustin

    2008-01-01

    Numerous nuclear proteins bind to chromatin by targeting unique DNA sequences or specific histone modifications. In contrast, HMGN proteins recognize the generic structure of the 147-bp nucleosome core particle. HMGNs alter the structure and activity of chromatin by binding to nucleosomes; however, the determinants of the specific interaction of HMGNs with chromatin are not known. Here we use systematic mutagenesis,

  18. Asymmetric condensed dark matter

    E-print Network

    Aguirre, Anthony

    2015-01-01

    We explore the viability of a boson dark matter candidate with an asymmetry between the number densities of particles and antiparticles. A simple thermal field theory analysis confirms that, under certain general conditions, this component would develop a Bose-Einstein condensate in the early universe that, for appropriate model parameters, could survive the ensuing cosmological evolution until now. The condensation of a dark matter component in equilibrium with the thermal plasma is a relativistic process, hence the amount of matter dictated by the charge asymmetry is complemented by a hot relic density frozen out at the time of decoupling. Contrary to the case of ordinary WIMPs, dark matter particles in a condensate can be very light, $10^{-22}\\,{\\rm eV} \\lesssim m \\lesssim 10^2\\,{\\rm eV}$; the lower limit arises from constraints on small-scale structure formation, while the upper bound ensures that the density from thermal relics is not too large. Big-Bang nucleosynthesis constrains the temperature of deco...

  19. Bovine Papillomavirus Clastogenic Effect Analyzed in Comet Assay

    PubMed Central

    Araldi, R. P.; Melo, T. C.; Diniz, N.; Mazzuchelli-de-Souza, J.; Carvalho, R. F.; Beçak, W.; Stocco, R. C.

    2013-01-01

    Bovine papillomavirus (BPV) is an oncogenic virus related to serious livestock diseases. Oncoproteins encoded by BPV are involved in several steps of cellular transformation and have been reported as presenting clastogenic effects in peripheral lymphocytes and primary culture cells. The aim of this study was to evaluate the clastogenic potential of BPV types 1, 2, and 4 by comet assay. Peripheral blood was collected from 37 bovines, 32 infected with different levels of papillomatosis (12 animals have no affection) and five calves, virus free (negative control). The viral identification showed presence of more than one virus type in 59.375% of the infected animals. Comet assay was performed according to alkaline technique. The Kruskal-Wallis test showed statistical difference between the negative control group and infected animals (P = 0.0015). The Dunn post hoc test showed difference comparing the infected animals with calves. Mann-Whitney U test verified no difference between animals infected with only one viral type and animals presenting more than one viral type. The comet assay is considered an efficient tool for assessment of damage in the host chromatin due to viral action, specifically highlighting viral activity in blood cells. PMID:23956996

  20. Rejoining and misrejoining of radiation-induced chromatin breaks. III. Hypertonic treatment

    NASA Technical Reports Server (NTRS)

    Durante, M.; George, K.; Wu, H. L.; Yang, T. C.

    1998-01-01

    It has been shown that treatment in anisotonic medium modifies rejoining of radiation-induced breaks in interphase chromosomes. In previous work, we have demonstrated that formation of exchanges in human lymphocytes has a slow component (half-time of 1-2 h), but a fraction of exchanges are also observed in samples assayed soon after exposure. In this paper we studied the effect of hypertonic treatment on rejoining and misrejoining of radiation-induced breaks using fluorescence in situ hybridization of prematurely condensed chromosomes in human lymphocytes. Isolated lymphocytes were irradiated with 7 Gy gamma rays, fused to mitotic hamster cells and incubated in hypertonic solution (0.5 M NaCl) for the period normally allowed for interphase chromosome condensation to occur. The data from hypertonic treatment experiments indicate the presence of a class of interphase chromosome breaks that rejoin and misrejoin very quickly (half-time of 5-6 min). The fast misrejoining of these lesions is considered to be responsible for the initial level of exchanges which we reported previously. No significant effect of hypertonic treatment on the yield of chromosome aberrations scored at the first postirradiation mitosis was detected.

  1. Poor Homologous Synapsis 1 Interacts with Chromatin but Does Not Colocalise with ASYnapsis 1 during Early Meiosis in Bread Wheat.

    PubMed

    Khoo, Kelvin H P; Able, Amanda J; Able, Jason A

    2012-01-01

    Chromosome pairing, synapsis, and DNA recombination are three key processes that occur during early meiosis. A previous study of Poor Homologous Synapsis 1 (PHS1) in maize suggested that PHS1 has a role in coordinating these three processes. Here we report the isolation of wheat (Triticum aestivum) PHS1 (TaPHS1), and its expression profile during and after meiosis. While the TaPHS1 protein has sequence similarity to other plant PHS1/PHS1-like proteins, it also possesses a unique region of oligopeptide repeat units. We show that TaPHS1 interacts with both single- and double-stranded DNA in vitro and provide evidence of the protein region that imparts the DNA-binding ability. Immunolocalisation data from assays conducted using antisera raised against TaPHS1 show that TaPHS1 associates with chromatin during early meiosis, with the signal persisting beyond chromosome synapsis. Furthermore, TaPHS1 does not appear to colocalise with the asynapsis protein (TaASY1) suggesting that these proteins are probably independently coordinated. Significantly, the data from the DNA-binding assays and 3-dimensional immunolocalisation of TaPHS1 during early meiosis indicates that TaPHS1 interacts with DNA, a function not previously observed in either the Arabidopsis or maize PHS1 homologues. As such, these results provide new insight into the function of PHS1 during early meiosis in bread wheat. PMID:22518114

  2. Poor Homologous Synapsis 1 Interacts with Chromatin but Does Not Colocalise with ASYnapsis 1 during Early Meiosis in Bread Wheat

    PubMed Central

    Khoo, Kelvin H. P.; Able, Amanda J.; Able, Jason A.

    2012-01-01

    Chromosome pairing, synapsis, and DNA recombination are three key processes that occur during early meiosis. A previous study of Poor Homologous Synapsis 1 (PHS1) in maize suggested that PHS1 has a role in coordinating these three processes. Here we report the isolation of wheat (Triticum aestivum) PHS1 (TaPHS1), and its expression profile during and after meiosis. While the TaPHS1 protein has sequence similarity to other plant PHS1/PHS1-like proteins, it also possesses a unique region of oligopeptide repeat units. We show that TaPHS1 interacts with both single- and double-stranded DNA in vitro and provide evidence of the protein region that imparts the DNA-binding ability. Immunolocalisation data from assays conducted using antisera raised against TaPHS1 show that TaPHS1 associates with chromatin during early meiosis, with the signal persisting beyond chromosome synapsis. Furthermore, TaPHS1 does not appear to colocalise with the asynapsis protein (TaASY1) suggesting that these proteins are probably independently coordinated. Significantly, the data from the DNA-binding assays and 3-dimensional immunolocalisation of TaPHS1 during early meiosis indicates that TaPHS1 interacts with DNA, a function not previously observed in either the Arabidopsis or maize PHS1 homologues. As such, these results provide new insight into the function of PHS1 during early meiosis in bread wheat. PMID:22518114

  3. Kinetic Tetrazolium Microtiter Assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Stowe, Raymond; Koenig, David

    1993-01-01

    Kinetic tetrazolium microtiter assay (KTMA) involves use of tetrazolium salts and Triton X-100 (or equivalent), nontoxic, in vitro color developer solubilizing colored metabolite formazan without injuring or killing metabolizing cells. Provides for continuous measurement of metabolism and makes possible to determine rate of action of antimicrobial agent in real time as well as determines effective inhibitory concentrations. Used to monitor growth after addition of stimulatory compounds. Provides for kinetic determination of efficacy of biocide, greatly increasing reliability and precision of results. Also used to determine relative effectiveness of antimicrobial agent as function of time. Capability of generating results on day of test extremely important in treatment of water and waste, disinfection of hospital rooms, and in pharmaceutical, agricultural, and food-processing industries. Assay also used in many aspects of cell biology.

  4. Lifespan Assay Reagents needed

    E-print Network

    Lamitina, Todd

    with a 50µl spot of OP50 Lifespan assay using RNAi · 6cm NGM/FUDR RNAi plates - standard NGM recipe + 50µg/ml FUDR + 1mM IPTG + 25µg/ml carbenicillin) seeded with a 50µl spot of appropriate RNAi bacteria · 6cm NGM RNAi plates - standard NGM recipe + 1mM IPTG + 25µg/ml carbenicillin) seeded with a 50µl spot

  5. Theory and simulations of toroidal and rod-like structures in single-molecule DNA condensation

    E-print Network

    Cortini, Ruggero; Victor, Jean-Marc; Barbi, Maria

    2015-01-01

    DNA condensation by multivalent cations plays a crucial role in genome packaging in viruses and sperm heads, and has been extensively studied using single-molecule experimental methods. In those experiments, the values of the critical condensation forces have been used to estimate the amplitude of the attractive DNA-DNA interactions. Here, to describe these experiments, we developed an analytical model and a rigid body Langevin dynamics assay to investigate the behavior of a polymer with self-interactions, in the presence of a traction force applied at its extremities. We model self-interactions using a pairwise attractive potential, thereby treating the counterions implicitly. The analytical model allows to accurately predict the equilibrium structures of toroidal and rod-like condensed structures, and the dependence of the critical condensation force on the DNA length. We find that the critical condensation force depends strongly on the length of the DNA, and finite-size effects are important for molecules ...

  6. Insect vector transmission assays.

    PubMed

    Bosco, Domenico; Tedeschi, Rosemarie

    2013-01-01

    Phytoplasmas are transmitted in a persistent propagative manner by phloem-feeding vectors belonging to the order Hemiptera, suborder Homoptera. Following acquisition from the infected source plant, there is a latent period before the vector can transmit, so transmission assays consist of three basic steps: acquisition, latency, and inoculation. More than 90 vector species (plant-, leafhoppers, and psyllids) have been discovered so far but many others are still undiscovered, and their role in spreading economically important crop diseases is neglected. Therefore, screening for vectors is an essential step in developing rational control strategies targeted against the actual vectors for phytoplasma-associated diseases. The mere detection of a phytoplasma in an insect does not imply that the insect is a vector; a transmission assay is required to provide conclusive evidence. Transmission experiments can be carried out using insects from phytoplasma-free laboratory colonies or field-collections. Moreover, transmission assays can be performed by feeding vectors on an artificial diet through Parafilm(®), after which phytoplasmas can be detected in the sucrose feeding medium by PCR. Transmission trials involve the use of different techniques according to the biology of the different vector species; planthoppers, leafhoppers, and psyllids. PMID:22987407

  7. The Chromatin Assembly Factor 1 Promotes Rad51-Dependent Template Switches at Replication Forks by Counteracting D-Loop Disassembly by the RecQ-Type Helicase Rqh1

    PubMed Central

    Hardy, Julien; Costes, Audrey; Iraqui, Ismail; Ochsenbein, Françoise; Lambert, Sarah A.E.

    2014-01-01

    At blocked replication forks, homologous recombination mediates the nascent strands to switch template in order to ensure replication restart, but faulty template switches underlie genome rearrangements in cancer cells and genomic disorders. Recombination occurs within DNA packaged into chromatin that must first be relaxed and then restored when recombination is completed. The chromatin assembly factor 1, CAF-1, is a histone H3-H4 chaperone involved in DNA synthesis-coupled chromatin assembly during DNA replication and DNA repair. We reveal a novel chromatin factor-dependent step during replication-coupled DNA repair: Fission yeast CAF-1 promotes Rad51-dependent template switches at replication forks, independently of the postreplication repair pathway. We used a physical assay that allows the analysis of the individual steps of template switch, from the recruitment of recombination factors to the formation of joint molecules, combined with a quantitative measure of the resulting rearrangements. We reveal functional and physical interplays between CAF-1 and the RecQ-helicase Rqh1, the BLM homologue, mutations in which cause Bloom's syndrome, a human disease associating genome instability with cancer predisposition. We establish that CAF-1 promotes template switch by counteracting D-loop disassembly by Rqh1. Consequently, the likelihood of faulty template switches is controlled by antagonistic activities of CAF-1 and Rqh1 in the stability of the D-loop. D-loop stabilization requires the ability of CAF-1 to interact with PCNA and is thus linked to the DNA synthesis step. We propose that CAF-1 plays a regulatory role during template switch by assembling chromatin on the D-loop and thereby impacting the resolution of the D-loop. PMID:25313826

  8. The chromatin assembly factor 1 promotes Rad51-dependent template switches at replication forks by counteracting D-loop disassembly by the RecQ-type helicase Rqh1.

    PubMed

    Pietrobon, Violena; Fréon, Karine; Hardy, Julien; Costes, Audrey; Iraqui, Ismail; Ochsenbein, Françoise; Lambert, Sarah A E

    2014-10-01

    At blocked replication forks, homologous recombination mediates the nascent strands to switch template in order to ensure replication restart, but faulty template switches underlie genome rearrangements in cancer cells and genomic disorders. Recombination occurs within DNA packaged into chromatin that must first be relaxed and then restored when recombination is completed. The chromatin assembly factor 1, CAF-1, is a histone H3-H4 chaperone involved in DNA synthesis-coupled chromatin assembly during DNA replication and DNA repair. We reveal a novel chromatin factor-dependent step during replication-coupled DNA repair: Fission yeast CAF-1 promotes Rad51-dependent template switches at replication forks, independently of the postreplication repair pathway. We used a physical assay that allows the analysis of the individual steps of template switch, from the recruitment of recombination factors to the formation of joint molecules, combined with a quantitative measure of the resulting rearrangements. We reveal functional and physical interplays between CAF-1 and the RecQ-helicase Rqh1, the BLM homologue, mutations in which cause Bloom's syndrome, a human disease associating genome instability with cancer predisposition. We establish that CAF-1 promotes template switch by counteracting D-loop disassembly by Rqh1. Consequently, the likelihood of faulty template switches is controlled by antagonistic activities of CAF-1 and Rqh1 in the stability of the D-loop. D-loop stabilization requires the ability of CAF-1 to interact with PCNA and is thus linked to the DNA synthesis step. We propose that CAF-1 plays a regulatory role during template switch by assembling chromatin on the D-loop and thereby impacting the resolution of the D-loop. PMID:25313826

  9. Assembly of telomeric chromatin to create ALTernative endings.

    PubMed

    O'Sullivan, Roderick J; Almouzni, Genevieve

    2014-11-01

    Circumvention of the telomere length-dependent mechanisms that control the upper boundaries of cellular proliferation is necessary for the unlimited growth of cancer. Most cancer cells achieve cellular immortality by up-regulating the expression of telomerase to extend and maintain their telomere length. However, a small but significant number of cancers do so via the exchange of telomeric DNA between chromosomes in a pathway termed alternative lengthening of telomeres, or ALT. Although it remains to be clarified why a cell chooses the ALT pathway and how ALT is initiated, recently identified mutations in factors that shape the chromatin and epigenetic landscape of ALT telomeres are shedding light on these mechanisms. In this review, we examine these recent findings and integrate them into the current models of the ALT mechanism. PMID:25172551

  10. Paternal diet defines offspring chromatin state and intergenerational obesity.

    PubMed

    Öst, Anita; Lempradl, Adelheid; Casas, Eduard; Weigert, Melanie; Tiko, Theodor; Deniz, Merdin; Pantano, Lorena; Boenisch, Ulrike; Itskov, Pavel M; Stoeckius, Marlon; Ruf, Marius; Rajewsky, Nikolaus; Reuter, Gunter; Iovino, Nicola; Ribeiro, Carlos; Alenius, Mattias; Heyne, Steffen; Vavouri, Tanya; Pospisilik, J Andrew

    2014-12-01

    The global rise in obesity has revitalized a search for genetic and epigenetic factors underlying the disease. We present a Drosophila model of paternal-diet-induced intergenerational metabolic reprogramming (IGMR) and identify genes required for its encoding in offspring. Intriguingly, we find that as little as 2 days of dietary intervention in fathers elicits obesity in offspring. Paternal sugar acts as a physiological suppressor of variegation, desilencing chromatin-state-defined domains in both mature sperm and in offspring embryos. We identify requirements for H3K9/K27me3-dependent reprogramming of metabolic genes in two distinct germline and zygotic windows. Critically, we find evidence that a similar system may regulate obesity susceptibility and phenotype variation in mice and humans. The findings provide insight into the mechanisms underlying intergenerational metabolic reprogramming and carry profound implications for our understanding of phenotypic variation and evolution. PMID:25480298

  11. Chromatin architectures at fission yeast transcriptional promoters and replication origins.

    PubMed

    Givens, Robert M; Lai, William K M; Rizzo, Jason M; Bard, Jonathan E; Mieczkowski, Piotr A; Leatherwood, Janet; Huberman, Joel A; Buck, Michael J

    2012-08-01

    We have used micrococcal nuclease (MNase) digestion followed by deep sequencing in order to obtain a higher resolution map than previously available of nucleosome positions in the fission yeast, Schizosaccharomyces pombe. Our data confirm an unusually short average nucleosome repeat length, ?152 bp, in fission yeast and that transcriptional start sites (TSSs) are associated with nucleosome-depleted regions (NDRs), ordered nucleosome arrays downstream and less regularly spaced upstream nucleosomes. In addition, we found enrichments for associated function in four of eight groups of genes clustered according to chromatin configurations near TSSs. At replication origins, our data revealed asymmetric localization of pre-replication complex (pre-RC) proteins within large NDRs-a feature that is conserved in fission and budding yeast and is therefore likely to be conserved in other eukaryotic organisms. PMID:22573177

  12. Increased Polyamines Alter Chromatin and Stabilize Autoantigens in Autoimmune Diseases

    PubMed Central

    Brooks, Wesley H.

    2013-01-01

    Polyamines are small cations with unique combinations of charge and length that give them many putative interactions in cells. Polyamines are essential since they are involved in replication, transcription, translation, and stabilization of macro-molecular complexes. However, polyamine synthesis competes with cellular methylation for S-adenosylmethionine, the methyl donor. Also, polyamine degradation can generate reactive molecules like acrolein. Therefore, polyamine levels are tightly controlled. This control may be compromised in autoimmune diseases since elevated polyamine levels are seen in autoimmune diseases. Here a hypothesis is presented explaining how polyamines can stabilize autoantigens. In addition, the hypothesis explains how polyamines can inappropriately activate enzymes involved in NETosis, a process in which chromatin is modified and extruded from cells as extracellular traps that bind pathogens during an immune response. This polyamine-induced enzymatic activity can lead to an increase in NETosis resulting in release of autoantigenic material and tissue damage. PMID:23616785

  13. Histone target selection within chromatin: an exemplary case of teamwork

    PubMed Central

    Lalonde, Marie-Eve; Cheng, Xue; Côté, Jacques

    2014-01-01

    Histone modifiers like acetyltransferases, methyltransferases, and demethylases are critical regulators of most DNA-based nuclear processes, de facto controlling cell cycle progression and cell fate. These enzymes perform very precise post-translational modifications on specific histone residues, which in turn are recognized by different effector modules/proteins. We now have a better understanding of how these enzymes exhibit such specificity. As they often reside in multisubunit complexes, they use associated factors to target their substrates within chromatin structure and select specific histone mark-bearing nucleosomes. In this review, we cover the current understanding of how histone modifiers select their histone targets. We also explain how different experimental approaches can lead to conflicting results about the histone specificity and function of these enzymes. PMID:24831698

  14. Changes in chromatin state in donors subjected to physical stress

    E-print Network

    Yuriy Shckorbatov; Valeriy Samokhvalov; Dariya Bevziuk; Maxim Kovaliov

    2012-06-04

    The purpose of the present study is to evaluate changes in chromatin of human buccal epithelium under the influence of stressing factor - dosed physical activity. Investigations were performed in a group of students (13 men) of age 19-23. Cells were stained on a slide by a 2% orcein solution in 45% acetic acid during 1 h. The following physiological indexes were determined: arterial blood pressure, pulse frequency, and frequency of breathing. The physical stress produced by the dosed physical activity causes the considerable increase of degree of heterochromatinization in the cell nuclei of human buccal epithelium. As a rule, the level of heterochromatinization increases after first stage of training, but in some donors it increases significantly only after the second stage of training.

  15. Unexpected mobility of plant chromatin-associated HMGB proteins.

    PubMed

    Merkle, Thomas; Grasser, Klaus D

    2011-06-01

    High mobility group (HMG) proteins of the HMGB family containing a highly conserved HMG box are chromatin-associated proteins that interact with DNA and nucleosomes and catalyze changes in DNA topology, thereby facilitating important DNA-dependent processes. The genome of Arabidopsis thaliana encodes 15 different HMG-box proteins that are further subdivided into four groups: HMGB-type proteins, ARID-HMG proteins, 3xHMG proteins that contain three HMG boxes and the structure-specific recognition protein 1 (SSRP1). Typically, HMGB proteins are localized exclusively to the nucleus, like Arabidopsis HMGB1 and B5. However, these Arabidopsis HMGB proteins showed a very high mobility within the nuclear compartment. Recent studies revealed that Arabidopsis HMGB2/3 and B4 proteins are predominantly nuclear but also exist in the cytoplasm, suggesting an as yet unknown cytoplasmic function of these chromosomal HMG proteins. PMID:21543902

  16. Chromatin dynamics of plant telomeres and ribosomal genes.

    PubMed

    Dvo?á?ková, Martina; Fojtová, Miloslava; Fajkus, Ji?í

    2015-07-01

    Telomeres and genes encoding 45S ribosomal RNA (rDNA) are frequently located adjacent to each other on eukaryotic chromosomes. Although their primary roles are different, they show striking similarities with respect to their features and additional functions. Both genome domains have remarkably dynamic chromatin structures. Both are hypersensitive to dysfunctional histone chaperones, responding at the genomic and epigenomic levels. Both generate non-coding transcripts that, in addition to their epigenetic roles, may induce gross chromosomal rearrangements. Both give rise to chromosomal fragile sites, as their replication is intrinsically problematic. However, at the same time, both are essential for maintenance of genomic stability and integrity. Here we discuss the structural and functional inter-connectivity of telomeres and rDNA, with a focus on recent results obtained in plants. PMID:25752316

  17. Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

    PubMed Central

    Poh, Huay Mei; Peh, Su Qin; Ong, Chin Thing; Zhang, Jingyao; Ruan, Xiaoan; Ruan, Yijun

    2012-01-01

    Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video. PMID:22564980

  18. Fluorescence lifetime, precision calorimetry, and fluorescence energy transfer measurements in the study of normal and tumoral chromatin structure

    NASA Astrophysics Data System (ADS)

    Radu, Liliana; Preoteasa, Vasile; Radulescu, Irina; Radu, Serban

    1997-06-01

    Chromatin is a complex of deoxyribonucleic acid (DNA) with proteins, that exists in the nuclei of eukaryotic cells. Three methods have been used to study protein-DNA interactions in chromatin and to compare the chromatin from normal tissue with that from tumoral tissue: determination of the fluorescence lifetimes and measurement of the heats of reaction of complexation of the ligand ethidium bromide with chromatin, and evaluation of the fluorescence energy transfer between two ligands dansyl chloride and acridine orange when coupled with chromatin.

  19. In vivo dynamical behavior of yeast chromatin modeled as an entangled polymer network with constraint release

    NASA Astrophysics Data System (ADS)

    Wang, Chenxi; Kilfoil, Maria L.

    2013-03-01

    The high fidelity segregation of chromatin is the central problem in cell mitosis. The role of mechanics underlying this, however, is undetermined. Work in this area has largely focused on cytoskeletal elements of the process. Preliminary work in our lab suggests the mechanical properties of chromatin are fundamental in this process. Nevertheless, the mechanical properties of chromatin in the cellular context are not well-characterized. For better understanding of the role of mechanics in this cellular process, and of the chromatin mechanics in vivo generally, a systematic dynamical description of chromatin in vivo is required. Accordingly, we label specific sites on chromatin with fluorescent proteins of different wave lengths, enabling us to detect multiple spots separately in 3D and track their displacements in time inside living yeast cells. We analyze the pairwise cross-correlated motion between spots as a function of relative distance along the DNA contour. Comparison between the reptation model and our data serves to test our conjecture that chromatin in the cell is basically an entangled polymer network under constraints to thermal motion, and removal of constraints by non-thermal cellular processes is expected to affect its dynamic behavior.

  20. How the cell cycle impacts chromatin architecture and influences cell fate

    PubMed Central

    Ma, Yiqin; Kanakousaki, Kiriaki; Buttitta, Laura

    2015-01-01

    Since the earliest observations of cells undergoing mitosis, it has been clear that there is an intimate relationship between the cell cycle and nuclear chromatin architecture. The nuclear envelope and chromatin undergo robust assembly and disassembly during the cell cycle, and transcriptional and post-transcriptional regulation of histone biogenesis and chromatin modification is controlled in a cell cycle-dependent manner. Chromatin binding proteins and chromatin modifications in turn influence the expression of critical cell cycle regulators, the accessibility of origins for DNA replication, DNA repair, and cell fate. In this review we aim to provide an integrated discussion of how the cell cycle machinery impacts nuclear architecture and vice-versa. We highlight recent advances in understanding cell cycle-dependent histone biogenesis and histone modification deposition, how cell cycle regulators control histone modifier activities, the contribution of chromatin modifications to origin firing for DNA replication, and newly identified roles for nucleoporins in regulating cell cycle gene expression, gene expression memory and differentiation. We close with a discussion of how cell cycle status may impact chromatin to influence cell fate decisions, under normal contexts of differentiation as well as in instances of cell fate reprogramming. PMID:25691891

  1. The bromodomain protein Brd4 insulates chromatin from DNA damage signalling.

    PubMed

    Floyd, Scott R; Pacold, Michael E; Huang, Qiuying; Clarke, Scott M; Lam, Fred C; Cannell, Ian G; Bryson, Bryan D; Rameseder, Jonathan; Lee, Michael J; Blake, Emily J; Fydrych, Anna; Ho, Richard; Greenberger, Benjamin A; Chen, Grace C; Maffa, Amanda; Del Rosario, Amanda M; Root, David E; Carpenter, Anne E; Hahn, William C; Sabatini, David M; Chen, Clark C; White, Forest M; Bradner, James E; Yaffe, Michael B

    2013-06-13

    DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response. Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage. PMID:23728299

  2. hiHMM: Bayesian non-parametric joint inference of chromatin state maps

    PubMed Central

    Sohn, Kyung-Ah; Ho, Joshua W. K.; Djordjevic, Djordje; Jeong, Hyun-hwan; Park, Peter J.; Kim, Ju Han

    2015-01-01

    Motivation: Genome-wide mapping of chromatin states is essential for defining regulatory elements and inferring their activities in eukaryotic genomes. A number of hidden Markov model (HMM)-based methods have been developed to infer chromatin state maps from genome-wide histone modification data for an individual genome. To perform a principled comparison of evolutionarily distant epigenomes, we must consider species-specific biases such as differences in genome size, strength of signal enrichment and co-occurrence patterns of histone modifications. Results: Here, we present a new Bayesian non-parametric method called hierarchically linked infinite HMM (hiHMM) to jointly infer chromatin state maps in multiple genomes (different species, cell types and developmental stages) using genome-wide histone modification data. This flexible framework provides a new way to learn a consistent definition of chromatin states across multiple genomes, thus facilitating a direct comparison among them. We demonstrate the utility of this method using synthetic data as well as multiple modENCODE ChIP-seq datasets. Conclusion: The hierarchical and Bayesian non-parametric formulation in our approach is an important extension to the current set of methodologies for comparative chromatin landscape analysis. Availability and implementation: Source codes are available at https://github.com/kasohn/hiHMM. Chromatin data are available at http://encode-x.med.harvard.edu/data_sets/chromatin/. Contact: peter_park@harvard.edu or juhan@snu.ac.kr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25725496

  3. Cell Cycle-Dependent Binding Modes of the Ran Exchange Factor RCC1 to Chromatin

    PubMed Central

    Bierbaum, Martin; Bastiaens, Philippe I.H.

    2013-01-01

    The formation of an activity gradient of the small G-protein Ran around chromatin depends on the differential partitioning of the opposing enzyme activities of the Ran guanine nucleotide exchange factor RCC1 that resides on chromatin, and the cytoplasmic Ran GTPase activating protein RanGAP. We studied the time-dependent interaction kinetics between RCC1 and chromatin and the mobility of the Ran-RCC1 complex in living cells by fluorescence correlation spectroscopy to investigate whether binding of RCC1 to chromatin regulates the exchange activity of RCC1, and whether the stability of the RCC1-chromatin interaction is regulated during the cell cycle. We found that RCC1 mobility is dominated by two states: a highly mobile state that is trapped within chromatin, and a transiently immobilized state that is stabilized during mitosis. We show that only the immobilized state of RCC1 interacts with Ran and conclude that its guanine nucleotide exchange activity is restricted to specific sites on chromatin. PMID:23601311

  4. The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins.

    PubMed

    Orphanides, G; Wu, W H; Lane, W S; Hampsey, M; Reinberg, D

    1999-07-15

    The regulation of gene expression depends critically upon chromatin structure. Transcription of protein-coding genes can be reconstituted on naked DNA with only the general transcription factors and RNA polymerase II. This minimal system cannot transcribe DNA packaged into chromatin, indicating that accessory factors may facilitate access to DNA. Two classes of accessory factor, ATP-dependent chromatin-remodelling enzymes and histone acetyltransferases, facilitate transcription initiation from chromatin templates. FACT (for facilitates chromatin transcription) is a chromatin-specific elongation factor required for transcription of chromatin templates in vitro. Here we show that FACT comprises a new human homologue of the Saccharomyces cerevisiae Spt16/Cdc68 protein and the high-mobility group-1-like protein structure-specific recognition protein-1. Yeast SPT16/CDC68 is an essential gene that has been implicated in transcription and cell-cycle regulation. Consistent with our biochemical analysis of FACT, we provide evidence that Spt16/Cdc68 is involved in transcript elongation in vivo. Moreover, FACT specifically interacts with nucleosomes and histone H2A/H2B dimers, indicating that it may work by promoting nucleosome disassembly upon transcription. In support of this model, we show that FACT activity is abrogated by covalently crosslinking nucleosomal histones. PMID:10421373

  5. Targeted INO80 enhances subnuclear chromatin movement and ectopic homologous recombination

    PubMed Central

    Neumann, Frank R.; Dion, Vincent; Gehlen, Lutz R.; Tsai-Pflugfelder, Monika; Schmid, Roger; Taddei, Angela; Gasser, Susan M.

    2012-01-01

    Chromatin in the interphase nucleus moves in a constrained random walk. Despite extensive study, the molecular causes of such movement and its impact on DNA-based reactions are unclear. Using high-precision live fluorescence microscopy in budding yeast, we quantified the movement of tagged chromosomal loci to which transcriptional activators or nucleosome remodeling complexes were targeted. We found that local binding of the transcriptional activator VP16, but not of the Gal4 acidic domain, enhances chromatin mobility. The increase in movement did not correlate strictly with RNA polymerase II (PolII) elongation, but could be phenocopied by targeting the INO80 remodeler to the locus. Enhanced chromatin mobility required Ino80's ATPase activity. Consistently, the INO80-dependent remodeling of nucleosomes upon transcriptional activation of the endogenous PHO5 promoter enhanced chromatin movement locally. Finally, increased mobility at a double-strand break was also shown to depend in part on the INO80 complex. This correlated with increased rates of spontaneous gene conversion. We propose that local chromatin remodeling and nucleosome eviction increase large-scale chromatin movements by enhancing the flexibility of the chromatin fiber. PMID:22345518

  6. Regulation of the Chlamydomonas Cell Cycle by a Stable, Chromatin-Associated Retinoblastoma Tumor Suppressor Complex[W

    PubMed Central

    Olson, Bradley J.S.C.; Oberholzer, Michael; Li, Yubing; Zones, James M.; Kohli, Harjivan S.; Bisova, Katerina; Fang, Su-Chiung; Meisenhelder, Jill; Hunter, Tony; Umen, James G.

    2010-01-01

    We examined the cell cycle dynamics of the retinoblastoma (RB) protein complex in the unicellular alga Chlamydomonas reinhardtii that has single homologs for each subunit—RB, E2F, and DP. We found that Chlamydomonas RB (encoded by MAT3) is a cell cycle–regulated phosphoprotein, that E2F1-DP1 can bind to a consensus E2F site, and that all three proteins interact in vivo to form a complex that can be quantitatively immunopurified. Yeast two-hybrid assays revealed the formation of a ternary complex between MAT3, DP1, and E2F1 that requires a C-terminal motif in E2F1 analogous to the RB binding domain of plant and animal E2Fs. We examined the abundance of MAT3/RB and E2F1-DP1 in highly synchronous cultures and found that they are synthesized and remain stably associated throughout the cell cycle with no detectable fraction of free E2F1-DP1. Consistent with their stable association, MAT3/RB and DP1 are constitutively nuclear, and MAT3/RB does not require DP1-E2F1 for nuclear localization. In the nucleus, MAT3/RB remains bound to chromatin throughout the cell cycle, and its chromatin binding is mediated through E2F1-DP1. Together, our data show that E2F-DP complexes can regulate the cell cycle without dissociation of their RB-related subunit and that other changes may be sufficient to convert RB-E2F-DP from a cell cycle repressor to an activator. PMID:20978220

  7. Biosensors: Viruses for ultrasensitive assays

    NASA Astrophysics Data System (ADS)

    Donath, Edwin

    2009-04-01

    A three-dimensional assay based on genetically engineered viral nanoparticles and nickel nanohairs can detect much lower levels of protein markers associated with heart attacks than conventional assays.

  8. Cell cycle-dependent chromatin shuttling of HBO1–JADE1 histone acetyl transferase (HAT) complex

    PubMed Central

    Siriwardana, Nirodhini S; Meyer, Rosana; Havasi, Andrea; Dominguez, Isabel; Panchenko, Maria V

    2014-01-01

    HAT HBO1 interacts with 2 isoforms of JADE1: JADE1S and JADE1L. JADE1 promotes acetylation of nucleosomal histones by HBO1. HBO1–JADE1 complex facilitates cell proliferation by unclear mechanisms. Here we report intracellular chromatin shuttling of HBO1–JADE1 complex during mitosis coupled to phosphorylation of JADE1. In interphase of dividing cells JADE1S was localized to the nucleus and associated with chromatin. As cells approached mitosis, specifically prophase, JADE1S dissociated from chromatin and associated with cytoplasm. JADE1S chromatin re-association began in telophase and paralleled nuclear envelope membrane reassembly. By early G1, JADE1S was re-associated with chromatin and localized to the nucleus. Importantly, cytoplasmic but not chromatin-associated JADE1 protein was phosphorylated. Mass-Spectrometric analysis of JADE1S protein isolated from G2/M-arrested cells identified 6 phosphorylated amino acid residues: S89, T92, S102, S121, S392, and T468, including 3 novel sites. Temporally, JADE1S phosphorylation and dephosphorylation during mitosis correlated with JADE1S chromatin dissociation and recruitment. JADE1S chromatin recruitment was accompanied by the global histone H4 acetylation. Pharmacological inhibitor of Aurora A kinase prevented JADE1S protein band shift and chromatin dissociation, suggesting regulatory function for phosphorylation. In vivo experiments supported our in vitro results. In mouse kidneys, JADE1S transiently accumulated in the cytoplasm of tubular epithelial cells during kidney regeneration. The transient increase in the number of cells with cytoplasmic JADE1S directly correlated with activation of tubular cell proliferation and inversely correlated with the number of cells with nuclear JADE1S staining, supporting biological role of HBO1–JADE1 shuttling during organ regeneration. PMID:24739512

  9. Variation in chromatin accessibility in human kidney cancer links H3K36 methyltransferase loss with widespread RNA processing defects

    PubMed Central

    Simon, Jeremy M.; Hacker, Kathryn E.; Singh, Darshan; Brannon, A. Rose; Parker, Joel S.; Weiser, Matthew; Ho, Thai H.; Kuan, Pei-Fen; Jonasch, Eric; Furey, Terrence S.; Prins, Jan F.; Lieb, Jason D.; Rathmell, W. Kimryn; Davis, Ian J.

    2014-01-01

    Comprehensive sequencing of human cancers has identified recurrent mutations in genes encoding chromatin regulatory proteins. For clear cell renal cell carcinoma (ccRCC), three of the five commonly mutated genes encode the chromatin regulators PBRM1, SETD2, and BAP1. How these mutations alter the chromatin landscape and transcriptional program in ccRCC or other cancers is not understood. Here, we identified alterations in chromatin organization and transcript profiles associated with mutations in chromatin regulators in a large cohort of primary human kidney tumors. By associating variation in chromatin organization with mutations in SETD2, which encodes the enzyme responsible for H3K36 trimethylation, we found that changes in chromatin accessibility occurred primarily within actively transcribed genes. This increase in chromatin accessibility was linked with widespread alterations in RNA processing, including intron retention and aberrant splicing, affecting ?25% of all expressed genes. Furthermore, decreased nucleosome occupancy proximal to misspliced exons was observed in tumors lacking H3K36me3. These results directly link mutations in SETD2 to chromatin accessibility changes and RNA processing defects in cancer. Detecting the functional consequences of specific mutations in chromatin regulatory proteins in primary human samples could ultimately inform the therapeutic application of an emerging class of chromatin-targeted compounds. PMID:24158655

  10. Steam Condensation Induced Waterhammer 

    E-print Network

    Kirsner, W.

    2000-01-01

    of heat. Wayne was knocked down and stunned by the scalding water spraying from the valve. Egress via the manhole exit was cut offby steam spraying from the valve. The only way out appeared to be through the material passouts constructed into the roof... exceed 1000 psi. This is enough pressure to fracture a cast iron valve. blowout a steam gasket, or burst an accordion type expansion joint. And. in fact. failure ofeach ofthese compo nents in separate condensation induced water hammer accidents has...

  11. Confinement Contains Condensates

    SciTech Connect

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

    2012-03-12

    Dynamical chiral symmetry breaking and its connection to the generation of hadron masses has historically been viewed as a vacuum phenomenon. We argue that confinement makes such a position untenable. If quark-hadron duality is a reality in QCD, then condensates, those quantities that have commonly been viewed as constant empirical mass-scales that fill all spacetime, are instead wholly contained within hadrons; i.e., they are a property of hadrons themselves and expressed, e.g., in their Bethe-Salpeter or light-front wave functions. We explain that this paradigm is consistent with empirical evidence, and incidentally expose misconceptions in a recent Comment.

  12. Exhaled breath condensate: an overview.

    PubMed

    Davis, Michael D; Montpetit, Alison; Hunt, John

    2012-08-01

    Exhaled breath condensate (EBC) is a promising source of biomarkers of lung disease. EBC may be thought of either as a body fluid or as a condensate of exhaled gas. There are 3 principal contributors to EBC: variable-sized particles or droplets that are aerosolized from the airway lining fluid, distilled water that condenses from gas phase out of the nearly water-saturated exhalate, and water-soluble volatiles that are exhaled and absorbed into the condensing breath. The nonvolatile constituents and the water-soluble volatile constituents are of particular interest. Several key issues are discussed in this article. PMID:22877615

  13. TOTAL CULTURABLE VIRUS QUANTAL ASSAY

    EPA Science Inventory

    This chapter describes a quantal method for assaying culturable human enteric viruses from water matrices. The assay differs from the plaque assay described in Chapter 10 (December 1987 Revision) in that it is based upon the direct microscopic viewing of cells for virus-induced ...

  14. Estrogenic effects of marijuana smoke condensate and cannabinoid compounds

    SciTech Connect

    Lee, Soo Yeun [National Institute of Scientific Investigation, 331-1 Shinwol-7-dong, Yangcheon-gu, Seoul 158-707 (Korea, Republic of); Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746 (Korea, Republic of); Oh, Seung Min [Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746 (Korea, Republic of); Chung, Kyu Hyuck [Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746 (Korea, Republic of)]. E-mail: khchung@skku.edu

    2006-08-01

    Chronic exposure to marijuana produces adverse effects on the endocrine and reproductive systems in humans; however, the experimental evidence for this presented thus far has not been without controversy. In this study, the estrogenic effect of marijuana smoke condensate (MSC) was evaluated using in vitro bioassays, viz., the cell proliferation assay, the reporter gene assay, and the ER competitive binding assay. The results of these assays were compared with those of three major cannabinoids, i.e., THC, CBD, and CBN. The estrogenic effect of MSC was further confirmed by the immature female rat uterotrophic assay. MSC stimulated the estrogenicity related to the ER-mediated pathway, while neither THC, CBD, nor CBN did. Moreover, treatment with 10 and 25 mg/kg MSC induced significant uterine response, and 10 mg/kg MSC resulted in an obvious change in the uterine epithelial cell appearance. MSC also enhanced the IGFBP-1 gene expression in a dose-dependent manner. To identify the constituents of MSC responsible for its estrogenicity, the MSC fractionated samples were examined using another cell proliferation assay, and the estrogenic active fraction was analyzed using GC-MS. In the organic acid fraction that showed the strongest estrogenic activity among the seven fractions of MSC, phenols were identified. Our results suggest that marijuana abuse is considered an endocrine-disrupting factor. Furthermore, these results suggest that the phenolic compounds contained in MSC play a role in its estrogenic effect.

  15. Structural hierarchy of chromatin in chicken erythrocyte nuclei based on small-angle neutron scattering: Fractal nature of the large-scale chromatin organization

    NASA Astrophysics Data System (ADS)

    Lebedev, D. V.; Filatov, M. V.; Kuklin, A. I.; Islamov, A. Kh.; Stellbrink, J.; Pantina, R. A.; Denisov, Yu. Yu.; Toperverg, B. P.; Isaev-Ivanov, V. V.

    2008-01-01

    The chromatin organization in chicken erythrocyte nuclei was studied by small-angle neutron scattering in the scattering-vector range from 1.5 × 10-1 to 10-4 Å-1 with the use of the contrast-variation technique. This scattering-vector range corresponds to linear dimensions from 4 nm to 6 ?m and covers the whole hierarchy of chromatin structures, from the nucleosomal structure to the entire nucleus. The results of the present study allowed the following conclusions to be drawn: (1) both the chromatin-protein structure and the structure of the nucleic acid component in chicken erythrocyte nuclei have mass-fractal properties, (2) the structure of the protein component of chromatin exhibits a fractal behavior on scales extending over two orders of magnitude, from the nucleosomal size to the size of an entire nucleus, and (3) the structure of the nucleic acid component of chromatin in chicken erythrocyte nuclei is likewise of a fractal nature and has two levels of organization or two phases with the crossover point at about 300-400 nm.

  16. Structural hierarchy of chromatin in chicken erythrocyte nuclei based on small-angle neutron scattering: Fractal nature of the large-scale chromatin organization

    SciTech Connect

    Lebedev, D. V., E-mail: isaev@omrb.pnpi.spb.ru; Filatov, M. V. [Russian Academy of Sciences, Petersburg Nuclear Physics Institute (Russian Federation); Kuklin, A. I.; Islamov, A. Kh. [Joint Institute of Nuclear Research (Russian Federation); Stellbrink, J. [Research Centre Juelich (Germany); Pantina, R. A.; Denisov, Yu. Yu.; Toperverg, B. P.; Isaev-Ivanov, V. V. [Russian Academy of Sciences, Petersburg Nuclear Physics Institute (Russian Federation)

    2008-01-15

    The chromatin organization in chicken erythrocyte nuclei was studied by small-angle neutron scattering in the scattering-vector range from 1.5 x 10{sup -1} to 10{sup -4} A{sup -1} with the use of the contrast-variation technique. This scattering-vector range corresponds to linear dimensions from 4 nm to 6 {mu}m and covers the whole hierarchy of chromatin structures, from the nucleosomal structure to the entire nucleus. The results of the present study allowed the following conclusions to be drawn: (1) both the chromatin-protein structure and the structure of the nucleic acid component in chicken erythrocyte nuclei have mass-fractal properties, (2) the structure of the protein component of chromatin exhibits a fractal behavior on scales extending over two orders of magnitude, from the nucleosomal size to the size of an entire nucleus, and (3) the structure of the nucleic acid component of chromatin in chicken erythrocyte nuclei is likewise of a fractal nature and has two levels of organization or two phases with the crossover point at about 300-400 nm.

  17. Growth cone collapse assay.

    PubMed

    Cook, Geoffrey M W; Jareonsettasin, Prem; Keynes, Roger J

    2014-01-01

    The growth cone collapse assay has proved invaluable in detecting and purifying axonal repellents. Glycoproteins/proteins present in detergent extracts of biological tissues are incorporated into liposomes, added to growth cones in culture and changes in morphology are then assessed. Alternatively purified or recombinant molecules in aqueous solution may be added directly to the cultures. In both cases after a defined period of time (up to 1 h), the cultures are fixed and then assessed by inverted phase contrast microscopy for the percentage of growth cones showing a collapsed profile with loss of flattened morphology, filopodia, and lamellipodia. PMID:24838959

  18. Macropinosome quantitation assay

    PubMed Central

    Wang, Jack T.H.; Teasdale, Rohan D.; Liebl, David

    2014-01-01

    In contrast to phagocytosis, macropinocytosis is not directly initiated by interactions between cell surface receptors and cargo ligands, but is a result of constitutive membrane ruffling driven by dynamic remodelling of cortical actin cytoskeleton in response to stimulation of growth factor receptors. Wang et al. (2010) [13] developed a reliable assay that allows quantitative assessment of the efficiency and kinetics of macropinosome biogenesis and/or maturation in cells where the function of a targeted protein has been perturbed by pharmacological inhibitors or by knock-down or knock-out approaches. In this manuscript we describe a modified quantitative protocol to measure the rate and volume of fluid phase uptake in adherent cells. This assay:•uses fluorescent dextran, microscopy and semi-automated image analysis;•allows quantitation of macropinosomes within large numbers of individual cells;•can be applied also to non-homogenous cell populations including transiently transfected cell monolayers. We present the background necessary to consider when customising this protocol for application to new cell types or experimental variations.

  19. Assays for ?-synuclein aggregation.

    PubMed

    Giehm, Lise; Lorenzen, Nikolai; Otzen, Daniel E

    2011-03-01

    This review describes different ways to achieve and monitor reproducible aggregation of ?-synuclein, a key protein in the development of Parkinson's disease. For most globular proteins, aggregation is promoted by partially denaturing conditions which compromise the native state without destabilizing the intermolecular contacts required for accumulation of regular amyloid structure. As a natively disordered protein, ?-synuclein can fibrillate under physiological conditions and this process is actually stimulated by conditions that promote structure formation, such as low pH, ions, polyamines, anionic surfactants, fluorinated alcohols and agitation. Reproducibility is a critical issue since ?-synuclein shows erratic fibrillation behavior on its own. Agitation in combination with glass beads significantly reduces the variability of aggregation time curves, but the most reproducible aggregation is achieved by sub-micellar concentrations of SDS, which promote the rapid formation of small clusters of ?-synuclein around shared micelles. Although the fibrils produced this way have a different appearance and secondary structure, they are rich in cross-? structure and are amenable to high-throughput screening assays. Although such assays at best provide a very simplistic recapitulation of physiological conditions, they allow the investigator to focus on well-defined molecular events and may provide the opportunity to identify, e.g. small molecule inhibitors of aggregation that affect these steps. Subsequent experiments in more complex cellular and whole-organism environments can then validate whether there is any relation between these molecular interactions and the broader biological context. PMID:21163351

  20. Improving shuffler assay accuracy

    SciTech Connect

    Rinard, P.M.

    1995-07-01

    Drums of uranium waste should be disposed of in an economical and environmentally sound manner. The most accurate possible assays of the uranium masses in the drums are required for proper disposal. The accuracies of assays from a shuffler are affected by the type of matrix material in the drums. Non-hydrogenous matrices have little effect on neutron transport and accuracies are very good. If self-shielding is known to be a minor problem, good accuracies are also obtained with hydrogenous matrices when a polyethylene sleeve is placed around the drums. But for those cases where self-shielding may be a problem, matrices are hydrogenous, and uranium distributions are non-uniform throughout the drums, the accuracies are degraded. They can be greatly improved by determining the distributions of the uranium and then applying correction factors based on the distributions. This paper describes a technique for determining uranium distributions by using the neutron count rates in detector banks around the waste drum and solving a set of overdetermined linear equations. Other approaches were studied to determine the distributions and are described briefly. Implementation of this correction is anticipated on an existing shuffler next year.

  1. Antikaon condensation in neutron stars

    E-print Network

    Subrata Pal; Debades Bandyopadhyay; Walter Greiner

    2000-03-08

    We investigate the condensation of charged $K^-$ meson and neutral $\\bar K^0$ meson in dense neutron star matter. Calculations are performed in relativistic mean field models in which both the baryon-baryon and (anti)kaon-baryon interactions are mediated by meson exchange. It is found that $\\bar K^0$ condensation is quite sensitive to the antikaon optical potential and depends more strongly on the nucleonic equation of state. For moderate values of antikaon potential and a rather stiff equation of state, a significant region of maximum mass star will contain $\\bar K^0$ meson. The critical density of $\\bar K^0$ condensation is always higher than that of $K^-$ condensation. With the appearance of $K^-$ and $\\bar K^0$ condensates, pairs of $p-K^-$ and $n-\\bar K^0$ are produced with equal proportion leading to a perfectly symmetric matter of nucleons and antikaons in neutron stars. Along with $K^-$ condensate, $\\bar K^0$ condensate makes the equation of state much softer resulting in smaller maximum mass stars compared to the case without any condensate.

  2. Amine catalyzed condensation of tetraethylorthosilicate

    NASA Technical Reports Server (NTRS)

    Jones, S.

    2001-01-01

    The catalysis of the condensation of hydrolyzed metal alkoxides by amines has been mentioned in the literature, but there has been no systematic study of their influence on the rate of the condensation reaction of the alkoxide and the microstructure of the resultant gel.

  3. Condensed Matter Physics - Biology Resonance

    Microsoft Academic Search

    G. Baskaran

    2000-01-01

    The field of condensed matter physics had its genesis this century and it has had a remarkable evolution. A closer look at its growth reveals a hidden aim in the collective consciousness of the field - a part of the development this century is a kind of warm up exercise to understand the nature of living condensed matter, namely the

  4. Principles of Condensed Matter Physics

    Microsoft Academic Search

    P. M. Chaikin; T. C. Lubensky

    2000-01-01

    Now in paperback, this book provides an overview of the physics of condensed matter systems. Assuming a familiarity with the basics of quantum mechanics and statistical mechanics, the book establishes a general framework for describing condensed phases of matter based on symmetries and conservation laws. After surveying the structure and properties of materials with different symmetries, it explores the role

  5. The Chromatin Landscape of Kaposi’s Sarcoma-Associated Herpesvirus

    PubMed Central

    Toth, Zsolt; Brulois, Kevin; Jung, Jae U.

    2013-01-01

    Kaposi’s sarcoma-associated herpesvirus is an oncogenic ?-herpesvirus that causes latent infection in humans. In cells, the viral genome adopts a highly organized chromatin structure, which is controlled by a wide variety of cellular and viral chromatin regulatory factors. In the past few years, interrogation of the chromatinized KSHV genome by whole genome-analyzing tools revealed that the complex chromatin landscape spanning the viral genome in infected cells has important regulatory roles during the viral life cycle. This review summarizes the most recent findings regarding the role of histone modifications, histone modifying enzymes, DNA methylation, microRNAs, non-coding RNAs and the nuclear organization of the KSHV epigenome in the regulation of latent and lytic viral gene expression programs as well as their connection to KSHV-associated pathogenesis. PMID:23698402

  6. Classification of genes using clustering of chromatin state segmentations in human epigenomes

    E-print Network

    Kumar, Nischay

    2013-01-01

    Combinatorial patterns of chromatin marks have been shown to play a significant role in gene regulation activities by changing the landscape of the DNA through chemical means. Recent work has expanded on this observation ...

  7. Isolation of selected chromatin fragments from yeast by site-specific recombination in vivo.

    PubMed

    Ansari, A; Cheng, T H; Gartenberg, M R

    1999-02-01

    A burgeoning interest in the role of chromatin structure in a wide variety of chromosome functions has established a need for methods to obtain chromatin in its native form. Here we describe a simple and efficient method for biochemical isolation of selected chromatin fragments from yeast chromosomes. The approach involves three steps. First, site-specific recombination in vivo is used to excise a chromosomal domain of interest in the form of a small extrachromosomal ring. Second, whole cell lysate is prepared from cultures in which recombination has been induced. Third, differential centrifugation is used to separate excised chromatin rings from chromosomes and other cellular debris. Using this methodology, we show that rings containing the transcriptionally repressed HMR mating-type locus can be formed and isolated in high yield. Furthermore, we show that the isolation procedure results in significant enrichment of recombinant rings. Finally, we show that the nucleosomal organization of the recombined material is not altered during isolation. PMID:10075889

  8. The bromodomain protein Brd4 insulates chromatin from DNA damage signalling

    E-print Network

    Floyd, Scott R.

    DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death. Alterations in chromatin structure are implicated in the ...

  9. Phosphorylation of MeCP2 at Serine 80 regulates its chromatin association and neurological function

    E-print Network

    Tao, Jifang

    Mutations of MECP2 (Methyl-CpG Binding Protein 2) cause Rett syndrome. As a chromatin-associated multifunctional protein, how MeCP2 integrates external signals and regulates neuronal function remain unclear. Although ...

  10. Ordered Arrays of Native Chromatin Molecules for High-Resolution Imaging and Analysis

    PubMed Central

    Cerf, Aline; Tian, Harvey C.; Craighead, Harold G.

    2012-01-01

    Individual chromatin molecules contain valuable genetic and epigenetic information. To date, there have not been reliable techniques available for the controlled stretching and manipulation of individual chromatin fragments for high-resolution imaging and analysis of these molecules. We report the controlled stretching of single chromatin fragments extracted from two different cancerous cell types (M091 and HeLa) characterized through fluorescence microscopy and atomic force microscopy (AFM). Our method combines soft-lithography with molecular stretching to form ordered arrays of more than 250,000 individual chromatin fragments immobilized into a beads-on-a-string structure on a solid transparent support. Using fluorescence microscopy and AFM, we verified the presence of histone proteins after the stretching and transfer process. PMID:22816516

  11. Heritable Individual-Specific and Allele-Specific Chromatin Signatures in Humans

    PubMed Central

    McDaniell, Ryan; Lee, Bum-Kyu; Song, Lingyun; Liu, Zheng; Boyle, Alan P.; Erdos, Michael R.; Scott, Laura J.; Morken, Mario A.; Kucera, Katerina S.; Battenhouse, Anna; Keefe, Damian; Collins, Francis S.; Willard, Huntington F.; Lieb, Jason D.; Furey, Terrence S.; Crawford, Gregory E.; Iyer, Vishwanath R.; Birney, Ewan

    2010-01-01

    The extent to which variation in chromatin structure and transcription factor binding may influence gene expression, and thus underlie or contribute to variation in phenotype, is unknown. To address this question, we cataloged both individual-to-individual variation and differences between homologous chromosomes within the same individual (allele-specific variation) in chromatin structure and transcription factor binding in lymphoblastoid cells derived from individuals of geographically diverse ancestry. Ten percent of active chromatin sites were individual-specific; a similar proportion were allele-specific. Both individual-specific and allele-specific sites were commonly transmitted from parent to child, which suggests that they are heritable features of the human genome. Our study shows that heritable chromatin status and transcription factor binding differ as a result of genetic variation and may underlie phenotypic variation in humans. PMID:20299549

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

    PubMed Central

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

    2014-01-01

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

  13. Chromatin Structure and Gene Expression Programs of Human Embryonic and Induced Pluripotent Stem Cells

    E-print Network

    Guenther, Matthew G.

    Knowledge of both the global chromatin structure and the gene expression programs of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) should provide a robust means to assess whether the genomes ...

  14. Shedding light on large-scale chromatin reorganization in Arabidopsis thaliana.

    PubMed

    van Zanten, Martijn; Tessadori, Federico; Peeters, Anton J M; Fransz, Paul

    2012-05-01

    Plants need to respond quickly and appropriately to various types of light signals from the environment to optimize growth and development. The immediate response to shading, reduced photon flux (low light), and changes in spectral quality involves changes in gene regulation. In the case of more persistent shade, the plant shows a dramatic change in the organization of chromatin. Both plant responses are controlled via photoreceptor signaling proteins. Recently, several studies have revealed similar features of chromatin reorganization in response to various abiotic and biotic signals, while others have unveiled intricate molecular networks of light signaling towards gene regulation. This opinion paper briefly describes the chromatin (de)compaction response from a light-signaling perspective to provide a link between chromatin and the molecular network of photoreceptors and E3 ubiquitin ligase complexes. PMID:22528207

  15. Unlocking the milk protein gene loci during mammary gland development and differentiation; a role for chromatin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mammary gland development and differentiation occur mostly postnatally. Chromatin organization plays a key role in transcriptional and epigenetic regulation during development and differentiation. Considerable knowledge of the systemic hormones and local growth factors important for development and ...

  16. Mapping and Analysis of Chromatin State Dynamics in Nine Human Cell Types

    E-print Network

    Ernst, Jason M.

    Chromatin profiling has emerged as a powerful means of genome annotation and detection of regulatory activity. The approach is especially well suited to the characterization of non-coding portions of the genome, which ...

  17. Wilms Tumor Chromatin Profiles Highlight Stem Cell Properties and a Renal Developmental Network

    E-print Network

    Lander, Eric S.

    Wilms tumor is the most common pediatric kidney cancer. To identify transcriptional and epigenetic mechanisms that drive this disease, we compared genome-wide chromatin profiles of Wilms tumors, embryonic stem cells (ESCs), ...

  18. Application of the protein semisynthesis strategy to the generation of modified chromatin.

    PubMed

    Holt, Matthew; Muir, Tom

    2015-06-01

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

  19. Quantifying transient binding of ISWI chromatin remodelers in living cells by pixel-wise photobleaching

    E-print Network

    Rippe, Karsten

    nucleus, namely, the chromatin remod- elers Snf2H and Snf2L from the imitation switch (ISWI) protein with the associated histone proteins, to regulate DNA compaction and accessi- bility. Snf2H and Snf2L can change

  20. Discovery and Characterization of Chromatin States for Systematic Annotation of the Human Genome

    NASA Astrophysics Data System (ADS)

    Ernst, Jason; Kellis, Manolis

    A plethora of epigenetic modifications have been described in the human genome and shown to play diverse roles in gene regulation, cellular differentiation and the onset of disease. Although individual modifications have been linked to the activity levels of various genetic functional elements, their combinatorial patterns are still unresolved and their potential for systematic de novo genome annotation remains untapped. Here, we use a multivariate Hidden Markov Model to reveal chromatin states in human T cells, based on recurrent and spatially coherent combinations of chromatin marks.We define 51 distinct chromatin states, including promoter-associated, transcription-associated, active intergenic, largescale repressed and repeat-associated states. Each chromatin state shows specific enrichments in functional annotations, sequence motifs and specific experimentally observed characteristics, suggesting distinct biological roles. This approach provides a complementary functional annotation of the human genome that reveals the genome-wide locations of diverse classes of epigenetic function.