Sample records for chromatin condensation assay

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

    PubMed

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

    1997-01-01

    The quality of sperm chromatin is an important factor in fertilization and is especially critical where one spermatozoon is artificially selected for fertilizing an egg (as in intracytoplasmic sperm injection). In this study, flow cytometry after staining of human spermatozoa with Acridine Orange was used to study chromatin structure. A method is described for estimating the percentage of cells in a human sperm sample that have completed epididymal maturation in regard to chromatin condensation. Of the 121 samples of the semen that were examined, nine contained a higher percentage of hypocondensed spermatozoa and six samples contained elevated amounts of hypercondensed spermatozoa. In addition to aberrancies in chromatin condensation other defects showed up as satellite populations of spermatozoa with higher than normal ratios of red/green fluorescence after Acridine Orange staining. Such defects were found in 15 semen samples. The use of swim-up and Percoll gradient centrifugation methods was shown to improve the percentage of spermatozoa with normal chromatin structure in some samples with poor initial quality. PMID:9239707

  2. Ion binding and chromatin condensation.

    PubMed

    Jacobs, G A; Smith, J A; Watt, R A; Barry, J M

    1976-08-01

    1. The binding of 45Ca2+ to hen erythrocyte chromatin has been studied to help elucidate how cations induce a reversible condensation of this chromatin. 2. As the unbound Ca2+ of the medium rises from 0.5 to 4 mM, Ca2+ is bound to the chromatin with a stability constant of approx. 3.1 and a saturation value of 0.25 Ca2+ per DNA phosphate, or one-half the value for pure DNA. Condensation of the chromatin is half complete when this binding of calcium is roughly half complete. Hence the transition from the uncondensed to the condensed state occurs as repulsion between the free DNA phosphates of erythrocyte chromatin is neutralised by bound cations. Genetically active chromatin may be maintained in an uncondensed state in living cells by the presence of different negative groups that remain unneutralised at the unbound cation concentrations of the cell. 3. That only one-half of the calcium binding sites of DNA are masked in erythrocyte chromatin supports recent models of chromatin structure in which the DNA double helix is wound round a core of histones. 4. Competition for calcium binding sites in the chromatin by other cations was also studied. PMID:952998

  3. Activation of DNA damage response signaling by condensed chromatin.

    PubMed

    Burgess, Rebecca C; Burman, Bharat; Kruhlak, Michael J; Misteli, Tom

    2014-12-11

    The DNA damage response (DDR) occurs in the context of chromatin, and architectural features of chromatin have been implicated in DNA damage signaling and repair. Whereas a role of chromatin decondensation in the DDR is well established, we show here that chromatin condensation is integral to DDR signaling. We find that, in response to DNA damage chromatin regions transiently expand before undergoing extensive compaction. Using a protein-chromatin-tethering system to create defined chromatin domains, we show that interference with chromatin condensation results in failure to fully activate DDR. Conversely, forced induction of local chromatin condensation promotes ataxia telangiectasia mutated (ATM)- and ATR-dependent activation of upstream DDR signaling in a break-independent manner. Whereas persistent chromatin compaction enhanced upstream DDR signaling from irradiation-induced breaks, it reduced recovery and survival after damage. Our results demonstrate that chromatin condensation is sufficient for activation of DDR signaling and is an integral part of physiological DDR signaling. PMID:25464843

  4. Quantification of chromatin condensation level by image processing.

    PubMed

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

    2014-03-01

    The level of chromatin condensation is related to the silencing/activation of chromosomal territories and therefore impacts on gene expression. Chromatin condensation changes during cell cycle, progression and differentiation, and is influenced by various physicochemical and epigenetic factors. This study describes a validated experimental technique to quantify chromatin condensation. A novel image processing procedure is developed using Sobel edge detection to quantify the level of chromatin condensation from nuclei images taken by confocal microscopy. The algorithm was developed in MATLAB and used to quantify different levels of chromatin condensation in chondrocyte nuclei achieved through alteration in osmotic pressure. The resulting chromatin condensation parameter (CCP) is in good agreement with independent multi-observer qualitative visual assessment. This image processing technique thereby provides a validated unbiased parameter for rapid and highly reproducible quantification of the level of chromatin condensation. PMID:24099693

  5. Premature chromatin condensation upon accumulation of NIMA.

    PubMed Central

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

    1994-01-01

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

  6. DNA digestion and chromatin condensation during nuclear death in Tetrahymena.

    PubMed

    Mpoke, S; Wolfe, J

    1996-06-15

    DNA fragmentation and nuclear condensation are key features in the regulated cell death of higher animal cells. Nuclear death also occurs as part of a developmentally programmed process during the sexual life cycle of the unicellular organism Tetrahymena. We examined the regulation of nuclear death and the relationship between DNA fragmentation and chromatin condensation in this model system. Nuclear death is accompanied by DNA digestion to low-molecular-weight oligonucleosomal-length fragments, in agreement with a previous study, indicating an endonuclease-like activity typical of apoptosis in higher organisms. Actinomycin D and cycloheximide block DNA digestion as well as nuclear condensation suggesting that nuclear death is under genetic regulation. DNA digestion is completely blocked by aurin, a general nuclease inhibitor. In addition, when DNA fragmentation is blocked, nuclear condensation also fails to occur. Moreover, a kinetic analysis of DNA breakdown, using agarose gels, shows that some DNA digestion occurs before nuclear condensation has taken place. Thus the initiation of DNA digestion may provide conditions necessary for nuclear condensation. Temporary inhibition of nuclear death aborts the death program since after removal of inhibitors cells revert to a vegetative pathway without having eliminated the old or developed the new macronucleus. Zn2+ and EGTA, both of which inhibit apoptosis in some cell types, fail to prevent nuclear condensation or DNA digestion in Tetrahymena, suggesting a requirement here for an endonuclease which is Ca2+-independent and Zn2+-insensitive. With the TUNEL assay, DNA breakdown is detected exclusively in the condensed macronucleus (and occasional micronuclei identified as degenerating haploid products of meiosis), but not in precondensed macronuclei. These studies show that apoptotic-like DNA fragmentation occurs after condensation of the degenerating macronucleus. However, early DNA digestion may be critical for nuclear condensation and subsequent degeneration. PMID:8660924

  7. Proteome analysis of nuclear matrix proteins during apoptotic chromatin condensation

    Microsoft Academic Search

    C Gerner; J Gotzmann; U Fröhwein; C Schamberger; A Ellinger; G Sauermann

    2002-01-01

    The nuclear matrix (NM) is considered a proteinaceous scaffold spatially organizing the interphase nucleus, the integrity of which is affected during apoptosis. Caspase-mediated degradation of NM proteins, such as nuclear lamins, precedes apoptotic chromatin condensation (ACC). Nevertheless, other NM proteins remain unaffected, which most likely maintain a remaining nuclear structure devoid of chromatin. We, therefore, screened various types of apoptotic

  8. The superstructure of chromatin and its condensation mechanism

    Microsoft Academic Search

    M. H. J. Koch; Z. Sayers; A. M. Michon; P. Sicre; R. Marquet; C. Houssier

    1989-01-01

    Electric dichroism and X-ray scattering measurements on solutions of uncondensed and condensed chicken erythrocyte chromatin were interpreted on the basis of model calculations. Information about the state of uncondensed fibers in the conditions of electric dichroism measurements was obtained from scattering patterns recorded as a function of pH, in the presence of spermine and at very low monovalent cation concentrations.

  9. Chromatin immunoprecipitation assay of Piwi in Drosophila.

    PubMed

    Yin, Hang; Lin, Haifan

    2014-01-01

    The generation of high-resolution maps of the epigenome is crucial to research in epigenetics, developmental biology, and stem cell biology. In recent years, small RNA pathways have been implicated in epigenetic regulation. All small RNA pathways involve Argonaute proteins as their key biogenesis and effector components. In this chapter, we describe a chromatin immunoprecipitation method for whole-genome mapping of Drosophila Piwi, the defining member of the Argonaute protein family. This method should have general utility for mapping other chromatin-associated factors. PMID:24178552

  10. CHD5 is required for spermiogenesis and chromatin condensation.

    PubMed

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

    2014-02-01

    Haploid spermatids undergo extensive cellular, molecular and morphological changes to form spermatozoa during spermiogenesis. Abnormalities in these steps can lead to serious male fertility problems, from oligospermia to complete azoospermia. CHD5 is a chromatin-remodeling nuclear protein expressed almost exclusively in the brain and testis. Male Chd5 knockout (KO) mice have deregulated spermatogenesis, characterized by immature sloughing of spermatids, spermiation failure, disorganization of the spermatogenic cycle and abnormal head morphology in elongating spermatids. This results in the inappropriate placement and juxtaposition of germ cell types within the epithelium. Sperm that did enter the epididymis displayed irregular shaped sperm heads, and retained cytoplasmic components. These sperm also stained positively for acidic aniline, indicating improper removal of histones and lack of proper chromatin condensation. Electron microscopy showed that spermatids in the seminiferous tubules of Chd5 KO mice have extensive nuclear deformation, with irregular shaped heads of elongated spermatids, and lack the progression of chromatin condensation in an anterior-to-posterior direction. However, the mRNA expression levels of other important genes controlling spermatogenesis were not affected. Chd5 KO mice also showed decreased H4 hyperacetylation beginning at stage IX, step 9, which is vital for the histone-transition protein replacement in spermiogenesis. Our data indicate that CHD5 is required for normal spermiogenesis, especially for spermatid chromatin condensation. PMID:24252660

  11. CHD5 is Required for Spermiogenesis and Chromatin Condensation

    PubMed Central

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

    2014-01-01

    Haploid spermatids undergo extensive cellular, molecular and morphological changes to form spermatozoa during spermiogenesis. Abnormalities in these steps can lead to serious male fertility problems, from oligospermia to complete azoospermia. CHD5 is a chromatin-remodeling nuclear protein expressed almost exclusively in the brain and testis. Male Chd5 knockout (KO) mice have deregulated spermatogenesis, characterized by immature sloughing of spermatids, spermiation failure, disorganization of the spermatogenic cycle and abnormal head morphology in elongating spermatids. This results in the inappropriate placement and juxtaposition of germ cell types within the epithelium. Sperm that did enter the epididymis displayed irregular shaped sperm heads, and retained cytoplasmic components. These sperm also stained positively for acidic aniline, indicating improper removal of histones and lack of proper chromatin condensation. Electron microscopy showed that spermatids in the seminiferous tubules of Chd5 KO mice have extensive nuclear deformation, with irregular shaped heads of elongated spermatids, and lack the progression of chromatin condensation in an anterior-to-posterior direction. However, the mRNA expression levels of other important genes controlling spermatogenesis were not affected. Chd5 KO mice also showed decreased H4 hyperacetylation beginning at stage IX, step 9, which is vital for the histone-transition protein replacement in spermiogenesis. Our data indicate that CHD5 is required for normal spermiogenesis, especially for spermatid chromatin condensation. PMID:24252660

  12. Chromatin Immunoprecipitation Assays for Myc and N-Myc

    PubMed Central

    Barrilleaux, Bonnie L.; Cotterman, Rebecca; Knoepfler, Paul S.

    2015-01-01

    Myc and N-Myc have widespread impacts on the chromatin state within cells, both in a gene-specific and genome-wide manner. Our laboratory uses functional genomic methods including chromatin immunoprecipitation (ChIP), ChIP-chip, and, more recently, ChIP-seq to analyze the binding and genomic location of Myc. In this chapter, we describe an effective ChIP protocol using specific validated antibodies to Myc and N-Myc. We discuss the application of this protocol to several types of stem and cancer cells, with a focus on aspects of sample preparation prior to library preparation that are critical for successful Myc ChIP assays. Key variables are discussed and include the starting quantity of cells or tissue, lysis and sonication conditions, the quantity and quality of antibody used, and the identification of reliable target genes for ChIP validation. PMID:24006062

  13. Molecular analysis of mitotic chromosome condensation using a quantitative time-resolved fluorescence microscopy assay

    PubMed Central

    Maddox, Paul S.; Portier, Nathan; Desai, Arshad; Oegema, Karen

    2006-01-01

    Chromosomes condense during mitotic entry to facilitate their segregation. Condensation is typically assayed in fixed preparations, limiting analysis of contributing factors. Here, we describe a quantitative method to monitor condensation kinetics in living cells expressing GFP fused to a core histone. We demonstrate the utility of this method by using it to analyze the molecular requirements for the condensation of holocentric chromosomes during the first division of the Caenorhabditis elegans embryo. In control embryos, the fluorescence intensity distribution for nuclear GFP:histone changes during two distinct time intervals separated by a plateau phase. During the first interval, primary condensation converts diffuse chromatin into discrete linear chromosomes. After the plateau, secondary condensation compacts the curvilinear chromosomes to form shorter bar-shaped structures. We quantitatively compared the consequences on this characteristic profile of depleting the condensin complex, the mitosis-specific histone H3 kinase Aurora B, the centromeric histone CENP-A, and CENP-C, a conserved protein required for kinetochore assembly. Both condensin and CENP-A play critical but distinct roles in primary condensation. In contrast, depletion of CENP-C slows but does not prevent primary condensation. Finally, Aurora B inhibition has no effect on primary condensation, but slightly delays secondary condensation. These results provide insights into the process of condensation, help resolve apparent contradictions from prior studies, and indicate that CENP-A chromatin has an intrinsic role in the condensation of holocentric chromosomes that is independent of its requirement for kinetochore assembly. PMID:17005720

  14. Histone deacetylase 2 is required for chromatin condensation and subsequent enucleation of cultured mouse fetal erythroblasts

    E-print Network

    Ji, Peng

    Background: During the final stages of differentiation of mammalian erythroid cells, the chromatin is condensed and enucleated. We previously reported that Rac GTPases and their downstream target, mammalian homolog of ...

  15. Easy detection of chromatin binding proteins by the histone association assay

    PubMed Central

    Ricke, Robin

    2005-01-01

    The Histone Association Assay provides an easy approach for detecting proteins that bind chromatin in vivo. This technique is based on a chromatin immunoprecipitation protocol using histone H3-specific antibodies to precipitate bulk chromatin from crosslinked whole cell extracts. Proteins that co-precipitate with chromatin are subsequently detected by conventional SDS-PAGE and Western blot analysis. Unlike techniques that separate chromatin and non-chromatin interacting proteins by centrifugation, this method can be used to delineate whether a protein is chromatin associated regardless of its innate solubility. Moreover, the relative amount of protein bound to DNA can be ascertained under quantitative conditions. Therefore, this technique may be utilized for analyzing the chromatin association of proteins involved in diverse cellular processes. PMID:16136225

  16. Mitotic chromatin condensation in vitro using somatic cell extracts and nuclei with variable levels of endogenous topoisomerase II

    Microsoft Academic Search

    Edgar R. Wood; William C. Earnshaw

    1990-01-01

    We report the development of a new method for producing mitotic extracts from tissue cul- ture cells. These extracts reproducibly promote the condensation of chromatin in vitro when incubated with purified interphase nuclei. This condensation reaction is not species specific, since nuclei from chicken, human, and hamster cell lines all undergo chromatin condensation upon incubation with the ex- tract. We

  17. Dynamic condensation of linker histone C-terminal domain regulates chromatin structure

    PubMed Central

    Luque, Antoni; Collepardo-Guevara, Rosana; Grigoryev, Sergei; Schlick, Tamar

    2014-01-01

    The basic and intrinsically disordered C-terminal domain (CTD) of the linker histone (LH) is essential for chromatin compaction. However, its conformation upon nucleosome binding and its impact on chromatin organization remain unknown. Our mesoscale chromatin model with a flexible LH CTD captures a dynamic, salt-dependent condensation mechanism driven by charge neutralization between the LH and linker DNA. Namely, at low salt concentration, CTD condenses, but LH only interacts with the nucleosome and one linker DNA, resulting in a semi-open nucleosome configuration; at higher salt, LH interacts with the nucleosome and two linker DNAs, promoting stem formation and chromatin compaction. CTD charge reduction unfolds the domain and decondenses chromatin, a mechanism in consonance with reduced counterion screening in vitro and phosphorylated LH in vivo. Divalent ions counteract this decondensation effect by maintaining nucleosome stems and expelling the CTDs to the fiber exterior. Additionally, we explain that the CTD folding depends on the chromatin fiber size, and we show that the asymmetric structure of the LH globular head is responsible for the uneven interaction observed between the LH and the linker DNAs. All these mechanisms may impact epigenetic regulation and higher levels of chromatin folding. PMID:24906881

  18. Dynamic condensation of linker histone C-terminal domain regulates chromatin structure.

    PubMed

    Luque, Antoni; Collepardo-Guevara, Rosana; Grigoryev, Sergei; Schlick, Tamar

    2014-07-01

    The basic and intrinsically disordered C-terminal domain (CTD) of the linker histone (LH) is essential for chromatin compaction. However, its conformation upon nucleosome binding and its impact on chromatin organization remain unknown. Our mesoscale chromatin model with a flexible LH CTD captures a dynamic, salt-dependent condensation mechanism driven by charge neutralization between the LH and linker DNA. Namely, at low salt concentration, CTD condenses, but LH only interacts with the nucleosome and one linker DNA, resulting in a semi-open nucleosome configuration; at higher salt, LH interacts with the nucleosome and two linker DNAs, promoting stem formation and chromatin compaction. CTD charge reduction unfolds the domain and decondenses chromatin, a mechanism in consonance with reduced counterion screening in vitro and phosphorylated LH in vivo. Divalent ions counteract this decondensation effect by maintaining nucleosome stems and expelling the CTDs to the fiber exterior. Additionally, we explain that the CTD folding depends on the chromatin fiber size, and we show that the asymmetric structure of the LH globular head is responsible for the uneven interaction observed between the LH and the linker DNAs. All these mechanisms may impact epigenetic regulation and higher levels of chromatin folding. PMID:24906881

  19. Chromomycin A3 staining, sperm chromatin structure assay and hyaluronic acid binding assay as predictors for assisted reproductive outcome

    Microsoft Academic Search

    Martine Nijs; Eva Creemers; Annemie Cox; Kim Franssen; Mia Janssen; Elke Vanheusden; Christopher De Jonge; Willem Ombelet

    2009-01-01

    Functional sperm tests such as the sperm chromatin structure assay (SCSA), chromomycin A3 staining (CMA3) and hyaluronic acid binding assay (HBA) have been suggested as predictive tests of fertility invitro. This study aimed to define the clinical role of these functional parameters in assisted reproduction in a prospective cohort study. Conventional sperm diagnosis (motility, morphology and concentration) as well as

  20. 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

  1. Small molecule compound induces chromatin de-condensation and facilitates induced pluripotent stem cell generation.

    PubMed

    Wei, Xiaoyuan; Chen, Yueting; Xu, Yongyu; Zhan, Yang; Zhang, Ru; Wang, Min; Hua, Qiuhong; Gu, Haifeng; Nan, Fajun; Xie, Xin

    2014-10-01

    The revolutionary induced pluripotent stem cell (iPSC) technology provides a new means for cell replacement therapies and drug screening. Small molecule compounds have been found extremely useful to improve the generation of iPSCs and understand the reprogramming mechanism. Here we report the identification of a novel chemical, CYT296, which improves OSKM-mediated induction of iPSCs for >10 folds and enables efficient reprogramming with only Oct4 in combination with other small molecules. The derived iPSCs are genuinely pluripotent and support the development of two 'All-iPSC' mice by tetraploid complementation. CYT296 profoundly impacts heterochromatin formation without affecting cell viability. MEFs treated with CYT296 exhibit de-condensed chromatin structure with markedly reduced loci containing heterochromatin protein 1? (HP1?) and H3K9me3, which is very similar to the chromatin configuration in embryonic stem cells (ESCs). Given that an open chromatin structure serves as a hallmark of pluripotency and has to be acquired to fulfill reprogramming, we propose that CYT296 might facilitate this process by disrupting condensed chromatin, thereby creating a more favorable environment for reprogramming. In agreement of this idea, shRNA targeting HP1? also promotes the generation of iPSCs. Thus current findings not only provide a novel chemical for efficient iPSC induction, but also suggest a new approach to regulate somatic cell reprogramming by targeting chromatin de-condensation with small molecules. PMID:24838272

  2. The superstructure of chromatin and its condensation mechanism

    Microsoft Academic Search

    M. H. J. Koch; Z. Sayers; M. C. Vega; A. M. Michon

    1987-01-01

    Changes in the structure of chicken erythrocyte chromatin fibres at low ionic strength resulting from enzymatic digestion, thermal denaturation and binding of Netropsin and Distamycin were monitored by synchrotron X-ray solution scattering. Digestion with micrococcal nuclease confirms the previous assignment of the 0.05 nm-1 band to an interference between nucleosomes with an average distance of 23 nm. The results of

  3. Mitotic Transcription Repression in Vivo in the Absence of Nucleosomal Chromatin Condensation

    PubMed Central

    Spencer, Charlotte A.; Kruhlak, Michael J.; Jenkins, Heather L.; Sun, Xuejun; Bazett-Jones, David P.

    2000-01-01

    All nuclear RNA synthesis is repressed during the mitotic phase of the cell cycle. In addition, RNA polymerase II (RNAP II), nascent RNA and many transcription factors disengage from DNA during mitosis. It has been proposed that mitotic transcription repression and disengagement of factors are due to either mitotic chromatin condensation or biochemical modifications to the transcription machinery. In this study, we investigate the requirement for chromatin condensation in establishing mitotic transcription repression and factor loss, by analyzing transcription and RNAP II localization in mitotic cells infected with herpes simplex virus type 1. We find that virus-infected cells enter mitosis and that mitotic viral DNA is maintained in a nucleosome-free and noncondensed state. Our data show that RNAP II transcription is repressed on cellular genes that are condensed into mitotic chromosomes and on viral genes that remain nucleosome free and noncondensed. Although RNAP II may interact indirectly with viral DNA during mitosis, it remains transcriptionally unengaged. This study demonstrates that mitotic repression of transcription and loss of transcription factors from mitotic DNA can occur independently of nucleosomal chromatin condensation. PMID:10893252

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

    SciTech Connect

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

    2008-08-21

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

  5. Spermatozoa nucleus takes up lead during the epididymal maturation altering chromatin condensation.

    PubMed

    Hernández-Ochoa, I; Sánchez-Gutiérrez, M; Solís-Heredia, M J; Quintanilla-Vega, B

    2006-02-01

    Lead (Pb) alters sperm chromatin condensation (CC) and the mechanisms are investigated. During spermatogenesis, protamines replace histones and disulfide bonds formation during epididymal maturation condense the chromatin. We evaluated sperm Pb uptake in testis and epididymis and the effects on CC in mice (0.06% Pb(2+)/16 weeks/drinking water). Spermatozoa from caput epididymis (CP) and cauda epididymis-vas deferens (CE-VD) were obtained and CC was measured by SCSA. Lead levels in spermatozoa from CP were lower than those from CE-VD, and correlated with a decreased CC, while Pb in CE-VD spermatozoa correlated with an increased CC. Lead accumulation into the nucleus was observed and Pb binding to nuclear sulfhydryl groups decreased chromatin decondensation in vitro. Our results suggest that spermatozoa take up Pb during testicular development and epididymal transport and alter CC, depending of the timing of Pb incorporation into the sperm nucleus, which finally may interfere with the chromatin decondensation process after fertilization. PMID:16198534

  6. 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

  7. Seed maturation in Arabidopsis thaliana is characterized by nuclear size reduction and increased chromatin condensation

    PubMed Central

    van Zanten, Martijn; Koini, Maria A.; Geyer, Regina; Liu, Yongxiu; Brambilla, Vittoria; Bartels, Dorothea; Koornneef, Maarten; Fransz, Paul; Soppe, Wim J. J.

    2011-01-01

    Most plant species rely on seeds for their dispersal and survival under unfavorable environmental conditions. Seeds are characterized by their low moisture content and significantly reduced metabolic activities. During the maturation phase, seeds accumulate storage reserves and become desiccation-tolerant and dormant. Growth is resumed after release of dormancy and the occurrence of favorable environmental conditions. Here we show that embryonic cotyledon nuclei of Arabidopsis thaliana seeds have a significantly reduced nuclear size, which is established at the beginning of seed maturation. In addition, the chromatin of embryonic cotyledon nuclei from mature seeds is highly condensed. Nuclei regain their size and chromatin condensation level during germination. The reduction in nuclear size is controlled by the seed maturation regulator ABSCISIC ACID-INSENSITIVE 3, and the increase during germination requires two predicted nuclear matrix proteins, LITTLE NUCLEI 1 and LITTLE NUCLEI 2. Our results suggest that the specific properties of nuclei in ripe seeds are an adaptation to desiccation, independent of dormancy. We conclude that the changes in nuclear size and chromatin condensation in seeds are independent, developmentally controlled processes. PMID:22123962

  8. The Dynamics of Individual Nucleosomes Controls the Chromatin Condensation Pathway: Direct Atomic Force Microscopy Visualization of Variant Chromatin

    PubMed Central

    Montel, Fabien; Menoni, Hervé; Castelnovo, Martin; Bednar, Jan; Dimitrov, Stefan; Angelov, Dimitar; Faivre-Moskalenko, Cendrine

    2009-01-01

    Abstract Chromatin organization and dynamics is studied at scales ranging from single nucleosome to nucleosomal array by using a unique combination of biochemical assays, single molecule imaging technique, and numerical modeling. We show that a subtle modification in the nucleosome structure induced by the histone variant H2A.Bbd drastically modifies the higher order organization of the nucleosomal arrays. Importantly, as directly visualized by atomic force microscopy, conventional H2A nucleosomal arrays exhibit specific local organization, in contrast to H2A.Bbd arrays, which show “beads on a string” structure. The combination of systematic image analysis and theoretical modeling allows a quantitative description relating the observed gross structural changes of the arrays to their local organization. Our results suggest strongly that higher-order organization of H1-free nucleosomal arrays is determined mainly by the fluctuation properties of individual nucleosomes. Moreover, numerical simulations suggest the existence of attractive interactions between nucleosomes to provide the degree of compaction observed for conventional chromatin fibers. PMID:19619469

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

    EPA Science Inventory

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

  10. Influence of chromatin condensation on the number of direct DSB damages induced by ions studied using a Monte Carlo code.

    PubMed

    Dos Santos, M; Clairand, I; Gruel, G; Barquinero, J F; Incerti, S; Villagrasa, C

    2014-10-01

    The purpose of this work is to evaluate the influence of the chromatin condensation on the number of direct double-strand break (DSB) damages induced by ions. Two geometries of chromosome territories containing either condensed or decondensed chromatin were implemented as biological targets in the Geant4 Monte Carlo simulation code and proton and alpha irradiation was simulated using the Geant4-DNA processes. A DBSCAN algorithm was used in order to detect energy deposition clusters that could give rise to single-strand breaks or DSBs on the DNA molecule. The results of this study show an increase in the number and complexity of DNA DSBs in condensed chromatin when compared with decondensed chromatin. PMID:24615262

  11. Photobleaching Assays (FRAP & FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

    PubMed Central

    Nissim-Rafinia, Malka; Meshorer, Eran

    2011-01-01

    Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3. PMID:21730953

  12. 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

  13. Visualization of chromatin events associated with repair of ultraviolet light-induced damage by premature chromosome condensation

    SciTech Connect

    Hittelman, W.N.; Pollard, M.

    1984-10-01

    The purpose of this study was to characterize a system with which to study chromatin events associated with the repair of u.v. light-induced damage. Quiescent normal human fibroblasts were irradiated with u.v. and the ensuing chromatin events were visualized by inducing premature chromosome condensation in the treated cells. Treatment with u.v. induced the following two types of chromatin changes reflected in the morphology of G1 premature condensed chromosomes (PCC): (i) a generalized elongation of the G1 PCC and (ii) regions of localized elongation or gaps. The degree of chromatin change was dose dependent and could be seen immediately after irradiation. The generalized elongation process continued to increase for 24 h after irradiation, suggesting it represented a cellular reaction to the u.v.-induced damage, rather than a direct physical distortion. The localized decondensation reaction was associated with the site of unscheduled DNA synthesis. Posttreatment incubation of cells in the presence of cytosine arabinoside and hydroxyurea resulted in an accumulation of gaps. The inhibitor novobiocin predominantly inhibited the formation of gap regions, suggesting that a topoisomerase-like reaction might be important in their formation. The presence of cycloheximide after u.v. irradiation had no effect on the chromatin changes, suggesting that no new protein synthesis is required for these chromatin processes associated with repair. These results suggest that the PCC technique is useful in elucidating chromatin changes associated with DNA repair after u.v. treatment and can be used to elucidate chromatin events associated with the repair of other DNA-damaging agents.

  14. Assays for chromatin remodeling during nucleotide excision repair in Saccharomyces cerevisiae

    Microsoft Academic Search

    Ling Zhang; Kristi Jones; Michael J. Smerdon; Feng Gong

    2009-01-01

    How DNA repair proteins interact with the dynamic structure of chromatin is an emerging question. Chromatin structure impedes the access of repair proteins to sites of DNA damage. Several recent studies have implicated chromatin remodeling complexes in DNA repair. In this report we summarize the methods we used to investigate chromatin remodeling during nucleotide excision repair (NER) in vivo. We

  15. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic

    Microsoft Academic Search

    D. P. Evenson; L. K. Jost; D. Marshall; M. J. Zinaman; E. Clegg; K. Purvis; P. de Angelis; O. P. Claussen

    1999-01-01

    The sperm chromatin structure assay (SCSA) was used to measure over 500 human semen samples from two independent studies: Study I, 402 samples from 165 presumably fertile couples wishing to achieve pregnancy over 12 menstrual cycles; Study II, samples from 115 patients seeking fertility counselling. The SCSA measures susceptibility to DNA denaturation in situ in spermatozoa exposed to acid for

  16. Assessment of Chromatin Maturity in Human Spermatozoa: Useful Aniline Blue Assay for Routine Diagnosis of Male Infertility

    PubMed Central

    Chakroun, Nozha; Ben Zarrouk, Soumaya; Sellami, Hanen; Kebaili, Sahbi; Rebai, Tarek; Keskes, Leila

    2013-01-01

    During spermatogenesis, sperm chromatin undergoes structural changes and results in a high condensation. This nuclear compaction would be useful as a predictor of sperm fertilization capacity and pregnancy outcome. We purpose to evaluate firstly the relationship among chromatin maturity assessed by aniline blue staining (AB) and the semen parameters in infertile men. Secondly, we analyzed whether the sperm gradient density centrifugation is effective to select mature spermatozoa. Fifty-one ejaculates were investigated by semen analysis and stained for chromatin condensation with AB to distinguish between unstained mature sperm and stained immature sperm. AB was applied also on 12 ejaculates which proceeded by density gradient centrifugation to compare the rates of immature sperm before and after selection. Neat semen were divided into two groups: G1 (n = 31): immature sperm <20% and G2 (n = 20): immature sperm ?20%. No significant differences were detected in sperm concentration, motility, and normal morphology between G1 and G2. However, the rates of some morphology abnormalities were higher in G2: head abnormalities (P = 0.01) and microcephalic sperm (P = 0.02). We founded significant correlation between sperm immaturity and acrosome abnormalities (r = 0.292; P = 0.03). Sperm selection has significantly reduced the rates of immature sperm. A better understanding of chromatin structure and its impact on the sperm potential is needed to explore male infertility. PMID:24198830

  17. Nickel resistance and chromatin condensation in Saccharomyces cerevisiae expressing a maize high mobility group I/Y protein.

    PubMed

    Forzani, C; Loulergue, C; Lobréaux, S; Briat, J F; Lebrun, M

    2001-05-18

    Expression of a maize cDNA encoding a high mobility group (HMG) I/Y protein enables growth of transformed yeast on a medium containing toxic nickel concentrations. No difference in the nickel content was measured between yeast cells expressing either the empty vector or the ZmHMG I/Y2 cDNA. The ZmHMG I/Y2 protein contains four AT hook motifs known to be involved in binding to the minor groove of AT-rich DNA regions. HMG I/Y proteins may act as architectural elements modifying chromatin structure. Indeed, a ZmHMG I/Y2-green fluorescent protein fusion protein was observed in yeast nuclei. Nickel toxicity has been suggested to occur through an epigenetic mechanism related to chromatin condensation and DNA methylation, leading to the silencing of neighboring genes. Therefore, the ZmHMG I/Y2 protein could prevent nickel toxicity by interfering with chromatin structure. Yeast cell growth in the presence of nickel and yeast cells expressing the ZmHMG I/Y2 cDNA increased telomeric URA3 gene silencing. Furthermore, ZmHMG I/Y2 restored a wild-type level of nickel sensitivity to the yeast (Delta)rpd3 mutant. Therefore, nickel resistance of yeast cells expressing the ZmHMG I/Y2 cDNA is likely achieved by chromatin structure modification, restricting nickel accessibility to DNA. PMID:11278346

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  19. An Improved Restriction Enzyme Accessibility Assay (REAA) for Analyzing Changes in Chromatin Structure in Samples of Limited Cell Number

    PubMed Central

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

    2014-01-01

    Studies investigating mechanisms that control gene regulation frequently examine the accessibility of specific DNA sequences to nuclease cleavage. In general, sequences that are sensitive to nuclease cleavage are considered to be in an “open” chromatin conformation that is associated with regulatory factor binding, while sequences resistant to nuclease cleavage are considered to be in a “closed” conformation commonly associated with chromatin that is neither poised for transcription nor being actively transcribed. Changes in nuclease accessibility at specific genomic sequences reflect changes in the local chromatin structure that can occur as a result of signaling cues in the extracellular environment. These changes in chromatin structure usually precede or are coincident with changes in gene expression patterns and are therefore a useful marker of regulatory events controlling transcription. We describe a method to perform restriction enzyme accessibility assays (REAA) that utilizes ligation-mediated polymerase chain reaction (LM-PCR) technology and that permits assessment of samples from any source containing as few as 1,000 cells. Use of this modified REAA protocol will enhance analysis of chromatin structural changes at specific DNA sequences of interest by making it possible to analyze samples where unrestricted amounts of sample are not readily available. PMID:22130859

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

    PubMed Central

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

    2014-01-01

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

  1. Sperm chromatin structure assay (scsa®) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles

    Microsoft Academic Search

    Michael R Virro; Kjersten L Larson-Cook; Donald P Evenson

    2004-01-01

    ObjectiveTo determine the relationship between sperm chromatin structure assay (SCSA) parameters (DNA fragmentation index [DFI] and high DNA stainability [HDS]), and conventional IVF and IVF\\/intracytoplasmic sperm injection (ICSI) outcomes.

  2. The maternally expressed Drosophila gene encoding the chromatin-binding protein BJ1 is a homolog of the vertebrate gene Regulator of Chromatin Condensation, RCC1.

    PubMed Central

    Frasch, M

    1991-01-01

    Using monoclonal antibodies I have identified a nuclear protein of Drosophila, BJ1 (Mr approximately 68 kd), and isolated its gene. Biochemical analysis demonstrates that the BJ1 protein is associated with nucleosomes and is released from chromatin by agents which intercalate into DNA, as previously shown for the high mobility group proteins (HMGs). On polytene chromosomes the protein is localized in all bands, with no preference for particular loci. Both the BJ1 protein and in particular the BJ1 mRNA are strongly expressed maternally. In early embryos all nuclei contain equal amounts of BJ1. During neuroblast formation, BJ1 mRNA becomes restricted to cells of the central nervous system, and higher protein levels are found in the nuclei of this tissue. In late embryonic stages, the mRNA almost completely disappears, but significant amounts of BJ1 protein persist until morphogenesis. The BJ1 gene encodes a 547 amino acid polypeptide featuring two different types of internal repeats. The sequence from amino acids 46 to 417 containing seven repeats of the first type has been highly conserved in evolution. 45% of the amino acids in this region are conserved in seven similar tandem repeats of the human gene Regulator of Chromatin Condensation, RCC1. The phenotype of a cell line carrying a mutation of RCC1 suggested a main function for this gene in cell cycle control. A yeast gene, SRM1/PRP20, also contains these repeats and shows 30% amino acid identity to BJ1 in this region. Mutations in this gene perturb mRNA metabolism, disrupt nuclear structure and alter the signal transduction pathway for the mating pheromone. Complementation experiments argue for a common function of these genes in the different species. I propose that their gene products bind to the chromatin to establish or maintain a proper higher order structure as a prerequisite for a regulated gene expression. Disruption of this structure could cause both mis-expression and default repression of genes, which might explain the pleiotropic phenotypes of the mutants. Images PMID:2022188

  3. Sequential Recruitment of HAT and SWI\\/SNF Components to Condensed Chromatin by VP16

    Microsoft Academic Search

    Sevinci Memedula; Andrew S Belmont

    2003-01-01

    Eukaryotic transcription initiation requires the complex dynamics of hundreds of proteins, many of which are found in large multisubunit complexes [1]. Recent experiments have suggested stepwise recruitment of preassembled complexes, including chromatin remodeling, general transcription factor, mediator, and polymerase complexes [1], in which the actual order of recruitment may vary for different promoters [2]. How do these complexes access target

  4. Sperm chromatin structure assay (SCSA): a tool in diagnosis and treatment of infertility

    Microsoft Academic Search

    Mona Bungum; Leif Bungum; Aleksander Giwercman

    2011-01-01

    Diagnosis of male infertility has mainly been based on the World Health Organization (WHO) manual-based semen parameter's concentration, motility and morphology. It has, however, become apparent that none of these parameters are reliable markers for evaluation of the fertility potential of a couple. A search for better markers has led to an increased focus on sperm chromatin integrity testing in

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

    PubMed

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

    2014-12-01

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

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

    EPA Science Inventory

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

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

    PubMed

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

    2015-03-13

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

  8. 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

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

    SciTech Connect

    Komura, Jun-ichiro, E-mail: junkom@med.tohoku.ac.jp [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan)] [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Ikehata, Hironobu [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan)] [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Mori, Toshio [Radioisotope Research Center, Nara Medical University, Kashihara, Nara 634-8521 (Japan)] [Radioisotope Research Center, Nara Medical University, Kashihara, Nara 634-8521 (Japan); Ono, Tetsuya [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan)] [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan)

    2012-03-10

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

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

    EPA Science Inventory

    ABSTRACT A measure of sperm chromatin integrity was added to the routine semen end points evaluated in the Teplice Program male reproductive health studies. To address the hypothesis that exposure to periods of elevated air pollution may be associated with abnormalities in sp...

  11. Assessing the Applicability of FISH-based Prematurely Condensed Dicentric Chromosome Assay in Triage Biodosimetry.

    PubMed

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

    2015-03-01

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

  12. 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...

  13. Pyknotic cell death induced by Clostridium difficile TcdB: chromatin condensation and nuclear blister are induced independently of the glucosyltransferase activity.

    PubMed

    Wohlan, Katharina; Goy, Sebastian; Olling, Alexandra; Srivaratharajan, Sangar; Tatge, Helma; Genth, Harald; Gerhard, Ralf

    2014-11-01

    TcdA and TcdB are the main pathogenicity factors of Clostridium difficile-associated diseases. Both toxins inhibit Rho GTPases, and consequently, apoptosis is induced in the affected cells. We found that TcdB at higher concentrations exhibits cytotoxic effects that are independent on Rho glucosylation. TcdB and the glucosyltransferase-deficient mutant TcdB D286/288N induced pyknotic cell death which was associated with chromatin condensation and reduced H3 phosphorylation. Affected cells showed ballooning of the nuclear envelope and loss of the integrity of the plasma membrane. Furthermore, pyknotic cells were positively stained with dihydroethidium indicating production of reactive oxygen species. In line with this, pyknosis was reduced by apocynin, an inhibitor of the NADPH oxidase. Bafilomycin A1 prevented cytotoxic effects showing that the newly observed pyknosis depends on intracellular action of TcdB rather than on a receptor-mediated effect. Blister formation and chromatin condensation was specifically induced by the glucosyltransferase domain of TcdB from strain VPI10473 since neither TcdBF from cdi1470 nor the chimera of TcdB harbouring the glucosyltransferase domain of TcdBF was able to induce these effects. In summary, TcdB induces two different and independent phenotypes: (i) cell rounding due to glucosylation of Rho GTPases and (ii) shrinkage of cells and nuclear blister induced by the high concentrations of TcdB independent of Rho glucosylation. PMID:24898616

  14. Etoposide-induced apoptosis in lymphoblastoid leukaemic MOLT-4 cells: evidence that chromatin condensation, loss of phosphatidylserine asymmetry and apoptotic body formation can occur independently.

    PubMed

    Ramirez, C D; Catchpoole, D R

    2002-02-01

    Apoptosis is characterised by a series of typical morphological features, such as nuclear and cellular convolution, chromatin condensation and the final disintegration of the cell into membrane-bound apoptotic bodies, which are phagocytosed, by neighbouring cells. Relocation of phosphatidylserine residues from the inner leaflet of the cellular membrane to being exposed on the cell surface is a necessary event for the phagocytic elimination of apoptotic cell debris. Using the MOLT-4 lymphoblastoid leukaemic cell line we investigated whether the formation of apoptotic bodies and loss of phosphatidylserine asymmetry were causally related. We have previously demonstrated that classical apoptotic morphology, including production of apoptotic bodies, was only possible in etoposide-treated MOLT-4 cells when administered in the presence of non-cytotoxic doses (200 microM) of aurin tricarboxylic acid (ATA). Electron microscopic analysis, followed by the quantitation of the ultrastructural morphological features of apoptotic MOLT-4 cells, demonstrated that the etoposide and ATA co-treatment, which caused the cellular fragmentation into apoptotic bodies, was closely associated with extensive chromatin condensation in individual cells. In this model however, the addition of ATA to frank cytotoxic doses of etoposide (50 microM), which we confirmed lead to formation of apoptotic bodies, caused no further increase in externalisation of phosphatidylserine moieties as determined by staining with fluorescence labelled annexin V. Consequently, in MOLT-4 cells undergoing etoposide-induced apoptosis, the mole-cular mechanisms leading to loss of phosphatidylserine asymmetry and the formation of apoptotic bodies are not causally related. PMID:11773706

  15. 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.

  16. Condensation

    NSDL National Science Digital Library

    James H. Kessler

    2007-01-01

    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.

  17. nAture methods | VOL.10 NO.12 | DECEMBER2013 | 1213 We describe an assay for transposase-accessible chromatin

    E-print Network

    Cai, Long

    into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis. AtAc-seq captures, could tolerate or tended to overlap with nucleosomes. using AtAc-seq maps of human cd4+ t cells from's epigenome on a timescale compatible with clinical decision-making. Eukaryotic genomes are hierarchically

  18. nAture methods | VOL.10 NO.2 | FEBRUARY2013 | 171 chromatin immunoprecipitation assays have contributed greatly

    E-print Network

    Cai, Long

    regulate SMC differentiation in vivo14,15 under normal as well as pathological conditions. Moreover, it is impossible to obtain chromatin derived exclusively from SMCs from tissue sources because all SMC-containing tissues contain multiple other cell types. Conversely, all non-SMC tissues contain large numbers of SMCs

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

    SciTech Connect

    Mannioui, Abdelkrim [EMI-0013 Institut National de la Sante et de la Recherche Medicale-Universite Paris 7, 75010 Paris (France) and Laboratoire d'Immunologie Cellulaire et Immunopathologie de l'Ecole Pratique des Hautes Etudes, Institut Universitaire d'Hematologie, hopital Saint-Louis, 75010 Paris (France) and Laboratoire de Biochimie and JFR 3012 Associee a l'Agence Universitaire Francophone (AUPELF-UREF), Faculte des Sciences, Rabat (Morocco)]. E-mail: karim.mannioui@chu-stlouis.fr; Schiffer, Cecile [EMI-0013 Institut National de la Sante et de la Recherche Medicale-Universite Paris 7, 75010 Paris (France) and Laboratoire d'Immunologie Cellulaire et Immunopathologie de l'Ecole Pratique des Hautes Etudes, Institut Universitaire d'Hematologie, hopital Saint-Louis, 75010 Paris (France)]. E-mail: cecile.schiffer@voila.fr; Felix, Nathalie [EMI-0013 Institut National de la Sante et de la Recherche Medicale-Universite Paris 7, 75010 Paris (France) and Laboratoire d'Immunologie Cellulaire et Immunopathologie de l'Ecole Pratique des Hautes Etudes, Institut Universitaire d'Hematologie, hopital Saint-Louis, 75010 Paris (France)]. E-mail: nathalie.felix@chu-stlouis.fr [and others

    2004-11-10

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

  20. 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...

  1. Chromatin Remodeling

    NSDL National Science Digital Library

    Neal Lue (Department of Microbiology and Immunology, Weill Medical College of Cornell University; )

    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.

  2. Ectopically tethered CP190 induces large-scale chromatin decondensation

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

  3. Comparison between two kinds of cigarette smoke condensates (CSCs) of the cytogenotoxicity and protein expression in a human B-cell lymphoblastoid cell line using CCK8 assay, comet assay and protein microarray

    Microsoft Academic Search

    Jianlin Lou; Guohai Chu; Guojun Zhou; Jian Jiang; Fangfang Huang; Juanjuan Xu; Shu Zheng; Wei Jiang; Yezhen Lu; Xiaoxue Li; Zhijian Chen; Jiliang He

    2010-01-01

    The differences of the cytogenotoxicity and proteins expression of human B-cell lymphoblastoid cells exposed to cigarette smoke condensates (CSCs) from two kinds of cigarettes were detected with CCK-8 assay, comet assay, protein microarray and western blot assay in vitro. Human B-cell lymphoblastoid cell line was exposed to CSCs from two cigarettes (which delivers approximately 3mg tar, 0.3mg nicotine, 3mg CO

  4. Reprogramming chromatin.

    PubMed

    Ehrensberger, Andreas Hasso; Svejstrup, Jesper Qualmann

    2012-09-01

    Cellular reprogramming involves the artificial dedifferentiation of somatic cells to a pluripotent state. When affected by overexpressing specific transcription factors, the process is highly inefficient, as only 0.1-1% of cells typically undergo the transformation. This low efficiency has been attributed to high kinetic barriers that affect all cells equally and can only be overcome by rare stochastic events. The barriers to reprogramming are likely to involve transformations of chromatin state because (i) inhibitors of chromatin-modifying enzymes can enhance the efficiency of reprogramming and (ii) knockdown or knock-out of chromatin-modifying enzymes can lower the efficiency of reprogramming. Here, we review the relationship between chromatin state transformations (chromatin reprogramming) and cellular reprogramming, with an emphasis on transcription factors, chromatin remodeling factors, histone modifications and DNA methylation. PMID:22757592

  5. Chromatin Dynamics during Cellular Reprogramming

    PubMed Central

    Apostolou, Effie; Hochedlinger, Konrad

    2014-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. 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

  8. 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

  9. Cell-Type-Specific Profiling of Gene Expression and Chromatin Binding without Cell Isolation: Assaying RNA Pol II Occupancy in Neural Stem Cells

    PubMed Central

    Southall, Tony D.; Gold, Katrina S.; Egger, Boris; Davidson, Catherine M.; Caygill, Elizabeth E.; Marshall, Owen J.; Brand, Andrea H.

    2013-01-01

    Summary Cell-type-specific transcriptional profiling often requires the isolation of specific cell types from complex tissues. We have developed “TaDa,” a technique that enables cell-specific profiling without cell isolation. TaDa permits genome-wide profiling of DNA- or chromatin-binding proteins without cell sorting, fixation, or affinity purification. The method is simple, sensitive, highly reproducible, and transferable to any model system. We show that TaDa can be used to identify transcribed genes in a cell-type-specific manner with considerable temporal precision, enabling the identification of differential gene expression between neuroblasts and the neuroepithelial cells from which they derive. We profile the genome-wide binding of RNA polymerase II in these adjacent, clonally related stem cells within intact Drosophila brains. Our data reveal expression of specific metabolic genes in neuroepithelial cells, but not in neuroblasts, and highlight gene regulatory networks that may pattern neural stem cell fates. PMID:23792147

  10. The Drosophila MI-2 chromatin-remodeling factor regulates higher-order chromatin structure and cohesin dynamics in vivo.

    PubMed

    Fasulo, Barbara; Deuring, Renate; Murawska, Magdalena; Gause, Maria; Dorighi, Kristel M; Schaaf, Cheri A; Dorsett, Dale; Brehm, Alexander; Tamkun, John W

    2012-01-01

    dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. Here we report an unanticipated role for dMi-2 in the regulation of higher-order chromatin structure in Drosophila. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells. PMID:22912596

  11. Chromatin remodeling and cancer, part I: covalent histone modifications

    Microsoft Academic Search

    Gang G. Wang; C. David Allis; Ping Chi

    2007-01-01

    Dynamic chromatin remodeling underlies many, if not all, DNA-templated biological processes, including gene transcription; DNA replication and repair; chromosome condensation; and segregation and apoptosis. Disrup- tion of these processes has been linked to the develop- ment and progression of cancer. The mechanisms of dynamic chromatin remodeling include the use of covalent histone modifications, histone variants, ATP- dependent complexes and DNA

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  13. Nanostructure-induced DNA condensation

    NASA Astrophysics Data System (ADS)

    Zhou, Ting; Llizo, Axel; Wang, Chen; Xu, Guiying; Yang, Yanlian

    2013-08-01

    The control of the DNA condensation process is essential for compaction of DNA in chromatin, as well as for biological applications such as nonviral gene therapy. This review endeavours to reflect the progress of investigations on DNA condensation effects of nanostructure-based condensing agents (such as nanoparticles, nanotubes, cationic polymer and peptide agents) observed by using atomic force microscopy (AFM) and other techniques. The environmental effects on structural characteristics of nanostructure-induced DNA condensates are also discussed.

  14. Characterization of Chromatin Distribution in Cell Nuclei

    Microsoft Academic Search

    Ian T. Young; P. W. Verbeek; Brian H. Mayall

    1986-01-01

    In this paper we develop four measures to describe the distribution of nuclear chroma- tin. These measures attempt to describe in an objective and meaningful way the heteroge- neity, granularity, condensation, and margin- ation of chromatin in cell nuclei. Starting with a high-resolution digitized image of a cell where the nuclear pixels have been iden- tified, the four measures may

  15. Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate

    PubMed Central

    1994-01-01

    Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template were investigated. Extracts were separated by ultracentrifugation into cytosol, membrane, and gelatinous pellet fractions. It was found that, in addition to the cytosolic and membrane fractions, a component of the gelatinous pellet fraction was required for the assembly of functional nuclei around a naked DNA template. In the absence of this component, membrane-bound but functionally inert spheres of lambda DNA were formed. Purification of the active pellet factor unexpectedly demonstrated the component to be glycogen. The assembly of functionally active nuclei, as assayed by DNA replication and nuclear transport, required that glycogen be pre-incubated with the lambda DNA and cytosol during the period of chromatin and higher order intermediate formation, before the addition of membranes. Hydrolysis of glycogen with alpha- amylase in the extract blocked nuclear formation. Upon analysis, chromatin formed in the presence of cytosol and glycogen alone appeared highly condensed, reminiscent of the nuclear assembly intermediate described by Newport in crude extracts (Newport, J. 1987. Cell. 48:205- 217). In contrast, chromatin formed from phage lambda DNA in cytosol lacking glycogen formed "fluffy chromatin-like" structures. Using sucrose gradient centrifugation, the highly condensed intermediates formed in the presence of glycogen could be isolated and were now able to serve as nuclear assembly templates in extracts lacking glycogen, arguing that the requirement for glycogen is temporally restricted to the time of intermediate formation and function. Glycogen does not act simply by inducing condensation of the chromatin, since similarly isolated mitotically condensed chromatin intermediates do not form functional nuclei. However, both mitotic and fluffy interphase chromatin intermediates formed in the absence of glycogen can be rescued to form functional nuclei when added to a second extract which contains glycogen. This study presents a novel role for a carbohydrate in nuclear assembly, a role which involves the formation of a particular chromatin intermediate. Potential models for the role of glycogen are discussed. PMID:8294509

  16. 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

  17. 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

  18. Neutron scattering studies on chromatin higher-order structure

    SciTech Connect

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

    1994-12-31

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

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

    SciTech Connect

    Terry, Samantha Y.A. [CR-UK/MRC Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Oxford (United Kingdom); Vallis, Katherine A., E-mail: katherine.vallis@oncology.ox.ac.uk [CR-UK/MRC Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Oxford (United Kingdom)

    2012-07-15

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

  20. Structure of active chromatin: higher-order folding of transcriptionally active chromatin in control and hypothyroid rat liver.

    PubMed Central

    Tikoo, K; Hamid, Q A; Ali, Z

    1997-01-01

    Investigation have been carried out into the salt-induced higher-order folding in the transcriptionally active chromatin region of rat liver nuclei by nuclease digestion, sedimentation and CD. The sensitivity of active chromatin in nuclei to micrococcal nuclease was suppressed by raising the ionic strength from 25 to 90 mM, indicating the occurrence of salt-induced condensation. The rate of sedimentation of fractionated inactive chromatin fragments of both moderate (approximately 3.5 kbp) and large (approximately 8.8 kbp) size increased maximally to the same extent, while that of active chromatin fragments was dependent on their size. The rate of sedimentation of moderately sized active chromatin fragments (approximately 5.5 kbp) showed a maximal 15% increase at 90 mM ionic strength. In contrast, a large increase (at least 60%) in the sedimentation rate of large active chromatin fragments (approximately 21 kbp) was observed at 65 mM ionic strength. A reasonable degree of higher-order folding was observed in large active chromatin fragment even at 25 mM ionic strength. On considering the percentage increase in sedimentation rate as a measure of the higher-order folding of chromatin, a different type of higher-order folding was observed in active chromatin fragments. Although the percentage increase in sedimentation decreased from 40 to 24% with an increase in the size of active chromatin from approximately 3 to approximately 9 kbp, a further increase in size up to 16 kbp brought the percentage increase back to 40%. CD studies agreed with the conclusions drawn from sedimentation studies. Active chromatin from hypothyroid rats showed similar folding behaviour, but the order of folding was slightly lower than for control active chromatin, at all sizes. PMID:9078275

  1. Shaping chromatin for repair.

    PubMed

    Gospodinov, Anastas; Herceg, Zdenko

    2013-01-01

    To counteract the adverse effects of various DNA lesions, cells have evolved an array of diverse repair pathways to restore DNA structure and to coordinate repair with cell cycle regulation. Chromatin changes are an integral part of the DNA damage response, particularly with regard to the types of repair that involve assembly of large multiprotein complexes such as those involved in double strand break (DSB) repair and nucleotide excision repair (NER). A number of phosphorylation, acetylation, methylation, ubiquitylation and chromatin remodeling events modulate chromatin structure at the lesion site. These changes demarcate chromatin neighboring the lesion, afford accessibility and binding surfaces to repair factors and provide on-the-spot means to coordinate repair and damage signaling. Thus, the hierarchical assembly of repair factors at a double strand break is mostly due to their regulated interactions with posttranslational modifications of histones. A large number of chromatin remodelers are required at different stages of DSB repair and NER. Remodelers physically interact with proteins involved in repair processes, suggesting that chromatin remodeling is a requisite for repair factors to access the damaged site. Together, recent findings define the roles of histone post-translational modifications and chromatin remodeling in the DNA damage response and underscore possible differences in the requirements for these events in relation to the chromatin context. PMID:23085398

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

    PubMed

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

    2012-01-01

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

  3. Keeping chromatin quiet

    PubMed Central

    Rowbotham, Samuel P; Varga-Weisz, Patrick D

    2011-01-01

    Disruption of chromatin during replication poses a major challenge to the maintenance and integrity of genome organization. It creates the need to accurately reconstruct the chromatin landscape following DNA duplication but there is little mechanistic understanding of how chromatin based modifications are restored on newly synthesized DNA. ATP-dependent chromatin remodeling activities serve multiple roles during replication and recent work underscores their requirement in the maintenance of proper chromatin organization. A new component of chromatin replication, the SWI/SNF-like chromatin remodeler SMARCAD1, acts at replication sites to facilitate deacetylation of newly assembled histones. Deacetylation is a pre-requisite for the restoration of epigenetic signatures in heterochromatin regions following replication. In this way, SMARCAD1, in concert with histone modifying activities and transcriptional repressors, reinforces epigenetic instructions to ensure that silenced loci are correctly perpetuated in each replication cycle. The emerging concept is that remodeling of nucleosomes is an early event imperative to promote the re-establishment of histone modifications following DNA replication. PMID:22101266

  4. Chromatin replication and epigenome maintenance

    Microsoft Academic Search

    Constance Alabert; Anja Groth

    2012-01-01

    Stability and function of eukaryotic genomes are closely linked to chromatin structure and organization. During cell division the entire genome must be accurately replicated and the chromatin landscape reproduced on new DNA. Chromatin and nuclear structure influence where and when DNA replication initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms

  5. Analysis of Chromatin Organisation

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

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

  6. 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

  7. [Genotoxicity of stack gas condensates of Bavarian waste incineration plants. III. Emission monitoring with a simple UDS assay using the human lung cell lines NCI-H 322 and 358].

    PubMed

    Raabe, F; Wichmann, G; Dautzenberg, D; Lierse, C; Zluticky, J; Metzner, G; Mücke, W

    1999-02-01

    For the validation of the genotoxicity testing on stack gas condensates from waste incineration plants using bacterial short time tests (15), a modified UDS assay with the lung cell lines NCI-H 322 and 358 was developed. The UDS assay is more sensitive than the SOS chromotest and discriminates better between the negative or weakly positive and the clearly positive samples. It has a high sensitive and specificity and also accuracy, is practicable in a comparatively simple, speedy and reasonably priced manner and is therefore appropriate for an emission monitoring similar to simple bacterial short time tests. Especially in strongly concentrated crude and clean gas condensates, maximal induction factors were seen in the range of strong UDS inducers. From 55 samples on 16 incineration plants tested in the years 1992 to 1995, in 48 we found weak to strong UDS inductions in at least one of the two test cell lines. From three plants examined continuously in this period only two emitted stack gases with constantly low genotoxicity at the end of sampling. 5 clean gas condensates, that were taken in random samples from 3 other plants in the period 1994 to 1995, proved to be non-genotoxic in the UDS assay. However, one of these plants emitted stack gases with high cytotoxicity, which might have masked UDS-inducing single substances. It is not possible to make a statement on the human toxicological relevance. However, a clearly positive development towards more harmless stack gas condensates was established. A definite correlation could not be shown between the chemical analysis of the detected cancerogenic organic single substances of the samples and the detected UDS inductions. Further investigations for finding strong UDS inducers from the substance spectrum of municipal stack gas emissions are necessary. PMID:10084205

  8. Nuclear size as estrogen-responsive chromatin quality parameter of mouse spermatozoa.

    PubMed

    Cacciola, Giovanna; Chioccarelli, Teresa; Altucci, Lucia; Viggiano, Andrea; Fasano, Silvia; Pierantoni, Riccardo; Cobellis, Gilda

    2013-11-01

    Recently, we have investigated the endocannabinoid involvement in chromatin remodeling events occurring in male spermatids. Indeed, we have demonstrated that genetic inactivation of the cannabinoid receptor type 1 (Cnr1) negatively influences chromatin remodeling mechanisms, by reducing histone displacement and indices of sperm chromatin quality (chromatin condensation and DNA integrity). Conversely, Cnr1 knock-out (Cnr1(-/-)) male mice, treated with estrogens, replaced histones and rescued chromatin condensation as well as DNA integrity. In the present study, by exploiting Cnr1(+/+), Cnr(+/-) and Cnr1(-/-) epididymal sperm samples, we show that histone retention directly correlates with low values of sperm chromatin quality indices determining sperm nuclear size elongation. Moreover, we demonstrate that estrogens, by promoting histone displacement and chromatin condensation rescue, are able to efficiently reduce the greater nuclear length observed in Cnr1(-/-) sperm. As a consequence of our results, we suggest that nucleus length may be used as a morphological parameter useful to screen out spermatozoa with low chromatin quality. PMID:23973938

  9. Epigenomics and chromatin dynamics

    PubMed Central

    2012-01-01

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

  10. DNA repair in a small yeast plasmid folded into chromatin.

    PubMed Central

    Smerdon, M J; Bedoyan, J; Thoma, F

    1990-01-01

    The question of whether excision repair of yeast plasmids accurately reflects the repair of yeast genomic chromatin has yielded conflicting answers. These conflicts could have arisen from differences in the conformation of plasmid molecules used during these studies. We have examined excision repair of UV photoproducts in a small (2619 bp) autonomously replicating plasmid (YRp-TRURAP), known to be folded into chromatin with positioned nucleosomes in vivo, in the yeast Saccharomyces cerevisiae. A quantitative assay was used to measure the yield of cyclobutane pyrimidine dimers (PD) in plasmid DNA by measuring the fraction of Form I molecules resistant to T4 endonuclease V. After a UV dose of 100 J/m2, which yields 1.2 PD/plasmid in irradiated cells, radiation insensitive (wt) cells repair approximately 70% of the PD in TRURAP chromatin in 2 hr (a rate comparable to that of genomic chromatin). On the other hand, no measurable repair occurs in TRURAP chromatin in radiation sensitive cells (rad1) during the same time period. Thus, this small plasmid contains sufficient chromatin structure in vivo to reflect the incompetent repair of genomic chromatin seen in a rad mutant, while maintaining the competent repair level in wt cells. Images PMID:2186374

  11. Conformational change in the chromatin remodelling protein MENT.

    PubMed

    Ong, Poh Chee; Golding, Sarah J; Pearce, Mary C; Irving, James A; Grigoryev, Sergei A; Pike, Debbie; Langendorf, Christopher G; Bashtannyk-Puhalovich, Tanya A; Bottomley, Stephen P; Whisstock, James C; Pike, Robert N; McGowan, Sheena

    2009-01-01

    Chromatin condensation to heterochromatin is a mechanism essential for widespread suppression of gene transcription, and the means by which a chromatin-associated protein, MENT, induces a terminally differentiated state in cells. MENT, a protease inhibitor of the serpin superfamily, is able to undergo conformational change in order to effect enzyme inhibition. Here, we sought to investigate whether conformational change in MENT is 'fine-tuned' in the presence of a bound ligand in an analogous manner to other serpins, such as antithrombin where such movements are reflected by a change in intrinsic tryptophan fluorescence. Using this technique, MENT was found to undergo structural shifts in the presence of DNA packaged into nucleosomes, but not naked DNA. The contribution of the four Trp residues of MENT to the fluorescence change was mapped using deconvolution analysis of variants containing single Trp to Phe mutations. The analysis indicated that the overall emission spectra is dominated by a helix-H tryptophan, but this residue did not dominate the conformational change in the presence of chromatin, suggesting that other Trp residues contained in the A-sheet and RCL regions contribute to the conformational change. Mutagenesis revealed that the conformational change requires the presence of the DNA-binding 'M-loop' and D-helix of MENT, but is independent of the protease specificity determining 'reactive centre loop'. The D-helix mutant of MENT, which is unable to condense chromatin, does not undergo a conformational change, despite being able to bind chromatin, indicating that the conformational change may contribute to chromatin condensation by the serpin. PMID:19266095

  12. Chromatin fiber allostery and the epigenetic code

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  13. 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

  14. Pulling the chromatin.

    PubMed

    Claudet, C; Bednar, J

    2006-03-01

    Nucleosome is the basic subunit of the chromatin, which organizes the genomic DNA within the cell nucleus. It was understood in the last decade that beside the DNA compaction it plays an important role in the regulation of the gene expression. In its intact form, the nucleosome represents an important mechanical barrier and, among others, it prevents access to the DNA and blocks the transcription elongation. Therefore, it has become important to know the forces and energies necessary to destabilize the nucleosome in order to understand the DNA-related processes. Stretching the chromatin fibre using micromanipulation techniques (e.g. optical tweezers) is an ideal approach to study the nucleosomal stability and the parameters that can modify it. In this short review we will discuss the existing data and potential difficulties that this state-of-the-art technique still has to overcome. PMID:16534544

  15. A modified neutral comet assay: elimination of lysis at high temperature and validation of the assay with anti-single-stranded DNA antibody.

    PubMed

    Wojewódzka, Maria; Buraczewska, Iwona; Kruszewski, Marcin

    2002-06-27

    Comet assay under neutral conditions allows the detection of DNA double-strand breaks (DSB), considered to be the biologically relevant radiation-induced lesion. In this report, we describe modifications of the neutral comet method, which simplify and facilitate its use for estimation of DNA DSB in X-irradiated mammalian cells in culture. The analysis carried out according to this protocol takes less time than those most often applied. Also, the use of lysis at 50 degrees C is avoided; this is important in view of the presence of heat-labile sites in the chromatin of irradiated cells, recently reported by Rydberg [Radiation-induced heat-labile sites that convert into DNA double-strand breaks, Radiation Research 153 (2000) 805-812]. The comets have well-defined, sharp limits, suitable for image analysis. The chromatin of the hydrogen peroxide-treated or UV-C-irradiated cell remains condensed similarly to that of the control cells. We checked the neutral comets for the presence of single-stranded DNA by means of a specific antibody. The results point to a satisfactory sensitivity of the modified neutral comet assay and its specificity for DSB. The minimum detection level of the modified neutral comet assay is about 5 Gy. PMID:12063063

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

    Microsoft Academic Search

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

    2008-01-01

    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

  17. Open and closed domains in the mouse genome are configured as 10-nm chromatin fibres

    PubMed Central

    Fussner, Eden; Strauss, Mike; Djuric, Ugljesa; Li, Ren; Ahmed, Kashif; Hart, Michael; Ellis, James; Bazett-Jones, David P

    2012-01-01

    The mammalian genome is compacted to fit within the confines of the cell nucleus. DNA is wrapped around nucleosomes, forming the classic ‘beads-on-a-string' 10-nm chromatin fibre. Ten-nanometre chromatin fibres are thought to condense into 30-nm fibres. This structural reorganization is widely assumed to correspond to transitions between active and repressed chromatin, thereby representing a chief regulatory event. Here, by combining electron spectroscopic imaging with tomography, three-dimensional images are generated, revealing that both open and closed chromatin domains in mouse somatic cells comprise 10-nm fibres. These findings indicate that the 30-nm chromatin model does not reflect the true regulatory structure in vivo. PMID:22986547

  18. Chromatin-tethered MAPKs

    PubMed Central

    Klein, Aileen M.; Zaganjor, Elma; Cobb, Melanie H.

    2013-01-01

    Mitogen-activated protein kinases (MAPKs) are a family of protein kinases that are essential nodes in many cellular regulatory circuits including those that take place on DNA. Most members of the four MAPK subgroups that exist in canonical three kinase cascades - extracellular signal-regulated kinases 1 and 2 (ERK1/2), ERK5, c-Jun N-terminal kinases (JNK1-3), and p38 (?, ?, ?, and ?) families - have been shown to perform regulatory functions on chromatin. This review offers a brief update on the variety of processes that involve MAPKs and available mechanisms garnered in the last two years. PMID:23434067

  19. What determines the folding of the chromatin fiber?

    PubMed Central

    van Holde, K; Zlatanova, J

    1996-01-01

    In this review, we attempt to summarize, in a critical manner, what is currently known about the processes of condensation and decondensation of chromatin fibers. We begin with a critical analysis of the possible mechanisms for condensation, considering both old and new evidence as to whether the linker DNA between nucleosomes bends or remains straight in the condensed structure. Concluding that the preponderance of evidence is for straight linkers, we ask what other fundamental process might allow condensation, and argue that there is evidence for linker histone-induced contraction of the internucleosome angle, as salt concentration is raised toward physiological levels. We also ask how certain specific regions of chromatin can become decondensed, even at physiological salt concentration, to allow transcription. We consider linker histone depletion and acetylation of the core histone tails, as possible mechanisms. On the basis of recent evidence, we suggest a unified model linking targeted acetylation of specific genomic regions to linker histone depletion, with unfolding of the condensed fiber as a consequence. Images Fig. 2 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:8855215

  20. Chromatin, DNA methylation and neuron gene regulation — the purpose of the package

    PubMed Central

    Sharma, Rajiv P.; Grayson, Dennis R.; Guidotti, Alessandro; Costa, Erminio

    2005-01-01

    The accessibility of cognate binding sites within a gene promoter can be modified by the condensation or relaxation of local chromatin structure. Local chromatin structure is in turn programmed by covalent modifications of cytosine bases in DNA and amino acid residues in histone protein tails. These chemical and physical adaptations around gene promoters can significantly change levels of mRNA expression. Furthermore, linear patterns of covalent modification of histone protein tails are emerging as a distinct regulatory code — another form of cellular memory. Because chromatin structure can be modified by conventional pharmacologic therapy, a novel approach to the regulation of neuronal gene expression in clinical populations is possible. PMID:16049569

  1. Dissection of open chromatin domain formation by site-specific recombination in Drosophila.

    PubMed

    Zielke, Thomas; Saumweber, Harald

    2014-05-15

    Drosophila polytene interphase chromosomes provide an ideal test system to study the rules that define the structure of chromatin domains. We established a transgenic condensed chromatin domain cassette for the insertion of large pieces of DNA by site-specific recombination. Insertion of this cassette into open chromatin generated a condensed domain, visible as an extra band on polytene chromosomes. Site-specific recombination of DNA sequence variants into this ectopic band allowed us to compare their capacity for open chromatin formation by cytogenetic methods. We demonstrate that the 61C7-8 interband DNA maintains its open chromatin conformation and epigenetic state at an ectopic position. By deletion analysis, we mapped the sequences essential for open chromatin formation to a 490-bp fragment in the proximal part of the 17-kb interband sequence. This fragment overlaps binding sites for the chromatin protein Chriz (also known as Chro), the histone kinase Jil-1 and the boundary element protein CP190. It also overlaps a promoter region that locates between the Rev1 and Med30 transcription units. PMID:24639466

  2. 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

  3. A Physical Model for the Condensation and Decondensation of Eukaryotic Chromosomes

    E-print Network

    Julien Mozziconacci; Christophe Lavelle; Maria Barbi; Annick Lesne; Jean-Marc Victor

    2007-09-03

    During the eukaryotic cell cycle, chromatin undergoes several conformational changes, which are believed to play key roles in gene expression regulation during interphase, and in genome replication and division during mitosis. In this paper, we propose a scenario for chromatin structural reorganization during mitosis, which bridges all the different scales involved in chromatin architecture, from nucleosomes to chromatin loops. We build a model for chromatin, based on available data, taking into account both physical and topological constraints DNA has to deal with. Our results suggest that the mitotic chromosome condensation/decondensation process is induced by a structural change at the level of the nucleosome itself.

  4. 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.

  5. Hormone action and chromatin remodelling.

    PubMed

    Robyr, D; Wolffe, P

    1998-02-01

    Current attention in transcriptional regulation is focused on the properties of coactivators and corepressors that mediate communication between sequence-specific transcription factors, the basal transcriptional machinery and the chromatin environment. Nuclear and steroid hormone receptors represent the best-understood transcription factors that utilize coactivators and corepressors. This review considers the access of these receptors to chromatin, the modifications of chromatin structure that the receptors instigate and the implications for transcriptional control. Nucleosome positioning and targeted histone modification emerge as central controlling elements for gene expression. PMID:9539951

  6. Chromatin, Cancer and Drug Therapies

    PubMed Central

    Cortez, Connie C.; Jones, Peter A.

    2008-01-01

    The structure and organization of chromatin have attracted a great deal of attention recently because of their implications for the field of epigenetics. DNA methylation and the post-translational modifications that occur on histones can specify transcriptional competency. During cancer development, tumor suppressor genes become silenced by DNA hypermethylation and chromatin modifiers no longer perform in their usual manner. Current epigenetic therapy has been able to take advantage of the reversibility of these epimutations. Progress has been made in the treatment of hematological malignancies and some solid tumors. As the knowledge of how chromatin regulates gene expression is enhanced, improvements in the treatment of cancer can be made. PMID:18691602

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

    PubMed Central

    Mehra, Sonal; Fritzler, Marvin J.

    2014-01-01

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

  8. Chromatin structure as a mediator of aging

    PubMed Central

    Feser, Jason; Tyler, Jessica

    2014-01-01

    The aging process is characterized by gradual changes to an organism's macromolecules, which negatively impacts biological processes. The complex macromolecular structure of chromatin regulates all nuclear processes requiring access to the DNA sequence. As such, maintenance of chromatin structure is an integral component to deter premature aging. In this review, we describe current research that links aging to chromatin structure. Histone modifications influence chromatin compaction and gene expression and undergo many changes during aging. Histone protein levels also decline during aging, dramatically affecting chromatin structure. Excitingly, lifespan can be extended by manipulations that reverse the age-dependent changes to chromatin structure, indicating the pivotal role chromatin structure plays during aging. PMID:21081125

  9. ATP-dependent Chromatin Remodelling

    Microsoft Academic Search

    Parul Choudhary; Patrick Varga-Weisz

    Alterations of chromatin structure play an important role in gene regulation. One way of doing so involves ATP-dependent chromatin\\u000a remodelling enzymes that act as molecular machines coupling ATP-hydrolysis to structural changes of the nucleosome. Several\\u000a recent studies shed important insights into the mechanism of these factors and indicate that they couple DNA translocation\\u000a within the nucleosome to DNA loop propagation

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

    PubMed

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

    2014-01-01

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

  11. Sudemycin E influences alternative splicing and changes chromatin modifications

    PubMed Central

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

    2014-01-01

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

  12. Open, repair and close again: Chromatin dynamics and the response to UV-induced DNA damage

    Microsoft Academic Search

    Zoraya Palomera-Sanchez; Mario Zurita

    2011-01-01

    Due to its link with human pathologies, including cancer, the mechanism of Nucleotide Excision Repair (NER) has been extensively studied. Most of the pathway and players have been defined using in vitro reconstitution experiments. However, in vivo, the NER machinery must deal with the presence of organized chromatin, which in some regions, such as heterochromatin, is highly condensed but still

  13. Differential Association of Chromatin Proteins Identifies BAF60a/SMARCD1 as a Regulator of Embryonic Stem Cell Differentiation.

    PubMed

    Alajem, Adi; Biran, Alva; Harikumar, Arigela; Sailaja, Badi Sri; Aaronson, Yair; Livyatan, Ilana; Nissim-Rafinia, Malka; Sommer, Andreia Gianotti; Mostoslavsky, Gustavo; Gerbasi, Vincent R; Golden, Daniel E; Datta, Arnab; Sze, Siu Kwan; Meshorer, Eran

    2015-03-31

    Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation. PMID:25818293

  14. When repair meets chromatin. First in series on chromatin dynamics.

    PubMed

    Green, Catherine M; Almouzni, Geneviève

    2002-01-01

    In eukaryotic cells, the inheritance of both the DNA sequence and its organization into chromatin is critical to maintain genome stability. This maintenance is challenged by DNA damage. To fully understand how the cell can tolerate genotoxic stress, it is necessary to integrate knowledge of the nature of DNA damage, its detection and its repair within the chromatin environment of a eukaryotic nucleus. The multiplicity of the DNA damage and repair processes, as well as the complex nature of chromatin, have made this issue difficult to tackle. Recent progress in each of these areas enables us to address, both at a molecular and a cellular level, the importance of inter-relationships between them. In this review we revisit the 'access, repair, restore' model, which was proposed to explain how the conserved process of nucleotide excision repair operates within chromatin. Recent studies have identified factors potentially involved in this process and permit refinement of the basic model. Drawing on this model, the chromatin alterations likely to be required during other processes of DNA damage repair, particularly double-strand break repair, are discussed and recently identified candidates that might perform such alterations are highlighted. PMID:11799057

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

    PubMed

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

    2015-02-18

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  17. 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.

  18. Global Quantitative Modeling of Chromatin Factor Interactions

    PubMed Central

    Zhou, Jian; Troyanskaya, Olga G.

    2014-01-01

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

  19. Global quantitative modeling of chromatin factor interactions.

    PubMed

    Zhou, Jian; Troyanskaya, Olga G

    2014-03-01

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

  20. Upstream binding factor association induces large-scale chromatin decondensation

    PubMed Central

    Chen, Danyang; Belmont, Andrew S.; Huang, Sui

    2004-01-01

    The function of upstream binding factor (UBF), an essential component of the RNA polymerase (pol) I preinitiation complex, is unclear. Recently, UBF was found distributed throughout ribosomal gene repeats rather than being restricted to promoter regions. This observation has led to the speculation that one role of UBF binding may be to induce chromatin remodeling. To directly evaluate the impact of UBF on chromatin structure, we used an in vivo assay in which UBF is targeted via a lac repressor fusion protein to a heterochromatic, amplified chromosome region containing lac operator repeats. We show that the association of UBF with this locus induces large-scale chromatin decondensation. This process does not appear to involve common remodeling complexes, including SWI/SNF and histone acetyltransferases, and is independent of histone H3 lysine 9 acetylation. However, UBF recruits the pol I-specific, TATA box-binding protein containing complex SL1 and pol I subunits. Our results suggest a working hypothesis in which the dynamic association of UBF with ribosomal DNA clusters recruits the pol I transcription machinery and maintains these loci in a transcriptionally competent configuration. These studies also provide an in vivo model simulating ribosomal DNA transactivation outside the nucleolus, allowing temporal and spatial analyses of chromatin remodeling and assembly of the pol I transcription machinery. PMID:15477594

  1. T cell development: better living through chromatin

    Microsoft Academic Search

    Michael S Krangel

    2007-01-01

    T lymphocyte development is directed by a gene-expression program that occurs in the complex nucleoprotein environment of chromatin. This review examines basic principles of chromatin regulation and evaluates ongoing progress toward understanding how the chromatin template is manipulated to control gene expression and gene recombination in developing thymocytes. Special attention is devoted to the loci encoding T cell receptors ?

  2. Chromatin rearrangements during nucleotide excision repair

    Microsoft Academic Search

    Jonathan G. Moggs; Geneviève Almouzni

    1999-01-01

    The removal of DNA damage from the eukaryotic genome requires DNA repair enzymes to operate within the complex environment of chromatin. We review the evidence for chromatin rearrangements during nucleotide excision repair and discussthe extent and possible molecular mechanisms of these rearrangements, focusing on events at the nucleosome level of chromatin structure.

  3. Effects of divalent metal cations on composition and neoplasia-specific antigenicity of chromatins.

    PubMed

    Dupere, S L; Holland, S; Gawne, S; Cancelliere, K E; Sedita, B A; Dale, P J; Jarrell, E D; O'Connor, T E

    1983-10-01

    The antigenicity and composition of chromatins differ markedly in chromatin preparations obtained by different procedures. Rat Novikoff hepatoma chromatin (NC) obtained by the "salt precipitation" and the micrococcal nuclease digestion procedures using significant levels of EDTA and NaCl each shows a common complement fixation (CF) capacity, exceeding chromatin preparations obtained from normal rat liver when tested with rabbit antisera raised to dehistonized NC. In contrast, "structured" NC preparations, which have been postulated to retain a native physical conformation, show minimal CF capacity when tested with the same antiserum but show high CF following elution of histones. While further progressive elution of non-histone proteins (NHPs) did not alter the CF capacity per unit DNA, the completely separated DNA and NHP fractions each showed minimal CF. The data suggest that the antigens detected in the CF assay predominantly represent an artifactual but specific complex of DNA and NHP arising from a denaturation of the native chromatin following elution of metal ions or histones. A qualitatively similar profile of NHPs in salt-precipitated NCs shows a range of total protein/DNA ratios, suggesting that the NHPs found in chromatin preparations may not be intrinsic to the native chromatin structure. PMID:6883342

  4. Micromechanics of chromatin and chromosomes

    Microsoft Academic Search

    John F. Marko; Michael G. Poirier

    2003-01-01

    The enzymes that transcribe, recombine, package, and duplicate the eukaryotic genome all are highly processive and capable of generating large forces. Understanding chromosome function therefore will require analysis of mechanics as well as biochemistry. Here we review development of new biophysical-biochemical techniques for studying the mechanical properties of isolated chromatin fibers and chromosomes. We also discuss microscopy-based experiments on cells

  5. Neutron scatter studies of chromatin structures related to functions

    SciTech Connect

    Bradbury, E.M.

    1992-01-01

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

  6. Neutron scatter studies of chromatin structures related to functions

    SciTech Connect

    Bradbury, E.M.

    1992-01-01

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

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

    E-print Network

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

    2008-01-01

    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.

  8. 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.

  9. The impact of chromatin dynamics on plant light responses and circadian clock function.

    PubMed

    Barneche, Fredy; Malapeira, Jordi; Mas, Paloma

    2014-06-01

    Research on the functional properties of nucleosome structure and composition dynamics has revealed that chromatin-level regulation is an essential component of light signalling and clock function in plants, two processes that rely extensively on transcriptional controls. In particular, several types of histone post-translational modifications and chromatin-bound factors act sequentially or in combination to establish transcriptional patterns and to fine-tune the transcript abundance of a large repertoire of light-responsive genes and clock components. Cytogenetic approaches have also identified light-induced higher-order chromatin changes that dynamically organize the condensation of chromosomal domains into sub-nuclear foci containing silenced repeat elements. In this review, we report recently identified molecular actors that establish chromatin state dynamics in response to light signals such as photoperiod, intensity, and spectral quality. We also highlight the chromatin-dependent mechanisms that contribute to the 24-h circadian gene expression and its impact on plant physiology and development. The commonalities and contrasts of light- and clock-associated chromatin-based mechanisms are discussed, with particular emphasis on their impact on the selective regulation and rapid modulation of responsive genes. PMID:24520020

  10. 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

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

    SciTech Connect

    Tone, Shigenobu [Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 (Japan)], E-mail: tone@med.kawasaki-m.ac.jp; Sugimoto, Kenji [Laboratory of Applied Molecular Biology, Division of Bioscience and Informatics, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531 (Japan); Tanda, Kazue [Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 (Japan); Suda, Taiji; Uehira, Kenzo [Electron Microscope Center, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 (Japan); Kanouchi, Hiroaki [Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 (Japan); Samejima, Kumiko [Wellcome Trust Centre for Cell Biology, ICMB, King's Buildings, The University of Edinburgh, Edinburgh, EH93JR, Scotland (United Kingdom); Minatogawa, Yohsuke [Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 (Japan); Earnshaw, William C. [Wellcome Trust Centre for Cell Biology, ICMB, King's Buildings, The University of Edinburgh, Edinburgh, EH93JR, Scotland (United Kingdom)], E-mail: Bill.Earnshaw@ed.ac.uk

    2007-10-01

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

  12. Plant chromatin warms up in Madrid

    PubMed Central

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

    2014-01-01

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

  13. Chromatin fluorescence after carmine staining.

    PubMed

    Stockert, J C; Llorente, A R; Del Castillo, P; Gómez, A

    1990-01-01

    After staining with dilute solutions (0.1 mg/ml in distilled water) of commercial carmine, a strong reddish orange fluorescence was observed in nuclei from cell smears and frozen and paraffin tissue sections. Optimal exciting light was 436 nm (violet-blue) or 450-490 nm (blue). Compact chromatin from interphase nuclei, mitotic and meiotic chromosomes and the kinetoplast of Trypanosoma cruzi showed the highest fluorescence, while the basophilic cytoplasm appeared weakly fluorescent. No emission was observed in cartilage matrix, mast cell granules or goblet cell mucin. This selective method could be valuable in microscopic and cytochemical studies on chromatin because the carmine fluorescence is stable and preparations can be dehydrated and mounted permanently without changes in the fluorescence pattern. PMID:2080525

  14. 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

  15. Transcription factors, chromatin and cancer.

    PubMed

    Thorne, James L; Campbell, Moray J; Turner, Bryan M

    2009-01-01

    Transcription factors, chromatin and chromatin-modifying enzymes are key components in a complex network through which the genome interacts with its environment. For many transcription factors, binding motifs are found adjacent to the promoter regions of a large proportion of genes, requiring mechanisms that confer binding specificity in any given cell type. These include association of the factor with other proteins and packaging of DNA, as chromatin, at the binding sequence so as to inhibit or facilitate binding. Recent evidence suggests that specific post-translational modifications of the histones packaging promoter DNA can help guide transcription factors to selected sites. The enzymes that put such modifications in place are dependent on metabolic components (e.g. acetyl CoA, S-adenosyl methionine) and susceptible to inhibition or activation by environmental factors. Local patterns of histone modification can be altered or maintained through direct interaction between the transcription factor and histone modifying enzymes. The functional consequences of transcription factor binding are also dependent on protein modifying enzymes, particularly those that alter lysine methylation at selected residues. Remarkably, the role of these enzymes is not limited to promoter-proximal events, but can be linked to changes in the intranuclear location of target genes. In this review we describe results that begin to define how transcription factors, chromatin and environmental variables interact and how these interactions are subverted in cancer. We focus on the nuclear receptor family of transcription factors, where binding of ligands such as steroid hormones and dietary derived factors provides an extra level of environmental input. PMID:18804550

  16. 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

  17. Localization of DNA in the condensed interphase chromosomes of Euglena

    Microsoft Academic Search

    K. Ueda; Y. Hayashi-Ishimaru

    1996-01-01

    The localization of DNA in the condensed interphase chromosomes of Euglena was determined by immunoelectron microscopy. Deposits of gold particles that coincided with the localization of DNA followed threads that corresponded to the chromatin fibers. The threads were 55–80 nm in diameter and were assumed to be supersolenoids. The localization of gold deposits on chromosomes that had been sectioned in

  18. Proteomics of a fuzzy organelle: interphase chromatin

    PubMed Central

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

    2014-01-01

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

  19. A dual role of H4K16 acetylation in the establishment of yeast silent chromatin.

    PubMed

    Oppikofer, Mariano; Kueng, Stephanie; Martino, Fabrizio; Soeroes, Szabolcs; Hancock, Susan M; Chin, Jason W; Fischle, Wolfgang; Gasser, Susan M

    2011-07-01

    Discrete regions of the eukaryotic genome assume heritable chromatin structure that is refractory to transcription. In budding yeast, silent chromatin is characterized by the binding of the Silent Information Regulatory (Sir) proteins to unmodified nucleosomes. Using an in vitro reconstitution assay, which allows us to load Sir proteins onto arrays of regularly spaced nucleosomes, we have examined the impact of specific histone modifications on Sir protein binding and linker DNA accessibility. Two typical marks for active chromatin, H3K79(me) and H4K16(ac) decrease the affinity of Sir3 for chromatin, yet only H4K16(ac) affects chromatin structure, as measured by nuclease accessibility. Surprisingly, we found that the Sir2-4 subcomplex, unlike Sir3, has higher affinity for chromatin carrying H4K16(ac). NAD-dependent deacetylation of H4K16(ac) promotes binding of the SIR holocomplex but not of the Sir2-4 heterodimer. This function of H4K16(ac) cannot be substituted by H3K56(ac). We conclude that acetylated H4K16 has a dual role in silencing: it recruits Sir2-4 and repels Sir3. Moreover, the deacetylation of H4K16(ac) by Sir2 actively promotes the high-affinity binding of the SIR holocomplex. PMID:21666601

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

    PubMed Central

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

    2010-01-01

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

  1. The Prefoldin Complex Regulates Chromatin Dynamics during Transcription Elongation

    PubMed Central

    Millán-Zambrano, Gonzalo; Rodríguez-Gil, Alfonso; Peñate, Xenia; de Miguel-Jiménez, Lola; Morillo-Huesca, Macarena; Krogan, Nevan; Chávez, Sebastián

    2013-01-01

    Transcriptional elongation requires the concerted action of several factors that allow RNA polymerase II to advance through chromatin in a highly processive manner. In order to identify novel elongation factors, we performed systematic yeast genetic screening based on the GLAM (Gene Length-dependent Accumulation of mRNA) assay, which is used to detect defects in the expression of long transcription units. Apart from well-known transcription elongation factors, we identified mutants in the prefoldin complex subunits, which were among those that caused the most dramatic phenotype. We found that prefoldin, so far involved in the cytoplasmic co-translational assembly of protein complexes, is also present in the nucleus and that a subset of its subunits are recruited to chromatin in a transcription-dependent manner. Prefoldin influences RNA polymerase II the elongation rate in vivo and plays an especially important role in the transcription elongation of long genes and those whose promoter regions contain a canonical TATA box. Finally, we found a specific functional link between prefoldin and histone dynamics after nucleosome remodeling, which is consistent with the extensive network of genetic interactions between this factor and the machinery regulating chromatin function. This study establishes the involvement of prefoldin in transcription elongation, and supports a role for this complex in cotranscriptional histone eviction. PMID:24068951

  2. 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.

  3. A histone code for chromatin assembly.

    PubMed

    Fillingham, Jeffrey; Greenblatt, Jack F

    2008-07-25

    In this issue, two papers implicate histone H3 lysine 56 acetylation in histone deposition in chromatin. Li et al. (2008) show that acetylation of H3K56 promotes S phase chromatin assembly that is mediated by the histone chaperones CAF-1 and Rtt106. Chen et al. (2008) establish that the acetylation mark promotes chromatin reassembly following DNA double-strand break repair. PMID:18662534

  4. ChromDB: the chromatin database.

    PubMed

    Gendler, Karla; Paulsen, Tara; Napoli, Carolyn

    2008-01-01

    The ChromDB website (http://www.chromdb.org) displays chromatin-associated proteins, including RNAi-associated proteins, for a broad range of organisms. Our primary focus is to display sets of highly curated plant genes predicted to encode proteins associated with chromatin remodeling. Our intent is to make this intensively curated sequence information available to the research and teaching communities in support of comparative analyses toward understanding the chromatin proteome in plants, especially in important crop species such as corn and rice. Model animal and fungal proteins are included in the database to facilitate a complete, comparative analysis of the chromatin proteome and to make the database applicable to all chromatin researchers and educators. Chromatin biology and chromatin remodeling are complex processes involving a multitude of proteins that regulate the dynamic changes in chromatin structure which either repress or activate transcription. We strive to organize ChromDB data in a straightforward and comparative manner to help users understand the complement of proteins involved in packaging DNA into chromatin. PMID:17942414

  5. The Chd Family of Chromatin Remodelers

    PubMed Central

    Marfella, Concetta G.A.; Imbalzano, Anthony N.

    2007-01-01

    Chromatin remodeling enzymes contribute to the dynamic changes that occur in chromatin structure during cellular processes such as transcription, recombination, repair, and replication. Members of the chromodomain helicase DNA-binding (Chd) family of enzymes belong to the SNF2 superfamily of ATP-dependent chromatin remodelers. The Chd proteins are distinguished by the presence of two N-terminal chromodomains that function as interaction surfaces for a variety of chromatin components. Genetic, biochemical, and structural studies demonstrate that Chd proteins are important regulators of transcription and play critical roles during developmental processes. Numerous Chd proteins are also implicated in human disease. PMID:17350655

  6. Genome-Wide Views of Chromatin Structure

    PubMed Central

    Rando, Oliver J.; Chang, Howard Y.

    2010-01-01

    Eukaryotic genomes are packaged into a nucleoprotein complex known as chromatin, which affects most processes that occur on DNA. Along with genetic and biochemical studies of resident chromatin proteins and their modifying enzymes, mapping of chromatin structure in vivo is one of the main pillars in our understanding of how chromatin relates to cellular processes. In this review, we discuss the use of genomic technologies to characterize chromatin structure in vivo, with a focus on data from budding yeast and humans. The picture emerging from these studies is the detailed chromatin structure of a typical gene, where the typical behavior gives insight into the mechanisms and deep rules that establish chromatin structure. Important deviation from the archetype is also observed, usually as a consequence of unique regulatory mechanisms at special genomic loci. Chromatin structure shows substantial conservation from yeast to humans, but mammalian chromatin has additional layers of complexity that likely relate to the requirements of multicellularity such as the need to establish faithful gene regulatory mechanisms for cell differentiation. PMID:19317649

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

    SciTech Connect

    Aoki, Ryuta; Inui, Masafumi; Hayashi, Yohei; Sedohara, Ayako [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan)] [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Okabayashi, Koji [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan) [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); ICORP Organ Regeneration Project, Japan Science and Technology Agency (JST), 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Ohnuma, Kiyoshi, E-mail: kohnuma@vos.nagaokaut.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan)] [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Murata, Masayuki [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan)] [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Asashima, Makoto, E-mail: asashi@bio.c.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan) [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); ICORP Organ Regeneration Project, Japan Science and Technology Agency (JST), 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Organ Development Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan)

    2010-09-17

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

  8. Imaging of DNA/Nanosphere Condensates

    NASA Astrophysics Data System (ADS)

    Krishnan, R.

    2005-03-01

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

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

    PubMed Central

    2011-01-01

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

  10. Open, repair and close again: chromatin dynamics and the response to UV-induced DNA damage.

    PubMed

    Palomera-Sanchez, Zoraya; Zurita, Mario

    2011-02-01

    Due to its link with human pathologies, including cancer, the mechanism of Nucleotide Excision Repair (NER) has been extensively studied. Most of the pathway and players have been defined using in vitro reconstitution experiments. However, in vivo, the NER machinery must deal with the presence of organized chromatin, which in some regions, such as heterochromatin, is highly condensed but still susceptible to DNA damage. A series of events involving different chromatin-remodeling factors and histone-modifying enzymes target chromatin regions that contain DNA lesions. CPDs change the structure of the nucleosome, allowing access to factors that can recognize the lesion. Next, DDB1-DDB2 protein complexes, which mono-ubiquitinate histones H2A, H3, and H4, recognize nucleosomes containing DNA lesions. The ubiquitinated nucleosome facilitates the recruitment of ATP-dependent chromatin-remodeling factors and the XPC-HR23B-Centrin 2 complex to the target region. Different ATP-dependent chromatin-remodeling factors, such as SWI/SNF and INO80, have been identified as having roles in the UV irradiation response prior to the action of the NER machinery. Subsequently, remodeling of the nucleosome allows enzymatic reactions by histone-modifying factors that may acetylate, methylate or demethylate specific histone residues. Intriguingly, some of these histone modifications are dependent on p53. These histone modifications and the remodeling of the nucleosome allow the entrance of TFIIH, XPC and other NER factors that remove the damaged strand; then, gap-filling DNA synthesis and ligation reactions are carried out after excision of the oligonucleotide with the lesion. Finally, after DNA repair, the initial chromatin structure has to be reestablished. Therefore, factors that modulate chromatin dynamics contribute to the NER mechanism, and they are significant in the future design of treatments for human pathologies related to genome instability and the appearance of drug-resistant tumors. PMID:21130713

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

    PubMed Central

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

    2009-01-01

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

  12. The role of chromatin insulators in nuclear architecture and genome function

    PubMed Central

    Van Bortle, Kevin; Corces, Victor G.

    2013-01-01

    Eukaryotic genomes are intricately arranged into highly organized yet dynamic structures that underlie patterns of gene expression and cellular identity. The recent adaptation of novel genomic strategies for assaying nuclear architecture has significantly extended and accelerated our ability to query the nature of genome organization and the players involved. In particular, recent explorations of physical arrangements and chromatin landscapes in higher eukaryotes have demonstrated that chromatin insulators, which mediate functional interactions between regulatory elements, appear to play an important role in these processes. Here we reflect on current findings and our rapidly expanding understanding of insulators and their role in nuclear architecture and genome function. PMID:23298659

  13. The Chromatin Remodeler SPLAYED Regulates Specific Stress Signaling Pathways

    E-print Network

    Plotkin, Joshua B.

    the necrotrophic pathogen Botrytis cinerea but not the biotrophic pathogen Pseudomonas syringae. These findings mechanism of transcriptional control involves dynamic changes in chromatin structure. Alterations in chromatin structure are brought about by a number of different mechanisms, including chromatin modifications

  14. 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

  15. Chromatin modification and muscle differentiation.

    PubMed

    Yahi, Hakima; Philipot, Ophélie; Guasconi, Valentina; Fritsch, Lauriane; Ait-Si-Ali, Slimane

    2006-12-01

    Skeletal muscle differentiation is a multistep process, which begins with the commitment of multi-potent mesodermal precursor cells to the muscle fate. These committed cells, the myoblasts, then differentiate and fuse into multinucleated myotubes. The final step of muscle differentiation is the maturation of differentiated myotubes into myofibres. Skeletal muscle development requires the coordinated expression of various transcription factors like the members of the myocyte enhancer binding-factor 2 family and the muscle regulatory factors. These transcription factors, in collaboration with chromatin-remodelling complexes, act in specific combinations and within complex transcriptional regulatory networks to achieve skeletal myogenesis. Additional factors involved in the epigenetic regulation of this process continue to be discovered. In this review, the authors discuss the recent discoveries in the epigenetic regulation of myogenesis. They also summarise the role of chromatin-modifying enzymes regulating muscle gene expression. These different factors are often involved in multiple steps of muscle differentiation and have redundant activities. Altogether, the recent findings have allowed a better understanding of myogenesis and have raised new hopes for the pharmacological development of new therapies aimed at muscle degeneration diseases, such as myotonic dystrophy or Duchenne muscular dystrophy. PMID:17105377

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

    PubMed

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

    2014-12-01

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

  17. 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

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

    PubMed Central

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

    2014-01-01

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

  19. 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

  20. Comparative analysis of metazoan chromatin organization.

    PubMed

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

    2014-08-28

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

  1. Chromatin insulators: lessons from the fly

    E-print Network

    Corces, Victor G.

    Chromatin insulators: lessons from the fly B.V.Gurudatta and Victor G.Corces Abstract Chromatin insulators are DNA^protein complexes with broad functions in nuclear biology. Drosophila has at least five different types of insulators; recent results suggest that these different insulators share some components

  2. A native chromatin extraction method based on salicylic acid coated magnetic nanoparticles and characterization of chromatin.

    PubMed

    Zhou, Zhongwu; Irudayaraj, Joseph

    2015-02-01

    Native chromatin contains valuable genetic, epigenetic and structural information. Though DNA and nucleosome structures are well defined, less is known about the higher-order chromatin structure. Traditional chromatin extraction methods involve fixation, fragmentation and centrifugation, which might distort the higher-order structural information of native chromatin. We present a simple approach to isolate native chromatin from cultured mammalian cells using salicylic acid coated magnetic nanoparticles (SAMNPs). Chromatin is magnetically separated from cell lysates without any filtration or high-speed centrifugation. The purified chromatin is suitable for the examination of histone modifications and other chromatin associated proteins as confirmed by western blotting analysis. The structure of chromatin was determined by confocal fluorescence microscopy, transmission electron microscopy (TEM) and atomic force microscopy (AFM). High-resolution AFM and TEM images clearly show a classical bead-on-a-string structure. The higher-order chromatin structure is also determined via electron microscopy. Our method provides a simple, inexpensive and an environmentally friendly means to extract native chromatin not possible before, suitable for both biochemical and structural analysis. PMID:25475154

  3. Identification of New Human Origins of DNA Replication by an Origin-Trapping Assay

    Microsoft Academic Search

    Jeannine Gerhardt; Samira Jafar; Mark-Peter Spindler; Elisabeth Ott; Aloys Schepers

    2006-01-01

    Metazoan genomes contain thousands of replication origins, but only a limited number have been charac- terized so far. We developed a two-step origin-trapping assay in which human chromatin fragments associated with origin recognition complex (ORC) in vivo were first enriched by chromatin immunoprecipitation. In a second step, these fragments were screened for transient replication competence in a plasmid-based assay utilizing

  4. Overlapping chromatin-remodeling systems collaborate genome wide at dynamic chromatin transitions.

    PubMed

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

    2014-01-01

    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 substantially 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

  5. 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

  6. 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

  7. 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.

  8. Polariton condensates

    SciTech Connect

    Snoke, David; Littlewood, Peter [University of Pittsburgh, Pennsylvania (United States); University of Cambridge (United Kingdom)

    2010-08-15

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

  9. Chromatin structure analysis of spermatozoa from reciprocal chromosome translocation (RCT) carriers with known meiotic segregation patterns.

    PubMed

    Olszewska, Marta; Fraczek, Monika; Huleyuk, Nataliya; Czernikiewicz, Anna; Wiland, Ewa; Boksa, Magdalena; Zastavna, Danuta; Panasiuk, Barbara; Midro, Alina T; Kurpisz, Maciej

    2013-09-01

    The presence of reciprocal chromosome translocations (RCTs), as well as sperm chromatin disturbances, is known to exert negative influence on male fertility. The aim of this study was to identify an association between chromosome structural rearrangements in male RCT carriers and sperm seminological parameters (concentration, motility, morphology), chromatin status (fragmentation and maturity), meiotic segregation pattern and observed chromosomal hyperhaploidy. Sperm samples originated from ten male RCT carriers with reproductive failure/success. TUNEL assay (DNA fragmentation) and chromomycin A3 (CMA3)/aniline blue (AB) staining (chromatin maturity) were used to analyze sperm chromatin status while fluorescent in situ hybridization (FISH) was applied to observe meiotic segregation patterns and hyperhaploidy in spermatozoa. We found that the mean level of sperm DNA fragmentation in the RCT carrier group (18.0 ± 11.9%) was significantly higher (p=0.0006) than the mean of the control group (7.5 ± 4.3%). There was no correlation observed between sperm DNA fragmentation levels (5.6-38.0%) and the frequency of genetically normal/balanced gametes (34.3-62.4%), sperm seminological quality or revealed reproductive failure. In contrast, a correlation between the frequencies of genetically normal/balanced spermatozoa and of gametes with mature chromatin was observed (CMA3: R=0.4524, p=0.2604; AB: R=0.5238, p=0.1827). A statistically significant increase in the hyperhaploidy level of selected chromosomes in all analyzed RCT carriers was documented but was not correlated to sperm seminology or fertility status. Further evaluation and additional assays toward sperm chromatin quality assessment in RCT carriers is suggested to explain the complexity of genomic structural rearrangements and its possible relevance to reproductive success or failure. PMID:24011192

  10. Chromatin modifications associated with diabetes.

    PubMed

    Keating, Samuel T; El-Osta, Assam

    2012-08-01

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

  11. Electron microscopy and atomic force microscopy studies of chromatin and metaphase chromosome structure.

    PubMed

    Daban, Joan-Ramon

    2011-12-01

    The folding of the chromatin filament and, in particular, the organization of genomic DNA within metaphase chromosomes has attracted the interest of many laboratories during the last five decades. This review discusses our current understanding of chromatin higher-order structure based on results obtained with transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), and different atomic force microscopy (AFM) techniques. Chromatin isolated from different cell types in buffers without cations form extended filaments with nucleosomes visible as separated units. In presence of low concentrations of Mg(2+), chromatin filaments are folded into fibers having a diameter of ? 30 nm. Highly compact fibers were obtained with isolated chromatin fragments in solutions containing 1-2mM Mg(2+). The high density of these fibers suggested that the successive turns of the chromatin filament are interdigitated. Similar results were obtained with reconstituted nucleosome arrays under the same ionic conditions. This led to the proposal of compact interdigitated solenoid models having a helical pitch of 4-5 nm. These findings, together with the observation of columns of stacked nucleosomes in different liquid crystal phases formed by aggregation of nucleosome core particles at high concentration, and different experimental evidences obtained using other approaches, indicate that face-to-face interactions between nucleosomes are very important for the formation of dense chromatin structures. Chromatin fibers were observed in metaphase chromosome preparations in deionized water and in buffers containing EDTA, but chromosomes in presence of the Mg(2+) concentrations found in metaphase (5-22 mM) are very compact, without visible fibers. Moreover, a recent cryo-electron microscopy analysis of vitreous sections of mitotic cells indicated that chromatin has a disordered organization, which does not support the existence of 30-nm fibers in condensed chromosomes. TEM images of partially denatured chromosomes obtained using different procedures that maintain the ionic conditions of metaphase showed that bulk chromatin in chromosomes is organized forming multilayered plate-like structures. The structure and mechanical properties of these plates were studied using cryo-EM, electron tomography, AFM imaging in aqueous media, and AFM-based nanotribology and force spectroscopy. The results obtained indicated that the chromatin filament forms a flexible two-dimensional network, in which DNA is the main component responsible for the mechanical strength observed in friction force measurements. The discovery of this unexpected structure based on a planar geometry has opened completely new possibilities for the understanding of chromatin folding in metaphase chromosomes. It was proposed that chromatids are formed by many stacked thin chromatin plates oriented perpendicular to the chromatid axis. Different experimental evidences indicated that nucleosomes in the plates are irregularly oriented, and that the successive layers are interdigitated (the apparent layer thickness is 5-6 nm), allowing face-to-face interactions between nucleosomes of adjacent layers. The high density of this structure is in agreement with the high concentration of DNA observed in metaphase chromosomes of different species, and the irregular orientation of nucleosomes within the plates make these results compatible with those obtained with mitotic cell cryo-sections. The multilaminar chromatin structure proposed for chromosomes allows an easy explanation of chromosome banding and of the band splitting observed in stretched chromosomes. PMID:21703860

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

    PubMed Central

    Periši?, Ognjen; Collepardo-Guevara, Rosana; Schlick, Tamar

    2010-01-01

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

  13. 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

  14. 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

  15. RNA traffic control of chromatin complexes

    PubMed Central

    Koziol, Magdalena J; Rinn, John L.

    2010-01-01

    It is widely accepted that the genome is regulated by histone modifications that induce epigenetic changes on the genome. However, it is still not understood how ubiquitously expressed chromatin modifying complexes are “guided” to specific genomic sites to induce intricate patterns of epigenetic modifications. Previously believed to represent “genome junk”, it is now becoming increasingly clear that large non-coding RNAs associate with chromatin modifying complexes. Here we explore an intriguing hypothesis that large non-coding RNA molecules might represent a molecular trafficking system that modulate chromatin modifying complexes to establish specific epigenetic landscapes. PMID:20362426

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

    USGS Publications Warehouse

    Jenkins, Jill A.

    2013-01-01

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

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

    PubMed

    Schoborg, Todd; Labrador, Mariano

    2014-11-01

    Of the numerous classes of elements involved in modulating eukaryotic chromosome structure and function, chromatin insulators arguably remain the most poorly understood in their contribution to these processes in vivo. Indeed, our view of chromatin insulators has evolved dramatically since their chromatin boundary and enhancer blocking properties were elucidated roughly a quarter of a century ago as a result of recent genome-wide, high-throughput methods better suited to probing the role of these elements in their native genomic contexts. The overall theme that has emerged from these studies is that chromatin insulators function as general facilitators of higher-order chromatin loop structures that exert both physical and functional constraints on the genome. In this review, we summarize the result of recent work that supports this idea as well as a number of other studies linking these elements to a diverse array of nuclear processes, suggesting that chromatin insulators exert master control over genome organization and behavior. PMID:25012699

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

    PubMed Central

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

    2014-01-01

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

  19. 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 ...

  20. Cell Reports Chromatin Modifications as Determinants

    E-print Network

    Brunet, Anne

    in quiescent stem cells. These findings highlight the importance of chromatin map- ping in understanding unique of distinct types of cells is established at the epigenetic level. The epigenome determines the pattern

  1. Chromatin modifiers: regulators of cellular differentiation

    PubMed Central

    Chen, Taiping; Dent, Sharon Y. R.

    2014-01-01

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

  2. Cellulase Assays

    NASA Astrophysics Data System (ADS)

    Zhang, Y. H. Percival; Hong, Jiong; Ye, Xinhao

    Cellulose is a heterogeneous polysaccharide, and its enzymatic hydrolysis requires endoglucanase, exoglucanase (cellobiohydrolase), and ?-glucosidase to work together. We summarize the most commonly used assays for individual enzymes and cellulase mixture.

  3. Automation of the buccal micronucleus cytome assay using laser scanning cytometry.

    PubMed

    Leifert, Wayne R; François, Maxime; Thomas, Philip; Luther, Ed; Holden, Elena; Fenech, Michael

    2011-01-01

    Laser scanning cytometry (LSC) can be used to quantify the fluorescence intensity or laser light loss (absorbance) of localized molecular targets within nuclear and cytoplasmic structures of cells while maintaining the morphological features of the examined tissue. It was aimed to develop an automated LSC protocol to study cellular and nuclear anomalies and DNA damage events in human buccal mucosal cells. Since the buccal micronucleus cytome assay has been used to measure biomarkers of DNA damage (micronuclei and/or nuclear buds), cytokinesis defects (binucleated cells), proliferative potential (basal cell frequency), and/or cell death (condensed chromatin, karyorrhexis, and pyknotic and karyolytic cells), the following automated LSC protocol describes scoring criteria for these same parameters using an automated imaging LSC. In this automated LSC assay, cells derived from the buccal mucosa were harvested from the inside of patient's mouths using a small-headed toothbrush. The cells were washed to remove any debris and/or bacteria, and a single-cell suspension prepared and applied to a microscope slide using a cytocentrifuge. Cells were fixed and stained with Feulgen and Light Green stain allowing both chromatic and fluorescent analysis to be undertaken simultaneously with the use of an LSC. PMID:21704845

  4. Long-Range Chromatin Interactions in Cells

    Microsoft Academic Search

    Guo Ling Zhou; Li Xin; Pei Liu

    \\u000a Interactions between long-range genetic elements play key roles in regulating gene expression in a spatially and temporally\\u000a restricted manner during differentiation and development in higher eukaryotic cells. With the aid of new technologies for\\u000a analyzing chromatin structural organization, new long-range chromatin interactions have been discovered and interaction networks\\u000a have been proposed. The underlying mechanisms by which these interactions influence gene

  5. 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

  6. 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

  7. Highly Compacted Chromatin Formed in vitro Reflect the Dynamics of Transcription Activation in vivo

    PubMed Central

    Li, Guohong; Margueron, Raphael; Hu, Guobin; Stokes, David; Wang, Yuh-Hwa; Reinberg, Danny

    2013-01-01

    Summary High order chromatin was reconstituted in vitro. This species reflects the criteria associated with transcriptional regulation in vivo. Histone H1 was determinant to formation of condensed structures, with deacetylated histones giving rise to highly compacted chromatin that approximated 30-nm fibers as evidenced by electron microscopy. Using the model PEPCK promoter, we validated the integrity of these templates that were refractory to transcription by attaining transcription through the progressive action of the pertinent factors. The retinoic acid receptor binds to highly compacted chromatin, but the NF1 transcription factor binds only after histone acetylation by p300 and SWI/SNF-mediated nucleosome mobilization, reflecting the in vivo case, as evidenced by ChIP analyses. Mapping studies revealed the same pattern of nucleosomal repositioning on the PEPCK promoter in vitro and in vivo, correlating with NF1 binding and transcription. The reconstitution of such highly compacted “30-nm” chromatin that mimics in vivo characteristics should advance studies of its conversion to a transcriptionally active form, as well as the relevant function(s) of histone posttranslational modifications. PMID:20385088

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  10. Time-lapse dynamics of the mouse oocyte chromatin organisation during meiotic resumption.

    PubMed

    Belli, Martina; Vigone, Giulia; Merico, Valeria; Redi, Carlo Alberto; Garagna, Silvia; Zuccotti, Maurizio

    2014-01-01

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

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

    PubMed Central

    Redi, Carlo Alberto; Zuccotti, Maurizio

    2014-01-01

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

  12. Pin1 promotes histone H1 dephosphorylation and stabilizes its binding to chromatin

    PubMed Central

    Raghuram, Nikhil; Strickfaden, Hilmar; McDonald, Darin; Williams, Kylie; Fang, He; Mizzen, Craig; Hayes, Jeffrey J.; Th’ng, John

    2013-01-01

    Histone H1 plays a crucial role in stabilizing higher order chromatin structure. Transcriptional activation, DNA replication, and chromosome condensation all require changes in chromatin structure and are correlated with the phosphorylation of histone H1. In this study, we describe a novel interaction between Pin1, a phosphorylation-specific prolyl isomerase, and phosphorylated histone H1. A sub-stoichiometric amount of Pin1 stimulated the dephosphorylation of H1 in vitro and modulated the structure of the C-terminal domain of H1 in a phosphorylation-dependent manner. Depletion of Pin1 destabilized H1 binding to chromatin only when Pin1 binding sites on H1 were present. Pin1 recruitment and localized histone H1 phosphorylation were associated with transcriptional activation independent of RNA polymerase II. We thus identify a novel form of histone H1 regulation through phosphorylation-dependent proline isomerization, which has consequences on overall H1 phosphorylation levels and the stability of H1 binding to chromatin. PMID:24100296

  13. Kinase-mediated changes in nucleosome conformation trigger chromatin decondensation via poly(ADP-ribosyl)ation.

    PubMed

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

    2014-03-01

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

  14. Macroautophagy-aided elimination of chromatin

    PubMed Central

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

    2012-01-01

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

  15. Understanding Condensation

    NSDL National Science Digital Library

    Monica Hartman

    2007-12-01

    Monica Hartman, Assistant Director for Science in St. Clair County, Michigan, conducted this research while she was the learning specialist in a small suburban district just outside a large Midwestern city. While teaching full time in this district she was also completing her doctoral program in education at the University of Michigan. In this chapter, she tells the story of a "science talk" about condensation among fifth graders. She acted as a source and facilitator of change as she and the fifth-grade teacher worked collaboratively to help students share responsibility for their own learning. She describes their continual assessment of student understanding that occurred as their students struggled to explain observations and as they, the teachers, carefully resisted the temptation to end the struggle by saying "that's right!"

  16. Sequential changes in macrostructural state and RNA-synthesizing activity of nucleolar and extranucleolar chromatin of rat liver cells during induction of DNA synthesis

    SciTech Connect

    Shevchenko, N.A.; Boikov, P.Ya.; Todorov, I.N.

    1986-04-10

    The dynamics of the structural changes in the RNA-synthesizing activity of rat liver cell chromatin was studied under conditions of sharp variations in the rate of protein synthesis. Inhibition of protein synthesis by the administration to animals of a single dose of cycloheximide (0.3 mg per 100 g weight) increased the total degree of condensation of the chromatin. Against this background the sequential activation of RNA polymerases I and II correlates with decondensation of the chromatin. In the 6th-12th h after the injection of cycloheximide a chromatin fraction enriched with RNA polymerase I and a fraction rich in RNA polymerase II were isolated from liver cell nuclei.

  17. Cryopreservation of human spermatozoa decreases the number of motile normal spermatozoa, induces nuclear vacuolization and chromatin decondensation.

    PubMed

    Boitrelle, Florence; Albert, Martine; Theillac, Claire; Ferfouri, Fatma; Bergere, Marianne; Vialard, François; Wainer, Robert; Bailly, Marc; Selva, Jacqueline

    2012-01-01

    Even though cryopreservation of human spermatozoa is known to alter sperm motility and viability, it may also induce nuclear damages. The present study set out to determine whether or not cryopreservation alters motile sperm morphology under high magnification and/or is associated with chromatin decondensation. For 25 infertile men, we used high-magnification microscopy to determine the proportions of various types of motile spermatozoa before and after freezing-thawing: morphometrically normal spermatozoa with no vacuole (grade I), ? 2 small vacuoles (grade II), at least 1 large vacuole or >2 small vacuoles (grade III), and morphometrically abnormal spermatozoa (grade IV). The spermatozoa's chromatin condensation and viability were also assessed before and after freezing-thawing. Cryopreservation induced sperm nuclear vacuolization. It decreased the proportion of grade I + II spermatozoa (P < .001). It induced a decrease in the sperm viability rate (P < .001) and increased the proportion of sperm with noncondensed chromatin (P < .001). The latter parameter was strongly correlated with sperm viability (r = 0.71; P < .001). However, even motile sperm presented a failure of chromatin condensation after freezing-thawing, because the proportion of sperm with noncondensed chromatin was correlated with high-magnification morphology (r = -0.49 and 0.49 for the proportions of grade I + II and grades III + IV, respectively; P < .001). Cryopreservation alters the organelle morphology of motile human spermatozoa and induces sperm chromatin decondensation. High-magnification microscopy may be useful for evaluating frozen-thawed spermatozoa before use in assisted reproductive technology procedures (such as intrauterine insemination, in vitro fertilization, and intracytoplasmic sperm injection) and for performing research on cryopreservation methods. If frozen-thawed sperm is to be used for intracytoplasmic sperm injection, morphological selection under high magnification may be of particular value. PMID:22700764

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

    ERIC Educational Resources Information Center

    Eirin-Lopez, Jose M.

    2013-01-01

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

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

    SciTech Connect

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

    2010-11-11

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

  20. Acetylation of histone H4 and its role in chromatin structure and function.

    PubMed

    Chahal, S S; Matthews, H R; Bradbury, E M

    1980-09-01

    Histone h4 is a highly conserved structural component of the nucleosome subunit of chromatin. The activation of chromatin is accompanied by changes in structure which may be caused by histone modification or by interactions of specific non-histone proteins, or both. Histone H4 can be modified by acetylation and this modification has been correlated with chromosome assembly and with transcription. We have now tested these correlations by studying H4 acetate content as a function of the cell cycle using the naturally synchronous cell cycle in Physarum polycephalum. The results show two clear correlations: (1) tetra-acetylated H4 correlates with transcription; (2) highly acetylated H4 (2 to 4 acetates per molecule) is inversely correlated with H1 phosphorylation and initiation of chromosome condensation in prophase. The results are consistent with turnover of di-acetylated H4 during chromosome assembly in S phase. PMID:7412879

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

    PubMed Central

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

    2011-01-01

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

  2. Extraction of Histone H1 and Decondensation of Nuclear Chromatin with Various Mg-Dependent Organization Levels under Treatment with Polyglutamic Acid and Distamycin.

    PubMed

    Prusov, A N; Smirnova, T A; Kolomijtseva, G Ya

    2015-03-01

    Chromatin in rat liver nuclei under conditions of low ionic strength (20-25 mM) and [Mg2+] from 2 to 5 mM has a condensed structure (100-200 nm globules) and gives the same CD signal (320-340 nm) at interaction with the antibiotic distamycin A (DM). Reducing [Mg2+] to 1 mM leads to chromatin decondensation to 30 nm structures and increases the CD signal. Poly-L-glutamic acid (PG) at weight ratio PG/DNA = 6 and in the presence of 5 mM Mg2+ extracts only about 1/8 of nuclear histone H1, preserving a condensed chromatin structure. Removal of about 1/4 of H1 at 3 mM Mg2+ leads to chromatin decondensation to 30 nm fibrils. Extraction of about half of histone H1 at [Mg2+] ? 2 mM results in chromatin refolding to nucleosome fibrils. PG-decondensation leads to a significant increase in the CD signal. The main H1 extraction occurs in 1-2 min, but at all Mg2+ concentrations the more slowly PG extracted fraction is found comprising 5-7% of nuclear H1. About 25% of leaving nuclear H1 can be extracted by PG in the presence of saturating DM concentration (molar DM/DNA = 0.1). H1 release depends significantly on the PG concentration. However, even at high weight ratio PG/DNA = 30 and DM/DNA = 0.1, about 5-10% of histone H1 remained in the nuclei. Decondensation of chromatin in the nucleus is not always proportional to the yield of extracted histone H1 and is weakened in the presence of positively charged DM or high concentrations of PG. Our results show that the interaction of DM with chromatin depends primarily on chromatin packaging, while PG extraction depends on [Mg2+] supporting this packaging. PMID:25761689

  3. Effects of acrylamide on sperm parameters, chromatin quality, and the level of blood testosterone in mice

    PubMed Central

    Pourentezari, Majid; Talebi, Alireza; Abbasi, Abulghasem; Khalili, Mohammad Ali; Mangoli, Esmat; Anvari, Morteza

    2014-01-01

    Background: Acrylamide (AA) is an important industrial chemical primarily. AA is also found in carbohydrate-rich foods that are prepared at high temperatures, such as French fries and potato chips. It is demonstrated that AA is a carcinogen and reproductive toxin and has ability to induce sperm damage. Objective: The aim of this study was to observe the effects of AA on sperm parameters and evaluation of sperm chromatin quality and testosterone hormone in mice. Materials and Methods: Totally, 16 adult male mice were divided into two groups. Mice of group A fed on basal diet; group B received basal diet and AA (10 mg/kg, water solution) for 35 days. The right cauda epididymis was incised and then placed in Ham’s F10 culture media at 37oC for 15 min. Released spermatozoa were used to analyze count, motility, morphology and viability. To determine the sperm DNA integrity and chromatin condensation, the cytochemical techniques including Aniline blue, Acridine orange and Chromomycin A3 staining were used. Results: AA-treated mice had poor parameters in comparison with control animals. In sperm chromatin assessments, except TB (p=0.16), significant differences were found in all of the tests between two groups. It was also seen a significant decrease in concentration of blood testosterone in AA-treated animals when compared to controls (p<0.001). Conclusion: According to our results, AA can affect sperm parameters as well as sperm chromatin condensation and DNA integrity in mice. These abnormalities may be related to the reduction in blood testosterone. PMID:25031578

  4. Activation of nuclear polypeptide synthesis in rat liver cells in the case of inhibition of template synthesis of proteins. Contribution of nuclear polypeptide synthesis to the change in the chromatin structure

    SciTech Connect

    Gutnikova, M.N.; Shevchenko, N.A.; Boikov, P.Ya.; Mitrokhin, Yu.I.; Todorov, I.N.

    1986-06-10

    The correlation between the activity of nuclear polypeptide synthesis (NPS) and template synthesis of proteins in rat liver cells was investigated. It was established that the NPS is activated under conditions of inhibition of protein synthesis in the cytoplasm. It was shown on model experiments that the systems of NPS and ADP-ribosylation of nuclear proteins compete in determining the general structure of chromatin: ADP-ribosylation promotes condensation, while NPS promotes decondensation of the chromatin.

  5. PROTOCOLS: Chromatin Immunoprecipitation from Arabidopsis Tissues

    PubMed Central

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

    2014-01-01

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

  6. Atomic force microscopy of mammalian sperm chromatin.

    PubMed

    Allen, M J; Lee, C; Lee, J D; Pogany, G C; Balooch, M; Siekhaus, W J; Balhorn, R

    1993-11-01

    We have used the atomic force microscope (AFM) to image the surfaces of intact bull, mouse and rat sperm chromatin and partially decondensed mouse sperm chromatin attached to coverglass. High resolution AFM imaging was performed in air and saline using uncoated, unfixed and unstained chromatin. Images of the surfaces of intact chromatin from all three species and of an AFM-dissected bull sperm nucleus have revealed that the DNA is organized into large nodular subunits, which vary in diameter between 50 and 100 nm. Other images of partially decondensed mouse sperm chromatin show that the nodules are arranged along thick fibers that loop out away from the nucleus upon decondensation. These fibers appear to stretch or unravel, generating narrow smooth fibers with thicknesses equivalent to a single DNA-protamine complex. High resolution AFM images of the nodular subunits suggest that they are discrete, ellipsoid-shaped DNA packaging units possibly only one level of packaging above the protamine-DNA complex. PMID:8306824

  7. Transcription elongation through a chromatin template.

    PubMed

    Lavelle, Christophe

    2007-04-01

    DNA transaction events occurring during cell life (replication, transcription, recombination, repair, cell division) are always linked to severe changes in the topological state of the double helix. However, since naked DNA almost does not exist in eukaryote nucleus but rather interacts with various proteins, including ubiquitous histones, these topological changes happen in a chromatin context. This review focuses on the role of chromatin fiber structure and dynamics in the regulation of transcription, with an almost exclusive emphasis on the elongation step. Beside a brief overview of our knowledge about transcribed chromatin, we will see how recent mechanistic and biochemical studies give us new insights into the way cell could modulate DNA supercoiling and chromatin conformational dynamics. The participation of topoisomerases in this complex ballet is discussed, since recent data suggest that their role could be closely related to the precise chromatin structure. Lastly, some future prospects to carry on are proposed, hoping this review will help in stimulating discussions and further investigations in the field. PMID:17070642

  8. Computational modeling of the chromatin fiber.

    PubMed

    Langowski, Jörg; Heermann, Dieter W

    2007-10-01

    The packing of the genomic DNA in the living cell is essential for its biological function. While individual aspects of the genome architecture, such as DNA and nucleosome structure or the arrangement of chromosome territories are well studied, much information is missing for a unified description of cellular DNA at all its structural levels. Computer modeling can contribute to such a description. We present here some typical approaches to models of the chromatin fiber, including different amounts of detail in the description of the local nucleosome structure. The main results from our simulations are that the physical properties of the chromatin fiber can be well described by a simplified model consisting of cylinder-like nucleosomes connected by flexible DNA segments, with a geometry determined by the bending and twisting angles between nucleosomes. Randomness in the local geometry - such as random absence of linker histone H1 - leads to a dramatic increase in the chromatin fiber flexibility. Furthermore, we show that chromatin is much more flexible to bending than to stretching, and that the structure of the chromatin fiber favors the formation of sharp bends. PMID:17936653

  9. Condensation model for the ESBWR passive condensers

    SciTech Connect

    Revankar, S. T. [Pohang Univ. of Science and Technology, 400 Central Drive, West Lafayette, IN 47906 (United States); Zhou, W.; Wolf, B.; Oh, S. [Purdue Univ., West Lafayette, IN 47906 (United States)

    2012-07-01

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

  10. Aberrant Chromatin Remodeling by Retinoic Acid Receptor ? Fusion Proteins Assessed at the Single-Cell Level

    PubMed Central

    Qiu, Jihui; Huang, Ying; Chen, Guoqiang; Chen, Zhu

    2007-01-01

    Acute promyelocytic leukemia (APL) is characterized by specific chromosomal translocations, which generate fusion proteins such as promyelocytic leukemia (PML)-retinoic acid receptor (RAR)? and promyelocytic leukemia zinc finger (PLZF)-RAR? (X-RAR?). In this study, we have applied lac operator array systems to study the effects of X-RAR? versus wild-type RAR? on large-scale chromatin structure. The targeting of these enhanced cyan fluorescent protein-lac repressor-tagged RAR?-containing proteins to the gene-amplification chromosomal region by lac operator repeats led to local chromatin condensation, recruitment of nuclear receptor corepressor, and histone deacetylase complex. The addition of retinoic acid (RA) induced large-scale chromatin decondensation in cells expressing RAR?; however, cells expressing X-RAR?, especially PML-RAR?, demonstrated insensitive response to this effect of all-trans retinoic acid (ATRA). Although we did not reveal differences in RA-dependent colocalization of either silencing mediator for retinoid and thyroid or steroid receptor coactivator (SRC)-1 with RAR? versus X-RAR?, the hormone-independent association between SRC-1 and X-RAR? on the array has been identified. Rather, compared with cells expressing RAR?, fluorescence recovery after photobleaching of live transfected cells, demonstrated decreased mobility of SRC-1 on the X-RAR?–bound chromatin. Thus, the impaired ability of APL fusion proteins to activate gene transcription in response to ATRA corresponds to their reduced ability to remodel chromatin, which may link to their ability to impair the mobility of key nuclear receptor coregulators. PMID:17671166

  11. The effects of age on DNA fragmentation, chromatin packaging and conventional semen parameters in spermatozoa of oligoasthenoteratozoospermic patients

    Microsoft Academic Search

    Konstantina Plastira; Pavlos Msaouel; Roxani Angelopoulou; Kyriaki Zanioti; Aris Plastiras; Alexios Pothos; Stamatis Bolaris; Nikolaos Paparisteidis; Dimitris Mantas

    2007-01-01

    Purpose  To investigate the effects of male ageing on DNA fragmentation and chromatin packaging in the spermatozoa of oligoasthenoteratozoospermic\\u000a (OAT) patients.\\u000a \\u000a \\u000a \\u000a Methods  Sixty-one OAT patients and 49 men with proven fertility (controls) were included in the present study. DNA fragmentation was\\u000a detected by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labelling (TUNEL) assay, while chromatin packaging\\u000a was assessed by chromomycin A3 (CMA3) staining.\\u000a \\u000a \\u000a \\u000a Results  In

  12. Doxorubicin, DNA torsion, and chromatin dynamics

    PubMed Central

    Yang, Fan; Teves, Sheila S.; Kemp, Christopher J.; Henikoff, Steven

    2014-01-01

    Doxorubicin is one of the most important anti-cancer chemotherapeutic drugs, being widely used for the treatment of solid tumors and acute leukemias. The action of doxorubicin and other anthracycline drugs has been intensively investigated during the last several decades, but the mechanisms that have been proposed for cell killing remain disparate and controversial. In this review, we examine the proposed models for doxorubicin action from the perspective of the chromatin landscape, which is altered in many types of cancer due to recurrent mutations in chromatin modifiers. We highlight recent evidence for effects of anthracyclines on DNA torsion and chromatin dynamics that may underlie basic mechanisms of doxorubicin-mediated cell death and suggest new therapeutic strategies for cancer treatment. PMID:24361676

  13. 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

  14. Classifying leukemia types with chromatin conformation data

    PubMed Central

    2014-01-01

    Background Although genetic or epigenetic alterations have been shown to affect the three-dimensional organization of genomes, the utility of chromatin conformation in the classification of human disease has never been addressed. Results Here, we explore whether chromatin conformation can be used to classify human leukemia. We map the conformation of the HOXA gene cluster in a panel of cell lines with 5C chromosome conformation capture technology, and use the data to train and test a support vector machine classifier named 3D-SP. We show that 3D-SP is able to accurately distinguish leukemias expressing MLL-fusion proteins from those expressing only wild-type MLL, and that it can also classify leukemia subtypes according to MLL fusion partner, based solely on 5C data. Conclusions Our study provides the first proof-of-principle demonstration that chromatin conformation contains the information value necessary for classification of leukemia subtypes. PMID:24995990

  15. Chromatin Remodeling, DNA Damage Repair and Aging

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  17. Hexosaminidase assays

    Microsoft Academic Search

    Michaela Wendeler; Konrad Sandhoff

    2009-01-01

    ?-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal ?-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the\\u000a diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited ?-hexosaminidase deficiency.\\u000a More recently, aberrant hexosaminidase levels have also been found to be associated with a variety

  18. Chromatin Structure Regulates Gene W. Jason Cummings1

    E-print Network

    Maizels, Nancy

    -directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin. Citation: Cummings WJ, Yabuki M, Ordinario EC, Bednarski DW, Quay S, et al. (2007) Chromatin structure

  19. Chromatin as an expansive canvas for chemical biology

    PubMed Central

    Fierz, Beat; Muir, Tom W

    2014-01-01

    Chromatin is extensively chemically modified and thereby acts as a dynamic signaling platform controlling gene function. Chromatin regulation is integral to cell differentiation, lineage commitment and organism development, whereas chromatin dysregulation can lead to age-related and neurodegenerative disorders as well as cancer. Investigating chromatin biology presents a unique challenge, as the issue spans many disciplines, including cell and systems biology, biochemistry and molecular biophysics. In recent years, the application of chemical biology methods for investigating chromatin processes has gained considerable traction. Indeed, chemical biologists now have at their disposal powerful chemical tools that allow chromatin biology to be scrutinized at the level of the cell all the way down to the single chromatin fiber. Here we present recent examples of how this rapidly expanding palette of chemical tools is being used to paint a detailed picture of chromatin function in organism development and disease. PMID:22510649

  20. Biased chromatin signatures around polyadenylation sites and exons

    E-print Network

    Spies, Noah Walter Benjamin

    Core RNA-processing reactions in eukaryotic cells occur cotranscriptionally in a chromatin context, but the relationship between chromatin structure and pre-mRNA processing is poorly understood. We observed strong nucleosome ...

  1. UT MD Anderson scientists discover secret life of chromatin:

    Cancer.gov

    Chromatin--the intertwined histone proteins and DNA that make up chromosomes--constantly receives messages that pour in from a cell’s intricate signaling networks... But chromatin also talks back, scientists at The University of Texas M.D. Anderson Cancer Center report today in the journal Cell, issuing orders affecting a protein that has nothing to do with chromatin’s central role in gene transcription--the first step in protein formation.

  2. 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

  3. Nucleotide excision repair in chromatin and the right of entry

    Microsoft Academic Search

    Feng Gong; YoungHo Kwon; Michael J. Smerdon

    2005-01-01

    DNA is packaged with histones and other accessory proteins into chromatin in eukaryotic cells. It is well established that the assembly of DNA into chromatin affects induction of DNA damage as well as repair of the damage. How the DNA repair machinery detects a lesion and ‘fixes it’ in chromatin has been an intriguing question since the dawn of understanding

  4. The Chromatin Remodeler SPLAYED Regulates Specific Stress Signaling Pathways

    Microsoft Academic Search

    Justin W. Walley; Heather C. Rowe; Yanmei Xiao; E. Wassim Chehab; Daniel J. Kliebenstein; Doris Wagner; Katayoon Dehesh

    2008-01-01

    Organisms are continuously exposed to a myriad of environmental stresses. Central to an organism's survival is the ability to mount a robust transcriptional response to the imposed stress. An emerging mechanism of transcriptional control involves dynamic changes in chromatin structure. Alterations in chromatin structure are brought about by a number of different mechanisms, including chromatin modifications, which covalently modify histone

  5. Chromatin regulation: how complex does it get?

    PubMed

    Meier, Karin; Brehm, Alexander

    2014-01-01

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

  6. Factors affecting chromatin stability of bovine spermatozoa

    Microsoft Academic Search

    T. A. A. Khalifa; C. A. Rekkas; A. G. Lymberopoulos; A. Sioga; I. Dimitriadis; Th. Papanikolaou

    2008-01-01

    The structural stability of transcriptionally inert paternal chromatin is of vital importance for the fertilization process and early embryonic development. Accordingly, a series of eight experiments were conducted during a 7-month period to investigate: (1) effects of bull breed, individuality, successive ejaculations, semen quality characteristics (SQC), semen dilution rates and hypothermic storage of semen in a Tris-egg yolk extender on

  7. Transcriptional transgene silencing and chromatin components.

    PubMed

    Meyer, P

    2000-06-01

    Contrary to simplistic views that have long prevailed in genetics textbooks, gene transcription in higher organisms cannot be fully understood by analysing binding of transcription factors to DNA target sites within the promoter regions, just as it would be inappropriate to picture the genetic information within a nucleus as a simple string of DNA. Instead, DNA is embedded in a highly complex chromatin structure that controls the location and accessibility of individual genetic regions in a way we are still far from understanding in detail. What has become obvious, mainly due to ground-breaking research in yeast and animal systems, is that the packaging of certain genes into a chromosomal matrix is regulated via sophisticated chromatin remodelling mechanisms that define whether and when a gene becomes accessible to the transcription machinery. In plants, especially the analysis of transgenes and transposable elements has reminded us of the presence of epigenetic control mechanisms, which can affect the reliable expression of transgenes. There is increasing evidence that chromatin components play an important part in plant epigenetics. The purpose of this review is to describe the main general principles of chromatin remodelling as they have been elucidated in non-plant systems and to discuss their relevance for the control of gene expression in plants. PMID:10999406

  8. Recruitment of Phosphorylated Chromatin Assembly Factor 1to Chromatin after UV Irradiation of Human Cells

    Microsoft Academic Search

    Emmanuelle Martini; Danièle M. J. Roche; Kathrin Marheineke; Alain Verreault; Geneviève Almouzni

    1998-01-01

    The subcellular distribution and posttransla- tional modification of human chromatin assembly fac- tor 1 (CAF-1) have been investigated after UV irradia- tion of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-asso- ciated fraction. This fraction is most abundant during S phase in

  9. Factors affecting chromatin stability of bovine spermatozoa.

    PubMed

    Khalifa, T A A; Rekkas, C A; Lymberopoulos, A G; Sioga, A; Dimitriadis, I; Papanikolaou, Th

    2008-03-01

    The structural stability of transcriptionally inert paternal chromatin is of vital importance for the fertilization process and early embryonic development. Accordingly, a series of eight experiments were conducted during a 7-month period to investigate: (1) effects of bull breed, individuality, successive ejaculations, semen quality characteristics (SQC), semen dilution rates and hypothermic storage of semen in a Tris-egg yolk extender on incidence of sperm nuclear chromatin instability (NCI), and (2) effects of the interaction between variation of NCI within a frozen ejaculate and variation of oocytes quality due to maturation time and/or season on the efficiency of in vitro embryo production (IVEP). Semen samples were collected once a week from six bulls using an AV and only ejaculates (n=220) of >0.30x10(9) sperm/ml and >or=60% motility were used. NCI was measured by: (1) detection of lysine-rich histones in sperm chromatin using aniline blue staining, (2) sperm susceptibility to acid-induced nuclear DNA denaturation in situ using acridine orange test, and (3) sperm susceptibility to nuclear chromatin decondensation (NCD). Bovine oocytes (n=695) were matured in vitro for 18 or 24 h, fertilized after sperm selection through a swim-up procedure and cultured for 72 h. The results showed that the 2nd ejaculates were superior to the 1st ones with respect to chromatin stability. Dilution of semen to 49.67+/-8.56x10(6) sperm/ml (1:19) decreased resistance of sperm to NCD. Cooling of semen had no significant effect on chromatin stability. Cryopreservation of semen augmented sperm vulnerability to DNA denaturation. Improvement of SQC (semen volume, sperm motility, velocity, viability and morphological normalcy) was generally concomitant with increase of sperm resistance to NCI. While Blonde d'Aquitaine bulls had a resistance to NCD higher than Limousine bulls in fresh semen, the former showed a greater susceptibility to DNA denaturation than the latter in cooled semen. Individuality significantly influenced NCI. The variability of NCI within a frozen ejaculate affected efficiency of IVEP. Significant negative correlations were observed between incidence of NCI and both fertilization rate and developmental capacity of embryos after maturation of oocytes for 18 h. The significant variation in IVEP traits due to season was independent of the effect of sperm chromatin instability. PMID:17398042

  10. The PHD and chromo domains regulate the ATPase activity of the human chromatin remodeler CHD4.

    PubMed

    Watson, Aleksandra A; Mahajan, Pravin; Mertens, Haydyn D T; Deery, Michael J; Zhang, Wenchao; Pham, Peter; Du, Xiuxia; Bartke, Till; Zhang, Wei; Edlich, Christian; Berridge, Georgina; Chen, Yun; Burgess-Brown, Nicola A; Kouzarides, Tony; Wiechens, Nicola; Owen-Hughes, Tom; Svergun, Dmitri I; Gileadi, Opher; Laue, Ernest D

    2012-09-01

    The NuRD (nucleosome remodeling and deacetylase) complex serves as a crucial epigenetic regulator of cell differentiation, proliferation, and hematopoietic development by coupling the deacetylation and demethylation of histones, nucleosome mobilization, and the recruitment of transcription factors. The core nucleosome remodeling function of the mammalian NuRD complex is executed by the helicase-domain-containing ATPase CHD4 (Mi-2?) subunit, which also contains N-terminal plant homeodomain (PHD) and chromo domains. The mode of regulation of chromatin remodeling by CHD4 is not well understood, nor is the role of its PHD and chromo domains. Here, we use small-angle X-ray scattering, nucleosome binding ATPase and remodeling assays, limited proteolysis, cross-linking, and tandem mass spectrometry to propose a three-dimensional structural model describing the overall shape and domain interactions of CHD4 and discuss the relevance of these for regulating the remodeling of chromatin by the NuRD complex. PMID:22575888

  11. Integrative analysis of chromatin states in Arabidopsis identified potential regulatory mechanisms for natural antisense transcript production.

    PubMed

    Luo, Chongyuan; Sidote, David J; Zhang, Yi; Kerstetter, Randall A; Michael, Todd P; Lam, Eric

    2012-09-11

    Genome-wide analyses of epigenomic and transcriptomic profiles provide extensive resources for discovering epigenetic regulatory mechanisms. However, the construction of functionally relevant hypotheses from correlative patterns and the rigorous testing of these hypotheses may be challenging. We combined bioinformatics-driven hypothesis building with mutant analyses to identify potential epigenetic mechanisms using the model plant Arabidopsis thaliana. Genome-wide maps of nine histone modifications produced by ChIP-seq were used together with a strand-specific RNA-seq dataset to profile the epigenome and transcriptome of Arabidopsis. Combinatorial chromatin patterns were described by 42 major chromatin states with selected states validated using the re-ChIP assay. The functional relevance of chromatin modifications was analyzed using the ANchored CORrelative Pattern (ANCORP) method and a newly developed state-specific effects analysis (SSEA) method, which interrogates individual chromatin marks in the context of combinatorial chromatin states. Based on results from these approaches, we propose the hypothesis that cytosine methylation (5mC) and histone methylation H3K36me may synergistically repress production of natural antisense transcripts (NATs) in the context of actively expressed genes. Mutant analyses supported this proposed model at a significant proportion of the tested loci. We further identified polymerase-associated factor as a potential repressor for NAT abundance. Although the majority of tested NATs were found to localize to the nucleus, we also found evidence for cytoplasmically partitioned NATs. The significance of the subcellular localization of NATs and their biological functions remain to be defined. PMID:22962860

  12. Sperm count and chromatin structure in men exposed to inorganic lead: lowest adverse effect levels

    PubMed Central

    Bonde, J; Joffe, M; Apostoli, P; Dale, A; Kiss, P; Spano, M; Caruso, F; Giwercman, A; Bisanti, L; Porru, S; Vanhoorne, M; Comhaire, F; Zschiesche, W

    2002-01-01

    Objectives: To obtain knowledge on male reproductive toxicity of inorganic lead at current European exposure levels and to establish lowest adverse effect levels, if any. Methods: A cross sectional survey of the semen of 503 men employed by 10 companies was conducted in the United Kingdom, Italy, and Belgium. The mean blood lead concentration was 31.0 µg/dl (range 4.6–64.5) in 362 workers exposed to lead and 4.4 µg/dl (range below the detection limit of 19.8) in 141 reference workers. Semen volume and sperm concentration were determined in a fresh semen sample according to an agreed protocol subject to quality assurance. The sperm chromatin structure assay (SCSA) was performed at a centralised laboratory. Extraneous determinants including centre, period of sexual abstinence, and age were taken into account in the statistical analysis. If appropriate, possible thresholds were examined by iterative threshold slope linear regression. Results: The median sperm concentration was reduced by 49% in men with blood lead concentration above 50 µg/dl. There was no indication of a linear trend of lower sperm concentration with increasing blood lead values, but threshold slope least square regression identified a blood lead concentration of 44 µg/dl (ß=-0.037, F=4.35, p=0.038) as a likely threshold. Abnormal sperm chromatin structure was not related to blood lead concentration, but some indications of deterioration of sperm chromatin was found in men with the highest concentrations of lead within spermatozoa. Biological monitoring data did not indicate long term effects of lead on semen quantity or sperm chromatin. Conclusion: Adverse effects of lead on sperm concentration and susceptibility to acid induced denaturation of sperm chromatin are unlikely at blood lead concentrations below 45 µg/dl. Effects of low level exposure to lead on other measures of testicular function cannot be ruled out. PMID:11934950

  13. 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

  14. Involvement of the SATB1/F-actin complex in chromatin reorganization during active cell death.

    PubMed

    Grzanka, Dariusz; Gagat, Maciej; Izdebska, Magdalena

    2014-06-01

    Over the past years, confirmations on the presence of actin and/or its polymerized form, F-actin, in the cell nucleus are progressively accumulating. Nevertheless, the function and localization of F-actin in the nucleus is still not fully characterized. Thus, the aim of the present study was to evaluate the association between F-actin and sequence-binding protein 1 (SATB1) and their involvement in chromatin remodeling associated with active cell death. Both SATB1 and F-actin were colocalized in the transcriptional active regions of the cell nucleus and a functional interaction was observed between SATB1 and higher-organized nuclear F-actin structures at the border between condensed and decondensed chromatin. These results extend the knowledge on the role of SATB1 and nuclear F-actin in three-dimensional chromatin organization and their functions during active cell death. Additionally, this study opens the discussion on the involvement of the SATB1/F-actin functional complex in active cell death; further studies are required to fully elucidate these issues. PMID:24676287

  15. Involvement of the SATB1/F-actin complex in chromatin reorganization during active cell death

    PubMed Central

    GRZANKA, DARIUSZ; GAGAT, MACIEJ; IZDEBSKA, MAGDALENA

    2014-01-01

    Over the past years, confirmations on the presence of actin and/or its polymerized form, F-actin, in the cell nucleus are progressively accumulating. Nevertheless, the function and localization of F-actin in the nucleus is still not fully characterized. Thus, the aim of the present study was to evaluate the association between F-actin and sequence-binding protein 1 (SATB1) and their involvement in chromatin remodeling associated with active cell death. Both SATB1 and F-actin were colocalized in the transcriptional active regions of the cell nucleus and a functional interaction was observed between SATB1 and higher-organized nuclear F-actin structures at the border between condensed and decondensed chromatin. These results extend the knowledge on the role of SATB1 and nuclear F-actin in three-dimensional chromatin organization and their functions during active cell death. Additionally, this study opens the discussion on the involvement of the SATB1/F-actin functional complex in active cell death; further studies are required to fully elucidate these issues. PMID:24676287

  16. Condensate Mixtures and Tunneling

    SciTech Connect

    Timmermans, E.

    1998-09-14

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

  17. Chromatin signature discovery via histone modification profile alignments.

    PubMed

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

    2012-11-01

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

  18. 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

  19. Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

    SciTech Connect

    Sigala, Barbara [Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT (United Kingdom); Edwards, Mina [Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT (United Kingdom); Puri, Teena [Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT (United Kingdom); Tsaneva, Irina R. [Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT (United Kingdom)]. E-mail: tsaneva@biochem.ucl.ac.uk

    2005-11-01

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

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

    PubMed Central

    2014-01-01

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

  1. Chromatin dynamics in DNA double-strand break repair

    PubMed Central

    Shi, Lei; Oberdoerffer, Philipp

    2012-01-01

    DNA double-strand breaks (DSBs) occur in the context of a highly organized chromatin environment and are, thus, a significant threat to the epigenomic integrity of eukaryotic cells. Changes in break-proximal chromatin structure are thought to be a prerequisite for efficient DNA repair and may help protect the structural integrity of the nucleus. Unlike most bona fide DNA repair factors, chromatin influences the repair process at several levels: the existing chromatin context at the site of damage directly affects the access and kinetics of the repair machinery; DSB induced chromatin modifications influence the choice of repair factors, thereby modulating repair outcome; lastly, DNA damage can have a significant impact on chromatin beyond the site of damage. We will discuss recent findings that highlight both the complexity and importance of dynamic and tightly orchestrated chromatin reorganization to ensure efficient DSB repair and nuclear integrity. PMID:22285574

  2. The molecular basis of chromatin dynamics during nucleotide excision repair.

    PubMed

    Zhang, Ling; Jones, Kristi; Gong, Feng

    2009-02-01

    The assembly of DNA into chromatin in eukaryotic cells affects all DNA-related cellular activities, such as replication, transcription, recombination, and repair. Rearrangement of chromatin structure during nucleotide excision repair (NER) was discovered more than 2 decades ago. However, the molecular basis of chromatin dynamics during NER remains undefined. Pioneering studies in the field of gene transcription have shown that ATP-dependent chromatin-remodeling complexes and histone-modifying enzymes play a critical role in chromatin dynamics during transcription. Similarly, recent studies have demonstrated that the SWI/SNF chromatin-remodeling complex facilitates NER both in vitro and in vivo. Additionally, histone acetylation has also been linked to the NER of ultraviolet light damage. In this article, we will discuss the role of these identified chromatin-modifying activities in NER. PMID:19234540

  3. Steam Condensation Induced Waterhammer

    E-print Network

    Kirsner, W.

    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..., TX, April 5-6, 2000 Condensation Induced Waterhammer A condensation induced water hammer is a rapid condensation event. It could also be aptly termed a "rapid steam bubble collapse". It occurs when a steam pocket becomes totally entrapped in sub...

  4. in Condensed Matter Physics

    E-print Network

    van der Torre, Leon

    Master in Condensed Matter Physics ­ Master académique #12;2 #12;3 Students at the University. Condensed matter physics is about explaining and predicting the relationship between the atomic, and broad education in the field of condensed matter physics · introduce you to current research topics

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

    NASA Astrophysics Data System (ADS)

    Krishnan, Rajagopal; Sandhu, Tejdev; Nordlund, Thomas

    2004-11-01

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

  6. Regulation of chromatin by histone modifications

    PubMed Central

    Bannister, Andrew J; Kouzarides, Tony

    2011-01-01

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

  7. 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

  8. Effects of chromatin decondensation on alternative NHEJ.

    PubMed

    Moscariello, Mario; Iliakis, George

    2013-11-01

    In cells of higher eukaryotes, repair of DNA double strand breaks (DSBs) utilizes different forms of potentially error-prone non-homologous end joining (NHEJ): canonical DNA-PK-dependent (C-NHEJ) and alternative backup pathways (A-NHEJ). In contrast to C-NHEJ, A-NHEJ shows pronounced efficiency fluctuations throughout the cell cycle and is severely compromised as cells cease proliferating and enter the plateau phase (Windhofer et al., 2007 [23]). The molecular mechanisms underpinning this response remain unknown but changes in chromatin structure are prime candidate-A-NHEJ-modulators. Since parameters beyond chromatin acetylation appear to determine A-NHEJ efficiency (Manova et al., 2012 [42,76]), we study here the role of chromatin decondensation mediated either by treatment with 5'-aza-2'-deoxycytidine (AzadC) or growth in hypotonic conditions, on A-NHEJ. We report that both treatments have no detectable effect on C-NHEJ but provoke, specifically for A-NHEJ, cell-growth-dependent effects. These results uncover for the first time a link between A-NHEJ and chromatin organization and provide means for understanding the regulatory mechanisms underpinning the growth-state dependency of A-NHEJ. A-NHEJ is implicated in the formation of chromosomal translocations and in chromosome fusions that underlie genomic instability and carcinogenesis. The observations reported here may therefore contribute to the development of drug-based A-NHEJ suppression-strategies aiming at optimizing cancer treatment outcomes and possibly also at suppressing carcinogenesis. PMID:24051048

  9. The polymorphisms of the chromatin fiber

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  10. SWI\\/SNF chromatin remodeling and cancer

    Microsoft Academic Search

    Agnès Klochendler-Yeivin; Christian Muchardt; Moshe Yaniv

    2002-01-01

    The SWI\\/SNF complex contributes to the regulation of gene expression by altering the chromatin structure. Depending on the context, it can be involved in either transcriptional activation or repression. Growing genetic and molecular evidence indicate that subunits of the SWI\\/SNF complex act as tumor suppressors in human and mice. Results from biochemical and transfection studies suggest also that SWI\\/SNF participates

  11. Chromatin maps, histone modifications and leukemia

    Microsoft Academic Search

    T Neff; S A Armstrong

    2009-01-01

    Recent years have seen great advances in the understanding of epigenetic gene regulation. Many of the molecular players involved have recently been identified and are rapidly being characterized in detail. Genome scale studies, using chromatin immunoprecipitation followed by expression arrays (‘ChIP-Chip’) or next generation sequencing (‘ChIP-Seq’), have been applied to the study of transcription factor binding, DNA methylation, alternative histone

  12. Visualizing Chromatin Dynamics in Interphase Nuclei

    NSDL National Science Digital Library

    Susan Gasser (University of Geneva; Department of Molecular Biology)

    2002-05-31

    Real-time fluorescence microscopy has emerged as a powerful tool for examining chromatin dynamics. The initial lesson is that much of the genome, particularly in yeast, is highly dynamic. Its movement within the interphase nucleus is correlated with metabolic activity. Nonetheless, the nucleus is an organelle with conserved rules of organization. Determining the distribution and regulation of mobile domains in interphase chromosomes, and characterizing sites of anchorage, will undoubtedly shed new light on the function of nuclear order.

  13. Chromatin remodeling enzymes: who's on first?

    Microsoft Academic Search

    Christopher J Fry; Craig L Peterson

    2001-01-01

    A central problem in the regulation of eukaryotic gene expression is understanding how gene-specific transcriptional activators orchestrate the recruitment of the myriad proteins that are required for transcription initiation. An emerging view indicates that activators must first target two types of chromatin remodeling enzyme to the promoter region: an ATP-dependent SWI\\/SNF-like complex and a histone acetyltransferase. These two enzymes appear

  14. Subfractionation of soluble macronuclear chromatin and enrichment of specific genes as chromatin from Euplotes eurystomus.

    PubMed

    Cadilla, C L; Roberson, A E; Harp, J; Olins, A L; Olins, D E

    1986-11-11

    Euplotes eurystomus is a hypotrichous ciliated protozoan possessing within one cytoplasm a transcriptionally-inactive micronucleus with chromosomal-size DNA and a transcriptionally active macronucleus with "gene-size" DNA fragments. The chromatin in the macronucleus can be isolated in a soluble form without prior treatment by nucleases. In this study, macronuclear, soluble chromatin was subfractionated using isokinetic sucrose density gradient ultracentrifugation in a buffer consisting of 50 mM NaCl, 1 mM Na2 EDTA, 1 mM TEA HCl, pH 7.0, 0.1 mM TLCK and 0.1 mM PMSF. Fractions were collected and analyzed by DNA and protein gel electrophoresis, dot blot hybridization with specific gene probes, and modified Miller chromatin spreads. Analysis of the chromatin spreads showed that the sizes of the chromatin fragments in the various fractions correlate with the DNA size of the fragments. When dot blots of the fractions were hybridized with 5S rRNA, tubulin and rRNA gene probes we obtained about a 6 to 14-fold enrichment of hybridizable sequences in individual fractions. There appear to be differences in the non-histones present on each fraction as well as some overall similarities in histone and non-histone proteins. PMID:3786133

  15. Genetic factors underlying discordance in chromatin accessibility between monozygotic twins

    PubMed Central

    2014-01-01

    Background Open chromatin is implicated in regulatory processes; thus, variations in chromatin structure may contribute to variations in gene expression and other phenotypes. In this work, we perform targeted deep sequencing for open chromatin, and array-based genotyping across the genomes of 72 monozygotic twins to identify genetic factors regulating co-twin discordance in chromatin accessibility. Results We show that somatic mutations cause chromatin discordance mainly via the disruption of transcription factor binding sites. Structural changes in DNA due to C:G to A:T transversions are under purifying selection due to a strong impact on chromatin accessibility. We show that CpGs whose methylation is specifically regulated during cellular differentiation appear to be protected from high mutation rates of 5?-methylcytosines, suggesting that the spectrum of CpG variations may be shaped fully at the developmental level but not through natural selection. Based on the association mapping of within-pair chromatin differences, we search for cases in which twin siblings with a particular genotype had chromatin discordance at the relevant locus. We identify 1,325 chromatin sites that are differentially accessible, depending on the genotype of a nearby locus, suggesting that epigenetic differences can control regulatory variations via interactions with genetic factors. Poised promoters present high levels of chromatin discordance in association with either somatic mutations or genetic-epigenetic interactions. Conclusion Our observations illustrate how somatic mutations and genetic polymorphisms may contribute to regulatory, and ultimately phenotypic, discordance. PMID:24887574

  16. The Chromatin Remodeler SPLAYED Regulates Specific Stress Signaling Pathways

    PubMed Central

    Walley, Justin W.; Rowe, Heather C.; Xiao, Yanmei; Chehab, E. Wassim; Kliebenstein, Daniel J.; Wagner, Doris; Dehesh, Katayoon

    2008-01-01

    Organisms are continuously exposed to a myriad of environmental stresses. Central to an organism's survival is the ability to mount a robust transcriptional response to the imposed stress. An emerging mechanism of transcriptional control involves dynamic changes in chromatin structure. Alterations in chromatin structure are brought about by a number of different mechanisms, including chromatin modifications, which covalently modify histone proteins; incorporation of histone variants; and chromatin remodeling, which utilizes ATP hydrolysis to alter histone-DNA contacts. While considerable insight into the mechanisms of chromatin remodeling has been gained, the biological role of chromatin remodeling complexes beyond their function as regulators of cellular differentiation and development has remained poorly understood. Here, we provide genetic, biochemical, and biological evidence for the critical role of chromatin remodeling in mediating plant defense against specific biotic stresses. We found that the Arabidopsis SWI/SNF class chromatin remodeling ATPase SPLAYED (SYD) is required for the expression of selected genes downstream of the jasmonate (JA) and ethylene (ET) signaling pathways. SYD is also directly recruited to the promoters of several of these genes. Furthermore, we show that SYD is required for resistance against the necrotrophic pathogen Botrytis cinerea but not the biotrophic pathogen Pseudomonas syringae. These findings demonstrate not only that chromatin remodeling is required for selective pathogen resistance, but also that chromatin remodelers such as SYD can regulate specific pathways within biotic stress signaling networks. PMID:19079584

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

    PubMed

    Walley, Justin W; Rowe, Heather C; Xiao, Yanmei; Chehab, E Wassim; Kliebenstein, Daniel J; Wagner, Doris; Dehesh, Katayoon

    2008-12-01

    Organisms are continuously exposed to a myriad of environmental stresses. Central to an organism's survival is the ability to mount a robust transcriptional response to the imposed stress. An emerging mechanism of transcriptional control involves dynamic changes in chromatin structure. Alterations in chromatin structure are brought about by a number of different mechanisms, including chromatin modifications, which covalently modify histone proteins; incorporation of histone variants; and chromatin remodeling, which utilizes ATP hydrolysis to alter histone-DNA contacts. While considerable insight into the mechanisms of chromatin remodeling has been gained, the biological role of chromatin remodeling complexes beyond their function as regulators of cellular differentiation and development has remained poorly understood. Here, we provide genetic, biochemical, and biological evidence for the critical role of chromatin remodeling in mediating plant defense against specific biotic stresses. We found that the Arabidopsis SWI/SNF class chromatin remodeling ATPase SPLAYED (SYD) is required for the expression of selected genes downstream of the jasmonate (JA) and ethylene (ET) signaling pathways. SYD is also directly recruited to the promoters of several of these genes. Furthermore, we show that SYD is required for resistance against the necrotrophic pathogen Botrytis cinerea but not the biotrophic pathogen Pseudomonas syringae. These findings demonstrate not only that chromatin remodeling is required for selective pathogen resistance, but also that chromatin remodelers such as SYD can regulate specific pathways within biotic stress signaling networks. PMID:19079584

  18. Chromatin modifications remodel cardiac gene expression.

    PubMed

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

    2014-07-01

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

  19. Remodeling is at the heart of chromatin

    PubMed Central

    2011-01-01

    Chromatin modifications are integral elements of chromosome structure and its function and the vasculature depends on tissue-specific genome regulation for its development. A general concept for the deregulation of chromatin modifications in cardiac and vascular disease is also emerging. The recognition that metabolic memory contributes to disease persistence highlights the benefit of early and aggressive treatment. As for the importance of memory, we do know that good metabolic control delays the onset of long-term diabetic complications. There are striking parallels between the timing of disease and the development of complications. Landmark multicenter clinical trials on diabetes patients have popularized the concept that glucose is also a demonstrable determinant for the development of complications, indicating the prolonged benefit of intensive treatment and the lasting damage of conventional therapy. Each cell type experiences thousands of modifications to the epigenome in response to environmental changes it is exposed to. Therefore, history is neither lost nor forgotten and previous experiences and exposure may form future memories. There is now a strong resurgence in research trying to understand gene-environment interactions and to determine what commits vascular cell types to specific memories. Recent insights show that cardiac gene expression is distinguished by specific chromatin remodeling events and histone modifications that are associated with heart disease. PMID:21646860

  20. An examination of models for chromatin transcription.

    PubMed Central

    Gould, H J; Cowling, G J; Harborne, N R; Allan, J

    1980-01-01

    Structural studies have revealed that chromatin is composed of repeating units or nucleosomes having two distinct domains, the nucleosome core and the linker region. The nucleosome core comprises 146 base pairs of DNA wound in one and three quarter turns around an octamer of histones made up of two symmetrical tetramers (1). It may be inferred on topological grounds that this structure must be perturbed during chromatin transcription and replication since the histone core bridges the supercoil which blocks the passage of polymerase along the template and prevents the unwinding of DNA required for enzymatic copying. A number of mechanisms for freeing the DNA template may be envisaged, and one detailed model, based on symmetrical dissociation of the histone tetramers, has been proposed (2). Here we present evidence against such unpairing or indeed any detachment of histones from the octamer during chromatin transcription, and we give reasons for favouring a transcriptional mechanism based upon the separation of the octamer from at least one of the DNA. Images PMID:7465413

  1. Chromatin Structure in Situ: The Contribution of DNA Ultrastructural Cytochemistry

    PubMed Central

    Derenzini, M.; Olins, A.L.; Olins, D.E.

    2014-01-01

    Ultrastructural studies conducted in situ using conventional transmission electron microscopy have had relatively little impact on defining the structural organization of chromatin. This is due to the fact that in routine transmission electron microscopy, together with the deoxyribonucleoprotein, many different intermingled substances are contrasted, masking the ultrastructure of chromatin. By selective staining of DNA in thin sections, using the Feulgen-like osmium-ammine reaction, these drawbacks have been overcome and worthwhile data have been obtained both on the gross morphology and the ultrastructural-functional organization of chromatin in situ. In the present study these results are reviewed and discussed in light of recent achievements in both interphase nuclear chromatin compartmentalization in interphase nuclei and in the structural organization of chromatin fibers in transcriptionally active and inactive chromatin. PMID:24704998

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

    PubMed

    Kuo, Wei-Hung W; Wang, Yemin; Wong, Ronald P C; Campos, Eric I; Li, Gang

    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. PMID:17379210

  3. 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.

  4. Proceedings: Condenser technology conference

    SciTech Connect

    Tsou, J.L. (ed.)(Electric Power Research Inst., Palo Alto, CA (United States)); Mussalli, Y.G. (comp.)(Stone and Webster Engineering Corp., Boston, MA (United States))

    1991-08-01

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

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

    PubMed Central

    Linnemann, Amelia K.; Krawetz, Stephen A.

    2010-01-01

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

  6. Dynamics of the higher-order structure of chromatin

    Microsoft Academic Search

    Ping Chen; Guohong Li

    2010-01-01

    Eukaryotic DNA is hierarchically packaged into chromatin to fit inside the nucleus. Dynamics of the chromatin structure plays\\u000a a critical role in transcriptional regulation and other biological processes that involve DNA, such as DNA replication and\\u000a DNA repair. Many factors, including histone variants, histone modification, DNA methylation and the binding of non-histone\\u000a architectural proteins regulate the structure of chromatin. Although

  7. Snapshots: Chromatin control of viral infection David M. Knipe a

    E-print Network

    Knipe, David M.

    Adenovirus Papillomavirus Human Immunodeficiency Virus Influenza virus a b s t r a c t Like their cellular . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Association of papillomavirus genomes with host chromatin . . . . . . . . . . . . . . . . . . . .

  8. The Centromere: Chromatin Foundation for the Kinetochore Machinery

    PubMed Central

    Fukagawa, Tatsuo; Earnshaw, William C.

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Hada, Megumi; Wu, Honglu

    2014-01-01

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

  11. Effects of airport screening X-irradiation on bovine sperm chromatin integrity and embryo development.

    PubMed

    Hendricks, K E M; Penfold, L M; Evenson, D P; Kaproth, M T; Hansen, P J

    2010-01-15

    Biological samples, including cryopreserved sperm, are routinely X-rayed during air shipment. The goal was to investigate the impact of X-irradiation used for checked and carry-on luggage on bovine sperm chromatin integrity and postfertilization in vitro embryonic development. Frozen domestic bull sperm (Bos taurus) (n=9 bulls) stored in a dry shipper (-160 degrees C) was screened by X-irradiation 0, 1, 2, and 3 times as either carry-on or checked luggage. Duplicate straws were thawed, and sperm were assessed for chromatin damage using the sperm chromatin structure assay (SCSA) and by postfertilization in vitro developmental competence of mature oocytes. Multiple exposure to X-rays did not significantly affect sperm chromatin integrity assessed by SCSA. There were lower proportions of oocytes cleaved (P=0.07; 21.6+/-3.1% vs. 29.4+/-3.1%, 24.9+/-3.1%, and 25.7+/-3.3% for 3 vs. 0, 1, and 2 times, respectively; least-squares means+/-SEM) and that developed to blastocysts (P=0.06; 9.0+/-1.7% vs. 13.8+/-1.7%, 11.5+/-1.7%, and 12.6+/-1.9%, respectively) when fertilization was performed with sperm X-rayed 3 times using checked luggage irradiation; developmental competence (percentage cleaved embryos becoming blastocysts) was unaffected. There were no deleterious effects of other X-irradiation treatments on embryo development. We inferred that screening by X-irradiation may reduce the ability of sperm to activate oocyte cleavage after multiple exposures at the checked luggage dose. However, there was no evidence that competence of embryos to become blastocysts was reduced by X-irradiation (45.4+/-5.7%, 40.4+/-5.7%, 46.4+/-6.1%, and 41.8+/-5.7% for 0, 1, 2, and 3 doses, respectively), but potential long-term epigenetic effects are unknown. PMID:19864012

  12. Economical Condensing Turbines?

    E-print Network

    Dean, J. E.

    Economical Condensing Turbines? by J.E.Dean, P.E. Steam turbines have long been used at utilities and in industry to generate power. There are three basic types of steam turbines: condensing, letdown 1 and extraction/condensing. ? Letdown... turbines reduce the pressure of the incoming steam to one or more pressures and generate power very efficiently, assuming that all the letdown steam has a use. Two caveats: ? Letdown turbines produce power based upon steam requirements and not based upon...

  13. Steam and Condensate Systems

    E-print Network

    Yates, W.

    1980-01-01

    types of traps. Our data shows that most types have low losses when sized and in stalled properly. Virtually any condensate removel device will waste steam or fail prematurely if impro perly sized or installed. When selecting steam traps, factors... year and it can fail prematurely due to improper selection or installation. What is a steam trap? It is an automatic control valve that opens on condensate, air and non-condensable gases and closes on steam or hot condensate. The basic types are...

  14. Condensed Matter Physics

    NSDL National Science Digital Library

    Founded in 1993 by the Institute for Condensed Matter Physics of the National Academy of Sciences of Ukraine, the journal Condensed Matter Physics is a peer-reviewed, English-language journal covering such aspects of condensed matter as phase transition theory, statistical mechanics of spin and spin-electron systems, metals and alloys, liquids, solutions, electrolytes, surface phenomena, and plasma physics. Selected issues of Condensed Matter Physics from January 1994 to March 2000 are now available free, online in LaTeX format.

  15. Of Matters Condensed

    E-print Network

    Shulman, Michael

    2015-01-01

    The American Physical Society (APS) March Meeting of condensed matter physics has grown to nearly 10,000 participants, comprises 23 individual APS groups, and even warrants its own hashtag (#apsmarch). Here we analyze the text and data from March Meeting abstracts of the past nine years and discuss trends in condensed matter physics over this time period. We find that in comparison to atomic, molecular, and optical physics, condensed matter changes rapidly, and that condensed matter appears to be moving increasingly toward subject matter that is traditionally in materials science and engineering.

  16. Stem cell factors in plants: chromatin connections.

    PubMed

    Kornet, N; Scheres, B

    2008-01-01

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

  17. Chromatin insulators: regulatory mechanisms and epigenetic inheritance

    PubMed Central

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

    2008-01-01

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

  18. Role of chromatin states in transcriptional memory

    PubMed Central

    Kundu, Sharmistha; Peterson, Craig L.

    2009-01-01

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

  19. 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.

  20. Global chromatin fibre compaction in response to DNA damage

    SciTech Connect

    Hamilton, Charlotte [Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom)] [Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom); Hayward, Richard L. [Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom) [Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom); Breakthrough Research Unit, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom); Gilbert, Nick, E-mail: Nick.Gilbert@ed.ac.uk [Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom) [Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom); Breakthrough Research Unit, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom)

    2011-11-04

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

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

    PubMed Central

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

    2011-01-01

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

  2. Postsynthetic acetylation of histones during the cell cycle: a general function for the displacement of histones during chromatin rearrangements.

    PubMed

    Loidl, P; Gröbner, P

    1987-10-26

    Postsynthetic acetylation of core histones exhibits a peak during S-phase of the Physarum cell cycle. The maximum 3H-acetate incorporation precedes the maximum of histone synthesis. Acetate is incorporated into all core histones during S-phase, but only into H2A and H2B during G2-period. Resolution of acetylated H4-subspecies reveals acetate incorporation into preexisting H4, but not into newly synthesized molecules during mitosis and early S-phase. In a protamine competition assay histones from S-phase chromatin are released at lower protamine concentrations as compared to the lower acetylated G2-chromatin. We demonstrate a preferential release of highly acetylated H4-subspecies at low protamine concentrations. Our results fit into a general model of the relationship between histone acetylation and chromatin assembly. According to this model acetylation of core histones would serve as a signal for displacement of histones from nucleosomes by modulating histone-protein or histone-DNA interactions. We propose that this mechanism operates during DNA-replication and transcription, as well as during other chromatin rearrangements. PMID:3118335

  3. 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

  4. Targeting aberrant chromatin structure in colorectal carcinomas.

    PubMed

    Konishi, Kazuo; Issa, Jean-Pierre J

    2007-01-01

    Epigenetic processes such as DNA methylation and histone modifications are now recognized as critical events for regulation of gene expression in mammalian cells and affect gene function without a change in coding sequence. Neoplastic cells often show profound epigenetic alterations that contribute to tumorigenesis by altering expression of critical genes. In colorectal tumorigenesis, detailed analysis led to a hypothesis on a critical role for epigenetic changes in age-related cancer susceptibility and separately identified a distinct phenotype termed the CpG island methylator phenotype. CpG island methylator phenotype-positive colorectal cancers have significant associations with female sex, older age, proximal location, mucinous histology, KRAS and BRAF mutations, wild-type p53, and microsatellite instability. Histone modifications that affect chromatin structures are also closely implicated in tumor suppressor gene inactivation and DNA methylation and histone modifications seem to form reinforcing networks for stable gene silencing. Much of the excitement in this field relates to the possibility of therapeutic reversal of epigenetic changes by chromatin-modifying drugs. In CpG island methylator phenotype-positive colorectal cancers, DNA methylation inhibitors restore key silenced pathways in vivo (eg, mismatch repair defects), and hypomethylation can largely abolish tumorigenesis in a mouse model. Drugs that inhibit DNA methylation and histone deacetylation are in use in the clinic and should be tested in colorectal malignancy. PMID:17464246

  5. 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

  6. SUMO conjugation attenuates the activity of the gypsy chromatin insulator

    E-print Network

    Corces, Victor G.

    suggesting that two protein components of the gypsy chromatin insulator of Dorsophila melanogaster, Mod(mdg4. Sumoylation does not affect the ability of CP190 and Mod(mdg4)2.2 to bind chromatin, but instead appears consists of a B350 bp DNA sequence that binds a protein complex of at least three components, Su(Hw), Mod(mdg

  7. CHD chromatin remodelling enzymes and the DNA damage response.

    PubMed

    Stanley, Fintan K T; Moore, Shaun; Goodarzi, Aaron A

    2013-10-01

    The protein and DNA complex known as chromatin is a dynamic structure, adapting to alter the spatial arrangement of genetic information within the nucleus to meet the ever changing demands of life. Following decades of research, a dizzying array of regulatory factors is now known to control the architecture of chromatin at nearly every level. Amongst these, ATP-dependent chromatin remodelling enzymes play a key role, required for the establishment, maintenance and re-organization of chromatin through their ability to adjust the contact points between DNA and histones, the spacing between individual nucleosomes and the over-arching chromatin superstructure. Utilizing energy from ATP hydrolysis, these enzymes serve as the gatekeepers of genomic access and are essential for transcriptional regulation, DNA replication and cell division. In recent years, a vital role in DNA Double Strand Break (DSB) repair has emerged, particularly within complex chromatin environments such as heterochromatin, or regions undergoing energetic transactions such as transcription or DNA replication. Here, we will provide an overview of what is understood about ATP-dependent chromatin remodelling enzymes in the context of the DNA damage response. We will first touch upon all four major chromatin remodelling enzyme families and then focus chiefly on the nine members of the Chromodomain, Helicase, DNA-binding (CHD) family, particularly CHD3, CHD4, CHD5 and CHD6. These four proteins have established and emerging roles in DNA repair, the oxidative stress response, the maintenance of genomic stability and/or cancer prevention. PMID:23954449

  8. Chromatin dynamics: interplay between remodeling enzymes and histone modifications.

    PubMed

    Swygert, Sarah G; Peterson, Craig L

    2014-08-01

    Chromatin dynamics play an essential role in regulating the accessibility of genomic DNA for a variety of nuclear processes, including gene transcription and DNA repair. The posttranslational modification of the core histones and the action of ATP-dependent chromatin remodeling enzymes represent two primary mechanisms by which chromatin dynamics are controlled and linked to nuclear events. Although there are examples in which a histone modification or a remodeling enzyme may be sufficient to drive a chromatin transition, these mechanisms typically work in concert to integrate regulatory inputs, leading to a coordinated alteration in chromatin structure and function. Indeed, site-specific histone modifications can facilitate the recruitment of chromatin remodeling enzymes to particular genomic regions, or they can regulate the efficiency or the outcome of a chromatin remodeling reaction. Conversely, chromatin remodeling enzymes can also influence, and sometimes directly modulate, the modification state of histones. These functional interactions are generally complex, frequently transient, and often require the association of myriad additional factors. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function. PMID:24583555

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

    Microsoft Academic Search

    Anjanabha Saha; Jacqueline Wittmeyer; Bradley R. Cairns

    2006-01-01

    Chromatin remodellers are specialized multi-protein machines that enable access to nucleosomal DNA by altering the structure, composition and positioning of nucleosomes. All remodellers have a catalytic ATPase subunit that is similar to known DNA-translocating motor proteins, suggesting DNA translocation as a unifying aspect of their mechanism. Here, we explore the diversity and specialization of chromatin remodellers, discuss how nucleosome modifications

  10. Genome-wide approaches to studying chromatin modifications

    Microsoft Academic Search

    Dustin E. Schones; Keji Zhao

    2008-01-01

    Over two metres of DNA is packaged into each nucleus in the human body in a manner that still allows for gene regulation. This remarkable feat is accomplished by the wrapping of DNA around histone proteins in repeating units of nucleosomes to form a structure known as chromatin. This chromatin structure is subject to various modifications that have profound influences

  11. Genome-wide approaches to studying yeast chromatin modifications.

    PubMed

    Schones, Dustin E; Cui, Kairong; Cuddapah, Suresh

    2011-01-01

    The genomes of eukaryotic organisms are packaged into nuclei by wrapping DNA around proteins in a structure known as chromatin. The most basic unit of chromatin, the nucleosome, consists of approximately 146 bp of DNA wrapped around an octamer of histone proteins. The placement of nucleosomes relative to a gene can influence the regulation of the transcription of this gene. Furthermore, the N-terminal tails of histone proteins are subjected to numerous post-translational modifications that are also known to influence gene regulation. In recent years, a number of genome-scale approaches to identify modifications to chromatin have been developed. Techniques combining chromatin immunoprecipitation (ChIP) with microarrays (ChIP-chip) and second-generation sequencing (ChIP-Seq) have led to great advances in our understanding of how chromatin modifications contribute to gene regulation. Many excellent protocols related to ChIP-chip have been published recently (Lieb, J. D. (2003) Genome-wide mapping of protein-DNA interactions by chromatin immunoprecipitation and DNA microarray hybridization. Methods Mol. Biol. 224, 99-109.). For this reason, we will focus our attention here on the application of second-generation sequencing platforms to the study of chromatin modifications in yeast. As these genome-scale experiments require both wet-lab and bioinformatic components to reach their full potential, we will detail both the wet-lab protocols and bioinformatic steps necessary to fully conduct genome-scale studies of chromatin modifications. PMID:21863481

  12. 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

  13. Chromatin Higher Order Structure and Chromosomal Positioning in the Nucleus

    Microsoft Academic Search

    Matthew Schmerer

    While it has been known for some time that chromatin is highly packaged, it has only recently been discovered that chromatin is attached to the nuclear matrix. It has also been recently shown that interphase chromosomes remain separate from one another in so called, \\

  14. Cooperation between Complexes that Regulate Chromatin Structure and Transcription

    Microsoft Academic Search

    Geeta J. Narlikar; Hua-Ying Fan; Robert E. Kingston

    2002-01-01

    Chromatin structure creates barriers for each step in eukaryotic transcription. Here we discuss how the activities of two major classes of chromatin-modifying complexes, ATP-dependent remodeling complexes and HAT or HDAC complexes, might be coordinated to create a DNA template that is accessible to the general transcription apparatus.

  15. Comprehensive analysis of the chromatin landscape in Drosophila

    PubMed Central

    Kharchenko, Peter V.; Alekseyenko, Artyom A.; Schwartz, Yuri B.; Minoda, Aki; Riddle, Nicole C.; Ernst, Jason; Sabo, Peter J.; Larschan, Erica; Gorchakov, Andrey A.; Gu, Tingting; Linder-Basso, Daniela; Plachetka, Annette; Shanower, Gregory; Tolstorukov, Michael Y.; Luquette, Lovelace J.; Xi, Ruibin; Jung, Youngsook L.; Park, Richard W.; Bishop, Eric P.; Canfield, Theresa P.; Sandstrom, Richard; Thurman, Robert E.; MacAlpine, David M.; Stamatoyannopoulos, John A.; Kellis, Manolis; Elgin, Sarah C. R.; Kuroda, Mitzi I.; Pirrotta, Vincenzo; Karpen, Gary H.; Park, Peter J.

    2011-01-01

    Summary Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which impact cell differentiation, gene regulation and other key cellular processes. We present a genome-wide chromatin landscape for Drosophila melanogaster based on 18 histone modifications, summarized by 9 prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNaseI hypersensitivity, GRO-seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements, and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions, and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function. PMID:21179089

  16. 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

  17. Connecting Chromatin Modifying Factors to DNA Damage Response

    PubMed Central

    Lai, Weiwei; Li, Hongde; Liu, Shuang; Tao, Yongguang

    2013-01-01

    Cells are constantly damaged by factors that can induce DNA damage. Eukaryotic cells must rapidly load DNA repair proteins onto damaged chromatin during the DNA damage response (DDR). Chromatin-remodeling complexes use the energy from ATP hydrolysis to remodel nucleosomes and have well-established functions in transcription. Emerging lines of evidence indicate that chromatin-remodeling complexes are important and may remodel nucleosomes during DNA damage repair. New studies also reveal that ATP-dependent chromatin remodeling is involved in cell cycle progression, signal transduction pathways, and interaction and modification of DDR-related proteins that are specifically and intimately connected with the process of DNA damage. This article summarizes the recent advances in our understanding of the interplay between chromatin remodeling and DNA damage response. PMID:23348929

  18. Chromatin regulators of the AID induced DNA damage response

    PubMed Central

    Daniel, Jeremy A.; Nussenzweig, André

    2013-01-01

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

  19. 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

  20. Chromatin fluorescence characteristics and standard semen analysis parameters: correlations observed in andrology testing among 136 males referred for infertility evaluation.

    PubMed

    Sills, E Scott; Fryman, Julie T; Perloe, M; Michels, Karin B; Tucker, M J

    2004-01-01

    This paper aims to describe the relation between standard semen analysis parameters (concentration, motility and morphology) and sperm chromatin structure assay (SCSA) results among patients referred for infertility evaluation. Healthy males (n=136) seeking infertility consultation were evaluated prospectively by semen analysis and sperm chromatin structure assay (SCSA). Significant inverse correlations were observed between high sperm concentration and DNA fragmentation index (DFI) and high DNA stainability (HDS) (r=- 0.45; P<0.001, and r=- 0.40; P<0.001, respectively). Both progressive motility and normal morphology were also strongly inversely correlated with DFI and HDS. However, in stratified analysis the correlation between concentration < or =20 M/ml and DFI, and concentration < or =20 M/ml and HDS were not significant (P=0.31 and 0.38, respectively). For men with sperm motility < or =40% the correlation between motility and HDS was not significant (P=0.22), but between motility and DFI the correlation remained significant (P=0.04). Although strong correlations between DFI, HDS and semen analysis findings were noted in the overall study population, when oligozoospermic and asthenozoospermic patients were analysed separately the correlation between concentration and sperm chromatin fragmentation was not significant. For such men, SCSA appears to be a diagnostic variable independent of the semen analysis, providing information about nuclear abnormalities not readily apparent from standard semen analysis alone. Additionally, SCSA data may offer explanations for previous miscarriage, providing closure for some couples contemplating future use of anonymous donor sperm. PMID:14675988

  1. NAP1 Modulates Binding of Linker Histone H1 to Chromatin and Induces an Extended Chromatin Fiber Conformation*S

    E-print Network

    Rippe, Karsten

    NAP1 Modulates Binding of Linker Histone H1 to Chromatin and Induces an Extended Chromatin Fiber Heidelberg, Germany NAP1 (nucleosome assembly protein 1) is a histone chaperone that has been described into the nucleus, nucleosome assembly/remodeling, and transcription. Here it was examined how NAP1 interacts

  2. The mobile nucleoporin Nup2p and chromatin-bound Prp20p function in endogenous NPC-mediated transcriptional control

    PubMed Central

    Dilworth, David J.; Tackett, Alan J.; Rogers, Richard S.; Yi, Eugene C.; Christmas, Rowan H.; Smith, Jennifer J.; Siegel, Andrew F.; Chait, Brian T.; Wozniak, Richard W.; Aitchison, John D.

    2005-01-01

    Nuclear pore complexes (NPCs) govern macromolecular transport between the nucleus and cytoplasm and serve as key positional markers within the nucleus. Several protein components of yeast NPCs have been implicated in the epigenetic control of gene expression. Among these, Nup2p is unique as it transiently associates with NPCs and, when artificially tethered to DNA, can prevent the spread of transcriptional activation or repression between flanking genes, a function termed boundary activity. To understand this function of Nup2p, we investigated the interactions of Nup2p with other proteins and with DNA using immunopurifications coupled with mass spectrometry and microarray analyses. These data combined with functional assays of boundary activity and epigenetic variegation suggest that Nup2p and the Ran guanylyl-nucleotide exchange factor, Prp20p, interact at specific chromatin regions and enable the NPC to play an active role in chromatin organization by facilitating the transition of chromatin between activity states. PMID:16365162

  3. 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

  4. Chromatin Immunoprecipitation (ChIP) for Analysis of Histone Modifications and Chromatin-Associated Proteins

    PubMed Central

    Milne, Thomas A.; Zhao, Keji; Hess, Jay L.

    2014-01-01

    Summary Disruption of epigenetic regulators of transcription is a central mechanism of oncogenesis. Many of the advances in the understanding of these mechanisms are attributable to the successful development of chromatin immunoprecipitation (ChIP) for in vivo detection of histone modifications as well as chromatin binding regulatory proteins. This is a powerful technique for analyzing histone modifications as well as binding sites for proteins that bind either directly or indirectly to DNA. Here we present two ChIP protocols. The first is particularly useful for identifying histone modifications or binding at specific, known genomic sites. The second, employing serial analysis of gene expression, is particularly powerful for the discovery of previously unidentified sites of modification or binding. PMID:19277579

  5. Chromatin Composition Is Changed by Poly(ADP-ribosyl)ation during Chromatin Immunoprecipitation

    PubMed Central

    Beneke, Sascha; Meyer, Kirstin; Holtz, Anja; Hüttner, Katharina; Bürkle, Alexander

    2012-01-01

    Chromatin-immunoprecipitation (ChIP) employs generally a mild formaldehyde cross-linking step, which is followed by isolation of specific protein-DNA complexes and subsequent PCR testing, to analyze DNA-protein interactions. Poly(ADP-ribosyl)ation, a posttranslational modification involved in diverse cellular functions like repair, replication, transcription, and cell death regulation, is most prominent after DNA damage. Poly(ADP-ribose)polymerase-1 is activated upon binding to DNA strand-breaks and coordinates repair by recruitment or displacement of proteins. Several proteins involved in different nuclear pathways are directly modified or contain poly(ADP-ribose)-interaction motifs. Thus, poly(ADP-ribose) regulates chromatin composition. In immunofluorescence experiments, we noticed artificial polymer-formation after formaldehyde-fixation of undamaged cells. Therefore, we analyzed if the formaldehyde applied during ChIP also induces poly(ADP-ribosyl)ation and its impact on chromatin composition. We observed massive polymer-formation in three different ChIP-protocols tested independent on the cell line. This was due to induction of DNA damage signaling as monitored by ?H2AX formation. To abrogate poly(ADP-ribose) synthesis, we inhibited this enzymatic reaction either pharmacologically or by increased formaldehyde concentration. Both approaches changed ChIP-efficiency. Additionally, we detected specific differences in promoter-occupancy of tested transcription factors as well as the in the presence of histone H1 at the respective sites. In summary, we show here that standard ChIP is flawed by artificial formation of poly(ADP-ribose) and suppression of this enzymatic activity improves ChIP-efficiency in general. Also, we detected specific changes in promoter-occupancy dependent on poly(ADP-ribose). By preventing polymer synthesis with the proposed modifications in standard ChIP protocols it is now possible to analyze the natural chromatin-composition. PMID:22479348

  6. Reconstitution of chromatin: assembly of the nucleosome.

    PubMed Central

    Wilhelm, F X; Wilhelm, M L; Erard, M; Duane, M P

    1978-01-01

    The order of reassociation of the four histones H2a, H2b, H3 and H4 to the DNA during the reconstitution of chromatin was determined. At each step of the reconstitution the DNA and associated histones were separated from the free histones by centrifugation in a glycerol gradient. The unbound and reassociated histones were analysed by gel electrophoresis and the histone-DNA complexes characterized by circular dichroism and electron microscopy. We show that H3 and H4 bind first to the DNA between 1.2 M NaCl and 0.85 M NaCl and impose a nucleosome like structure; in a second step histones H2a and H2b are placed around this kernel to complete the nucleosome. Images PMID:634796

  7. Cohesinopathies, gene expression, and chromatin organization

    PubMed Central

    Bose, Tania

    2010-01-01

    The cohesin protein complex is best known for its role in sister chromatid cohesion, which is crucial for accurate chromosome segregation. Mutations in cohesin proteins or their regulators have been associated with human diseases (termed cohesinopathies). The developmental defects observed in these diseases indicate a role for cohesin in gene regulation distinct from its role in chromosome segregation. In mammalian cells, cohesin stably interacts with specific chromosomal sites and colocalizes with CTCF, a protein that promotes long-range DNA interactions, implying a role for cohesin in genome organization. Moreover, cohesin defects compromise the subnuclear position of chromatin. Therefore, defects in the cohesin network that alter gene expression and genome organization may underlie cohesinopathies. PMID:20404106

  8. On the mechanochemical machinery underlying chromatin remodeling

    NASA Astrophysics Data System (ADS)

    Yusufaly, Tahir I.

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

  9. Polariton Condensate Transistor Switch

    E-print Network

    Gao, T; Liew, T C H; Tsintzos, S I; Stavrinidis, G; Deligeorgis, G; Hatzopoulos, Z; Savvidis, P G

    2012-01-01

    A polariton condensate transistor switch is realized through optical excitation of a microcavity ridge with two beams. The ballistically ejected polaritons from a condensate formed at the source are gated using the 20 times weaker second beam to switch on and off the flux of polaritons. In the absence of the gate beam the small built-in detuning creates potential landscape in which ejected polaritons are channelled toward the end of the ridge where they condense. The low loss photon-like propagation combined with strong nonlinearities associated with their excitonic component makes polariton based transistors particularly attractive for the implementation of all-optical integrated circuits.

  10. Electrolyte vapor condenser

    DOEpatents

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

    1983-02-08

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

  11. Electrolyte vapor condenser

    DOEpatents

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

    1983-01-01

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

  12. Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent Chromatin Assembly Dynamics

    PubMed Central

    Yan, Jie; Maresca, Thomas J.; Skoko, Dunja; Adams, Christian D.; Xiao, Botao; Christensen, Morten O.; Heald, Rebecca

    2007-01-01

    We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening–closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy difference between core histone octamers on and off DNA. Addition of ATP led to highly dynamic behavior with time courses exhibiting processive runs of assembly and disassembly not observed in the ATP-depleted case. With ATP present, application of forces of 2 pN led to nearly complete fiber disassembly. Our study suggests that ATP hydrolysis plays a major role in nucleosome rearrangement and removal and that chromatin in vivo may be subject to highly dynamic assembly and disassembly processes that are modulated by DNA tension. PMID:17108322

  13. Remodeling is at the heart of chromatin: the heartaches of chromatin.

    PubMed

    El-Osta, Assam

    2011-07-01

    Chromatin modifications are integral elements of chromosome structure and its function and the vasculature depends on tissue-specific genome regulation for its development. A general concept for the de-regulation of chromatin modifications in cardiac and vascular disease is also emerging. The recognition that metabolic memory contributes to disease persistence highlights the benefit of early and aggressive treatment. As for the importance of memory, we do know that good metabolic control delays the onset of long-term diabetic complications. There are striking parallels between the timing of disease and the development of complications. Landmark multicenter clinical trials on diabetes patients have popularized the concept that glucose is also a demonstrable determinant for the development of complications, indicating the prolonged benefit of intensive therapy and the lasting damage of conventional therapy. Each cell type experiences thousands of modifications to the epigenome in response to environmental changes it is exposed to. Therefore, history is neither lost nor forgotten and previous experiences and exposure may form future memories. There is now a strong resurgence in research trying to understand gene-environment interactions and to determine what commits specific vascular cell types to specific memories. Recent insights show that cardiac gene expression is distinguished by specific chromatin remodeling events and histone modifications that are associated with heart disease. PMID:21646860

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

    SciTech Connect

    Bradbury, E.M.

    1992-11-01

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

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

    SciTech Connect

    Bradbury, E.M.

    1992-06-01

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

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

    PubMed

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

    2013-11-01

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

  17. A role for CARM1-mediated histone H3 arginine methylation in protecting histone acetylation by releasing corepressors from chromatin.

    PubMed

    Wu, Jing; Cui, Nan; Wang, Rui; Li, Jiwen; Wong, Jiemin

    2012-01-01

    Arginine methylation broadly occurs in histones and has been linked to transcriptional regulation, cell cycle regulation and DNA repair. While numerous proteins (histone code effectors) that specifically recognize or read the methylated lysine residues in core histones have been identified, little is known for effectors specific for methylated arginines in histones. In this study, we attempted to identify effector(s) recognizing asymmetrically methylated R17 and R26 in H3, which are catalyzed by CARM1/PRMT4, through an unbiased biochemical approach. Although we have yet to identify such effector using this approach, we find that these modifications function cooperatively with histone acetylation to inhibit the binding of the nucleosome remodeling and deacetylase complex (NuRD) and TIF1 family corepressors to H3 tail in vitro. In support of this finding, we show that overexpression of CARM1 in 293 T cells leads to reduced association of NuRD with chromatin, whereas knockdown of CARM1 in HeLa cells leads to increased association of NuRD with chromatin and decreased level of histone acetylation. Furthermore, in the Carm1-/- MEF cells there is an increased association of NuRD and TIF1? with chromatin and a global decrease in histone acetylation. By chromatin immunoprecipitation assay, we show that overexpression of CARM1 results in reduced association of NuRD complex and TIF1? with an episomal reporter and that CARM1 is required in MEF cells for LPS-induced dissociation of NuRD from a NF-?b target gene. Taking together, our study provides evidence for a role of CARM1-mediated arginine methylation in regulation of histone acetylation and transcription: facilitating transcription by discharging corepressors from chromatin. PMID:22723830

  18. 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

  19. 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

  20. 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

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

    PubMed

    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

  2. A novel role for the Aurora B kinase in epigenetic marking of silent chromatin in differentiated postmitotic cells.

    PubMed

    Sabbattini, Pierangela; Canzonetta, Claudia; Sjoberg, Marcela; Nikic, Svetlana; Georgiou, Andrew; Kemball-Cook, Geoffrey; Auner, Holger W; Dillon, Niall

    2007-11-14

    Combinatorial modifications of the core histones have the potential to fine-tune the epigenetic regulation of chromatin states. The Aurora B kinase is responsible for generating the double histone H3 modification tri-methylated K9/phosphorylated S10 (H3K9me3/S10ph), which has been implicated in chromosome condensation during mitosis. In this study, we have identified a novel role for Aurora B in epigenetic marking of silent chromatin during cell differentiation. We find that phosphorylation of H3 S10 by Aurora B generates high levels of the double H3K9me3/S10ph modification in differentiated postmitotic cells and also results in delocalisation of HP1beta away from heterochromatin in terminally differentiated plasma cells. Microarray analysis of the H3K9me3/S10ph modification shows a striking increase in the modification across repressed genes during differentiation of mesenchymal stem cells. Our results provide evidence that the Aurora B kinase has a role in marking silent chromatin independently of the cell cycle and suggest that targeting of Aurora B-mediated phosphorylation of H3 S10 to repressed genes could be a mechanism for epigenetic silencing of gene expression. PMID:17948062

  3. A novel role for the Aurora B kinase in epigenetic marking of silent chromatin in differentiated postmitotic cells

    PubMed Central

    Sabbattini, Pierangela; Canzonetta, Claudia; Sjoberg, Marcela; Nikic, Svetlana; Georgiou, Andrew; Kemball-Cook, Geoffrey; Auner, Holger W; Dillon, Niall

    2007-01-01

    Combinatorial modifications of the core histones have the potential to fine-tune the epigenetic regulation of chromatin states. The Aurora B kinase is responsible for generating the double histone H3 modification tri-methylated K9/phosphorylated S10 (H3K9me3/S10ph), which has been implicated in chromosome condensation during mitosis. In this study, we have identified a novel role for Aurora B in epigenetic marking of silent chromatin during cell differentiation. We find that phosphorylation of H3 S10 by Aurora B generates high levels of the double H3K9me3/S10ph modification in differentiated postmitotic cells and also results in delocalisation of HP1? away from heterochromatin in terminally differentiated plasma cells. Microarray analysis of the H3K9me3/S10ph modification shows a striking increase in the modification across repressed genes during differentiation of mesenchymal stem cells. Our results provide evidence that the Aurora B kinase has a role in marking silent chromatin independently of the cell cycle and suggest that targeting of Aurora B-mediated phosphorylation of H3 S10 to repressed genes could be a mechanism for epigenetic silencing of gene expression. PMID:17948062

  4. Nucleosome positioning and composition modulate in silico chromatin flexibility

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

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

    PubMed

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

    2015-02-18

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

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

    PubMed

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

    1991-05-01

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

  7. Trifluorophenylethylidene Condensation Polyimides

    NASA Technical Reports Server (NTRS)

    Alston, William B.; Gratz, Roy F.

    1987-01-01

    Report describes synthesis and properties of trifluorophenylethylidene (3F) condensation polyimides. Properties of these new polymers make suitable thermal-control coatings, interlayer dielectrics in advanced wafer chips, and electronic-multilevel-interconnection layers.

  8. THE COLOR GLASS CONDENSATE.

    SciTech Connect

    MCLERRAN,L.

    2001-08-26

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

  9. 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 ...

  10. Nanoscale squeezing in elastomeric nanochannels for single chromatin linearization

    PubMed Central

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

    2012-01-01

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

  11. THE ORIENTATION OF DNA WITHIN 80-ANGSTROM CHROMATIN FIBERS

    PubMed Central

    Dusenbery, David B.; Uretz, Robert B.

    1972-01-01

    Squashing salivary glands of Chironomus thummi larvae, Amblystoma tigrinum erythrocytes, or Spirostromum frequently results in stretched chromatin having highly oriented DNA as determined by polarized fluorescence microscopy of acridine orange-stained preparations. The examination of such material from C. thummi in the electron microscope indicates that the individual chromatin fibers have an average thickness of 80 A as is usually found in embedded and sectioned material. It is thus concluded that the DNA lies nearly parallel to the axis of these chromatin fibers. Detailed calculations of the polarization expected from various models of DNA packing are contained in an appendix. PMID:4621586

  12. Histone Post-Translation Modifications Influence Chromatin Mechanical Stability

    NASA Astrophysics Data System (ADS)

    Poirier, Michael

    2011-03-01

    Histone proteins organize the human genome into chromatin fibers while their post-translation modification (PTM) regulates genome replication, expression and repair. The mechanistic connections between histone PTMs and biological functions remain enigmatic. We find with a combination of magnetic tweezers mechanical measurements and biochemical studies that a number of histone PTMs influence the DNA mismatch repair process by mechanically destabilizing chromatin. The location of the PTM within the chromatin structure appears to determine the mechanism by which it alters the mechanical stability. These findings have direct implications for understanding the repair of the human genome.

  13. Mapping regulatory factors by immunoprecipitation from native chromatin.

    PubMed

    Orsi, Guillermo A; Kasinathan, Sivakanthan; Zentner, Gabriel E; Henikoff, Steven; Ahmad, Kami

    2015-01-01

    Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin (ORGANIC) is a high-resolution method that can be used to quantitatively map protein-DNA interactions with high specificity and sensitivity. This method uses micrococcal nuclease (MNase) digestion of chromatin and low-salt solubilization to preserve protein-DNA complexes, followed by immunoprecipitation and paired-end sequencing for genome-wide mapping of binding sites. In this unit, we describe methods for isolation of nuclei and MNase digestion of unfixed chromatin, immunoprecipitation of protein-DNA complexes, and high-throughput sequencing to map sites of bound factors. © 2015 by John Wiley & Sons, Inc. PMID:25827087

  14. THE ORIENTATION OF DNA WITHIN 80-ANGSTROM CHROMATIN FIBERS

    E-print Network

    David B Dusenbery; Robert B Uretz

    Squashing salivary glands of Chironomus thummi larvae, Amblystoma tigrinum erythrocytes, or Spirostromum frequently results in stretched chromatin having highly oriented DNA as determined by polarized fluorescence microscopy of acridine orange-stained preparations. The examination of such material from C. thummi in the electron microscope indicates that the individual chromatin fibers have an average thickness of 80 A as is usually found in embedded and sectioned material. It is thus concluded that the DNA lies nearly parallel to the axis of these chromatin fibers. Detailed calculations of the polarization expected from various models of DNA packing are contained in an appendix.

  15. Measure Guideline: Evaporative Condensers

    SciTech Connect

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

    2012-03-01

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

  16. Open Problems in $?$ Particle Condensation

    E-print Network

    Y. Funaki; M. Girod; H. Horiuchi; G. Roepke; P. Schuck; A. Tohsaki; T. Yamada

    2010-03-05

    $\\alpha$ particle condensation is a novel state in nuclear systems. We briefly review the present status on the study of $\\alpha$ particle condensation and address the open problems in this research field: $\\alpha$ particle condensation in heavier systems other than the Hoyle state, linear chain and $\\alpha$ particle rings, Hoyle-analogue states with extra neutrons, $\\alpha$ particle condensation related to astrophysics, etc.

  17. Condensate dark matter stars

    SciTech Connect

    Li, X.Y.; Harko, T.; Cheng, K.S., E-mail: lixinyu@hku.hk, E-mail: harko@hkucc.hku.hk, E-mail: hrspksc@hkucc.hku.hk [Department of Physics and Center for Theoretical and Computational Physics, The University of Hong Kong, Pok Fu Lam Road, Hong Kong (China)

    2012-06-01

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

  18. 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

  19. Relationships between chromatin organization and DNA methylation in determining gene expression

    Microsoft Academic Search

    Peter L. Jones; Alan P. Wolffe

    1999-01-01

    Chromatin is the natural substrate for the control of gene expression. Chromatin contains DNA, the transcriptional machinery and structural proteins such as histones. Recent advances demonstrate that transcriptional activity of a gene is largely controlled by the packaging of the template within chromatin. The covalent modification of chromatin provides an attractive mechanism for establishing and maintaining stable states of gene

  20. Condensed tannins act against cattle nematodes.

    PubMed

    Novobilský, Adam; Mueller-Harvey, Irene; Thamsborg, Stig Milan

    2011-12-15

    The use of natural plant anthelmintics was suggested as a possible alternative control of gastrointestinal nematodes (GIN) in ruminants. Direct anthelmintic effects of tannin-containing plants have already been shown in sheep and goat GIN. These anthelmintic properties are mainly associated with condensed tannins. In the present study, we evaluated possible in vitro effects of three tannin-containing plants against bovine GIN. Effects of Onobrychis viciifolia, Lotus pedunculatus and Lotus corniculatus condensed tannin (CT) extracts on Cooperia oncophora and Ostertagia ostertagi were determined by a larval feeding inhibition assay (LFIA) and a larval exsheathment assay (LEA). In the LFIA, all three plant extracts significantly inhibited larval feeding behaviour of both C. oncophora and O. ostertagi first stage larvae in a dose-dependent manner. The L. pedunculatus extract, based on EC(50) (effective concentration for 50% inhibition), was the most effective against both nematodes, followed by O. viciifolia and L. corniculatus. The effect of CT extracts upon larval feeding behaviour correlates with CT content and procyanidin/prodelphidin ratio. Larval exsheathment of C. oncophora and O. ostertagi L3 larvae (third stage larvae) was also affected by CT extracts from all three plants. In both in vitro assays, extracts with added polyvinylpolypyrrolidone, an inhibitor of tannins, generated almost the same values as the negative control; this confirms the role of CT in the anthelmintic effect of these plant extracts. Our results, therefore, indicated that tannin-containing plants could act against cattle nematodes. PMID:21726942

  1. [The condensation mechanism of sodium new houttuyfonate and determination of the chemical structure of condensation products].

    PubMed

    Xu, Rui; Jiang, Ling-Min; He, Jiu-Ming; Liu, Yu-Ling

    2009-06-01

    To study the condensation mechanism of sodium new houttuyfonate, and determinate the chemical structure of condensation products, dimer was prepared, and LC-DAD-MS/MS multiple techniques were employed to investigate the ultraviolet absorption feature and mass spectrum of transformation solution of dimer, and the transformation kinetics and half-life were studied by ultraviolet spectrophotometry. The pure substance of stable condensation product was obtained by extracting with organic solvent and purifying with column chromatography, the chemical structure of this substance was identified by assaying of IR, HR-ESI-MS and NMR, and the data of LC-MS/MS were compared with that of transformation products of dimer. The results indicated that the dimer is unstable, it will be rapidly dissociated in aqueous solution to form free new houttuyfonate and then cycloaddition reaction will occur and followed by an in situ dehydration to generate 1, 3, 5-tri (dodecanoyl) benzene (trimer) with a six-ring which is stable in aqueous solution. The transformation process may fit second-order kinetics, and the half-times were found to be 3.17 hours at 25 degrees C (298 K) and 6.39 min at 100 degrees C (373 K), separately. It suggests that dimer is an intermediate in condensation reaction, and the end condensation product of sodium new houttuyfonate injection may exist as trimer. PMID:19806891

  2. Chromatin dynamics at the Saccharomyces cerevisiae PHO5 promoter

    E-print Network

    Jessen, Walter Joseph

    2006-04-12

    In eukaryotes, the organization of DNA into chromatin is a primary determinant of gene expression. Positioned nucleosomes in promoter regions are frequently found to regulate gene expression by obstructing the accessibility of cis...

  3. METHODOLOGY Open Access Protocol: Chromatin immunoprecipitation (ChIP)

    E-print Network

    Paris-Sud XI, Université de

    species Phaeodactylum tricornutum and Thalassiosira pseudonana. This protocol allows the extraction. Keywords: Phaeodactylum tricornutum, Thalassiosira pseudonana, Histone modifications, Chromatin environments. Completed whole genome sequences from three species, the centric diatom Thalassiosira pseudonana

  4. Chromatin modifying agents - the cutting edge of anticancer therapy.

    PubMed

    Kwa, Faith A A; Balcerczyk, Aneta; Licciardi, Paul; El-Osta, Assam; Karagiannis, Tom C

    2011-07-01

    Chromatin modifying compounds are emerging as the next generation of anticancer therapies. By altering gene expression they could be able to correct uncontrolled proliferation and, in certain cases, aberrant apoptotic pathways, which are hallmarks of malignant cells. The modulation of gene expression is regulated via chromatin remodelling processes that include DNA methylation and chromatin modifications. The identification of aberrant methylation of genes and dysregulated histone acetylation status in cancer cells provides a basis for novel epigenetic therapies. Currently available chromatin modifying agents, a group that includes DNA methyltransferase and histone deacetylase inhibitors, exert anticancer effects by reactivating tumour suppressor genes, inhibiting proliferation and inducing apoptosis. It is anticipated that massive parallel sequencing will identify new epigenetic targets for drug development. PMID:21664485

  5. From neural development to cognition: unexpected roles for chromatin

    PubMed Central

    Ronan, Jehnna L.; Wu, Wei

    2014-01-01

    Recent genome-sequencing studies in human neurodevelopmental and psychiatric disorders have uncovered mutations in many chromatin regulators. These human genetic studies, along with studies in model organisms, are providing insight into chromatin regulatory mechanisms in neural development and how alterations to these mechanisms can cause cognitive deficits, such as intellectual disability. We discuss several implicated chromatin regulators, including BAF (also known as SWI/SNF) and CHD8 chromatin remodellers, HDAC4 and the Polycomb component EZH2. Interestingly, mutations in EZH2 and certain BAF complex components have roles in both neurodevelopmental disorders and cancer, and overlapping point mutations are suggesting functionally important residues and domains. We speculate on the contribution of these similar mutations to disparate disorders. PMID:23568486

  6. KAT5 tyrosine phosphorylation couples chromatin sensing to ATM signalling.

    PubMed

    Kaidi, Abderrahmane; Jackson, Stephen P

    2013-06-01

    The detection of DNA lesions within chromatin represents a critical step in cellular responses to DNA damage. However, the regulatory mechanisms that couple chromatin sensing to DNA-damage signalling in mammalian cells are not well understood. Here we show that tyrosine phosphorylation of the protein acetyltransferase KAT5 (also known as TIP60) increases after DNA damage in a manner that promotes KAT5 binding to the histone mark H3K9me3. This triggers KAT5-mediated acetylation of the ATM kinase, promoting DNA-damage-checkpoint activation and cell survival. We also establish that chromatin alterations can themselves enhance KAT5 tyrosine phosphorylation and ATM-dependent signalling, and identify the proto-oncogene c-Abl as a mediator of this modification. These findings define KAT5 tyrosine phosphorylation as a key event in the sensing of genomic and chromatin perturbations, and highlight a key role for c-Abl in such processes. PMID:23708966

  7. Transcription complex disruption caused by a transition in chromatin structure.

    PubMed Central

    Almouzni, G; Méchali, M; Wolffe, A P

    1991-01-01

    Chromatin structure is known to influence class III gene expression in vitro. We describe the active transcription of Xenopus class III genes following replication and assembly into chromatin by using Xenopus egg extracts. Changes in the structure of this active chromatin dependent on the presence of exogeneous Mg2+ ATP or on the addition of a mixture of histones H2A and H2B are shown to lead to the selective repression of Xenopus 5S RNA genes. Preexisting transcription complexes on 5S DNA are disrupted following the reorganization of a "disordered" histone-DNA complex into a structure consisting of physiologically spaced nucleosomes. Thus, we demonstrate that chromatin structural transitions can have dominant and specific effects on transcription. Images PMID:1990277

  8. Genome-wide analysis of chromatin status using tiling microarrays

    PubMed Central

    Shivaswamy, Sushma; Iyer, Vishwanath R

    2007-01-01

    The eukaryotic genome is packaged into chromatin, and chromatin modification and remodeling play an important role in transcriptional regulation, DNA replication, recombination and repair. Recent findings have shown that various post-translational histone modifications cooperate to recruit different effector proteins that bring about mobilization of the nucleosomes and cause distinct downstream consequences. The combination of chromatin immunoprecipitation (ChIP) using antibodies directed against the core histones or specific histone modifications, with high-resolution tiling microarray analysis allows the examination of nucleosome occupancy and histone modification status genome-wide. Comparing genome-wide chromatin status with global gene expression patterns can reveal causal connections between specific patterns of histone modifications and the resulting gene expression. Here, we describe current methods based on recent advances in microarray technology to conduct such studies. PMID:17309841

  9. Integrative annotation of chromatin elements from ENCODE data

    E-print Network

    Ernst, Jason

    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 ...

  10. Chromatin-modifying enzymes as modulators of reprogramming

    E-print Network

    Onder, Tamer T.

    Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic ...

  11. Reshaping the chromatin landscape after spinal cord injury

    PubMed Central

    WONG, Jamie K.; ZOU, Hongyan

    2014-01-01

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

  12. Insights into Chromatin Structure and Dynamics in Plants

    PubMed Central

    Rosa, Stefanie; Shaw, Peter

    2013-01-01

    The packaging of chromatin into the nucleus of a eukaryotic cell requires an extraordinary degree of compaction and physical organization. In recent years, it has been shown that this organization is dynamically orchestrated to regulate responses to exogenous stimuli as well as to guide complex cell-type-specific developmental programs. Gene expression is regulated by the compartmentalization of functional domains within the nucleus, by distinct nucleosome compositions accomplished via differential modifications on the histone tails and through the replacement of core histones by histone variants. In this review, we focus on these aspects of chromatin organization and discuss novel approaches such as live cell imaging and photobleaching as important tools likely to give significant insights into our understanding of the very dynamic nature of chromatin and chromatin regulatory processes. We highlight the contribution plant studies have made in this area showing the potential advantages of plants as models in understanding this fundamental aspect of biology. PMID:24833230

  13. Regulation of cellular chromatin state: insights from quiescence and differentiation.

    PubMed

    Srivastava, Surabhi; Mishra, Rakesh K; Dhawan, Jyotsna

    2010-01-01

    The identity and functionality of eukaryotic cells is defined not just by their genomic sequence which remains constant between cell types, but by their gene expression profiles governed by epigenetic mechanisms. Epigenetic controls maintain and change the chromatin state throughout development, as exemplified by the setting up of cellular memory for the regulation and maintenance of homeotic genes in proliferating progenitors during embryonic development. Higher order chromatin structure in reversibly arrested adult stem cells also involves epigenetic regulation and in this review we highlight common trends governing chromatin states, focusing on quiescence and differentiation during myogenesis. Together, these diverse developmental modules reveal the dynamic nature of chromatin regulation providing fresh insights into the role of epigenetic mechanisms in potentiating development and differentiation. PMID:20592864

  14. 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

  15. 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

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

    PubMed

    Bergmann, Jan H; Martins, Nuno M C; Larionov, Vladimir; Masumoto, Hiroshi; Earnshaw, William C

    2012-07-01

    The centromere is a specialized chromosomal region that serves as the assembly site of the kinetochore. At the centromere, CENP-A nucleosomes form part of a chromatin landscape termed centrochromatin. This chromatin environment conveys epigenetic marks regulating kinetochore formation. Recent work sheds light on the intricate relationship between centrochromatin state, the CENP-A assembly pathway and the maintenance of centromere function. Here, we review the emerging picture of how chromatin affects mammalian kinetochore formation. We place particular emphasis on data obtained from Human Artificial Chromosome (HAC) biology and the targeted engineering of centrochromatin using synthetic HACs. We discuss implications of these findings, which indicate that a delicate balance of histone modifications and chromatin state dictates both de novo centromere formation and the maintenance of centromere identity in dividing cell populations. PMID:22825423

  17. Sequence analysis of chromatin immunoprecipitation data for factors

    E-print Network

    MacIsaac, Kenzie Daniel

    Chromatin immunoprecipitation (ChIP) experiments allow the location of transcription factors to be determined across the genome. Subsequent analysis of the sequences of the identified regions allows binding to be localized ...

  18. Sperm FISH and chromatin integrity in spermatozoa from a t(6;10;11) carrier.

    PubMed

    Olszewska, Marta; Huleyuk, Nataliya; Fraczek, Monika; Zastavna, Danuta; Wiland, Ewa; Kurpisz, Maciej

    2014-05-01

    Complex chromosome rearrangements (CCRs) are structurally balanced or unbalanced aberrations involving more than two breakpoints on two or more chromosomes. CCRs can be a potential reason for genomic imbalance in gametes, which leads to a drastic reduction in fertility. In this study, the meiotic segregation pattern, aneuploidy of seven chromosomes uninvolved in the CCR and chromatin integrity were analysed in the ejaculated spermatozoa of a 46,XY,t(6;10;11)(q25.1;q24.3;q23.1)mat carrier with asthenozoospermia and a lack of conception. The frequency of genetically unbalanced spermatozoa was 78.8% with a prevalence of 4:2 segregants of 38.2%, while the prevalence of the adjacent 3:3 mode was 35.3%. Analysis of the aneuploidy of chromosomes 13, 15, 18, 21, 22, X and Y revealed an approximately fivefold increased level in comparison with that of the control group, indicating the presence of an interchromosomal effect. Sperm chromatin integrity status was evaluated using chromomycin A3 and aniline blue staining (deprotamination), acridine orange test and TUNEL assay (sperm DNA fragmentation). No differences were found when comparisons were made with a control group. We suggest that the accumulation of genetically unbalanced spermatozoa, significantly increased sperm aneuploidy level and decreased sperm motility (20%, progressive) were not responsible for the observed lack of reproductive success in the analysed infertile t(6;10;11) carrier. Interestingly, in the case described herein, a high level of sperm chromosomal imbalance appears not to be linked to sperm chromatin integrity status. PMID:24713394

  19. Ultraviolet radiation induces structural and chromatin damage in Mediterranean sea-urchin spermatozoa.

    PubMed

    Pruski, Audrey M; Nahon, Sarah; Escande, Marie-Line; Charles, François

    2009-02-19

    There is growing concern about the effects of enhanced levels of solar ultraviolet radiation on the living components of the biosphere (i.e. cancer, loss of biodiversity and productivity, etc.). In shallow coastal environments, many benthic species release their gametes directly in the water column where fertilisation occurs and the planktonic larvae remain for several weeks. Any effects on these early life stages could significantly impair reproductive input or alter the fitness of the community. The purpose of this paper is to provide new insights into the mechanisms of UV toxicity on sea-urchin spermatozoa in a cytological context, and to address the question of the potential ecological consequences of the damage. The Mediterranean sea-urchin Sphaerechinus granularis (Lamarck) was chosen as a model to study the effects of ecologically relevant doses of UV-R on the spermatozoa of marine invertebrates. Structural damage was visualised by use of transmission electron microscopy and the single-cell gel electrophoresis (Comet) assay was used to assess chromatin integrity in spermatozoa. The present results provide experimental evidence that irradiation with UV induces structural and chromatin damage in sea-urchin sperm. Almost 90% of spermatozoa exhibited morphological alterations and DNA strand breakage increased 2-fold. The observed alterations of the acrosome, plasma membrane and mitochondria can explain the concomitant impairment of fertilisation (23% decrease of fertilisation rate), which in turn may affect reproductive success. On the other hand, how DNA damage and fertilisation rate correlate remains unclear; however, when not repaired genetic lesions can lead to abnormal development and/or the transmission of heritable damage. The 3-fold decrease of the frequency of 2-celled embryos indicates a delay or inhibition of the first cell division, which may be ascribed to impairment of nuclear chromatin and/or other cellular targets. PMID:19146985

  20. SPERM CHROMATIN STRUCTURE ASSAY IS USEFUL FOR FERTILITY ASSESSMENT (R827019)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  1. SPERM CHROMATIN STRUCTURE ASSAY PARAMETERS AS PREDICTORS OF FAILED PREGNANCY FOLLOWING ASSISTED REPRODUCTIVE TECHNIQUES. (R827019)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  2. 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...

  3. 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

  4. An oestrogen-receptor-?-bound human chromatin interactome

    Microsoft Academic Search

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

    2009-01-01

    Genomes are organized into high-level three-dimensional structures, and DNA elements separated by long genomic distances can in principle interact functionally. Many transcription factors bind to regulatory DNA elements distant from gene promoters. Although distal binding sites have been shown to regulate transcription by long-range chromatin interactions at a few loci, chromatin interactions and their impact on transcription regulation have not

  5. Regulation of secondary metabolism by chromatin structure and epigenetic codes

    PubMed Central

    Strauss, Joseph; Reyes-Dominguez, Yazmid

    2012-01-01

    Chromatin, composed of DNA wrapped around an octamer of histones, is the relevant substrate for all genetic processes in eukaryotic nuclei. Changes in chromatin structure are associated with the activation and silencing of gene transcription and reversible post-translational modifications of histones are now known to direct chromatin structure transitions. Recent studies in several fungal species have identified a chromatin-based regulation of secondary metabolism (SM) gene clusters representing an upper-hierarchical level for the coordinated control of large chromosomal elements. Regulation by chromatin transition processes provides a mechanistic model to explain how different SM clusters located at dispersed genomic regions can be simultaneously silenced during primary metabolism. Activation of SM clusters has been shown to be associated with increased acetylation of histones H3 and H4 and, consequently, inhibition of histone de-acetylase activities also leads to increased production of secondary metabolites. New findings suggest that SM clusters are silenced by heterochromatic histone marks and that the “closed” heterochromatic structures are reversed during SM activation. This process is mediated by the conserved activator of SM, LaeA. Despite the increase in knowledge about these processes, much remains to be learned from chromatin-level regulation of SM. For example, which proteins “position” the chromatin restructuring signal onto SM clusters or how exactly LaeA works to mediate the low level of heterochromatic marks inside different clusters remain open questions. Answers to these and other chromatin-related questions would certainly complete our understanding of SM gene regulation and signaling and, because for many predicted SM clusters corresponding products have not been identified so far, anti-silencing strategies would open new ways for the identification of novel bioactive substances. PMID:20659575

  6. Schultheiss, Schiepe, & Rawolle Hormone assays 1 Running head: HORMONE ASSAYS

    E-print Network

    Schultheiss, Oliver C.

    Schultheiss, Schiepe, & Rawolle Hormone assays 1 Running head: HORMONE ASSAYS Hormone assays Oliver: Schultheiss, O. C., Schiepe, A., & Rawolle, M. (2012). Hormone assays. In H. Cooper, P. M. Camic, D. L. Long Association. #12;Schultheiss, Schiepe, & Rawolle Hormone assays 2 Hormone assays Hormones can be assayed from

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

    PubMed

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

    2015-02-18

    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. PMID:25563346

  8. 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.

  9. DC Protein Assay Instruction

    E-print Network

    Lebendiker, Mario

    DC Protein Assay Instruction Manual For Technical Service Call Your Local Bio-Rad Office and Principle The Bio-Rad DC Protein Assay is a colorimetric assay for protein concen- tration following preparation Spectrophotometer set to 750 nm 1 #12;Vortex mixer Plastic or glass cuvettes with 1 cm path length

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

    PubMed Central

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

    2014-01-01

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

  11. Accelerated Chromatin Biochemistry Using DNA-Barcoded Nucleosome Libraries

    PubMed Central

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

    2014-01-01

    Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone posttranslational modification (‘PTM’) signatures remains a daunting task in the epigenetics field. Here, we introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semi-synthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries treated with purified chromatin effectors or the combined chromatin recognizing and modifying activities of the nuclear proteome is followed by multiplexed DNA-barcode sequencing. This ultrasensitive workflow allowed us to collect thousands of biochemical data points revealing the binding preferences of various nuclear factors for PTM patterns and how pre-existing PTMs, alone or synergistically, affect further PTM deposition via crosstalk mechanisms. We anticipate that the high-throughput and -sensitivity of the technology will help accelerate the decryption of the diverse molecular controls that operate at the level of chromatin. PMID:24997861

  12. Chd5 orchestrates chromatin remodeling during sperm development

    PubMed Central

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

    2014-01-01

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

  13. 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.

  14. Ordered Nucleation and Spreading of Silenced Chromatin in Saccharomyces cerevisiae

    PubMed Central

    Rusché, Laura N.; Kirchmaier, Ann L.; Rine, Jasper

    2002-01-01

    In Saccharomyces cerevisiae, silencing at the HM loci depends on Sir proteins, which are structural components of silenced chromatin. To explore the structure and assembly of silenced chromatin, the associations of Sir proteins with sequences across the HMR locus were examined by chromatin immunoprecipitation. In wild-type cells, Sir2p, Sir3p, and Sir4p were spread throughout and coincident with the silenced region at HMR. Sir1p, in contrast, associated only with the HMR-E silencer, consistent with its role in establishment but not maintenance of silencing. Sir4p was required for the association of other Sir proteins with silencers. In contrast, in the absence of Sir2p or Sir3p, partial assemblies of Sir proteins could form at silencers, where Sir protein assembly began. Spreading across HMR required Sir2p and Sir3p, as well as the deacetylase activity of Sir2p. These data support a model for the spreading of silenced chromatin involving cycles of nucleosome deacetylation by Sir2p followed by recruitment of additional Sir2p, Sir3p, and Sir4p to the newly deacetylated nucleosome. This model suggests mechanisms for boundary formation, and for maintenance and inheritance of silenced chromatin. The principles are generalizable to other types of heritable chromatin states. PMID:12134062

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

    PubMed

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

    2014-08-01

    Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone post-translational modification (PTM) signatures remains a daunting task in the epigenetics field. We introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semisynthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries, once they have been treated with purified chromatin effectors or the combined chromatin recognizing and modifying activities of the nuclear proteome, is followed by multiplexed DNA-barcode sequencing. This ultrasensitive workflow allowed us to collect thousands of biochemical data points revealing the binding preferences of various nuclear factors for PTM patterns and how preexisting PTMs, alone or synergistically, affect further PTM deposition via cross-talk mechanisms. We anticipate that the high throughput and sensitivity of the technology will help accelerate the decryption of the diverse molecular controls that operate at the level of chromatin. PMID:24997861

  16. 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

  17. A map of open chromatin in human pancreatic islets

    PubMed Central

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

    2010-01-01

    Tissue-specific transcriptional regulation is central to human disease1. To identify regulatory DNA active in human pancreatic islets, we profiled chromatin by FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements)2–4 coupled with high-throughput sequencing. We identified ~80,000 open chromatin sites. Comparison of islet FAIRE-seq to five non-islet cell lines revealed ~3,300 physically linked clusters of islet-selective open chromatin sites, which typically encompassed single genes exhibiting islet-specific expression. We mapped sequence variants to open chromatin sites and found that rs7903146, a TCF7L2 intronic variant strongly associated with type 2 diabetes (T2D)5, is located in islet-selective open chromatin. We show that rs7903146 heterozygotes exhibit allelic imbalance in islet FAIRE signal, and that the variant alters enhancer activity, indicating that genetic variation at this locus acts in cis with local chromatin and regulatory changes. These findings illuminate the tissue-specific organization of cis-regulatory elements, and show that FAIRE-seq can guide identification of regulatory variants important for disease. PMID:20118932

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

    PubMed

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

    2012-01-01

    In order to survive, cells have evolved highly effective repair mechanisms to deal with the potentially lethal DNA damage produced by exposure to endogenous as well as exogenous agents. Ionizing radiation exposure induces highly lethal DNA damage, especially DNA double-strand breaks (DSBs), that is sensed by the cellular machinery and then subsequently repaired by either of two different DSB repair mechanisms: (1) non-homologous end joining, which re-ligates the broken ends of the DNA and (2) homologous recombination, that employs an undamaged identical DNA sequence as a template, to maintain the fidelity of DNA repair. Repair of DSBs must occur within the natural context of the cellular DNA which, along with specific proteins, is organized to form chromatin, the overall structure of which can impede DNA damage site access by repair proteins. The chromatin complex is a dynamic structure and is known to change as required for ongoing cellular processes such as gene transcription or DNA replication. Similarly, during the process of DNA damage sensing and repair, chromatin needs to undergo several changes in order to facilitate accessibility of the repair machinery. Cells utilize several factors to modify the chromatin in order to locally open up the structure to reveal the underlying DNA sequence but post-translational modification of the histone components is one of the primary mechanisms. In this review, we will summarize chromatin modifications by the respective chromatin modifying factors that occur during the DNA damage response. PMID:23346550

  19. A general method for preparing chromatin containing intact DNA.

    PubMed Central

    Jackson, D A; Cook, P R

    1985-01-01

    A simple and general method is described for preparing chromatin from eukaryotic cells using isotonic conditions. First, cells are encapsulated in agarose microbeads and then lysed using Triton X-100 in the presence of a chelating agent and a physiological concentration of salt. Most cytoplasmic proteins and RNA diffuse rapidly out through pores in the beads to leave encapsulated chromatin which is nevertheless completely accessible to enzymes and other probes. This chromatin can be manipulated freely without aggregation in a variety of different salt and detergent concentrations. It also contains intact DNA since removal of the histones releases superhelical DNA. Conditions are described for incubating this chromatin at 37 degrees C in the presence of Mg2+ ions without any nicking of the DNA. We illustrate the usefulness of this chromatin in investigations on the attachment of nascent RNA to the nucleoskeleton, the accessibility of the ribosomal locus to EcoRI and the properties of the endogenous RNA polymerase II. This type of chromatin preparation should prove useful for both structural and functional studies. Images Fig. 1. Fig. 2. Fig. 5. PMID:3894011

  20. 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

  1. Epigenetic regulation of open chromatin in pluripotent stem cells.

    PubMed

    Kobayashi, Hiroshi; Kikyo, Nobuaki

    2015-01-01

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

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

    PubMed Central

    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

    2009-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  4. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation.

    PubMed

    Wang, Yanming; Li, Ming; Stadler, Sonja; Correll, Sarah; Li, Pingxin; Wang, Danchen; Hayama, Ryo; Leonelli, Lauriebeth; Han, Hyunsil; Grigoryev, Sergei A; Allis, C David; Coonrod, Scott A

    2009-01-26

    Peripheral blood neutrophils form highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs), that have been implicated in innate immune response to bacterial infection. Neutrophils express high levels of peptidylarginine deiminase 4 (PAD4), which catalyzes histone citrullination. However, whether PAD4 or histone citrullination plays a role in chromatin structure in neutrophils is unclear. In this study, we show that the hypercitrullination of histones by PAD4 mediates chromatin decondensation. Histone hypercitrullination is detected on highly decondensed chromatin in HL-60 granulocytes and blood neutrophils. The inhibition of PAD4 decreases histone hypercitrullination and the formation of NET-like structures, whereas PAD4 treatment of HL-60 cells facilitates these processes. The loss of heterochromatin and multilobular nuclear structures is detected in HL-60 granulocytes after PAD4 activation. Importantly, citrullination of biochemically defined avian nucleosome arrays inhibits their compaction by the linker histone H5 to form higher order chromatin structures. Together, these results suggest that histone hypercitrullination has important functions in chromatin decondensation in granulocytes/neutrophils. PMID:19153223

  5. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation

    PubMed Central

    Li, Ming; Stadler, Sonja; Correll, Sarah; Li, Pingxin; Wang, Danchen; Hayama, Ryo; Leonelli, Lauriebeth; Han, Hyunsil; Grigoryev, Sergei A.; Allis, C. David; Coonrod, Scott A.

    2009-01-01

    Peripheral blood neutrophils form highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs), that have been implicated in innate immune response to bacterial infection. Neutrophils express high levels of peptidylarginine deiminase 4 (PAD4), which catalyzes histone citrullination. However, whether PAD4 or histone citrullination plays a role in chromatin structure in neutrophils is unclear. In this study, we show that the hypercitrullination of histones by PAD4 mediates chromatin decondensation. Histone hypercitrullination is detected on highly decondensed chromatin in HL-60 granulocytes and blood neutrophils. The inhibition of PAD4 decreases histone hypercitrullination and the formation of NET-like structures, whereas PAD4 treatment of HL-60 cells facilitates these processes. The loss of heterochromatin and multilobular nuclear structures is detected in HL-60 granulocytes after PAD4 activation. Importantly, citrullination of biochemically defined avian nucleosome arrays inhibits their compaction by the linker histone H5 to form higher order chromatin structures. Together, these results suggest that histone hypercitrullination has important functions in chromatin decondensation in granulocytes/neutrophils. PMID:19153223

  6. Chromatin structure in double strand break repair.

    PubMed

    Gospodinov, Anastas; Herceg, Zdenko

    2013-10-01

    Cells are under constant assault by endogenous and environmental DNA damaging agents. DNA double strand breaks (DSBs) sever entire chromosomes and pose a major threat to genome integrity as a result of chromosomal fragment loss or chromosomal rearrangements. Exogenous factors such as ionizing radiation, crosslinking agents, and topoisomerase poisons, contribute to break formation. DSBs are associated with oxidative metabolism, form during the normal S phase, when replication forks collapse and are generated during physiological processes such as V(D)J recombination, yeast mating type switching and meiosis. It is estimated that in mammalian cells ?10 DSBs per cell are formed daily. If left unrepaired DSBs can lead to cell death or deregulated growth, and cancer development. Cellular response to DSB damage includes mechanisms to halt the progression of the cell cycle and to restore the structure of the broken chromosome. Changes in chromatin adjacent to DNA break sites are instrumental to the DNA damage response (DDR) with two apparent ends: to control compaction and to bind repair and signaling molecules to the lesion. Here, we review the key findings related to each of these functions and examine their cross-talk. PMID:23919923

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

    PubMed

    Lin, Xin; Tirichine, Leïla; Bowler, Chris

    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

  8. Significance of extruded nuclear chromatin (regional nuclear shape malformation) in human spermatozoa: implications for ICSI.

    PubMed

    Mauri, A L; Oliveira, J B A; Baruffi, R L R; Petersen, C G; Vagnini, L D; Massaro, F C; Silva, L F I; Nicoletti, A P M; Franco, J G

    2011-12-01

    The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-strand or normal double-strand DNA in spermatozoa with extruded nuclear chromatin (ENC) selected by high magnification. Fresh semen samples from 55 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and ENC were selected at 8400× magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation was not significantly different (p = 0.86) between spermatozoa with ENC (19.6%) and those with NN (20%). However, the percentage of spermatozoa with detectable denatured-stranded DNA in the ENC spermatozoon group (59.1%) was significantly higher (p < 0.0001) than in the NN group (44.9%). The high level of denatured DNA in spermatozoa with ENC suggests premature decondensation and disaggregation of sperm chromatin fibres. The results show an association between ENC and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high-magnification intracytoplasmic sperm injection. PMID:20969600

  9. Evidence that non-caspase proteases are required for chromatin degradation during apoptosis.

    PubMed

    Hughes, F M; Evans-Storms, R B; Cidlowski, J A

    1998-12-01

    Chromatin degradation into oligonucleosomal and approximately 30-50 Kb fragments is a hallmark of apoptosis. Crude nuclear extract from apoptotic rat thymocytes is able to recapitulate both types of DNA fragmentation in an assay using HeLa cell nuclei as an exogenous substrate. Using size exclusion chromatography we have identified a novel activity (approximately 260 Kd) that produces only approximately 30-50 Kb DNA fragments, and a 25 Kd activity that generates both approximately 30-50 Kb and oligonucleosomal fragments. Both activities produced DNA fragments with 3'-OH termini, are dependent on Ca2+ and Mg2+ and are inhibited by N-ethyl-maleimide, sodium tetrathionate, aurintricarboxylic acid and sodium chloride, similar to other nucleases implicated in apoptosis. These activities were inhibited by the serine protease inhibitors N-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone, but not by the serine protease inhibitor diisopropyl fluorophosphate, or by calpain inhibitors I or II, or the capsase inhibitors Ac-Asp-Glu-Val-Asp-aldehyde, Ac-Tyr-Val-Ala-Asp-aldehyde, or Z-Val-Ala-Asp-fluoromethyl ketone. Both activities were insensitive to protease inhibitors when extracts were incubated with naked linear DNA, indicating the presence of both nuclease and protease activities in the preparation. Together, these observations suggest the involvement of non-caspase proteases in apoptosis which perhaps function by altering chromatin substructure and exposing it to nucleolytic attack. PMID:9894608

  10. Functional studies of MP62 during male chromatin decondensation in sea urchins.

    PubMed

    Iribarren, Claudio; Hermosilla, Viviana; Morin, Violeta; Puchi, Marcia

    2013-08-01

    In amphibians, sperm histone transition post-fertilization during male pronucleus formation is commanded by histone chaperone Nucleoplasmin (NPM). Here, we report the first studies to analyze the participation of a Nucleoplasmin-like protein on male chromatin remodeling in sea urchins. In this report, we present the molecular characterization of a nucleoplasmin-like protein that is present in non fertilized eggs and early zygotes in sea urchin specie Tetrapygus niger. This protein, named MP62 can interact with sperm histones in vitro. By male chromatin decondensation assays and immunodepletion experiments in vitro, we have demonstrated that this protein is responsible for sperm nucleosome disorganization. Furthermore, as amphibian nucleoplasmin MP62 is phosphorylated in vivo immediately post-fertilization and this phosphorylation is dependent on CDK-cyclin activities found after fertilization. As we shown, olomoucine and roscovitine inhibits male nucleosome decondensation, sperm histone replacement in vitro and MP62 phosphorylation in vivo. This is the first report of a nucleoplasmin-like activity in sea urchins participating during male pronucleus formation post-fecundation. PMID:23444173

  11. Chromatin Redistribution of the DEK Oncoprotein Represses hTERT Transcription in Leukemias12

    PubMed Central

    Karam, Maroun; Thenoz, Morgan; Capraro, Valérie; Robin, Jean-Philippe; Pinatel, Christiane; Lancon, Agnès; Galia, Perrine; Sibon, David; Thomas, Xavier; Ducastelle-Lepretre, Sophie; Nicolini, Franck; El-Hamri, Mohamed; Chelghoun, Youcef; Wattel, Eric; Mortreux, Franck

    2014-01-01

    Although numerous factors have been found to modulate hTERT transcription, the mechanism of its repression in certain leukemias remains unknown. We show here that DEK represses hTERT transcription through its enrichment on the hTERT promoter in cells from chronic and acute myeloid leukemias, chronic lymphocytic leukemia, but not acute lymphocytic leukemias where hTERT is overexpressed. We isolated DEK from the hTERT promoter incubated with nuclear extracts derived from fresh acute myelogenous leukemia (AML) cells and from cells expressing Tax, an hTERT repressor encoded by the human T cell leukemia virus type 1. In addition to the recruitment of DEK, the displacement of two potent known hTERT transactivators from the hTERT promoter characterized both AML cells and Tax-expressing cells. Reporter and chromatin immunoprecipitation assays permitted to map the region that supports the repressive effect of DEK on hTERT transcription, which was proportionate to the level of DEK-promoter association but not with the level of DEK expression. Besides hTERT repression, this context of chromatin redistribution of DEK was found to govern about 40% of overall transcriptional modifications, including those of cancer-prone genes. In conclusion, DEK emerges as an hTERT repressor shared by various leukemia subtypes and seems involved in the deregulation of numerous genes associated with leukemogenesis. PMID:24563617

  12. Chromatin remodeling of the interleukin-2 gene: distinct alterations in the proximal versus distal enhancer regions.

    PubMed Central

    Ward, S B; Hernandez-Hoyos, G; Chen, F; Waterman, M; Reeves, R; Rothenberg, E V

    1998-01-01

    Known transcription factor-DNA interactions in the minimal enhancer of the murine interleukin-2 gene (IL-2) do not easily explain the T cell specificity of IL-2 regulation. To seek additional determinants of cell type specificity, in vivo methodologies were employed to examine chromatin structure 5' and 3' of the 300 bp IL-2 proximal promoter/enhancer region. Restriction enzyme accessibility revealed that until stimulation the IL-2 proximal promoter/enhancer exists in a closed conformation in resting T and non-T cells alike. Within this promoter region, DMS and DNase I genomic footprinting also showed no tissue-specific differences prior to stimulation. However, DNase I footprinting of the distal -600 to -300 bp region revealed multiple tissue-specific and stimulation-independent DNase I hypersensitive sites. Gel shift assays detected T cell-specific complexes binding within this region, which include TCF/LEF or HMG family and probable Oct family components. Upon stimulation, new DNase I hypersensitive sites appeared in both the proximal and distal enhancer regions, implying that there may be a functional interaction between these two domains. These studies indicate that a region outside the established IL-2 minimal enhancer may serve as a stable nucleation site for tissue-specific factors and as a potential initiation site for activation-dependent chromatin remodeling. PMID:9611237

  13. Chromatin immunoprecipitation coupled by quantitative real-time PCR as a tool for analyzing epigenetic regulation of stem cell aging.

    PubMed

    Lee, Seunghee; Jung, Ji-Won; Kang, Kyung-Sun

    2013-01-01

    Epigenetic regulation is one of the major players in gene expression control. Histone modifications including acetylation or methylation are epigenetic mechanisms known to regulate senescence associated genes expression during adult stem cell aging. Chromatin immunoprecipitation (ChIP) assay is widely used to investigate histone modification patterns of genes of interest. For ChIP analysis, there are several conditions to be optimized empirically. In this article, not only a method of ChIP coupled by real-time quantitative PCR but also the detailed conditions is provided based on our previously published studies. PMID:23400441

  14. 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

  15. 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.

  16. Simple simulations of DNA condensation.

    PubMed Central

    Stevens, M J

    2001-01-01

    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 phenomenon 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. PMID:11159388

  17. 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 ...

  18. Condensation phenomena in plasmonics

    NASA Astrophysics Data System (ADS)

    Martikainen, J.-P.; Heikkinen, M. O. J.; Törmä, P.

    2014-11-01

    We study arrays of plasmonic nanoparticles combined with quantum emitters, quantum plasmonic lattices, as a platform for room-temperature studies of quantum many-body physics. We outline a theory to describe surface plasmon-polariton distributions when they are coupled to externally pumped molecules. The possibility of tailoring the dispersion in plasmonic lattices allows realization of a variety of distributions, including the Bose-Einstein distribution as in photon condensation [Klaers et al., Nature (London) 468, 545 (2010), 10.1038/nature09567]. We show that the presence of losses can relax some of the standard dimensionality restrictions for condensation.

  19. Containment condensing heat transfer

    NASA Astrophysics Data System (ADS)

    Gido, R. G.; Koestel, A.

    A mechanistic heat transfer model that is valid for large scale containment heat sinks was presented. The model development is based on the determination that the condensation is controlled by mass diffusion through the vapor-air boundary layer, and the application of the classic Reynolds' analog to formulate expressions for the transfer of heat and mass based on hydrodynamic measurements of the momentum transfer. As a result, the analysis depends on the quantification of the shear stress (momentum transfer) at the interface between the condensate film and the vapor-air boundary layer. In addition, the currently used Tagami and Uchida test observations and their range of applicability are explained.

  20. Galaxies as condensates

    E-print Network

    D. V. Bugg

    2012-12-21

    A novel interpretation of MOND is presented. For galactic data, in addition to Newtonian acceleration, there is an attractive acceleration peaking at Milgrom's parameter a_0. The peak lies within experimental error where a_0 = cH_0/2\\pi and H_0 is the present-time value of the Hubble constant. This peaking may be understood in terms of quantum mechanical mixing between Newtonian gravitation and the condensation mechanism. There are five pointers towards galaxies being Fermi-Dirac condensates.

  1. 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

  2. The NuRD chromatin-remodeling enzyme CHD4 promotes embryonic vascular integrity by transcriptionally regulating extracellular matrix proteolysis.

    PubMed

    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

  3. 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

  4. Caspase-activated DNase is necessary and sufficient for oligonucleosomal DNA breakdown, but not for chromatin disassembly during caspase-dependent apoptosis of LN-18 glioblastoma cells.

    PubMed

    Sánchez-Osuna, María; Garcia-Belinchón, Mercè; Iglesias-Guimarais, Victoria; Gil-Guiñón, Estel; Casanelles, Elisenda; Yuste, Victor J

    2014-07-01

    Caspase-dependent apoptosis is a controlled type of cell death characterized by oligonucleosomal DNA breakdown and major nuclear morphological alterations. Other kinds of cell death do not share these highly distinctive traits because caspase-activated DNase (DFF40/CAD) remains inactive. Here, we report that human glioblastoma multiforme-derived LN-18 cells do not hydrolyze DNA into oligonucleosomal fragments after apoptotic insult. Furthermore, their chromatin remains packaged into a single mass, with no signs of nuclear fragmentation. However, ultrastructural analysis reveals that nuclear disassembly occurs, although compacted chromatin does not localize into apoptotic nuclear bodies. Caspases become properly activated, and ICAD, the inhibitor of DFF40/CAD, is correctly processed. Using cell-free in vitro assays, we show that chromatin from isolated nuclei of LN-18 cells is suitable for hydrolysis into oligonuclesomal fragments by staurosporine-pretreated SH-SY5Y cytoplasms. However, staurosporine-pretreated LN-18 cytoplasms do not induce DNA laddering in isolated nuclei from either LN-18 or SH-SY5Y cells because LN-18 cells express lower amounts of DFF40/CAD. DFF40/CAD overexpression makes LN-18 cells fully competent to degrade their DNA into oligonucleosome-sized fragments, and yet they remain unable to arrange their chromatin into nuclear clumps after apoptotic insult. Indeed, isolated nuclei from LN-18 cells were resistant to undergoing apoptotic nuclear morphology in vitro. The use of LN-18 cells has uncovered a previously unsuspected cellular model, whereby a caspase-dependent chromatin package is DFF40/CAD-independent, and DFF40/CAD-mediated double-strand DNA fragmentation does not warrant the distribution of the chromatin into apoptotic nuclear bodies. The studies highlight a not-yet reported DFF40/CAD-independent mechanism driving conformational nuclear changes during caspase-dependent cell death. PMID:24838313

  5. Rejoining and misrejoining of radiation-induced chromatin breaks. IV. Charged particles

    NASA Technical Reports Server (NTRS)

    Durante, M.; Furusawa, Y.; George, K.; Gialanella, G.; Greco, O.; Grossi, G.; Matsufuji, N.; Pugliese, M.; Yang, T. C.

    1998-01-01

    We have recently reported the kinetics of chromosome rejoining and exchange formation in human lymphocytes exposed to gamma rays using the techniques of fluorescence in situ hybridization (FISH) and premature chromosome condensation (PCC). In this paper, we have extended previous measurements to cells exposed to charged particles. Our goal was to determine differences in chromatin break rejoining and misrejoining after exposure to low- and high-linear energy transfer (LET) radiation. Cells were irradiated with hydrogen, neon, carbon or iron ions in the LET range 0.3-140 keV/microm and were incubated at 37 degrees C for various times after exposure. Little difference was observed in the yield of early prematurely condensed chromosome breaks for the different ions. The kinetics of break rejoining was exponential for all ions and had similar time constants, but the residual level of unrejoined breaks after prolonged incubation was higher for high-LET radiation. The kinetics of exchange formation was also similar for the different ions, but the yield of chromosome interchanges measured soon after exposure was higher for high-LET particles, suggesting that a higher fraction of DNA breaks are misrejoined quickly. On the other hand, the rate of formation of complete exchanges was slightly lower for densely ionizing radiation. The ratios between the yields of different types of aberrations observed at 10 h postirradiation in prematurely condensed chromosome preparations were dependent on LET. We found significant differences between the yields of aberrations measured in interphase (after repair) and metaphase for densely ionizing radiation. This difference might be caused by prolonged mitotic delay and/or interphase death. Overall, the results point out significant differences between low- and high-LET radiation for the formation of chromosome aberrations.

  6. Analytical modeling of water condensation in condensing heat exchanger

    Microsoft Academic Search

    Kwangkook Jeong; Michael J. Kessen; Harun Bilirgen; Edward K. Levy

    2010-01-01

    An analytical model of heat and mass transfer processes in a flue gas condensing heat exchanger system was developed to predict the heat transferred from flue gas to cooling water and the condensation rate of water vapor in the flue gas. Flue gas exit temperature, cooling water outlet temperature, water vapor mole fraction, and condensation rate of water vapor were

  7. Exciton-polariton condensates

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

  8. The Color Glass Condensate

    E-print Network

    F. Gelis; E. Iancu; J. Jalilian-Marian; R. Venugopalan

    2010-02-01

    We provide a broad overview of the theoretical status and phenomenological applications of the Color Glass Condensate effective field theory describing universal properties of saturated gluons in hadron wavefunctions that are extracted from deeply inelastic scattering and hadron-hadron collision experiments at high energies.

  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. The Newcastle condensation survey

    Microsoft Academic Search

    G. K. Preston; C. J. Stephens; G. W. Brundrett

    1981-01-01

    The Housing Management Committee of the City of Newcastle wished to find some basis on which to establish priorities for remedial and up-grading work on existing houses. With the assistance of the Director of Housing three surveys were commissioned, two of which were specialist ones concentrating on over 600 dwellings sited in an area known to have condensation problems.The first

  11. 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...

  12. 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.

  13. Condenser biofouling control

    SciTech Connect

    Garey, J.F.; Jorden, R.M.; Aitken, A.H.; Burton, D.T.; Gray, R.H. (eds.)

    1980-01-01

    A compilation of most of the papers presented at the March 1979 symposium in Atlanta, GA on condensor biofouling control is presented. The following aspects of power plant condenser biofouling are discussed: effects on power plant equipment and operations; cost impact; biofilm formation; corrosive effects; control practices using chlorine or bromine chlorides, and the dechlorination of discharges of chlorinated coolants. (LCL)

  14. Diversity and evolution of chromatin proteins encoded by DNA viruses

    PubMed Central

    de Souza, Robson F.; Iyer, Lakshminarayan M.; Aravind, L.

    2011-01-01

    Double-stranded DNA viruses display a great variety of proteins that interact with host chromatin. Using the wealth of available genomic and functional information, we have systematically surveyed chromatin-related proteins encoded by dsDNA viruses. The distribution of viral chromatin-related proteins is primarily influenced by viral genome size and the superkingdom to which the host of the virus belongs. Smaller viruses usually encode multifunctional proteins that mediate several distinct interactions with host chromatin proteins and viral or host DNA. Larger viruses additionally encode several enzymes, which catalyze manipulations of chromosome structure, chromatin remodeling and covalent modifications of proteins and DNA. Among these viruses, it is also common to encounter transcription factors and DNA-packaging proteins such as histones and IHF/HU derived from cellular genomes, which might play a role in constituting virus-specific chromatin states. Through all size ranges a subset of domains in viral chromatin proteins appear to have been derived from those found in host proteins. Examples include the Zn-finger domains of the E6 and E7 proteins of papillomaviruses, SET-domain methyltransferases and Jumonji-related demethylases in certain nucleocytoplasmic large DNA viruses and BEN domains in poxviruses and polydnaviruses. In other cases, chromatin-interacting modules, such as the LxCxE motif, appear to have been widely disseminated across distinct viral lineages, resulting in similar retinoblastoma targeting strategies. Viruses, especially those with large linear genomes, have evolved a number of mechanisms to manipulate viral chromosomes in the process of replication-associated recombination. These include topoisomerases, Rad50/SbcC-like ABC ATPases and a novel recombinase system in bacteriophages utilizing RecA and Rad52 homologs. Larger DNA viruses also encode SWI2/SNF2 and A18-like ATPases which appear to play specialized roles in transcription and recombination. Finally, it also appears that certain domains of viral provenance have given rise to key functions in eukaryotic chromatin such as a HEH domain of chromosome tethering proteins and the TET/JBP-like cytosine and thymine hydroxylases. PMID:19878744

  15. Chromatin Insulator Factors Involved in Long-Range DNA Interactions and Their Role in the Folding of the Drosophila Genome

    PubMed Central

    Dejardin, Stephanie; Allemand, Frederic; Gamot, Adrien; Labesse, Gilles; Cuvier, Olivier; Nègre, Nicolas; Cohen-Gonsaud, Martin; Margeat, Emmanuel; Nöllmann, Marcelo

    2014-01-01

    Chromatin insulators are genetic elements implicated in the organization of chromatin and the regulation of transcription. In Drosophila, different insulator types were characterized by their locus-specific composition of insulator proteins and co-factors. Insulators mediate specific long-range DNA contacts required for the three dimensional organization of the interphase nucleus and for transcription regulation, but the mechanisms underlying the formation of these contacts is currently unknown. Here, we investigate the molecular associations between different components of insulator complexes (BEAF32, CP190 and Chromator) by biochemical and biophysical means, and develop a novel single-molecule assay to determine what factors are necessary and essential for the formation of long-range DNA interactions. We show that BEAF32 is able to bind DNA specifically and with high affinity, but not to bridge long-range interactions (LRI). In contrast, we show that CP190 and Chromator are able to mediate LRI between specifically-bound BEAF32 nucleoprotein complexes in vitro. This ability of CP190 and Chromator to establish LRI requires specific contacts between BEAF32 and their C-terminal domains, and dimerization through their N-terminal domains. In particular, the BTB/POZ domains of CP190 form a strict homodimer, and its C-terminal domain interacts with several insulator binding proteins. We propose a general model for insulator function in which BEAF32/dCTCF/Su(HW) provide DNA specificity (first layer proteins) whereas CP190/Chromator are responsible for the physical interactions required for long-range contacts (second layer). This network of organized, multi-layer interactions could explain the different activities of insulators as chromatin barriers, enhancer blockers, and transcriptional regulators, and suggest a general mechanism for how insulators may shape the organization of higher-order chromatin during cell division. PMID:25165871

  16. 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

  17. Chromatin is an ancient innovation conserved between Archaea and Eukarya

    PubMed Central

    Ammar, Ron; Torti, Dax; Tsui, Kyle; Gebbia, Marinella; Durbic, Tanja; Bader, Gary D; Giaever, Guri; Nislow, Corey

    2012-01-01

    The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps ?147 bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have historically been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-depleted regions at transcriptional start sites and conserved ?1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient. DOI: http://dx.doi.org/10.7554/eLife.00078.001 PMID:23240084

  18. Stepwise assembly of chromatin during DNA replication in vitro.

    PubMed Central

    Smith, S; Stillman, B

    1991-01-01

    A cell free system that supports replication-dependent chromatin assembly has been used to determine the mechanism of histone deposition during DNA replication. CAF-I, a human cell nuclear factor, promotes chromatin assembly on replicating SV40 DNA in the presence of a crude cytosol replication extract. Biochemical fractionation of the cytosol extract has allowed separation of the chromatin assembly reaction into two steps. During the first step, CAF-I targets the deposition of newly synthesized histones H3 and H4 to the replicating DNA. This reaction is dependent upon and coupled with DNA replication, and utilizes the newly synthesized forms of histones H3 and H4, which unlike bulk histone found in chromatin, do not bind to DNA by themselves. The H3/H4-replicated DNA complex is a stable intermediate which exhibits a micrococcal nuclease resistant structure and can be isolated by sucrose gradient sedimentation. In the second step, this replicated precursor is converted to mature chromatin by the addition of histones H2A and H2B in a reaction that can occur after DNA replication. The requirement for CAF-I in at least the first step of the reaction suggests a level of cellular control for this fundamental process. Images PMID:1849080

  19. 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

  20. Looking at plant cell cycle from the chromatin window

    PubMed Central

    Desvoyes, Bénédicte; Fernández-Marcos, María; Sequeira-Mendes, Joana; Otero, Sofía; Vergara, Zaida; Gutierrez, Crisanto

    2014-01-01

    The cell cycle is defined by a series of complex events, finely coordinated through hormonal, developmental and environmental signals, which occur in a unidirectional manner and end up in producing two daughter cells. Accumulating evidence reveals that chromatin is not a static entity throughout the cell cycle. In fact, there are many changes that include nucleosome remodeling, histone modifications, deposition and exchange, among others. Interestingly, it is possible to correlate the occurrence of several of these chromatin-related events with specific processes necessary for cell cycle progression, e.g., licensing of DNA replication origins, the E2F-dependent transcriptional wave in G1, the activation of replication origins in S-phase, the G2-specific transcription of genes required for mitosis or the chromatin packaging occurring in mitosis. Therefore, an emerging view is that chromatin dynamics must be considered as an intrinsic part of cell cycle regulation. In this article, we review the main features of several key chromatin events that occur at defined times throughout the cell cycle and discuss whether they are actually controlling the transit through specific cell cycle stages. PMID:25120553

  1. H4K20 methylation regulates quiescence and chromatin compaction

    PubMed Central

    Evertts, Adam G.; Manning, Amity L.; Wang, Xin; Dyson, Nicholas J.; Garcia, Benjamin A.; Coller, Hilary A.

    2013-01-01

    The transition between proliferation and quiescence is frequently associated with changes in gene expression, extent of chromatin compaction, and histone modifications, but whether changes in chromatin state actually regulate cell cycle exit with quiescence is unclear. We find that primary human fibroblasts induced into quiescence exhibit tighter chromatin compaction. Mass spectrometry analysis of histone modifications reveals that H4K20me2 and H4K20me3 increase in quiescence and other histone modifications are present at similar levels in proliferating and quiescent cells. Analysis of cells in S, G2/M, and G1 phases shows that H4K20me1 increases after S phase and is converted to H4K20me2 and H4K20me3 in quiescence. Knockdown of the enzyme that creates H4K20me3 results in an increased fraction of cells in S phase, a defect in exiting the cell cycle, and decreased chromatin compaction. Overexpression of Suv4-20h1, the enzyme that creates H4K20me2 from H4K20me1, results in G2 arrest, consistent with a role for H4K20me1 in mitosis. The results suggest that the same lysine on H4K20 may, in its different methylation states, facilitate mitotic functions in M phase and promote chromatin compaction and cell cycle exit in quiescent cells. PMID:23924899

  2. H4K20 methylation regulates quiescence and chromatin compaction.

    PubMed

    Evertts, Adam G; Manning, Amity L; Wang, Xin; Dyson, Nicholas J; Garcia, Benjamin A; Coller, Hilary A

    2013-10-01

    The transition between proliferation and quiescence is frequently associated with changes in gene expression, extent of chromatin compaction, and histone modifications, but whether changes in chromatin state actually regulate cell cycle exit with quiescence is unclear. We find that primary human fibroblasts induced into quiescence exhibit tighter chromatin compaction. Mass spectrometry analysis of histone modifications reveals that H4K20me2 and H4K20me3 increase in quiescence and other histone modifications are present at similar levels in proliferating and quiescent cells. Analysis of cells in S, G2/M, and G1 phases shows that H4K20me1 increases after S phase and is converted to H4K20me2 and H4K20me3 in quiescence. Knockdown of the enzyme that creates H4K20me3 results in an increased fraction of cells in S phase, a defect in exiting the cell cycle, and decreased chromatin compaction. Overexpression of Suv4-20h1, the enzyme that creates H4K20me2 from H4K20me1, results in G2 arrest, consistent with a role for H4K20me1 in mitosis. The results suggest that the same lysine on H4K20 may, in its different methylation states, facilitate mitotic functions in M phase and promote chromatin compaction and cell cycle exit in quiescent cells. PMID:23924899

  3. Histone modifications and chromatin dynamics: a focus on filamentous fungi

    PubMed Central

    Brosch, Gerald; Loidl, Peter; Graessle, Stefan

    2008-01-01

    The readout of the genetic information of eukaryotic organisms is significantly regulated by modifications of DNA and chromatin proteins. Chromatin alterations induce genome-wide and local changes in gene expression and affect a variety of processes in response to internal and external signals during growth, differentiation, development, in metabolic processes, diseases, and abiotic and biotic stresses. This review aims at summarizing the roles of histone H1 and the acetylation and methylation of histones in filamentous fungi and links this knowledge to the huge body of data from other systems. Filamentous fungi show a wide range of morphologies and have developed a complex network of genes that enables them to use a great variety of substrates. This fact, together with the possibility of simple and quick genetic manipulation, highlights these organisms as model systems for the investigation of gene regulation. However, little is still known about regulation at the chromatin level in filamentous fungi. Understanding the role of chromatin in transcriptional regulation would be of utmost importance with respect to the impact of filamentous fungi in human diseases and agriculture. The synthesis of compounds (antibiotics, immunosuppressants, toxins, and compounds with adverse effects) is also likely to be regulated at the chromatin level. PMID:18221488

  4. The molecular topography of silenced chromatin in Saccharomyces cerevisiae

    PubMed Central

    Thurtle, Deborah M.; Rine, Jasper

    2014-01-01

    Heterochromatin imparts regional, promoter-independent repression of genes and is epigenetically heritable. Understanding how silencing achieves this regional repression is a fundamental problem in genetics and development. Current models of yeast silencing posit that Sir proteins, recruited by transcription factors bound to the silencers, spread throughout the silenced region. To test this model directly at high resolution, we probed the silenced chromatin architecture by chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) of Sir proteins, histones, and a key histone modification, H4K16-acetyl. These analyses revealed that Sir proteins are strikingly concentrated at and immediately adjacent to the silencers, with lower levels of enrichment over the promoters at HML and HMR, the critical targets for transcriptional repression. The telomeres also showed discrete peaks of Sir enrichment yet a continuous domain of hypoacetylated histone H4K16. Surprisingly, ChIP-seq of cross-linked chromatin revealed a distribution of nucleosomes at silenced loci that was similar to Sir proteins, whereas native nucleosome maps showed a regular distribution throughout silenced loci, indicating that cross-linking captured a specialized chromatin organization imposed by Sir proteins. This specialized chromatin architecture observed in yeast informs the importance of a steric contribution to regional repression in other organisms. PMID:24493645

  5. Rad51 polymerization reveals a new chromatin remodeling mechanism.

    PubMed

    Dupaigne, Pauline; Lavelle, Christophe; Justome, Anthony; Lafosse, Sophie; Mirambeau, Gilles; Lipinski, Marc; Piétrement, Olivier; Le Cam, Eric

    2008-01-01

    Rad51 protein is a well known protagonist of homologous recombination in eukaryotic cells. Rad51 polymerization on single-stranded DNA and its role in presynaptic filament formation have been extensively documented. Rad51 polymerizes also on double-stranded DNA but the significance of this filament formation remains unclear. We explored the behavior of Saccharomyces cerevisiae Rad51 on dsDNA and the influence of nucleosomes on Rad51 polymerization mechanism to investigate its putative role in chromatin accessibility to recombination machinery. We combined biochemical approaches, transmission electron microscopy (TEM) and atomic force microscopy (AFM) for analysis of the effects of the Rad51 filament on chromatinized templates. Quantitative analyses clearly demonstrated the occurrence of chromatin remodeling during nucleoprotein filament formation. During Rad51 polymerization, recombinase proteins moved all the nucleosomal arrays in front of the progressing filament. This polymerization process had a powerful remodeling effect, as Rad51 destabilized the nucleosomes along considerable stretches of DNA. Similar behavior was observed with RecA. Thus, recombinase polymerization is a powerful mechanism of chromatin remodeling. These remarkable features open up new possibilities for understanding DNA recombination and reveal new types of ATP-dependent chromatin dynamics. PMID:18982066

  6. Chromatin: a tunable spring at work inside chromosomes

    E-print Network

    Eli Ben-Haïm; Annick Lesne; Jean-Marc Victor

    2001-10-11

    This paper focuses on mechanical aspects of chromatin biological functioning. Within a basic geometric modeling of the chromatin assembly, we give for the first time the complete set of elastic constants (twist and bend persistence lengths, stretch modulus and twist-stretch coupling constant) of the so-called 30-nm chromatin fiber, in terms of DNA elastic properties and geometric properties of the fiber assembly. The computation naturally embeds the fiber within a current analytical model known as the ``extensible worm-like rope'', allowing a straightforward prediction of the force-extension curves. We show that these elastic constants are strongly sensitive to the linker length, up to 1 bp, or equivalently to its twist, and might locally reach very low values, yielding a highly flexible and extensible domain in the fiber. In particular, the twist-stretch coupling constant, reflecting the chirality of the chromatin fiber, exhibits steep variations and sign changes when the linker length is varied. We argue that this tunable elasticity might be a key feature for chromatin function, for instance in the initiation and regulation of transcription.

  7. Forced unraveling of chromatin fibers with nonuniform linker DNA lengths

    NASA Astrophysics Data System (ADS)

    Ozer, Gungor; Collepardo-Guevara, Rosana; Schlick, Tamar

    2015-02-01

    The chromatin fiber undergoes significant structural changes during the cell's life cycle to modulate DNA accessibility. Detailed mechanisms of such structural transformations of chromatin fibers as affected by various internal and external conditions such as the ionic conditions of the medium, the linker DNA length, and the presence of linker histones, constitute an open challenge. Here we utilize Monte Carlo (MC) simulations of a coarse grained model of chromatin with nonuniform linker DNA lengths as found in vivo to help explain some aspects of this challenge. We investigate the unfolding mechanisms of chromatin fibers with alternating linker lengths of 26–62 bp and 44–79 bp using a series of end-to-end stretching trajectories with and without linker histones and compare results to uniform-linker-length fibers. We find that linker histones increase overall resistance of nonuniform fibers and lead to fiber unfolding with superbeads-on-a-string cluster transitions. Chromatin fibers with nonuniform linker DNA lengths display a more complex, multi-step yet smoother process of unfolding compared to their uniform counterparts, likely due to the existence of a more continuous range of nucleosome-nucleosome interactions. This finding echoes the theme that some heterogeneity in fiber component is biologically advantageous.

  8. Supervised learning method for predicting chromatin boundary associated insulator elements.

    PubMed

    Bednarz, Pawe?; Wilczy?ski, Bartek

    2014-12-01

    In eukaryotic cells, the DNA material is densely packed inside the nucleus in the form of a DNA-protein complex structure called chromatin. Since the actual conformation of the chromatin fiber defines the possible regulatory interactions between genes and their regulatory elements, it is very important to understand the mechanisms governing folding of chromatin. In this paper, we show that supervised methods for predicting chromatin boundary elements are much more effective than the currently popular unsupervised methods. Using boundary locations from published Hi-C experiments and modEncode tracks as features, we can tell the insulator elements from randomly selected background sequences with great accuracy. In addition to accurate predictions of the training boundary elements, our classifiers make new predictions. Many of them correspond to the locations of known insulator elements. The key features used for predicting boundary elements do not depend on the prediction method. Because of its miniscule size, chromatin state cannot be measured directly, we need to rely on indirect measurements, such as ChIP-Seq and fill in the gaps with computational models. Our results show that currently, at least in the model organisms, where we have many measurements including ChIP-Seq and Hi-C, we can make accurate predictions of insulator positions. PMID:25385081

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. A chromatin-based mechanism for limiting divergent noncoding transcription.

    PubMed

    Marquardt, Sebastian; Escalante-Chong, Renan; Pho, Nam; Wang, Jue; Churchman, L Stirling; Springer, Michael; Buratowski, Stephen

    2014-06-19

    In addition to their annotated transcript, many eukaryotic mRNA promoters produce divergent noncoding transcripts. To define determinants of divergent promoter directionality, we used genomic replacement experiments. Sequences within noncoding transcripts specified their degradation pathways, and functional protein-coding transcripts could be produced in the divergent direction. To screen for mutants affecting the ratio of transcription in each direction, a bidirectional fluorescent protein reporter construct was introduced into the yeast nonessential gene deletion collection. We identified chromatin assembly as an important regulator of divergent transcription. Mutations in the CAF-I complex caused genome-wide derepression of nascent divergent noncoding transcription. In opposition to the CAF-I chromatin assembly pathway, H3K56 hyperacetylation, together with the nucleosome remodeler SWI/SNF, facilitated divergent transcription by promoting rapid nucleosome turnover. We propose that these chromatin-mediated effects control divergent transcription initiation, complementing downstream pathways linked to early termination and degradation of the noncoding RNAs. PMID:24949978

  14. Argonaute proteins couple chromatin silencing to alternative splicing.

    PubMed

    Ameyar-Zazoua, Maya; Rachez, Christophe; Souidi, Mouloud; Robin, Philippe; Fritsch, Lauriane; Young, Robert; Morozova, Nadya; Fenouil, Romain; Descostes, Nicolas; Andrau, Jean-Christophe; Mathieu, Jacques; Hamiche, Ali; Ait-Si-Ali, Slimane; Muchardt, Christian; Batsché, Eric; Harel-Bellan, Annick

    2012-10-01

    Argonaute proteins play a major part in transcriptional gene silencing in many organisms, but their role in the nucleus of somatic mammalian cells remains elusive. Here, we have immunopurified human Argonaute-1 and Argonaute-2 (AGO1 and AGO2) chromatin-embedded proteins and found them associated with chromatin modifiers and, notably, with splicing factors. Using the CD44 gene as a model, we show that AGO1 and AGO2 facilitate spliceosome recruitment and modulate RNA polymerase II elongation rate, thereby affecting alternative splicing. Proper AGO1 and AGO2 recruitment to CD44 transcribed regions required the endonuclease Dicer and the chromobox protein HP1?, and resulted in increased histone H3 lysine 9 methylation on variant exons. Our data thus uncover a new model for the regulation of alternative splicing, in which Argonaute proteins couple RNA polymerase II elongation to chromatin modification. PMID:22961379

  15. HMGA proteins as modulators of chromatin structure during transcriptional activation

    PubMed Central

    Ozturk, Nihan; Singh, Indrabahadur; Mehta, Aditi; Braun, Thomas; Barreto, Guillermo

    2013-01-01

    High mobility group (HMG) proteins are the most abundant non-histone chromatin associated proteins. HMG proteins bind to DNA and nucleosome and alter the structure of chromatin locally and globally. Accessibility to DNA within chromatin is a central factor that affects DNA-dependent nuclear processes, such as transcription, replication, recombination, and repair. HMG proteins associate with different multi-protein complexes to regulate these processes by mediating accessibility to DNA. HMG proteins can be subdivided into three families: HMGA, HMGB, and HMGN. In this review, we will focus on recent advances in understanding the function of HMGA family members, specifically their role in gene transcription regulation during development and cancer. PMID:25364713

  16. Dynamical DNA accessibility induced by chromatin remodeling and protein binding

    NASA Astrophysics Data System (ADS)

    Montel, F.; Faivre-Moskalenko, C.; Castelnovo, M.

    2014-11-01

    Chromatin remodeling factors are enzymes being able to alter locally chromatin structure at the nucleosomal level and they actively participate in the regulation of gene expression. Using simple rules for individual nucleosome motion induced by a remodeling factor, we designed simulations of the remodeling of oligomeric chromatin, in order to address quantitatively collective effects in DNA accessibility upon nucleosome mobilization. Our results suggest that accessibility profiles are inhomogeneous thanks to borders effects like protein binding. Remarkably, we show that the accessibility lifetime of DNA sequence is roughly doubled in the vicinity of borders as compared to its value in bulk regions far from the borders. These results are quantitatively interpreted as resulting from the confined diffusion of a large nucleosome depleted region.

  17. 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.

  18. Footprint traversal by adenosine-triphosphate-dependent chromatin remodeler motor

    NASA Astrophysics Data System (ADS)

    Garai, Ashok; Mani, Jesrael; Chowdhury, Debashish

    2012-04-01

    Adenosine-triphosphate (ATP)-dependent chromatin remodeling enzymes (CREs) are biomolecular motors in eukaryotic cells. These are driven by a chemical fuel, namely, ATP. CREs actively participate in many cellular processes that require accessibility of specific segments of DNA which are packaged as chromatin. The basic unit of chromatin is a nucleosome where 146 bp ˜ 50 nm of a double-stranded DNA (dsDNA) is wrapped around a spool formed by histone proteins. The helical path of histone-DNA contact on a nucleosome is also called “footprint.” We investigate the mechanism of footprint traversal by a CRE that translocates along the dsDNA. Our two-state model of a CRE captures effectively two distinct chemical (or conformational) states in the mechanochemical cycle of each ATP-dependent CRE. We calculate the mean time of traversal. Our predictions on the ATP dependence of the mean traversal time can be tested by carrying out in vitro experiments on mononucleosomes.

  19. 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

  20. 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

  1. Regulation of the dynamic chromatin architecture of the Epidermal Differentiation Complex is mediated by a c-Jun/AP-1-modulated enhancer

    PubMed Central

    Oh, Inez Y.; Albea, Danielle M.; Goodwin, Zane A.; Quiggle, Ashley M.; Baker, Breeana P.; Guggisberg, Ann M.; Geahlen, Jessica H.; Kroner, Grace M.; de Guzman Strong, Cristina

    2014-01-01

    The Epidermal Differentiation Complex (EDC) locus comprises a syntenic and linear cluster of genes whose concomitant expression is a hallmark feature of differentiation in the developing skin epidermis. Many of the EDC proteins are cross-linked together to form the cornified envelope, an essential and discrete unit of the mammalian skin barrier. The mechanism underlying coordinate transcriptional activation of the EDC is unknown. Within the human EDC, we identified an epidermal-specific regulatory enhancer, 923, that responded to the developmental and spatio-temporal cues at the onset of epidermal differentiation in the mouse embryo. Comparative chromosomal conformation capture (3C) assays in proliferating and differentiated primary mouse keratinocytes revealed multiple chromatin interactions that were physiologically sensitive between the 923 enhancer and EDC gene promoters and thus depict the dynamic, chromatin topology of the EDC. We elucidate a mechanistic link between c-Jun/AP-1 and 923, whereby AP-1 and 923-mediated EDC chromatin remodeling is required for functional EDC gene activation. Thus, we identify a critical enhancer/transcription factor axis governing the dynamic regulation of the EDC chromatin architecture and gene expression and provide a framework for future studies towards understanding gene regulation in cutaneous diseases. PMID:24468747

  2. Condenser performance monitoring and cleaning

    SciTech Connect

    Walden, J.V. [Rochester Gas and Electric, Ontario, NY (United States)

    1998-12-31

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

  3. Direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep: in vitro and in vivo studies

    Microsoft Academic Search

    S Athanasiadou; I Kyriazakis; F Jackson; R. L Coop

    2001-01-01

    In vitro and in vivo studies were conducted to determine possible direct anthelmintic effects of condensed tannins towards different ovine gastrointestinal nematodes. A larval development\\/viability assay was used to investigate the effect of a condensed tannin extract (Quebracho) towards larvae of Haemonchuscontortus, Teladorsagiacircumcincta and Trichostrongylusvitrinus. The development to infective larvae and their viability was assessed in all three species and

  4. Interference of condensed tannin in lignin analyses of dry bean and forage crops.

    PubMed

    Marles, M A Susan; Coulman, Bruce E; Bett, Kirstin E

    2008-11-12

    Legumes with high concentrations of condensed tannin (pinto bean [Phaseolus vulgaris L.], sainfoin [Onobrychis viciifolia Scop.], and big trefoil [Lotus uliginosus Hoff.]), were compared to a selection of forages, with low or zero condensed tannin (smooth bromegrass [ Bromus inermis Leyss], Lotus japonicus [Regel] K. Larsen, and alfalfa [Medicago sativa L.]), using four methods to estimate fiber or lignin. Protocols were validated by using semipurified condensed tannin polymers in adulteration assays that tested low-lignin tissue with polyphenolic-enriched samples. The effect on lignin assay methods by condensed tannin concentration was interpreted using a multivariate analysis. There was an overestimation of fiber or lignin in the presence of condensed tannin in the acid detergent fiber (ADF) and Klason lignin (KL) assays compared to that in the thioglycolic acid (TGA) and acid detergent lignin (ADL) methods. Sulfite reagents (present in TGA lignin method) or sequential acidic digests at high temperatures (ADF followed by ADL) were required to eliminate condensed tannin. The ADF (alone) and KL protocols are not recommended to screen nonwoody plants, such as forages, where condensed tannin has accumulated in the tissue. PMID:18841900

  5. Discovery of MLL1 binding units, their localization to CpG Islands, and their potential function in mitotic chromatin

    PubMed Central

    2013-01-01

    Background Mixed Lineage Leukemia 1 (MLL1) is a mammalian ortholog of the Drosophila Trithorax. In Drosophila, Trithorax complexes transmit the memory of active genes to daughter cells through interactions with Trithorax Response Elements (TREs). However, despite their functional importance, nothing is known about sequence features that may act as TREs in mammalian genomic DNA. Results By analyzing results of reported DNA binding assays, we identified several CpG rich motifs as potential MLL1 binding units (defined as morphemes). We find that these morphemes are dispersed within a relatively large collection of human promoter sequences and appear densely packed near transcription start sites of protein-coding genes. Genome wide analyses localized frequent morpheme occurrences to CpG islands. In the human HOX loci, the morphemes are spread across CpG islands and in some cases tail into the surrounding shores and shelves of the islands. By analyzing results of chromatin immunoprecipitation assays, we found a connection between morpheme occurrences, CpG islands, and chromatin segments reported to be associated with MLL1. Furthermore, we found a correspondence of reported MLL1-driven “bookmarked” regions in chromatin to frequent occurrences of MLL1 morphemes in CpG islands. Conclusion Our results implicate the MLL1 morphemes in sequence-features that define the mammalian TREs and provide a novel function for CpG islands. Apparently, our findings offer the first evidence for existence of potential TREs in mammalian genomic DNA and the first evidence for a connection between CpG islands and gene-bookmarking by MLL1 to transmit the memory of highly active genes during mitosis. Our results further suggest a role for overlapping morphemes in producing closely packed and multiple MLL1 binding events in genomic DNA so that MLL1 molecules could interact and reside simultaneously on extended potential transcriptional maintenance elements in human chromosomes to transmit the memory of highly active genes during mitosis. PMID:24373511

  6. Measurement of local chromatin compaction by spectral precision distance microscopy

    NASA Astrophysics Data System (ADS)

    Rauch, Joachim; Hausmann, Michael; Solovei, Irina; Horsthemke, Bernhard; Cremer, Thomas; Cremer, Christoph G.

    2000-12-01

    Fluorescence in situ hybridization (FISH) offers an appropriate technique to specifically label any given chromatin region by multi spectrally labeled, specific DNA probes. Using confocal laser scanning microscopy, quantitative measurements on the spatial distribution of labeling sites can be performed in 3D conserved cell nuclei. Recently, 'Spectral Precision Distance Microscopy' has been developed that allows 3D distance measurements between point-like fluorescence objects of different spectral signatures far beyond the diffraction limited resolution. In a well characterized and sequenced DNA region, the Prader- Willi/Angelman region q11-13 on chromosome 15, geometric distances between the fluorescence intensity bary centers of four different 'point-like' labeling sites were measured. More than 300 cell nuclei were evaluated with a 3D resolution equivalent better than 100 nm. The geometric bary center distances in nanometers were compared with the genomic bary center distance in kilobases (kb). A direct correlation, for instance linear correlation between geometric and genomic distances was not observed. From the measured values, a local compaction factor for the high order chromatin folding in the analyzed genome region was calculated. Along the 1000 kb chromatin segment analyzed, which spans nearly the compete Prader-Willi/Angelman region, different compaction factors were found. The compaction factor 40 typical for a straight 30 nm chromatin fiber was not observed. This shows that chromatin folding and compaction in intact nuclei may be more complex. With SPDM, however, a microscopical technique is available that can sensitively analyze chromatin organization in the 100 nm range in 3D conserved cell nuclei.

  7. Gravity triggered neutrino condensates

    SciTech Connect

    Barenboim, Gabriela [Departament de Fisica Teorica and IFIC, Universitat de Valencia-CSIC, E-46100, Burjassot (Spain)

    2010-11-01

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

  8. Condensate phase microscopy.

    PubMed

    Kosior, Arkadiusz; Sacha, Krzysztof

    2014-01-31

    We show that the phase of a Bose-Einstein condensate wave function of ultracold atoms in an optical lattice potential in two dimensions can be detected. The time-of-flight images, obtained in a free expansion of initially trapped atoms, are related to the initial distribution of atomic momenta but the information on the phase is lost. However, the initial atomic cloud is bounded and this information, in addition to the time-of-flight images, is sufficient in order to employ the phase retrieval algorithms. We analyze the phase retrieval methods for model wave functions in a case of a Bose-Einstein condensate in a triangular optical lattice in the presence of artificial gauge fields. PMID:24580464

  9. 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

  10. Feshbach-Einstein Condensates

    SciTech Connect

    Rousseau, V. G.; Denteneer, P. J. H. [Instituut-Lorentz, LION, Universiteit Leiden, Postbus 9504, 2300 RA Leiden (Netherlands)

    2009-01-09

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

  11. Immunochromatographic assay on thread.

    PubMed

    Zhou, Gina; Mao, Xun; Juncker, David

    2012-09-18

    Lateral-flow immunochromatographic assays are low-cost, simple-to-use, rapid tests for point-of-care screening of infectious diseases, drugs of abuse, and pregnancy. However, lateral flow assays are generally not quantitative, give a yes/no answer, and lack multiplexing. Threads have recently been proposed as a support for transporting and mixing liquids in lateral-flow immunochromatographic assays, but their use for quantitative high-sensitivity immunoassays has yet to be demonstrated. Here, we introduce the immunochromatographic assay on thread (ICAT) in a cartridge format that is suitable for multiplexing. The ICAT is a sandwich assay performed on a cotton thread knotted to a nylon fiber bundle, both of which are precoated with recognition antibodies against one target analyte. Upon sample application, the assay results become visible to the eye within a few minutes and are quantified using a flatbed scanner. Assay conditions were optimized, the binding curves for C-reactive protein (CRP) in buffer and diluted serum were established and a limit of detection of 377 pM was obtained. The possibility of multiplexing was demonstrated using three knotted threads coated with antibodies against CRP, osteopontin, and leptin proteins. The performance of the ICAT was compared with that of the paper-based and conventional assays. The results suggest that thread is a suitable support for making low-cost, sensitive, simple-to-use, and multiplexed diagnostic tests. PMID:22889381

  12. Chromatin structure characteristics of pre-miRNA genomic sequences

    Microsoft Academic Search

    Shijia Zhu; Qinghua Jiang; Guohua Wang; Bo Liu; Mingxiang Teng; Yadong Wang

    2011-01-01

    Background  MicroRNAs (miRNAs) are non-coding RNAs with important roles in regulating gene expression. Recent studies indicate that transcription\\u000a and cleavage of miRNA are coupled, and that chromatin structure may influence miRNA transcription. However, little is known\\u000a about the relationship between the chromatin structure and cleavage of pre-miRNA from pri-miRNA.\\u000a \\u000a \\u000a \\u000a \\u000a Results  By analysis of genome-wide nucleosome positioning data sets from human and Caenorhabditis

  13. Structure and Function of Protein Modules in Chromatin Biology

    Microsoft Academic Search

    Kyoko L. Yap; Ming-Ming Zhou

    Chromatin-mediated gene transcription or silencing is a dynamic process in which binding\\u000a of various proteins or protein complexes can displace nucleosomal histones from DNA to relieve repression\\u000a or drive the gene into a highly repressed, silent state. Covalent modifications to DNA and histones\\u000a associated with chromatin structural change play a crucial role in transcriptional regulation,\\u000a with particular modifications on certain residues associated with

  14. Chromatin Changes in Reprogramming of Mammalian Somatic Cells

    PubMed Central

    Xu, Rong; Zhang, Shiqiang

    2014-01-01

    Abstract Somatic cell nuclear transfer (SCNT), cell fusion, and induced pluripotent stem cells (iPSCs) technologies are three strategies that allow reprogramming somatic cells into the pluripotent state; however, the efficiency is low and the mechanisms are not fully clear. In addition, there are reports that changes in chromatin play a critical role in these reprogramming strategies by modulating binding of transcription factors to their targets. In this review, we mainly discuss inactivation of the X chromosome, chromatin decondensation and remodeling, histone modifications, and histone variants in the three strategies. This review will provide an insight for future nuclear reprogramming research. PMID:23987213

  15. Chromatin isolation by RNA purification (ChIRP).

    PubMed

    Chu, Ci; Quinn, Jeffrey; Chang, Howard Y

    2012-01-01

    Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression (1,2,3,4,5,6,7). The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion (8,9). While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation (10,11). However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide( 3,12,13), but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex (14); HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex (13). Prior studies mapping RNA occupancy at chromatin have revealed substantial insights (15,16), but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution (17). This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains. PMID:22472705

  16. Phosphoinositides as Regulators of Protein-Chromatin Interactions

    NSDL National Science Digital Library

    Keijo Viiri (School of Medicine and Tampere University Hospital; University of Tampere REV)

    2012-05-01

    The molecular function of phospholipids in the nucleus has been only partially elucidated. The upsurge of epigenetic research has contributed to increased interest in nuclear phospholipids, such as phosphoinositides, and their involvement in gene transcription. However, the mechanisms by which phosphoinositides regulate transcription is still unknown at the molecular level. Certain phosphoinositide species can regulate protein-chromatin and protein–nucleic acid interactions, and specific nuclear target proteins link nuclear signaling lipids to gene expression. We propose that a phosphoinositide-mediated detachment of proteins from chromatin is a general biological mechanism that partly underlies the signaling effects of nuclear phosphoinositides.

  17. 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.

  18. 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 ...

  19. 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 ...

  20. Replication-Coupled Chromatin Assembly Generates a Neuronal Bilateral Asymmetry in C. elegans

    E-print Network

    Nakano, Shunji

    Although replication-coupled chromatin assembly is known to be important for the maintenance of patterns of gene expression through sequential cell divisions, the role of replication-coupled chromatin assembly in controlling ...

  1. Roles of HDACs in chromatin remodelling and response to chemotherapy in cancer 

    E-print Network

    Huang, Rui

    2014-07-05

    Background: The higher-order structure of chromatin changes in response to extracellular and environmental signals. We observed nuclear morphological changes in biopsied cancer tissue after chemotherapy. Since chromatin ...

  2. 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...

  3. Reconstitution of recombinant chromatin establishes a requirement for histone-tail modifications during chromatin assembly and transcription

    PubMed Central

    Loyola, Alejandra; LeRoy, Gary; Wang, Yuh-Hwa; Reinberg, Danny

    2001-01-01

    The human ISWI-containing factor RSF (remodeling and spacing factor) was found to mediate nucleosome deposition and, in the presence of ATP, generate regularly spaced nucleosome arrays. Using this system, recombinant chromatin was reconstituted with bacterially produced histones. Acetylation of the histone tails was found to play an important role in establishing regularly spaced nucleosome arrays. Recombinant chromatin lacking histone acetylation was impaired in directing transcription. Histone-tail modifications were found to regulate transcription from the recombinant chromatin. Acetylation of the histone tails by p300 was found to increase transcription. Methylation of the histone H3 tail by Suv39H1 was found to repress transcription in an HP1-dependent manner. The effects of histone-tail modifications were observed in nuclear extracts. A highly reconstituted RNA polymerase II transcription system was refractory to the effect imposed by acetylation and methylation. PMID:11691835

  4. 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.

  5. [The effect of chromatin remodeling and modification on RNA-polymerase-mediated transcription initiation].

    PubMed

    Shidlovski?, Iu V; Nabirochkina, E N

    2005-07-01

    As eukaryotes are characterized by the presence of chromatin (intricately packaged DNA), special mechanisms are required for preparing the DNA template for operation of the transcription machinery. Recently, a close association between the chromatin state and transcription was found and numerous transcription factors modifying the physical and chemical chromatin state were revealed. This review presents a brief description of transcription initiation on the DNA template within chromatin. PMID:16152792

  6. Ionic strength-dependent conformational transitions of chromatin. Circular dichroism and thermal denaturation studies

    Microsoft Academic Search

    Andrew W. Fulmer; Gerald D. Fasman

    1979-01-01

    High-molecular-weight chicken erythrocyte chromatin was prepared by mild digestion of nuclei with micrococcal nuclease. Samples of chromatin containing both core (H3, H4, and H2A, H2B) and lysine-rich (H1, H5) histone proteins (whole chromatin) or only core histone proteins (core chromatin) were examined by CD and thermal denaturation as a function of ionic strength between 0.75 and 7.0 x 10⁻³M Na\\/sup

  7. Chromatin profiling in model organisms Tony D. Southall and Andrea H. Brand

    E-print Network

    Brand, Andrea

    Chromatin profiling in model organisms Tony D. Southall and Andrea H. Brand Abstract The correct) and DamID (DNA adenine methyltransferase identification). Both of these methods, when combined chromatin structure and gene regulatory networks. In vivo chromatin profiling studies are now being

  8. 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

  9. Chromatin inheritance upon Zeste-mediated Brahma recruitment at a minimal cellular memory module

    Microsoft Academic Search

    Jérôme Déjardin; Giacomo Cavalli

    2004-01-01

    Polycomb group and trithorax group proteins maintain the memory of repressed and active chromatin states by reg- ulating chromatin of their target genes via DNA sequences termed Polycomb- and trithorax response elements. Since these elements often overlap and are able to convey the memory of both silent and active chromatin through cell division, they were also defined as cellular memory

  10. The INO80 chromatin remodeling complex is involved in the nucleotide excision repair pathway

    Microsoft Academic Search

    Yingjun Jiang

    2009-01-01

    The genomic DNA of eukaryotic cells is well organized into chromatin structures. However, these repressed structures present barriers that block the access of regulatory factors to the genome during various nuclear events. To overcome the obstacle, two major cellular processes, post-modification of histone tails and ATP-dependent chromatin remodeling, are involved in reconfiguring chromatin structure and creating accessible DNA. Despite the

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

    PubMed

    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

  12. Chromatin remodeling and transcriptional activation: the cast (in order of appearance)

    Microsoft Academic Search

    Fyodor D Urnov; Alan P Wolffe

    2001-01-01

    The number of chromatin modifying and remodeling complexes implicated in genome control is growing faster than our understanding of the functional roles they play. We discuss recent in vitro experiments with biochemically defined chromatin templates that illuminate new aspects of action by histone acetyltransferases and ATP-dependent chromatin remodeling engines in facilitating transcription. We review a number of studies that present

  13. Sperm chromatin dispersion test in the assessment of DNA fragmentation and aneuploidy in human spermatozoa.

    PubMed

    Balasuriya, A; Speyer, B; Serhal, P; Doshi, A; Harper, J C

    2011-05-01

    Sperm DNA damage is thought to be increased in men with male factor infertility. Previous studies suggest a correlation between sperm DNA fragmentation and aneuploidy. The sperm chromatin dispersion (SCD) test was modified to produce the Halosperm Kit. The SCD-fluorescent in-situ hybridization (FISH) test allows the simultaneous detection of DNA fragmentation and aneuploidy on the same sperm cell. The objectives of this study were to validate the SCD, SCD-FISH and Halosperm tests for the analysis of sperm DNA fragmentation and compare them to the sperm chromatin structure assay (SCSA). Semen samples from 20 males undergoing IVF/intracytoplasmic sperm injection were processed using FISH, SCD-FISH, SCD and Halosperm, and compared with SCSA results. There was a significant difference between FISH and SCD-FISH results in the detection of aneuploidy (P=0.000) and the level of sperm DNA fragmentation in the samples subjected to SCSA and SCD (P=0.001) or SCSA and SCD-FISH (P=0.001). There was no significant correlation between DNA fragmentation and aneuploidy. If sperm aneuploidy is to be determined, more reliable results will be obtained if FISH is performed rather than SCD-FISH. A lack of validation and unknown clinical significance question the value of DNA fragmentation assays. DNA damage in the male germ line may result in adverse clinical outcomes and the pathophysiology and clinical consequences of sperm DNA damage are being actively researched. Many DNA fragmentation assays such as the Halosperm Kit have been developed recently and are now available at a commercial level. Unfortunately, aimed at vulnerable couples with difficulty conceiving, many of these tests have not been clinically validated. Despite its plausible appeal and fervour of its supporters, the benefits of widespread DNA testing that only achieves the distressing of couples with the knowledge that effectual therapeutic strategies are absent are questionable. Commercially, however, it is no doubt lucrative. Analysis of gametes prior to the initiation of an IVF cycle may improve the quality of embryos transferred. The clinical and scientific community considers it a matter of urgency to translate the basic science behind how a cell prepares for fertilization into routine clinical practice. However, it is equally important, if not more, to allow the science behind such applications to draw level with its practice before its widespread implementation. PMID:21397561

  14. 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

  15. Chromatin immunoprecipitation to identify global targets of WRKY transcription factor family members involved in plant immunity.

    PubMed

    Roccaro, Mario; Somssich, Imre E

    2011-01-01

    The completion of the alfalfa, Arabidopsis, papaya, poplar, and rice genome sequences along with ongoing sequencing projects of various crop species, offers an excellent opportunity to study gene expression at the whole genome level and to unravel the complexity of gene networks underlying the reprogramming of plant defense toward pathogen challenge. Gene expression in eukaryotic cells is mainly controlled by regulatory elements that recruit transcription factors (TFs) to modulate transcriptional outputs. Therefore, methods allowing the identification of all cognate TF binding sites (TFBS) within the regulatory regions of target genes on a genome-wide basis are the next obvious step to elucidate the plant defense transcriptome. Chromatin immunoprecipitation (ChIP) is one such powerful technique for analyzing functional cis-regulatory DNA elements. The ChIP assay allows the identification of specific regulatory DNA regions associated with trans-acting regulatory factors in vivo. ChIP assays can provide spatial and temporal snapshots of the regulatory components involved in reprogramming host gene expression upon pathogen ingress. Moreover, the use of ChIP-enriched DNA for hybridization to tiling microarrays (ChIP-chip) or for direct sequencing (ChIP-Seq) by means of massively parallel sequencing technology has expanded this methodology to address global changes in gene expression. PMID:21359799

  16. Contributions of linker histones and histone H3 to chromatin structure: scanning force microscopy studies on trypsinized fibers.

    PubMed Central

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

    1998-01-01

    Little is known about the mechanisms that organize linear arrays of nucleosomes into the three-dimensional structures of extended and condensed chromatin fibers. We have earlier defined, from scanning force microscopy (SFM) and mathematical modeling, a set of simple structural determinants of extended fiber morphology, the critical parameters being the entry-exit angle between consecutive linkers and linker length. Here we study the contributions of the structural domains of the linker histones (LHs) and of the N-terminus of histone H3 to extended fiber morphology by SFM imaging of progressively trypsinized chromatin fibers. We find that cleavage of LH tails is associated with a lengthening of the internucleosomal center-to-center distance, and that the somewhat later cleavage of the N-terminus of histone H3 is associated with a flattening of the fiber. The persistence of the "zigzag" fiber morphology, even at the latest stages of trypsin digestion, can be attributed to the retention of the globular domain of LH in the fiber. PMID:9635736

  17. Lysosome-mediated processing of chromatin in senescence.

    PubMed

    Ivanov, Andre; Pawlikowski, Jeff; Manoharan, Indrani; van Tuyn, John; Nelson, David M; Rai, Taranjit Singh; Shah, Parisha P; Hewitt, Graeme; Korolchuk, Viktor I; Passos, Joao F; Wu, Hong; Berger, Shelley L; Adams, Peter D

    2013-07-01

    Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C-negative, but strongly ?-H2AX-positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression. PMID:23816621

  18. A Chromatin Insulator Determines the Nuclear Localization of DNA

    Microsoft Academic Search

    Tatiana I. Gerasimova; Keith Byrd; Victor G. Corces

    2000-01-01

    Chromatin insulators might regulate gene expression by controlling the subnuclear organization of DNA. We found that a DNA sequence normally located inside of the nucleus moved to the periphery when the gypsy insulator was placed within the sequence. The presence of the gypsy insulator also caused two sequences, normally found in different regions of the nucleus, to come together at

  19. Lysosome-mediated processing of chromatin in senescence

    PubMed Central

    Ivanov, Andre; Pawlikowski, Jeff; Manoharan, Indrani; van Tuyn, John; Nelson, David M.; Rai, Taranjit Singh; Shah, Parisha P.; Hewitt, Graeme; Korolchuk, Viktor I.; Passos, Joao F.; Wu, Hong; Berger, Shelley L.

    2013-01-01

    Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C–negative, but strongly ?-H2AX–positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression. PMID:23816621

  20. Cytoskeletal Control of Nuclear Morphology and Chromatin Organization.

    PubMed

    Ramdas, Nisha M; Shivashankar, G V

    2014-10-01

    The nucleus is sculpted toward various morphologies during cellular differentiation and development. Alterations in nuclear shape often result in changes to chromatin organization and genome function. This is thought to be reflective of its role as a cellular mechanotransducer. Recent evidence has highlighted the importance of cytoskeletal organization in defining how nuclear morphology regulates chromatin dynamics. However, the mechanisms underlying cytoskeletal control of chromatin remodeling are not well understood. We demonstrate here the differential influence of perinuclear actin- and microtubule-driven assemblies on nuclear architecture using pharmacological inhibitors and targeted RNA interference knockdown of cytoskeleton components in Drosophila cells. We find evidence that the loss of perinuclear actin assembly results in basolateral enhancement of microtubule organization and this is reflected functionally by enhanced nuclear dynamics. Cytoskeleton reorganization leads to nuclear lamina deformation that influences heterochromatin localization and core histone protein mobility. We also show that modulations in actin-microtubule assembly result in differential gene expression patterns. Taken together, we suggest that perinuclear actin and basolateral microtubule organization exerts mechanical control on nuclear morphology and chromatin dynamics. PMID:25281900

  1. Effect of saffron on rat sperm chromatin integrity

    PubMed Central

    Mardani, Mohammad; Vaez, Ahmad; Razavi, Shahnaz

    2014-01-01

    Background: Currently, relation between reactive oxygen species (ROS) ROS concentration and semen quality was indicated. Saffron has traditionally been not only considered as a food additive but also as a medicinal herb, which has a good antioxidant properties. Objective: The aim of this study was to evaluate the protection potency of saffron and vitamin E on sperm chromatin integrity. Materials and Methods: Thirty adult male Wistar rats divided equally into saffron (100 mg/kg), vitamin E (100 mg/kg) and control (0.5cc distilled water /day) groups. After 60 days, cauda epididymis dissected and sperm cells were used for analysis of sperm chromatin packaging by chromomycin A3 (CMA3) staining, and sperm chromatin susceptibility to acid denaturation by acridine orange (AO) staining. Results: The mean percentage of CMA3 positive sperm was significantly decreased in saffron and vitamin E groups relative to control group (p<0.001). Moreover, the AO staining results showed that the mean percentage of sperm with DNA damage was significantly decreased in saffron and vitamin E groups as compared with control group (p<0.001). Conclusion: Our results purposed that saffron can protect sperm against DNA damage and chromatin anomalies. PMID:25031579

  2. The SWI-SNF complex: a chromatin remodeling machine?

    Microsoft Academic Search

    Craig L. Peterson; John W. Tamkun

    1995-01-01

    The SWI-SNF complex plays a key role in the regulation of eukaryotic gene expression. Genetic studies in the yeast Saccharomyces cerevisiae suggest that one role for the complex is to antagonize chromatin-mediated repression of transcription. Recent biochemical studies indicate that S. cerevisiae and putative human SWI-SNF complexes use the energy of ATP hydrolysis to disrupt nucleosome structure.

  3. Porin new light onto chromatin and nuclear organization

    PubMed Central

    Finlan, Lee E; Bickmore, Wendy A

    2008-01-01

    A recent report identifies sites in the human genome that can associate with nucleoporin 93, a subunit of the nuclear pore complex. These associations are modulated by levels of global histone acetylation and highlight the dynamic nature of chromatin organization in the nucleus. PMID:18495049

  4. Ezh2 lines up the chromatin in T regulatory cells.

    PubMed

    Wu, Chuan; Chen, Zuojia; Kuchroo, Vijay K

    2015-02-17

    Epigenetic modulation is critical for regulating the development and function of T cells. In this issue of Immunity, DuPage et al. (2015) show that the chromatin-modifying enzyme Ezh2 induced by CD28 costimulation is essential for regulatory T (Treg) cell maintenance during activation and differentiation. PMID:25692693

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

    E-print Network

    Friedman, Nir

    From DNA Sequence to Chromatin Dynamics: Computational Analysis of Transcriptional Regulation an innovative algorithm for identifying the target genes of novel transcription factors, based on their protein sequence (Chapter 1). Second, I consider how several transcription factors cooperate to process external

  6. 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

  7. Altering the chromatin landscape for nucleotide excision repair

    Microsoft Academic Search

    Ronita Nag; Michael J. Smerdon

    2009-01-01

    DNA acts as a ‘workbench’ for various nuclear processes that occur inside living cells. In eukaryotic cells, DNA is highly compacted in a structural hierarchy with histones and other proteins into chromatin. This compaction affects DNA structure and coordinates the accessibility to site-specific nuclear factors during DNA processing events. DNA repair is no exception to this general rule and several

  8. Chromatin-regulating proteins as targets for cancer therapy.

    PubMed

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

    2014-07-01

    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

  9. 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

  10. Targeting chromatin remodelers: Signals and search mechanisms Fabian Erdel a

    E-print Network

    Rippe, Karsten

    16 June 2011 Keywords: Chromatin remodeling Nucleosome translocation Histone modifications Diffusion-controlled histones with histone variants [1]. Their ability to (re)position nucleosomes plays an important role for regulating gene expression as well as mediating access to the DNA during replication and repair [2

  11. Job Description Research Assistant Chromatin Sorghum Breeding Team Gainesville, FL

    E-print Network

    Mazzotti, Frank

    Job Description Research Assistant ­ Chromatin Sorghum Breeding Team Gainesville, FL Description The Research Assistant/field technician position will support day-to-day activities for the sorghum breeding will include providing support and assistance to sorghum breeders and research associates with all aspects

  12. Evolution of histone 2A for chromatin compaction in eukaryotes

    PubMed Central

    Macadangdang, Benjamin R; Oberai, Amit; Spektor, Tanya; Campos, Oscar A; Sheng, Fang; Carey, Michael F; Vogelauer, Maria; Kurdistani, Siavash K

    2014-01-01

    During eukaryotic evolution, genome size has increased disproportionately to nuclear volume, necessitating greater degrees of chromatin compaction in higher eukaryotes, which have evolved several mechanisms for genome compaction. However, it is unknown whether histones themselves have evolved to regulate chromatin compaction. Analysis of histone sequences from 160 eukaryotes revealed that the H2A N-terminus has systematically acquired arginines as genomes expanded. Insertion of arginines into their evolutionarily conserved position in H2A of a small-genome organism increased linear compaction by as much as 40%, while their absence markedly diminished compaction in cells with large genomes. This effect was recapitulated in vitro with nucleosomal arrays using unmodified histones, indicating that the H2A N-terminus directly modulates the chromatin fiber likely through intra- and inter-nucleosomal arginine–DNA contacts to enable tighter nucleosomal packing. Our findings reveal a novel evolutionary mechanism for regulation of chromatin compaction and may explain the frequent mutations of the H2A N-terminus in cancer. DOI: http://dx.doi.org/10.7554/eLife.02792.001 PMID:24939988

  13. 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

  14. 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.

  15. Citrullination regulates pluripotency and histone H1 binding to chromatin.

    PubMed

    Christophorou, Maria A; Castelo-Branco, Gonçalo; Halley-Stott, Richard P; Oliveira, Clara Slade; Loos, Remco; Radzisheuskaya, Aliaksandra; Mowen, Kerri A; Bertone, Paul; Silva, José C R; Zernicka-Goetz, Magdalena; Nielsen, Michael L; Gurdon, John B; Kouzarides, Tony

    2014-03-01

    Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction. PMID:24463520

  16. Citrullination regulates pluripotency and histone H1 binding to chromatin

    NASA Astrophysics Data System (ADS)

    Christophorou, Maria A.; Castelo-Branco, Gonçalo; Halley-Stott, Richard P.; Oliveira, Clara Slade; Loos, Remco; Radzisheuskaya, Aliaksandra; Mowen, Kerri A.; Bertone, Paul; Silva, José C. R.; Zernicka-Goetz, Magdalena; Nielsen, Michael L.; Gurdon, John B.; Kouzarides, Tony

    2014-03-01

    Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.

  17. Cosmological tachyon condensation

    SciTech Connect

    Bilic, Neven; Tupper, Gary B.; Viollier, Raoul D. [Rudjer Boskovic Institute, 10002 Zagreb (Croatia); Centre of Theoretical Physics and Astrophysics, University of Cape Town, Rondebosch 7701 (South Africa)

    2009-07-15

    We consider the prospects for dark matter/energy unification in k-essence type cosmologies. General mappings are established between the k-essence scalar field, the hydrodynamic and braneworld descriptions. We develop an extension of the general relativistic dust model that incorporates the effects of both pressure and the associated acoustic horizon. Applying this to a tachyon model, we show that this inhomogeneous 'variable Chaplygin gas' does evolve into a mixed system containing cold dark matter like gravitational condensate in significant quantities. Our methods can be applied to any dark energy model, as well as to mixtures of dark energy and traditional dark matter.

  18. Doped colorimetric assay liposomes

    DOEpatents

    Charych, Deborah (Albany, CA); Stevens, Raymond C. (Albany, CA)

    2001-01-01

    The present invention provides compositions comprising colorimetric assay liposomes. The present invention also provides methods for producing colorimetric liposomes and calorimetric liposome assay systems. In preferred embodiments, these calorimetric liposome systems provide high levels of sensitivity through the use of dopant molecules. As these dopants allow the controlled destabilization of the liposome structure, upon exposure of the doped liposomes to analyte(s) of interest, the indicator color change is facilitated and more easily recognized.

  19. Microfluidic DNA hybridization assays

    Microsoft Academic Search

    Xuan WengHai; Hai Jiang; Dongqing Li

    DNA hybridization is one of the most powerful techniques applied in diagnostic assays. Microfluidics provides a promising\\u000a means to analyse small sample volumes, reduce reagent consumption and cost, shorten processing time as well as develop fast,\\u000a sensitive and portable diagnostic tools. By coupling with the microfluidic technology, DNA hybridization assay can achieve\\u000a high sensitivity, enhance hybridization kinetics and decrease the

  20. Polariton condensates put in motion.

    PubMed

    Sanvitto, D; Amo, A; Laussy, F P; Lemaître, A; Bloch, J; Tejedor, C; Viña, L

    2010-04-01

    We present several examples of the interesting phenomenology shown by a moving polariton condensate in semiconductor microcavities. The superfluid behavior is probed by colliding the polariton condensate against physical obstacles in the form of natural defects of the sample, demonstrating a clear suppression of scattering when the speed of the flow lies below the critical velocity. At higher velocities Cerenkov-like shock waves around the defect and disruption of the condensate are also observed. PMID:20208103