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Sample records for chromatin condensation assay

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

    PubMed Central

    Irianto, Jerome; Swift, Joe; Martins, RuiP.; McPhail, GrahamD.; Knight, MartinM.; Discher, DennisE.; Lee, DavidA.

    2013-01-01

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

  2. Activation of DNA damage response signaling by condensed chromatin

    PubMed Central

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

    2014-01-01

    Summary The DNA damage response (DDR) occurs in the context of chromatin structure, and architectural features of chromatin contribute to DNA damage signaling and repair. While the role of chromatin decondensation in the DDR is established, we show here that chromatin condensation is integral to DDR signaling. We find that upon 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 ATM- and ATR-dependent activation of upstream DDR signaling in a break-independent manner. Finally, while 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

  3. Premature chromatin condensation upon accumulation of NIMA.

    PubMed Central

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

    1994-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  5. Incomplete Chromatin Condensation in Enlarged Rat Myelocytic Leukemia Cells

    PubMed Central

    Trencsenyi, Gyorgy; Nagy, Gabor; Bako, Fruzsina; Kertai, Pal

    2012-01-01

    The distinguishable morphologic features of nuclei of acute myelogenous leukemia cells with enlarged size and finely distributed nuclear chromatin indicate incomplete chromosome condensation that can be related to elevated gene expression. To confirm this, interphase chromosome structures were studied in exponentially growing rat myelomonocytic leukemia 1 cells isolated at the University of Debrecen (My1/De cells). This cell line was established from primary rat leukemia chemically induced by 7,12-dimethylbenz[a]anthracene treatment. The enlarged nuclei of My1/De cells allowed improved fluorescent visualization of chromosomal structures. Increased resolution revealed major interphase intermediates consisting of (1) veil-like chromatin, (2) chromatin ribbon, (3) chromatin funnel, (4) chromatin bodies, (5) elongated prechromosomes, (6) seal-ring, spiral shaped, and circular chromosomal subunits, (7) elongated, bent, u- and v-shaped prechromosomes, and (8) metaphase chromosomes. Results confirmed the existence of the chromatin funnel, the first visible interphase chromosome generated by the supercoiling of the chromatin ribbon. Other intermediates not seen previously included the spiral subunits that are involved in the chromonemic folding of metaphase chromosomes. The existence of spiral subunits favors the helical coil model of chromosome condensation. Incomplete chromatin condensation in leukemia cells throughout the cell cycle is an indication of euchromatization contributing to enhanced gene expression and is regarded as a leukemic factor. PMID:21942442

  6. PTEN Interacts with Histone H1 and Controls Chromatin Condensation

    PubMed Central

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

    2014-01-01

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

  7. 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

  8. CHD5 is Required for Spermiogenesis and Chromatin Condensation

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-01

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

  11. Osmotic stress alters chromatin condensation and nucleocytoplasmic transport

    SciTech Connect

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

    2011-05-06

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-05-19

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

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

    PubMed

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

    2016-01-01

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

  15. Condensation of interphase chromatin in nuclei of synchronized chinese hamster ovary (CHO-K1) cells.

    PubMed

    Gacsi, Mariann; Nagy, Gabor; Pinter, Gabor; Basnakian, Alexei G; Banfalvi, Gaspar

    2005-01-01

    Reversibly permeabilized cells have been used to visualize interphase chromatin structures in the presence and absence of biotinylated nucleotides. By reversing permeabilization, it was possible to confirm the existence of a flexible chromatin folding pattern through a series of transient geometric forms such as supercoiled, circular forms, chromatin bodies, thin and thick fibers, and elongated chromosomes. Our results show that the incorporation of biotin-11-dUTP interferes with chromatin condensation, leading to the accumulation of decondensed chromatin structures. Chromatin condensation without nucleotide incorporation was also studied in cell populations synchronized by centrifugal elutriation. After reversal of permeabilization, nuclei were isolated and chromatin structures were visualized after DAPI staining by fluorescent microscopy. Decondensed veil-like structures were observed in the early S phase (at an average C-value of 2.21), supercoiled chromatin later in the early S (2, 55 C), fibrous structures in the early mid S phase (2, 76 C), ribboned structures in the mid-S phase (2, 98 C), continuous chromatin strings later in the mid-S phase (3,28), elongated prechromosomes in the late S-phase (3, 72 C), precondensed chromosomes at the end and after the S phase (3, 99 C). Fluorescent microscopy revealed that neither interphase nor metaphase chromosomes are separate entities but form a linear array arranged in a semicircle. Linear arrangement was confirmed by computer image analysis. PMID:15684719

  16. Study of DNA accessibility in the condensed chromatin structures by resonance energy transfer.

    PubMed

    Labarbe, R; Flock, S; Colson, P; Houssier, C

    1994-12-01

    The linker DNA accessibility of chicken erythrocyte chromatin was studied by diffusion-enhanced resonance energy transfer (DERET). The 4″-{9‴-[((4-carboxy-3-hydroxyphenyl)-acetatamido)-3‴,6‴,9‴-(triacetyl)-3″,6‴,9‴-triazanonamido]-2″,6″-diazanonyl}-4,5',8-trimethyl psoralen-terbium complex was photocovalently bound to linker DNA and transferred its energy to fluorescein free in solution or bound on proteins of different sizes. We observed a diminution of linker DNA accessibility in chromatin as the protein size increased. Free fluorescein and proteins (up to a molecular weight of 24,000) labeled with fluorescein isothiocyanate (FITC) showed no variation in linker accessibility as chromatin condensation from 10- to 30-nm fibers was induced by an increase in ionic strength. We can conclude from these observations that linker DNA is located on the outside of the condensed chromatin fiber or, alternatively, that small proteins are free to diffuse toward an inside-located linker DNA, even in the condensed state of chromatin, possibly through the central cavity of the solenoïd model. PMID:24233605

  17. 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

  18. Characterization of chromatin accessibility with a transposome hypersensitive sites sequencing (THS-seq) assay.

    PubMed

    Sos, Brandon Chin; Fung, Ho-Lim; Gao, Derek Rui; Osothprarop, Trina Faye; Kia, Amirali; He, Molly Min; Zhang, Kun

    2016-01-01

    Chromatin accessibility captures in vivo protein-chromosome binding status, and is considered an informative proxy for protein-DNA interactions. DNase I and Tn5 transposase assays require thousands to millions of fresh cells for comprehensive chromatin mapping. Applying Tn5 tagmentation to hundreds of cells results in sparse chromatin maps. We present a transposome hypersensitive sites sequencing assay for highly sensitive characterization of chromatin accessibility. Linear amplification of accessible DNA ends with in vitro transcription, coupled with an engineered Tn5 super-mutant, demonstrates improved sensitivity on limited input materials, and accessibility of small regions near distal enhancers, compared with ATAC-seq. PMID:26846207

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

    PubMed

    Baron, Margaret H; Barminko, Jeffrey

    2016-03-01

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

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

    PubMed

    Spagnol, Stephen T; Dahl, Kris Noel

    2016-01-01

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

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

    PubMed Central

    Spagnol, Stephen T.; Dahl, Kris Noel

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  3. 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.

  4. 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

  5. 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...

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

    PubMed

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

    2005-09-01

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

  7. Progressive Chromatin Condensation and H3K9 Methylation Regulate the Differentiation of Embryonic and Hematopoietic Stem Cells.

    PubMed

    Ugarte, Fernando; Sousae, Rebekah; Cinquin, Bertrand; Martin, Eric W; Krietsch, Jana; Sanchez, Gabriela; Inman, Margaux; Tsang, Herman; Warr, Matthew; Passegué, Emmanuelle; Larabell, Carolyn A; Forsberg, E Camilla

    2015-11-10

    Epigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increased cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation. PMID:26489895

  8. Progressive Chromatin Condensation and H3K9 Methylation Regulate the Differentiation of Embryonic and Hematopoietic Stem Cells

    PubMed Central

    Ugarte, Fernando; Sousae, Rebekah; Cinquin, Bertrand; Martin, Eric W.; Krietsch, Jana; Sanchez, Gabriela; Inman, Margaux; Tsang, Herman; Warr, Matthew; Passegué, Emmanuelle; Larabell, Carolyn A.; Forsberg, E. Camilla

    2015-01-01

    Summary Epigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increased cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation. PMID:26489895

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

    PubMed Central

    2011-01-01

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

  10. HP1 knockdown is associated with abnormal condensation of almost all chromatin types in a grasshopper (Eyprepocnemis plorans).

    PubMed

    Ruiz-Estévez, Mercedes; Bakkali, Mohammed; Cabrero, Josefa; Camacho, Juan Pedro M; López-León, María Dolores

    2014-09-01

    Heterochromatin protein 1 (HP1) is a highly conserved family of eukaryotic proteins required for heterochromatic gene silencing and euchromatic gene transcription regulation. In addition, HP1 is involved in chromatin organization and protection of chromosome integrity during cell division. Here, we present a cytological and molecular analysis of the effects of HP1 knockdown in Eyprepocnemis plorans, a grasshopper species polymorphic for supernumerary heterochromatic chromosomes. Our results revealed contrasting effects of HP1 knockdown on gene activity. While the Bub1 gene decreased in expression level in HP1 knockdown animals, NOR activity, rRNA and, contrarily to previous reports in Drosophila, Hsp70 gene expression remained unchanged. Furthermore, HP1 knockdown resulted in abnormal chromatin condensation, chromosomal bridges, higher frequency of macrospermatids, loss of muscle mass and hemolymph amount as well as a low number of dividing cells and survival reduction. All these phenotypes are very likely due to the chromatin condensation disruption observed for almost all kinds of chromatin. PMID:24398928

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

    PubMed

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

    2009-01-01

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

  12. Optimization of a method for chromatin immunoprecipitation assays in the marine invertebrate chordate Ciona

    PubMed Central

    Aihara, Hitoshi; Katikala, Lavanya; Zeller, Robert W.; Di Gregorio, Anna; Nibu, Yutaka

    2013-01-01

    Chromatin immunoprecipitation (ChIP) assays allow the efficient characterization of the in vivo occupancy of genomic regions by DNA-binding proteins, and thus facilitate the prediction of cis-regulatory sequences in silico and guide their validation in vivo. For these reasons, these assays and their permutations (e.g., ChIP-on-chip, ChIP-Sequencing) are currently being extended to several non-mainstream model organisms, as the availability of specific antibodies increases. Here we describe the development of a polyclonal antibody against the Brachyury protein of the marine invertebrate chordate Ciona intestinalis and provide a detailed ChIP protocol that should be easily adaptable to other marine organisms. PMID:23592257

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

    PubMed

    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. PMID:22314931

  14. 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.

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

    PubMed

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

    2015-09-15

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    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 describe a procedure to analyze UV-induced chromatin remodeling at the silent mating-type locus HML using isolated nuclei from UV treated yeast cells. In addition, a method to capture transient protein-protein associations in chromatin is outlined. We have used the methods described here to demonstrate that the SWI/SNF chromatin remodeling complex is involved in chromatin rearrangement during NER. PMID:19336254

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

    PubMed

    Agnieszka, Wojtczak

    2014-10-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  2. Comparative sperm chromatin structure assay measurements on epiillumination and orthogonal axes flow cytometers

    SciTech Connect

    Evenson, D.; Jost, L.; Gandour, D.; Gandour, D.; Rhodes, L.

    1995-04-01

    The sperm chromatin structure assay (SCSA) measures the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ, and was developed on two Ortho flow cytometers, an FC200 and a cytofluorograf 30 (BDIS), both having orthogonal axes of fluorochrome excitation, emission, and sample flow. Sperm cells are first treated with a pH 1.4 buffer to denature DNA in situ and then stained with the metachromatic dye acridine orange (AO). The metachromatic fluorescence measured reflects relative amounts of denatured (red fluorescence) and native (green fluorescence) DNA present per cell. The extent of DNA denaturation is quantified by the calculated parameter alpha t [{alpha}{sub t} = red/(red + green) fluorescence]. Alpha t variables important for correlations with fertility and toxicant-induced chromatin damage include mean (X{alpha}{sub t}), standard deviation (SD{alpha}{sub t}), and cells outside the main population (COMP{alpha}{sub t}). This study showed that the SCSA can be successfully run on two epiillumination-type instruments, an Ortho ICP22A and Skatron Argus {trademark}, and two additional orthogonal axes instruments, a Becton Dickinson FACScan {trademark} and a Coulter Elite {trademark}. Epiillumination instruments produced a different fluorescence distribution than orthogonal instruments, but the resulting {alpha}{sub t} values showed strong conformity and interpretation of results was the same. SCSA values obtained on the Coultier Elite {trademark} were most similar to the Cytofluorograf 30; the FACScan {trademark} green fluorescence distribution was narrower and allowed resolution of cell doublets. Neither orthogonal instrument has the ability to directly calculate {alpha}{sub t} values. Listmode data from these instruments were transferred to an off-line personal computer (PC) for calculation of {alpha}{sub t} values using LIST-VIEW {trademark} software. 28 refs., 5 figs., 2 tabs.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. 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

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

    PubMed

    Oleszczuk, K; Giwercman, A; Bungum, M

    2016-03-01

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

  7. 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...

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

    SciTech Connect

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

    2015-03-13

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

  9. Nuclear DNA Methylation and Chromatin Condensation Phenotypes Are Distinct Between Normally Proliferating/Aging, Rapidly Growing/Immortal, and Senescent Cells

    PubMed Central

    Gertych, Arkadiusz; Tajbakhsh, Jian

    2013-01-01

    This study reports on probing the utility of in situ chromatin texture features such as nuclear DNA methylation and chromatin condensation patterns — visualized by fluorescent staining and evaluated by dedicated three-dimensional (3D) quantitative and high-throughput cell-by-cell image analysis — in assessing the proliferative capacity, i.e. growth behavior of cells: to provide a more dynamic picture of a cell population with potential implications in basic science, cancer diagnostics/prognostics and therapeutic drug development. Two types of primary cells and four different cancer cell lines were propagated and subjected to cell-counting, flow cytometry, confocal imaging, and 3D image analysis at various points in culture. Additionally a subset of primary and cancer cells was accelerated into senescence by oxidative stress. DNA methylation and chromatin condensation levels decreased with declining doubling times when primary cells aged in culture with the lowest levels reached at the stage of proliferative senescence. In comparison, immortal cancer cells with constant but higher doubling times mostly displayed lower and constant levels of the two in situ-derived features. However, stress-induced senescent primary and cancer cells showed similar levels of these features compared with primary cells that had reached natural growth arrest. With regards to global DNA methylation and chromatin condensation levels, aggressively growing cancer cells seem to take an intermediate level between normally proliferating and senescent cells. Thus, normal cells apparently reach cancer-cell equivalent stages of the two parameters at some point in aging, which might challenge phenotypic distinction between these two types of cells. Companion high-resolution molecular profiling could provide information on possible underlying differences that would explain benign versus malign cell growth behaviors. PMID:23562889

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

    SciTech Connect

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

    2012-03-10

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

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

    PubMed Central

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

    2009-01-01

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

  12. Long-term effects of triethylenemelamine exposure on mouse testis cells and sperm chromatin structure assayed by flow cytometry

    SciTech Connect

    Evenson, D.P.; Baer, R.K.; Jost, L.K. )

    1989-01-01

    The toxic and potentially mutagenic actions of triethylenemelamine (TEM) on mouse body and testis weights, testicular cell kinetics, sperm production, sperm head morphology, and sperm chromatin structure were assessed in two experiments. The first experiment examined effects of four dose levels of TEM, assayed 1, 4, or 10 wk after toxic exposure. In the second study, effects from five dosage levels were measured at 1, 4, and 10 wk, and the highest dosage level was evaluated over 44 wk. TEM produced an expected dose related loss of spermatogenic activity and subsequent recovery as determined by dual-parameter (DNA, RNA) flow cytometry (FCM) measurements of testicular cells. Both testicular weights and caudal sperm reserves remained generally below controls after 44 wk recovery following exposure to the highest dosage. Chromatin structure alterations, defined as increased susceptibility to DNA denaturation in situ, and sperm head morphology were highly correlated with dose and with each other. Sperm head morphology and sperm chromatic structure remained abnormal at 44 wk for the 1.0 mg/kg TEM dosage, suggesting that the abnormalities, present long after the initial toxic response, may be a result of mutation. This study demonstrates that flow cytometry provides a unique, rapid, and efficient means to measure effects of reproductive toxins and potential mutagens.

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2012-04-01

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

  15. 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...

  16. 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

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

    PubMed

    Dixon, D K; Burkholder, G D

    1985-03-01

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

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

    PubMed

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

    2014-10-01

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

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

    2004-11-10

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

  20. 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...

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

    PubMed Central

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

    2007-01-01

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

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

    SciTech Connect

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

    2005-09-01

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

  3. 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...

  4. Validation of the sperm chromatin dispersion (SCD) test in the amphibian Xenopus laevis using in situ nick translation and comet assay.

    PubMed

    Pollock, K; Gosálvez, J; Arroyo, F; López-Fernández, C; Guille, M; Noble, A; Johnston, S D

    2015-11-01

    The integrity of sperm DNA is becoming increasingly recognised as an important parameter of semen quality, but there are no published reports of this procedure for any amphibian. The primary aim of this study was to apply a modified sperm chromatin dispersion (SCD) test (Halomax) to an amphibian sperm model (African clawed frog; Xenopus laevis) and to validate the assay against in situ nick translation (ISNT) and the double-comet assay procedure. Inactivated spermatozoa were collected from fresh testes (n=3). Sperm DNA fragmentation (SDF) for each sperm sample was conducted immediately following activation (T0) and again after 1h (T1) and 24h (T24) of incubation at room temperature in order to produce a range of spermatozoa with differing levels of DNA damage. The SCD procedure resulted in the production of three nuclear morphotypes; amphibian sperm morphotype 1 (ASM-1) and ASM-2 showed no evidence of DNA damage, whereas ASM-3 spermatozoa were highly fragmented with large halos of dispersed DNA fragments and a reduced nuclear core. ISNT confirmed that ASM-3 nuclei contained damaged DNA. There was a significant correlation (r=0.9613) between the levels of ASM-3 detected by the SCD test and SDF revealed by the double-comet assay. PMID:25482041

  5. Ectopically tethered CP190 induces large-scale chromatin decondensation

    PubMed Central

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

    2014-01-01

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

  6. 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.

  7. Germline chromatin.

    PubMed Central

    Schaner, Christine E; Kelly, William G

    2006-01-01

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

  8. Role of histone modification in chromatin dynamics.

    PubMed

    Ito, Takashi

    2007-05-01

    The covalent modification of histone tails has emerged as a crucial step in controlling the eucaryotic genomes. Eucaryotic cells must possess mechanisms for condensing and decondensing chromatin. Moreover, chromatin condensation is particularly evident during mitosis and apoptotic cell death, whereas chromatin relaxation is necessary for replication, repair, recombination and transcription. The post-translational modifications of histone tails such as reversible acetylation, phosphorylation and methylation play a critical role in dynamic condensation/relaxation that occurs during the cell cycle. Histone phosphorylation is believed to play a direct role in mitosis, cell death, repair, replication and recombination. In this review, we discuss recent progress in studies of histone phosphorylation. PMID:17405795

  9. The Sperm Chromatin Structure Assay (SCSA(®)) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility.

    PubMed

    Evenson, Donald P

    2016-06-01

    Thirty-five years ago the pioneering paper in Science (240:1131) on the relationship between sperm DNA integrity and pregnancy outcome was featured as the cover issue showing a fluorescence photomicrograph of red and green stained sperm. The flow cytometry data showed a very significant difference in sperm DNA integrity between fertile and subfertile bulls and men. This study utilized heat (100°C, 5min) to denature DNA at sites of DNA strand breaks followed by staining with acridine orange (AO) and measurements of 5000 individual sperm of green double strand (ds) DNA and red single strand (ss) DNA fluorescence. Later, the heat protocol was changed to a low pH protocol to denature the DNA at sites of strand breaks; the heat and acid procedures produced the same results. SCSA data are very advantageously dual parameter with 1024 channels (degrees) of both red and green fluorescence. Hundreds of publications on the use of the SCSA test in animals and humans have validated the SCSA as a highly useful test for determining male breeding soundness. The SCSA test is a rapid, non-biased flow cytometer machine measurement providing robust statistical data with exceptional precision and repeatability. Many genotoxic experiments showed excellent dose response data with very low coefficient of variation that further validated the SCSA as being a highly powerful assay for sperm DNA integrity. Twelve years following the introduction of the SCSA test, the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labelling (TUNEL) test (1993) for sperm was introduced as the only other flow cytometric assay for sperm DNA fragmentation. However, the TUNEL test can also be done by light microscopy with much less statistical robustness. The COMET (1998) and Sperm Chromatin Dispersion (SCD; HALO) (2003) tests were introduced as light microscope tests that don't require a flow cytometer. Since these tests measure only 50-200 sperm per sample, they suffer from the lack of the statistical robustness of flow cytometric measurements. Only the SCSA test has an exact standardization of a fixed protocol. The many variations of the other tests make it very difficult to compare data and thresholds for risk of male factor infertility. Data from these four sperm DNA fragmentation tests plus the light microscope acridine orange test (AOT) are correlated to various degrees. PMID:26919909

  10. 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

  11. 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...

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

    NASA Astrophysics Data System (ADS)

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

    1998-11-01

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

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

    PubMed

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

    2007-05-01

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

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

    SciTech Connect

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

    1985-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

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

  16. The Drosophila Mi-2 Chromatin-Remodeling Factor Regulates Higher-Order Chromatin Structure and Cohesin Dynamics In Vivo

    PubMed Central

    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

  17. 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

  18. 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...

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

    EPA Science Inventory

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

  20. Common pathway of chromosome condensation in Mammalian cells.

    PubMed

    Banfalvi, Gaspar; Nagy, Gabor; Gacsi, Mariann; Roszer, Tamas; Basnakian, Alexei G

    2006-05-01

    Chromatin folding in the interphase nucleus is not known. We compared the pattern of chromatin condensation in Indian muntjac, Chinese hamster ovary, murine pre B, and K562 human erythroleukemia cells during the cell cycle. Fluorescent microscopy showed that chromosome condensation follows a general pathway. Synchronized cells were reversibly permeabilized and used to isolate interphase chromatin structures. Based on their structures two major categories of intermediates were distinguished: (1) decondensed chromatin and (2) condensed chromosomal forms. (1) Chromatin forms were found between the G1 and mid-S phase involving veil-like, supercoiled, fibrous, ribboned structures; (2) condensing chromosomal forms appeared in the late-S, G2, and M phase, including strings, chromatin bodies, elongated pre-chromosomes, pre-condensed chromosomes, and metaphase chromosomes. Results demonstrate that interphase chromosomes are clustered in domains; condensing interphase chromosomes are linearly arranged. Our results raise questions related to telomer sequences and to the chemical nature of chromosome connectivity. PMID:16716119

  1. Chromatin Higher-order Structure and Dynamics

    PubMed Central

    Woodcock, Christopher L.; Ghosh, Rajarshi P.

    2010-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  5. 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

  6. Epichromatin is conserved in Toxoplasma gondii and labels the exterior parasite chromatin throughout the cell cycle

    PubMed Central

    VANAGAS, LAURA; DALMASSO, MARIA C.; DUBREMETZ, JEAN F.; PORTIANSKY, ENRIQUE L.; OLINS, DONALD E.; ANGEL, SERGIO O.

    2014-01-01

    SUMMARY Toxoplasma gondii is an apicomplexan intracellular protozoan parasite responsible for toxoplasmosis, a disease with considerable medical and economic impact worldwide. Toxoplasma gondii cells never lose the nuclear envelope and their chromosomes do not condense. Here, we tested the murine monoclonal antibody PL2-6, which labels epichromatin (a conformational chromatin epitope based on histones H2A and H2B complexed with DNA), in T. gondii cultured in human fibroblasts. This epitope is present at the exterior chromatin surface of interphase nuclei and on the periphery of mitotic chromosomes in higher eukaryotes. PL2-6 reacted with T. gondii H2A and H2B histones in Western blot (WB) assays. In addition, the antibody reacted with the nuclear fraction of tachyzoites, as a single band coincident with H2B histone. In the T. gondii tachyzoite stage, PL2-6 also had peripheral nuclear localization, as observed by epifluorescence/confocal microscopy and immunoelectron microscopy. Confocal analysis showed that epichromatin is slightly polarized to one face of the parasite exterior chromatin surface. In replicating tachyzoites, PL2-6 also labels the exterior chromatin surface, covering the face of both segregating nuclei, facing the plasma membrane of the mother cell. The possible role of epichromatin in T. gondii is discussed. PMID:23701822

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

    PubMed

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

    1996-06-01

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

  8. Acetone enhances the direct analysis of procyanidin- and prodelphinidin-based condensed tannins in lotus species by the butanol-HCl-iron assay.

    PubMed

    Grabber, John H; Zeller, Wayne E; Mueller-Harvey, Irene

    2013-03-20

    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 improve CT quantitation, we tested various cosolvents with butanol-HCl and found that acetone increased anthocyanidin yields from two forage Lotus species having contrasting procyanidin and prodelphinidin compositions. A butanol-HCl-iron assay run with 50% (v/v) acetone gave linear responses with Lotus CT standards and increased estimates of total CT in Lotus herbage and leaves by up to 3.2-fold over the conventional method run without acetone. The use of thiolysis to determine the purity of CT standards further improved quantitation. Gel-state (13)C and (1)H-(13)C HSQC NMR spectra of insoluble residues collected after butanol-HCl assays revealed that acetone increased anthocyanidin yields by facilitating complete solubilization of CT from tissue. PMID:23383722

  9. Single-strand nuclease action on heat-denatured spermiogenic chromatin.

    PubMed

    Hunter, J D; Bodner, A J; Hatch, F T; Balhorn, R L; Mazrimas, J A; McQueen, A P; Gledhill, B L

    1976-08-01

    The aim of this study was to compare the sensitivity of chromatin from representative cellular stages of spermiogenesis to a single-strandeded nuclease after heat denaturation. Thermal denaturation of chromatin was assayed in situ in fixed round, elongating and elongated spermatids and in testicular sperm from mice. Production of single-stranded deoxyribonucleic acid (DNA) at elevated temperatures was monitored by digesting chromatin with endonuclease specific for single-stranded DNA (S1 nuclease), staining the residual DNA with gallocyanin-chrome alum (GAC) and measuring the stain content by absorption cytophotometry. Changes in GCA staining were minimal over the temperature range of 22-90 degrees C in each cell type not exposed to nuclease. Staining of undigested cells decreased progressively with advancing cell maturity. Nuclease had no effect on the GCA content of round spermatids below 60 degrees C, but above this temperature there was a progressive decrease in GCA-stainable chromatin. Both round and elongating spermatid stages showed a significantly greater sensitivity to nuclease digestion than did more mature stages; sperm showed no effects of nuclease action below 80 degrees C. Progressive chromatin condensation and a concomitant decrease in the number of available DNA phosphate groups during spermiogenic cell maturation may be responsible for the observed decline in sensitivity to nuclease and decreased GCA staining. Thermal denaturation of round spermatids labeled with 3H-thymidine produced no change in autoradiographic mean nuclear grain counts, indicating no loss of thymidine-labeled DNA from the slides during denaturation. When round spermatids and sperm were hydrolyzed with hot tricholoroacetic acid before staining, both nuclear GCA content and autoradiograph grain count were partially reduced, indicating incomplete DNA removal. Almost complete loss of Feulgen-stainable material occurred in these cells and may be due to depurination and elimination of Feulgren-reactant aldehyde groups. PMID:60438

  10. RNA meets chromatin.

    PubMed

    Bernstein, Emily; Allis, C David

    2005-07-15

    In the universe of science, two worlds have recently collided-those of RNA and chromatin. The intersection of these two fields has been impending, but evidence for such a meaningful collision has only recently become apparent. In this review, we discuss the implications for noncoding RNAs and the formation of specialized chromatin domains in various epigenetic processes as diverse as dosage compensation, RNA interference-mediated heterochromatin assembly and gene silencing, and programmed DNA elimination. While mechanistic details as to how the RNA and chromatin worlds connect remain unclear, intriguing parallels exist in the overall design and machinery used in model organisms from all eukaryotic kingdoms. The role of potential RNA-binding chromatin-associated proteins will be discussed as one possible link between RNA and chromatin. PMID:16024654

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

    PubMed

    Postnikov, Yuri V; Bustin, Michael

    2016-03-01

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

  12. Histone hyperacetylation has little effect on the higher order folding of chromatin.

    PubMed Central

    McGhee, J D; Nickol, J M; Felsenfeld, G; Rau, D C

    1983-01-01

    HeLa cells were grown in the presence of 10 mM sodium butyrate and soluble chromatin containing hyperacetylated histones was prepared by mild micrococcal nuclease digestion and sucrose gradient fractionation. Sedimentation and electric dichroism were used to study the cation-induced folding of this acetylated chromatin from the 10 nm filament to the 30 nm solenoid conformation. Although under some conditions acetylated chromatin appears slightly less condensed than control chromatin, the major conclusion is that hyperacetylation of histones does not in itself prevent the formation of the higher order chromatin solenoid. Images PMID:6866766

  13. 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,

  14. Chromatin deregulation in disease.

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    2001-01-01

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

  16. DNA damage induced by cigarette smoke condensate in vitro as assayed by 32P-postlabeling. Comparison with cigarette smoke-associated DNA adduct profiles in vivo.

    PubMed

    Randerath, E; Danna, T F; Randerath, K

    1992-07-01

    Cigarette smoke induces a multitude of bulky/aromatic DNA adducts in vivo as revealed by 32P-postlabeling assay. The formation of such adducts is thought to involve metabolic activation of aromatic chemicals especially polycyclic aromatic hydrocarbons (PAHs) present in tumor-initiating cigarette tar fractions, via cytochrome P450-associated monooxygenases. Because radicals are present in both the gas and particulate (tar) phase of cigarette smoke and in aqueous extracts of cigarette smoke condensate (CSC), we addressed the question as to whether cytochrome P450-independent, possibly free radical-mediated reactions may contribute, also, to formation of cigarette smoke-associated bulky DNA adducts. Rat-lung DNA was incubated with aqueous extracts of CSC in the absence of microsomes under various conditions and analyzed by 32P-postlabeling. Radioactively labeled bulky reaction products were found to accumulate in a time- and CSC concentration-dependent manner. The resulting chromatographic profiles resembled cigarette smoke-associated DNA-adduct patterns observed in vivo. Pretreatment of aqueous CSC extract with radical scavengers/reducing agents (ascorbic acid, glutathione) diminished adduct formation in a concentration-dependent manner. Adduct formation in vitro may involve oxygen-free radicals, which are known to be present in aqueous CSC extracts and could (i) attack DNA directly to produce bulky adducts, (ii) induce radical sites on DNA covalently binding CSC components, or (iii) convert CSC components to DNA-reactive electrophiles. In addition, DNA may react with direct-acting mutagens in CSC. Adduct fractions derived from in vitro and in vivo experiments showed similar chromatographic behavior, suggesting that metabolic activation as well as processes not involving metabolism lead to formation of smoking-induced bulky DNA adducts in vivo. PMID:1378180

  17. EBV latency types adopt alternative chromatin conformations.

    PubMed

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

    2011-07-01

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

  18. The Ino80 chromatin-remodeling complex restores chromatin structure during UV DNA damage repair

    PubMed Central

    Sarkar, Sovan; Kiely, Rhian

    2010-01-01

    Chromatin structure is modulated during deoxyribonucleic acid excision repair, but how this is achieved is unclear. Loss of the yeast Ino80 chromatin-remodeling complex (Ino80-C) moderately sensitizes cells to ultraviolet (UV) light. In this paper, we show that INO80 acts in the same genetic pathway as nucleotide excision repair (NER) and that the Ino80-C contributes to efficient UV photoproduct removal in a region of high nucleosome occupancy. Moreover, Ino80 interacts with the early NER damage recognition complex Rad4–Rad23 and is recruited to chromatin by Rad4 in a UV damage–dependent manner. Using a modified chromatin immunoprecipitation assay, we find that chromatin disruption during UV lesion repair is normal, whereas the restoration of nucleosome structure is defective in ino80 mutant cells. Collectively, our work suggests that Ino80 is recruited to sites of UV lesion repair through interactions with the NER apparatus and is required for the restoration of chromatin structure after repair. PMID:21135142

  19. Chromatin remodelling during male gametophyte development.

    PubMed

    Borg, Michael; Berger, Frédéric

    2015-07-01

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

  20. Mitotic checkpoint control and chromatin remodeling.

    PubMed

    Yao, Yixin; Dai, Wei

    2012-01-01

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

  1. Chromatin signatures of cancer

    PubMed Central

    Morgan, Marc A.; Shilatifard, Ali

    2015-01-01

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

  2. 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

  3. Chromatin immunoprecipitation using microarrays.

    PubMed

    Durand-Dubief, Mickaël; Ekwall, Karl

    2009-01-01

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

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

    PubMed Central

    Cognetti, G; Shaw, B R

    1981-01-01

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

  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. Histones: annotating chromatin.

    PubMed

    Campos, Eric I; Reinberg, Danny

    2009-01-01

    Chromatin is a highly regulated nucleoprotein complex through which genetic material is structured and maneuvered to elicit cellular processes, including transcription, cell division, differentiation, and DNA repair. In eukaryotes, the core of this structure is composed of nucleosomes, or repetitive histone octamer units typically enfolded by 147 base pairs of DNA. DNA is arranged and indexed through these nucleosomal structures to adjust local chromatin compaction and accessibility. Histones are subject to multiple covalent posttranslational modifications, some of which alter intrinsic chromatin properties, others of which present or hinder binding modules for non-histone, chromatin-modifying complexes. Although certain histone marks correlate with different biological outputs, we have yet to fully appreciate their effects on transcription and other cellular processes. Tremendous advancements over the past years have uncovered intriguing histone-related matters and raised important related questions. This review revisits past breakthroughs and discusses novel developments that pertain to histone posttranslational modifications and the affects they have on transcription and DNA packaging. PMID:19886812

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

    PubMed

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

    2004-06-01

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

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

    PubMed Central

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

    2015-01-01

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

  9. Nucleosome Shape Dictates Chromatin Fiber Structure

    PubMed Central

    Depken, Martin; Schiessel, Helmut

    2009-01-01

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

  10. 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.

  11. Nucleosomes arrangement in chromatin

    PubMed Central

    Marion, C.; Roux, B.

    1978-01-01

    The spatial arrangement of nucleosomes in rat liver chromatin has been examined using the electric birefringence technique. All chromatin subunits studied (up to 9 consecutive nucleosomes) contain their full complement of the five histone types associated with about 200 base pairs repeat length DNA. From the relaxation times and the orientation mechanisms, the nucleosome may be assimilated to an oblate ellipsoid of dimensions about 140 × 140 × 70 Å and the DNA superhelical axis is parallel to its shorter axis. The most important result is a sharp transition in the electro-optical properties of subunits when the number of nucleosomes in the chain is greater than 6: the initial negative birefringence, as for DNA, becomes positive and the relaxation time is multiplied by ten. The hexanucleosome, which presents no birefringence, has an helical symmetrical structure without preferential orientation axis. This structure is approximatively spherical of about 250 Å diameter and the chromatin appears as a periodic array of such a structure. PMID:724513

  12. Facilitation of base excision repair by chromatin remodeling.

    PubMed

    Hinz, John M; Czaja, Wioletta

    2015-12-01

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

  13. Chromatin decondensed by acetylation shows an elevated radiation response

    SciTech Connect

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

    1989-02-01

    V-79 Chinese hamster lung fibroblasts exposed to 5 mM n-sodium butyrate were irradiated with 60Co gamma rays and cell survival was determined by the cell colony assay. In a separate set of experiments the acetylated chromatin obtained from these cells was irradiated and the change of molecular weight of the DNA was evaluated by alkaline sucrose density centrifugation. At a survival level of 10(-2) to 10(-4) cells exposed to butyrate were found to be 1.3-1.4 times more radiosensitive than control cells. Exposure of isolated chromatin to 100 Gy of 60Co gamma irradiation generated 0.9 +/- 0.03 single-strand breaks (ssb) per 10 Gy per 10(8) Da and 2.0 +/- 0.3 ssb/10 Gy/10(8) Da for control and acetylated chromatin, respectively. The elevated radiation sensitivity of chromatin relaxed by acetylation is in good agreement with previous results on chromatin expanded by histone H1 depletion. Packing and accessibility of DNA in chromatin appear to be major factors which influence the radiation sensitivity. The intrinsic radiation sensitivity of chromatin in various packing states is discussed in light of the variation of radiation sensitivity of whole cells in the cell cycle which incorporates repair.

  14. Chromatin modifications in DNA repair.

    PubMed

    Morrison, Ashby J; Shen, Xuetong

    2006-01-01

    A requirement of nuclear processes that use DNA as a substrate is the manipulation of chromatin in which the DNA is packaged. Chromatin modifications cause alterations of histones and DNA, and result in a permissive chromatin environment for these nuclear processes. Recent advances in the fields of DNA repair and chromatin reveal that both histone modifications and chromatin-remodeling complexes are essential for the repair of DNA lesions, such as DNA double strand breaks (DSBs). In particular, chromatin-modifying complexes, such as the INO80, SWR1, RSC, and SWI/SNF ATP-dependent chromatin-remodeling complexes and the NuA4 and Tip60 histone acetyltransferase complexes are implicated in DNA repair. The activity of these chromatin-modifying complexes influences the efficiency of the DNA repair process, which ultimately affects genome integrity and carcinogenesis. Thus, the process of DNA repair requires the cooperative activities of evolutionarily conserved chromatin-modifying complexes that facilitate the dynamic chromatin alterations needed during repair of DNA damage. PMID:16909893

  15. Cas9 Functionally Opens Chromatin

    PubMed Central

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

    2016-01-01

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

  16. Linker histone variants control chromatin dynamics during early embryogenesis

    PubMed Central

    Saeki, Hideaki; Ohsumi, Keita; Aihara, Hitoshi; Ito, Takashi; Hirose, Susumu; Ura, Kiyoe; Kaneda, Yasufumi

    2005-01-01

    Complex transitions in chromatin structure produce changes in genome function during development in metazoa. Linker histones, the last component of nucleosomes to be assembled into chromatin, comprise considerably divergent subtypes as compared with core histones. In all metazoa studied, their composition changes dramatically during early embryogenesis concomitant with zygotic gene activation, leading to distinct functional changes that are still poorly understood. Here, we show that early embryonic linker histone B4, which is maternally expressed, is functionally different from somatic histone H1 in influencing chromatin structure and dynamics. We developed a chromatin assembly system with nucleosome assembly protein-1 as a linker histone chaperone. This assay system revealed that maternal histone B4 allows chromatin to be remodeled by ATP-dependent chromatin remodeling factor, whereas somatic histone H1 prevents this remodeling. Structural analysis shows that histone B4 does not significantly restrict the accessibility of linker DNA. These findings define the functional significance of developmental changes in linker histone variants. We propose a model that holds that maternally expressed linker histones are key molecules specifying nuclear dynamics with respect to embryonic totipotency. PMID:15821029

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

    PubMed

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

    2016-01-01

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

  18. Archaeal chromatin and transcription.

    PubMed

    Reeve, John N

    2003-05-01

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

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

    PubMed

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

    2006-11-01

    Abnormal chromatin condensation is not detected using classical techniques for sperm analysis. SCSA has demonstrated its usefulness in sperm chromatin analysis in several species (human, bull, stallion and boar). In this work, we studied sperm samples from red deer, ram and dog to analyze the differentiation of chromatin structure applying SCSA in epididymal and ejaculated spermatozoa. Epididymal samples were obtained from the caput, corpus and cauda by means of cuts, and ejaculated ones were obtained by electroejaculation (deer), artificial vagina (ram) and digital manipulation (dog). SCSA results suggested different critical points in sperm maturation (spermatozoa with loose chromatin to more condensed chromatin) among species: from corpus to cauda in ram and from caput to corpus in deer and dog. Moreover, we also detected differences in ruminants and dog, reflected in the appearance of SCSA plots. Indeed, ram and deer samples rendered two peaks within the sperm main population (sperm with condensed chromatin), whereas only one was detected in dog. Although some differences were observed between cauda and ejaculated samples, SCSA parameters indicated good chromatin condensation, making these samples suitable for germplasm banking. Some species-dependent modifications in the analysis of the results may be necessary to take full advantage of its analytical power. PMID:16790270

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

    PubMed

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

    2016-03-01

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

  1. Single Molecule Studies of Chromatin

    SciTech Connect

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

    2004-01-06

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

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

    PubMed

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

    2015-11-01

    Ochratoxin A (OTA) exposure during pregnancy in laboratory animals induces delayed/abnormal embryo development. Foetal adnexa-derived mesenchymal stem cells (MSCs) could help evaluate the developmental risk of exposure to chemicals in advanced gestational age. We tested the effects of OTA at concentrations ranging from 2.5×10(-4) to 25nM on growth parameters of canine umbilical cord matrix (UCM)-derived MSCs. The hypothesis that oxidative chromatin and DNA damage could underlie OTA-mediated cell toxicity was also investigated. After in vitro exposure, OTA significantly decreased cell density and increased doubling time in a passage- and concentration-dependent manner and no exposed cells survived beyond passage 5. Significantly higher rates of cells showed condensed and fragmented chromatin and oxidized DNA, as assessed by OxyDNA assay. These findings showed that in vitro exposure to OTA, at picomolar levels, perturbs UCM-MSC growth parameters through oxidative chromatin and DNA damage, suggesting possible consequences on canine foetal development. PMID:26055943

  3. Chromatin and Transcription in Yeast

    PubMed Central

    Rando, Oliver J.; Winston, Fred

    2012-01-01

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

  4. CONDENSATION CAN

    DOEpatents

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

    1962-03-01

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

  5. Regulation of cellular chromatin state

    PubMed Central

    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

  6. MNase titration reveals differences between nucleosome occupancy and chromatin accessibility.

    PubMed

    Mieczkowski, Jakub; Cook, April; Bowman, Sarah K; Mueller, Britta; Alver, Burak H; Kundu, Sharmistha; Deaton, Aimee M; Urban, Jennifer A; Larschan, Erica; Park, Peter J; Kingston, Robert E; Tolstorukov, Michael Y

    2016-01-01

    Chromatin accessibility plays a fundamental role in gene regulation. Nucleosome placement, usually measured by quantifying protection of DNA from enzymatic digestion, can regulate accessibility. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC (MNase accessibility), quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both nucleosome location and accessibility in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares histone and non-histone protection. We find that changes in accessibility at enhancers, promoters and other regulatory regions do not correlate with changes in nucleosome occupancy. Moreover, high nucleosome occupancy does not necessarily preclude high accessibility, which reveals novel principles of chromatin regulation. PMID:27151365

  7. MNase titration reveals differences between nucleosome occupancy and chromatin accessibility

    PubMed Central

    Mieczkowski, Jakub; Cook, April; Bowman, Sarah K.; Mueller, Britta; Alver, Burak H.; Kundu, Sharmistha; Deaton, Aimee M.; Urban, Jennifer A.; Larschan, Erica; Park, Peter J.; Kingston, Robert E.; Tolstorukov, Michael Y.

    2016-01-01

    Chromatin accessibility plays a fundamental role in gene regulation. Nucleosome placement, usually measured by quantifying protection of DNA from enzymatic digestion, can regulate accessibility. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC (MNase accessibility), quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both nucleosome location and accessibility in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares histone and non-histone protection. We find that changes in accessibility at enhancers, promoters and other regulatory regions do not correlate with changes in nucleosome occupancy. Moreover, high nucleosome occupancy does not necessarily preclude high accessibility, which reveals novel principles of chromatin regulation. PMID:27151365

  8. Painting a Clearer Picture of Chromatin.

    PubMed

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

    2016-02-22

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

  9. The effect of bacterial contamination of semen on sperm chromatin integrity and standard semen parameters in men from infertile couples.

    PubMed

    Rybar, R; Prinosilova, P; Kopecka, V; Hlavicova, J; Veznik, Z; Zajicova, A; Rubes, J

    2012-05-01

    A considerable proportion of male factor infertility cases are associated with inflammatory processes. The most common sexually transmissible agents causing sexually transmitted diseases in industrial countries are Chlamydia trachomatis, genital Ureaplasma and Mycoplasma spp. This study was undertaken to investigate whether these bacterial contaminants in semen affect sperm quality parameters and particularly DNA integrity (detected by sperm chromatin structure assay) in males from infertile couples (n = 293). The results showed that semen contaminations with the investigated bacterial species were not associated with sperm DNA fragmentation. However, contaminations with Mycoplasma spp. and C. trachomatis were associated with decreased sperm concentrations. Total sperm numbers in contaminated semen samples tended to be decreased, but not significantly. Mycoplasma had the highest adverse effect on sperm quality (concentration, motility, morphology and DNA condensation). Antibiotic therapy of the selected 47 men was successful in 55%, but semen quality parameters did not improve at least up to 3 months after the therapy. The presence of pathogenic bacteria in semen is primarily associated with low sperm production. Our data showed that Mycoplasma spp. contamination of semen had the highest adverse effect on sperm quality. Sperm chromatin integrity assessed by the presence of DNA breaks was not disturbed. PMID:21762193

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

    PubMed Central

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

    2015-01-01

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

  11. Spreading chromatin into chemical biology.

    PubMed

    Allis, C David; Muir, Tom W

    2011-01-24

    Epigenetics, broadly defined as the inheritance of non-Mendelian phenotypic traits, can be more narrowly defined as heritable alterations in states of gene expression ("on" versus "off") that are not linked to changes in DNA sequence. Moreover, these alterations can persist in the absence of the signals that initiate them, thus suggesting some kind of "memory" to epigenetic forms of regulation. How, for example, during early female mammalian development, is one X chromosome selected to be kept in an active state, while the genetically identical sister X chromosome is "marked" to be inactive, even though they reside in the same nucleus, exposed to the same collection of shared trans-factors? Once X inactivation occurs, how are these contrasting chromatin states maintained and inherited faithfully through subsequent cell divisions? Chromatin states, whether active (euchromatic) or silent (heterochromatic) are established, maintained, and propagated with remarkable precision during normal development and differentiation. However, mistakes made in establishing and maintaining these chromatin states, often executed by a variety of chromatin-remodeling activities, can lead to mis-expression or mis-silencing of critical downstream gene targets with far-reaching implications for human biology and disease, notably cancer. Though chromatin biologists have identified many of the "inputs" that are important for controlling chromatin states, the detailed mechanisms by which these processes work remain largely opaque, in part due to the staggering complexity of the chromatin polymer, the physiologically relevant form of our genome. The primary objective of this article is to serve as a "call to arms" for chemists to contribute to the development of the precision tools needed to answer pressing molecular problems in this rapidly moving field. PMID:21243714

  12. 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

  13. 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

  14. Chromatin structure and DNA damage

    SciTech Connect

    Gale, J.M.

    1987-01-01

    This dissertation examines the structure and structural transitions of chromatin in relation to DNA damage. The ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro was examined following DNA photodamage introduced by two different agents. (1) 254-nm UV radiation and (2) trimethylpsoralen (plus near-UV radiation). Both agents are highly specific for DNA and form adducts predicted to cause different degrees of distortion in the DNA helix. The salt-induced structural transitions of intact and histone H1 depleted chromatin fibers were monitored by both analytical ultracentrifugation and light scattering. Our results show that even in the presence of extremely large, nonphysiological amounts of photodamage by either agent the ability of chromatin to fold into higher ordered structures is not affected. The compact, 30 nm fiber must therefore be able to accommodate a large amount of DNA damage without any measurable changes in the overall size or degree of compaction of this structure. The distribution of pyrimidine dimers was mapped at the single nucleotide level in nucleosome core DNA from UV-irradiated mononucleosomes, chromatin fibers, and human cells in culture using the 3' ..-->.. 5' exonuclease activity of T4 DNA polymerase.

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

    Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells, and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell is sparse, comprising on the order of 1000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of sub-populations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone. PMID:26458175

  19. 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.

  20. Chromatin Decondensation and Nuclear Softening Accompany Nanog Downregulation in Embryonic Stem Cells

    PubMed Central

    Chalut, Kevin J.; Höpfler, Markus; Lautenschläger, Franziska; Boyde, Lars; Chan, Chii Jou; Ekpenyong, Andrew; Martinez-Arias, Alfonso; Guck, Jochen

    2012-01-01

    The interplay between epigenetic modification and chromatin compaction is implicated in the regulation of gene expression, and it comprises one of the most fascinating frontiers in cell biology. Although a complete picture is still lacking, it is generally accepted that the differentiation of embryonic stem (ES) cells is accompanied by a selective condensation into heterochromatin with concomitant gene silencing, leaving access only to lineage-specific genes in the euchromatin. ES cells have been reported to have less condensed chromatin, as they are capable of differentiating into any cell type. However, pluripotency itself—even prior to differentiation—is a split state comprising a naïve state and a state in which ES cells prime for differentiation. Here, we show that naïve ES cells decondense their chromatin in the course of downregulating the pluripotency marker Nanog before they initiate lineage commitment. We used fluorescence recovery after photobleaching, and histone modification analysis paired with a novel, to our knowledge, optical stretching method, to show that ES cells in the naïve state have a significantly stiffer nucleus that is coupled to a globally more condensed chromatin state. We link this biophysical phenotype to coinciding epigenetic differences, including histone methylation, and show a strong correlation of chromatin condensation and nuclear stiffness with the expression of Nanog. Besides having implications for transcriptional regulation and embryonic cell sorting and suggesting a putative mechanosensing mechanism, the physical differences point to a system-level regulatory role of chromatin in maintaining pluripotency in embryonic development. PMID:23200040

  1. 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

  2. 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

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

    PubMed

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

    2004-11-19

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

  4. Making the case for chromatin profiling: a new tool to investigate the immune-regulatory landscape.

    PubMed

    Winter, Deborah R; Jung, Steffen; Amit, Ido

    2015-09-15

    Recent technological advances have enabled researchers to accurately and efficiently assay the chromatin dynamics of scarce cell populations. In this Opinion article, we advocate the application of these technologies to central questions in immunology. Unlike changes to other molecular structures in the cell, chromatin features can reveal the past (developmental history), present (current activity) and future (potential response to challenges) of a given immune cell type; chromatin profiling is therefore an important new tool for studying the immune-regulatory networks of health and disease. PMID:26272294

  5. Structural and functional genome analysis using extended chromatin

    SciTech Connect

    Heaf, T.; Ward, D.C.

    1994-09-01

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

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

    PubMed Central

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

    1981-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  9. 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.

  10. Quantitative analysis of chromatin compaction in living cells using FLIMFRET

    PubMed Central

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

    2009-01-01

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

  11. Coming to terms with chromatin structure.

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    2007-01-01

    Background Histone H1 is involved in the formation and maintenance of chromatin higher order structure. H1 has multiple isoforms; the subtypes differ in timing of expression, extent of phosphorylation and turnover rate. In vertebrates, the amino acid substitution rates differ among subtypes by almost one order of magnitude, suggesting that each subtype might have acquired a unique function. We have devised a competitive assay to estimate the relative binding affinities of histone H1 mammalian somatic subtypes H1a-e and H1 for long chromatin fragments (3035 nucleosomes) in physiological salt (0.14 M NaCl) at constant stoichiometry. Results The H1 complement of native chromatin was perturbed by adding an additional amount of one of the subtypes. A certain amount of SAR (scaffold-associated region) DNA was present in the mixture to avoid precipitation of chromatin by excess H1. SAR DNA also provided a set of reference relative affinities, which were needed to estimate the relative affinities of the subtypes for chromatin from the distribution of the subtypes between the SAR and the chromatin. The amounts of chromatin, SAR and additional H1 were adjusted so as to keep the stoichiometry of perturbed chromatin similar to that of native chromatin. H1 molecules freely exchanged between the chromatin and SAR binding sites. In conditions of free exchange, H1a was the subtype of lowest affinity, H1b and H1c had intermediate affinities and H1d, H1e and H1 the highest affinities. Subtype affinities for chromatin differed by up to 19-fold. The relative affinities of the subtypes for chromatin were equivalent to those estimated for a SAR DNA fragment and a pUC19 fragment of similar length. Avian H5 had an affinity ~12-fold higher than H1e for both DNA and chromatin. Conclusion H1 subtypes freely exchange in vitro between chromatin binding sites in physiological salt (0.14 M NaCl). The large differences in relative affinity of the H1 subtypes for chromatin suggest that differential affinity could be functionally relevant and thus contribute to the functional differentiation of the subtypes. The conservation of the relative affinities for SAR and non-SAR DNA, in spite of a strong preference for SAR sequences, indicates that differential affinity alone cannot be responsible for the heterogeneous distribution of some subtypes in cell nuclei. PMID:17498293

  13. Dropwise condensation

    PubMed Central

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

    2008-01-01

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

  14. Irradiation damage in chromatin isolated from V-79 Chinese hamster lung fibroblasts

    SciTech Connect

    Heussen, C.; Nackerdien, Z.; Smit, B.J.; Boehm, L.

    1987-04-01

    The effect of chromatin structure on the extent of radiation damage induced by low doses of 100 KeV X rays was investigated using a fluorescent assay for DNA unwinding. Chromatin was isolated from V-79 Chinese hamster lung fibroblast nuclei by partial digestion with micrococcal nuclease. Gel electrophoresis of the isolated DNA showed the molecular weight of the chromatin preparation to be 10.6 X 10(6) with a size range of 6.6-21.7 X 10(6) Da while a size of 10.2 +/- 0.9 X 10(6) Da was found by sedimenting the DNA in alkaline sucrose gradients. The repeat length of V-79 chromatin was found to be 194 +/- 3 bp. The typical nucleosomal repeat structure of the isolated chromatin and that of intact nuclei was identical. Irradiation with 50 and 100 Gy of 100 KeV X rays and analysis by alkaline sucrose density centrifugation indicated that V-79 chromatin sustained 0.56 +/- 0.19 and 0.69 +/- 0.09 single-strand breaks per 10 Gy per 10(8) Da of DNA, respectively. Irradiation with doses of 0.5-3.0 Gy of 100 KeV X rays and analysis by the fluorometric assay showed that the radiation sensitivity of V-79 chromatin decreases sharply on compaction with MgCl/sub 2/. Histone H1 depletion, which inhibits compaction and causes chromatin to expand by increasing the linker from 26 to 48 bp, results in a considerable increase in the radiation sensitivity. It is concluded that radiation damage sustained by DNA is greatly influenced by chromatin structure.

  15. Chromatin-associated protein phosphatase 1 regulates aurora-B and histone H3 phosphorylation.

    PubMed

    Murnion, M E; Adams, R R; Callister, D M; Allis, C D; Earnshaw, W C; Swedlow, J R

    2001-07-13

    Proper chromosome condensation requires the phosphorylation of histone and nonhistone chromatin proteins. We have used an in vitro chromosome assembly system based on Xenopus egg cytoplasmic extracts to study mitotic histone H3 phosphorylation. We identified a histone H3 Ser(10) kinase activity associated with isolated mitotic chromosomes. The histone H3 kinase was not affected by inhibitors of cyclin-dependent kinases, DNA-dependent protein kinase, p90(rsk), or cAMP-dependent protein kinase. The activity could be selectively eluted from mitotic chromosomes and immunoprecipitated by specific anti-X aurora-B/AIRK2 antibodies. This activity was regulated by phosphorylation. Treatment of X aurora-B immunoprecipitates with recombinant protein phosphatase 1 (PP1) inhibited kinase activity. The presence of PP1 on chromatin suggested that PP1 might directly regulate the X aurora-B associated kinase activity. Indeed, incubation of isolated interphase chromatin with the PP1-specific inhibitor I2 and ATP generated an H3 kinase activity that was also specifically immunoprecipitated by anti-X aurora-B antibodies. Nonetheless, we found that stimulation of histone H3 phosphorylation in interphase cytosol does not drive chromosome condensation or targeting of 13 S condensin to chromatin. In summary, the chromosome-associated mitotic histone H3 Ser(10) kinase is associated with X aurora-B and is inhibited directly in interphase chromatin by PP1. PMID:11350965

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

    PubMed Central

    Rawlings, Jason S.

    2015-01-01

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

  17. Three-dimensional cell growth confers radioresistance by chromatin density modification.

    PubMed

    Storch, Katja; Eke, Iris; Borgmann, Kerstin; Krause, Mechthild; Richter, Christian; Becker, Kerstin; Schröck, Evelin; Cordes, Nils

    2010-05-15

    Cell shape and architecture are determined by cell-extracellular matrix interactions and have profound effects on cellular behavior, chromatin condensation, and tumor cell resistance to radiotherapy and chemotherapy. To evaluate the role of chromatin condensation for radiation cell survival, tumor cells grown in three-dimensional (3D) cell cultures as xenografts and monolayer cell cultures were compared. Here, we show that increased levels of heterochromatin in 3D cell cultures characterized by histone H3 deacetylation and induced heterochromatin protein 1alpha expression result in increased radiation survival and reduced numbers of DNA double strand breaks (DSB) and lethal chromosome aberrations. Intriguingly, euchromatin to heterochromatin-associated DSBs were equally distributed in irradiated 3D cell cultures and xenograft tumors, whereas irradiated monolayer cultures showed a 2:1 euchromatin to heterochromatin DSB distribution. Depletion of histone deacetylase (HDAC) 1/2/4 or application of the class I/II pharmacologic HDAC inhibitor LBH589 induced moderate or strong chromatin decondensation, respectively, which was translated into cell line-dependent radiosensitization and, in case of LBH589, into an increased number of DSBs. Neither growth conditions nor HDAC modifications significantly affected the radiation-induced phosphorylation of the important DNA repair protein ataxia telangiectasia mutated. Our data show an interrelation between cell morphology and cellular radiosensitivity essentially based on chromatin organization. Understanding the molecular mechanisms by which chromatin structure influences the processing of radiation-induced DNA lesions is of high relevance for normal tissue protection and optimization of cancer therapy. PMID:20442295

  18. Chromatin-directed Ribonucleic Acid Synthesis

    PubMed Central

    Niles, R. M.; Mount, M. S.

    1973-01-01

    Chromatin was extracted from healthy, avirulent Agrobacterium tumefaciens inoculated, and crown-gall tumor Vicia faba internodes of the same age. Chromatin from crown-gall tissue produced 5 times more RNA per 100 micrograms of DNA than chromatin from the healthy tissue. When template availability was compared using chromatin with saturating amounts of Escherichia coli RNA polymerase, chromatin from crown-gall tissue had 36% more available template than the controls. In addition, when γ-32P-ATP was incorporated into the RNA synthesizing reaction mixture, with saturating amounts of E. coli RNA polymerase, there were twice as many RNA chain starts in tumor as in control tissue. PMID:16658564

  19. Chromatin in embryonic stem cell neuronal differentiation.

    PubMed

    Meshorer, E

    2007-03-01

    Chromatin, the basic regulatory unit of the eukaryotic genetic material, is controlled by epigenetic mechanisms including histone modifications, histone variants, DNA methylation and chromatin remodeling. Cellular differentiation involves large changes in gene expression concomitant with alterations in genome organization and chromatin structure. Such changes are particularly evident in self-renewing pluripotent embryonic stem cells, which begin, in terms of cell fate, as a tabula rasa, and through the process of differentiation, acquire distinct identities. Here I describe the changes in chromatin that accompany neuronal differentiation, particularly of embryonic stem cells, and discuss how chromatin serves as the master regulator of cellular destiny. PMID:17163405

  20. Histone lysine methylation and chromatin replication.

    PubMed

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

    2014-12-01

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

  1. 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

  2. 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

  3. CpG island chromatin

    PubMed Central

    Blackledge, Neil P

    2011-01-01

    The majority of mammalian gene promoters are encompassed within regions of the genome called CpG islands that have an elevated level of non-methylated CpG dinucleotides. Despite over 20 years of study, the precise mechanisms by which CpG islands contribute to regulatory element function remain poorly understood. Recently it has been demonstrated that specific histone modifying enzymes are recruited directly to CpG islands through recognition of non-methylated CpG dinucleotide sequence. These enzymes then impose unique chromatin architecture on CpG islands that distinguish them from the surrounding genome. In the context of this work we discuss how CpG island elements may contribute to the function of gene regulatory elements through the utilization of chromatin and epigenetic processes. PMID:20935486

  4. Transcription factor access to chromatin.

    PubMed Central

    Beato, M; Eisfeld, K

    1997-01-01

    The question of how sequence-specific transcription factors access their cognate sites in nucleosomally organized DNA is discussed on the basis of genomic footprinting data and chromatin reconstitution experiments. A classification of factors into two categories is proposed: (i) initiator factors which are able to bind their target sequences within regular nucleosomes and initiate events leading to chromatin remodelling and transactivation; (ii) effector factors which are unable to bind regular nucleosomes and depend on initiator factors or on a pre-set nucleosomal structure for accessing their target sequences in chromatin. Studies with the MMTV promoter suggest that the extent and number of protein-DNA contacts determine whether a factor belongs to one or the other category. Initiator factors have only a few DNA contacts clustered on one side of the double helix, whereas effector factors have extensive contacts distributed throughout the whole circumference of the DNA helix. Thus, the nature of DNA recognition confers to sequence-specific factors their specific place in the sequential hierarchy of gene regulatory events. PMID:9278473

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

    PubMed

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

    2014-09-01

    The mammalian oocyte nucleus or germinal vesicle (GV) exhibits characteristic chromatin configurations, which are subject to dynamic modifications through oogenesis. Aim of this review is to highlight how changes in chromatin configurations are related to both functional and structural modifications occurring in the oocyte nuclear and cytoplasmic compartments. During the long phase of meiotic arrest at the diplotene stage, the chromatin enclosed within the GV is subjected to several levels of regulation. Morphologically, the chromosomes lose their individuality and form a loose chromatin mass. The decondensed configuration of chromatin then undergoes profound rearrangements during the final stages of oocyte growth that are tightly associated with the acquisition of meiotic and developmental competence. Functionally, the discrete stages of chromatin condensation are characterized by different level of transcriptional activity, DNA methylation and covalent histone modifications. Interestingly, the program of chromatin rearrangement is not completely intrinsic to the oocyte, but follicular cells exert their regulatory actions through gap junction mediated communications and intracellular messenger dependent mechanism(s). With this in mind and since oocyte growth mostly relies on the bidirectional interaction with the follicular cells, a connection between cumulus cells gene expression profile and oocyte developmental competence, according to chromatin configuration is proposed. This analysis can help in identifying candidate genes involved in the process of oocyte developmental competence acquisition and in providing non-invasive biomarkers of oocyte health status that can have important implications in treating human infertility as well as managing breeding schemes in domestic mammals. PMID:25028181

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

    SciTech Connect

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

    2007-10-01

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

  7. 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

  8. 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.

  9. 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.

  10. Mechanics of Viral Chromatin Reveals the Pressurization of Human Adenovirus.

    PubMed

    Ortega-Esteban, Alvaro; Condezo, Gabriela N; Pérez-Berná, Ana J; Chillón, Miguel; Flint, S Jane; Reguera, David; San Martín, Carmen; de Pablo, Pedro J

    2015-11-24

    Tight confinement of naked genomes within some viruses results in high internal pressure that facilitates their translocation into the host. Adenovirus, however, encodes histone-like proteins that associate with its genome resulting in a confined DNA-protein condensate (core). Cleavage of these proteins during maturation decreases core condensation and primes the virion for proper uncoating via unidentified mechanisms. Here we open individual, mature and immature adenovirus cages to directly probe the mechanics of their chromatin-like cores. We find that immature cores are more rigid than the mature ones, unveiling a mechanical signature of their condensation level. Conversely, intact mature particles demonstrate more rigidity than immature or empty ones. DNA-condensing polyamines revert the mechanics of mature capsid and cores to near-immature values. The combination of these experiments reveals the pressurization of adenovirus particles induced by maturation. We estimate a pressure of ∼30 atm by continuous elasticity, which is corroborated by modeling the adenovirus mini-chromosome as a confined compact polymer. We propose this pressurization as a mechanism that facilitates initiating the stepwise disassembly of the mature particle, enabling its escape from the endosome and final genome release at the nuclear pore. PMID:26491879

  11. Multiplex Single Cell Profiling of Chromatin Accessibility by Combinatorial Cellular Indexing

    PubMed Central

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

    2016-01-01

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

  12. 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

  13. Unreplicated DNA in mitosis precludes condensin binding and chromosome condensation in S. cerevisiae

    PubMed Central

    Dulev, Stanimir; Aragon, Luis; Strunnikov, Alexander

    2009-01-01

    Condensin is the core activity responsible for chromosome condensation in mitosis. In the yeast S. cerevisiae, condensin binding is enriched at the regions where DNA replication terminates. Therefore, we investigated whether DNA replication completion determines the condensin-binding proficiency of chromatin. In order to fulfill putative mitotic requirements for condensin activity we analyzed chromosome condensation and condensin binding to unreplicated chromosomes in mitosis. For this purpose we used pGAL:CDC6 cdc15-ts cells that are known to enter mitosis without DNA replication if CDC6 transcription is repressed prior to S-phase. Both the condensation of nucleolar chromatin and proper condensin targeting to rDNA sites failed when unreplicated chromosomes were driven in mitosis. We propose that the DNA replication results in structural and/or biochemical changes to replicated chromatin, which are required for two-phase condensin binding and proper chromosome condensation. PMID:18508626

  14. Chromatin state dynamics during blood formation

    PubMed Central

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

    2015-01-01

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

  15. Proteomics of a fuzzy organelle: interphase chromatin

    PubMed Central

    Kustatscher, Georg; Hgarat, 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

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

    PubMed

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

    2016-01-01

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

  17. Defining the chromatin landscape in demyelinating disorders

    PubMed Central

    Huynh, Jimmy Long; Casaccia, Patrizia

    2010-01-01

    An intricate network of epigenetic factors regulates cell differentiation by modulating the chromatin structure and ultimately affecting gene expression. This review describes the chromatin landscape defining oligodendrocyte progenitor differentiation during development and remyelination. We shall discuss the current knowledge regarding modifications of chromatin components during the progression of progenitors into myelinating cells and discuss the potential contribution of histone variants, microRNAs, and DNA methylation. We shall also briefly address how changes to this chromatin landscape can disturb this natural progression and alter the capacity to remyelinate. PMID:19853663

  18. Single cell correlation fractal dimension of chromatin

    PubMed Central

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

    2014-01-01

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

  19. Identification of lamin B–regulated chromatin regions based on chromatin landscapes

    PubMed Central

    Zheng, Xiaobin; Kim, Youngjo; Zheng, Yixian

    2015-01-01

    Lamins, the major structural components of the nuclear lamina (NL) found beneath the nuclear envelope, are known to interact with most of the nuclear peripheral chromatin in metazoan cells. Although NL–chromatin associations correlate with a repressive chromatin state, the role of lamins in tethering chromatin to NL and how such tether influences gene expression have remained challenging to decipher. Studies suggest that NL proteins regulate chromatin in a context-dependent manner. Therefore understanding the context of chromatin states based on genomic features, including chromatin–NL interactions, is important to the study of lamins and other NL proteins. By modeling genome organization based on combinatorial patterns of chromatin association with lamin B1, core histone modification, and core and linker histone occupancy, we report six distinct large chromatin landscapes, referred to as histone lamin landscapes (HiLands)-red (R), -orange (O), -yellow (Y), -green (G), -blue (B), and -purple (P), in mouse embryonic stem cells (mESCs). This HiLands model demarcates the previously mapped lamin-associated chromatin domains (LADs) into two HiLands, HiLands-B and HiLands-P, which are similar to facultative and constitutive heterochromatins, respectively. Deletion of B-type lamins in mESCs caused a reduced interaction between regions of HiLands-B and NL as measured by emerin–chromatin interaction. Our findings reveal the importance of analyzing specific chromatin types when studying the function of NL proteins in chromatin tether and regulation. PMID:25995381

  20. Fourier transform infrared spectroscopic analysis of sperm chromatin structure and DNA stability.

    PubMed

    Oldenhof, H; Schütze, S; Wolkers, W F; Sieme, H

    2016-05-01

    Sperm chromatin structure and condensation determine accessibility for damage, and hence success of fertilization and development. The aim of this study was to reveal characteristic spectral features coinciding with abnormal sperm chromatin packing (i.e., DNA-protein interactions) and decreased fertility, using Fourier transform infrared spectroscopy. Chromatin structure in spermatozoa obtained from different stallions was investigated. Furthermore, spermatozoa were exposed to oxidative stress, or treated with thiol-oxidizing and disulfide-reducing agents, to alter chromatin structure and packing. Spectroscopic studies were corroborated with flow cytometric analyses using the DNA-intercalating fluorescent dye acridine orange. Decreased fertility of individuals correlated with increased abnormal sperm morphology and decreased stability toward induced DNA damage. Treatment with the disulfide reducing agent dithiothreitol resulted in increased sperm chromatin decondensation and DNA accessibility, similar as found for less mature epididymal spermatozoa. In situ infrared spectroscopic analysis revealed that characteristic bands arising from the DNA backbone (ν1230, ν1086, ν1051 cm(-1) ) changed in response to induced oxidative damage, water removal, and decondensation. This coincided with changes in the amide-I region (intensity at ν1620 vs. ν1640 cm(-1) ) denoting concomitant changes in protein secondary structure. Reduction in protein disulfide bonds resulted in a decreased value of the asymmetric to symmetric phosphate band intensity (ν1230/ν1086 cm(-1) ), suggesting that this band ratio is sensitive for the degree of chromatin condensation. Moreover, when analyzing spermatozoa from different individuals, it was found that the asymmetric/symmetric phosphate band ratio negatively correlated with the percentage of morphologically abnormal spermatozoa. PMID:26916383

  1. Chromatin Regulatory Mechanisms in Pluripotency

    PubMed Central

    Lessard, Julie A.; Crabtree, Gerald R.

    2011-01-01

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

  2. Human tRNA genes function as chromatin insulators

    PubMed Central

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

    2012-01-01

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

  3. Tissue-Specific Regulation of Chromatin Insulator Function

    PubMed Central

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

    2012-01-01

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

  4. 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

  5. In vitro histone demethylase assays.

    PubMed

    Kokura, Kenji; Fang, Jia

    2009-01-01

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

  6. A comparison of the effect of lead nitrate on rat liver chromatin, DNA and histone proteins in solution.

    PubMed

    Rabbani-Chadegani, Azra; Abdosamadi, Sayeh; Fani, Nesa; Mohammadian, Shayesteh

    2009-06-01

    Although lead is widely recognized as a toxic substance in the environment and directly damage DNA, no studies are available on lead interaction with chromatin and histone proteins. In this work, we have examined the effect of lead nitrate on EDTA-soluble chromatin (SE chromatin), DNA and histones in solution using absorption and fluorescence spectroscopy, thermal denaturation and gel electrophoresis techniques. The results demonstrate that lead nitrate binds with higher affinity to chromatin than to DNA and produces an insoluble complex as monitored at 400 nm. Binding of lead to DNA decreases its Tm, increases its fluorescence intensity and exhibits hypochromicity at 210 nm which reveal that both DNA bases and the backbone participate in the lead-DNA interaction. Lead also binds strongly to histone proteins in the absence of DNA. The results suggest that although lead destabilizes DNA structure, in the chromatin, the binding of lead introduces some sort of compaction and aggregation, and the histone proteins play a key role in this aspect. This chromatin condensation, upon lead exposure, in turn may decrease fidelity of DNA, and inhibits DNA and RNA synthesis, the process that introduces lead toxicity at the chromatin level. PMID:18839148

  7. Condensate handling means for condensing furnace

    SciTech Connect

    Tomlinson, R. S.; Trent, B. O.

    1985-10-01

    An improved condensate neutralizer for use in a high efficiency furnace utilizing hydrocarbon gaseous fuel wherein acidic liquid condensate forms. The neutralizer is provided with a bypass for conducting the condensate from the furnace directly to a drain without passing through the neutralizer in the event condensate flow through the neutralizer becomes substantially impeded. Also disclosed is structure for indicating substantial impediment of flow of the condensate through the neutralizer for alerting the user to the need of servicing of the neutralizer. A separator/trap is provided for transferring the condensate from the furnace selectively to the neutralizer or bypass.

  8. 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

  9. Mechanisms for ATP-dependent chromatin remodelling.

    PubMed

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

    2000-01-01

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

  10. Modifying chromatin and concepts of cancer.

    PubMed

    Jacobson, S; Pillus, L

    1999-04-01

    A wave of biochemical and genetic progress has identified proteins that influence transcription by modifying chromatin. Many such factors are broadly conserved and, strikingly, occur in human diseases as partners in chromosomal translocations. Understanding the function of these factors will be key to defining mechanisms of transcriptional regulation and understanding cancers linked to aberrant chromatin modification. PMID:10322138

  11. Chromatin Remodelers: From Function to Dysfunction

    PubMed Central

    Längst, Gernot; Manelyte, Laura

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

    Summary In C. elegans, mRNA production is initially repressed in the embryonic germline by a protein unique to C. elegans germ cells, PIE-1. PIE-1 is degraded upon the birth of the germ cell precursors, Z2 and Z3. We have identified a chromatin-based mechanism that succeeds PIE-1 repression in these cells. A subset of nucleosomal histone modifications, methylated lysine 4 on histone H3 (H3meK4) and acetylated lysine 8 on histone H4 (H4acetylK8), are globally lost and the DNA appears more condensed. This coincides with PIE-1 degradation and requires that germline identity is not disrupted. Drosophila pole cell chromatin also lacks H3meK4, indicating that a unique chromatin architecture is a conserved feature of embryonic germ cells. Regulation of the germline-specific chromatin architecture requires functional nanos activity in both organisms. These results indicate that genome-wide repression via a nanos-regulated, germ cell-specific chromatin organization is a conserved feature of germline maintenance during embryogenesis. PMID:14602075

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

    PubMed Central

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

    1990-01-01

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

  14. Chromatin Dynamics of Circadian Transcription

    PubMed Central

    Aguilar-Arnal, Lorena; Sassone-Corsi, Paolo

    2015-01-01

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

  15. Inheritance of epigenetic chromatin silencing

    PubMed Central

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

    2010-01-01

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

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

    SciTech Connect

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

    2010-09-17

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

  17. 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

  18. Ribonucleic Acid Synthesis by Cucumber Chromatin

    PubMed Central

    Johnson, Kenneth D.; Purves, William K.

    1970-01-01

    When intact etiolated 2-day cucumber (Cucumis sativus) embryos were treated with indoleacetic acid (IAA), gibberellin A7 (GA7), or kinetin, chromatin derived from the embryonic axes exhibited an increased capacity to support RNA synthesis in either the presence or the absence of bacterial RNA polymerase. An IAA effect on cucumber RNA polymerase activity was evident after 4 hours of hormone treatment; the IAA effect on DNA template activity (bacterial RNA polymerase added) occurred after longer treatments (12 hours). GA7 also promoted template activity, but again only after a prior stimulation of endogenous chromatin activity. After 12 hours of kinetin treatment, both endogenous chromatin and DNA template activities were substantially above control values, but longer kinetin treatments caused these activities to decline in magnitude. When chromatin was prepared from hypocotyl segments that were floated on a GA7 solution, a GA-induced increase in endogenous chromatin activity occurred, but only if cotyledon tissue was left attached to the segments during the period of hormone treatment. Age of the seedling tissue had a profound influence on the chromatin characteristics. With progression of development from the 2-day to the 4-day stage, the endogenous chromatin activity declined while the DNA template activity increased. PMID:16657509

  19. Condensation model for the ESBWR passive condensers

    SciTech Connect

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

    2012-07-01

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

  20. Microfluidics Technologies for Low Cell Number Chromatin Immunoprecipitation.

    PubMed

    Wu, Angela R; Quake, Stephen R

    2016-01-01

    Protein-DNA interactions are responsible for numerous critical cellular events: For example, gene expression and silencing are mediated by transcription factor protein binding and histone protein modifications, and DNA replication and repair rely on site-specific protein binding. Chromatin immunoprecipitation (ChIP) is the only molecular assay that directly determines, in a living cell, the binding association between a protein of interest and specific genomic loci. It is an indispensible tool in the biologist's toolbox, but the many limitations of this technique prevent broad adoption of ChIP in biological studies. The typical ChIP assay can take up to 1 wk to complete, and the process is technically tricky, yet tedious. The ChIP assay yields are also low, thus requiring on the order of millions to billions of cells as starting material, which makes the assay unfeasible for studies using rare or precious samples. For example, fluorescence-activated cell sorting (FACS) of cancer stem cells (CSCs) obtained from primary tumors, rarely yields more than ~100,000 CSCs per tumor. This protocol describes a microfluidics-based strategy for performing ChIP, which uses automation and scalability to reduce both total and hands-on assay time, and improve throughput. It allows whole fixed cells as input, and enables automated ChIP from as few as 2000 cells. PMID:26700100

  1. Chromatin fiber dynamics under tension and torsion.

    PubMed

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

    2010-01-01

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

  2. Chromatin Evolution and Molecular Drive in Speciation

    PubMed Central

    Sawamura, Kyoichi

    2012-01-01

    Are there biological generalities that underlie hybrid sterility or inviability? Recently, around a dozen “speciation genes” have been identified mainly in Drosophila, and the biological functions of these genes are revealing molecular generalities. Major cases of hybrid sterility and inviability seem to result from chromatin evolution and molecular drive in speciation. Repetitive satellite DNAs within heterochromatin, especially at centromeres, evolve rapidly through molecular drive mechanisms (both meiotic and centromeric). Chromatin-binding proteins, therefore, must also evolve rapidly to maintain binding capability. As a result, chromatin binding proteins may not be able to interact with chromosomes from another species in a hybrid, causing hybrid sterility and inviability. PMID:22191063

  3. Nucleosome structure in chromatin from heated cells

    SciTech Connect

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

    1980-12-01

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

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

    SciTech Connect

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

    2005-01-15

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

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

    PubMed Central

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

    1980-01-01

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

  6. 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

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

    PubMed Central

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

    2010-01-01

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

  8. 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

  9. 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

  10. In vivo binding of retinol to chromatin

    SciTech Connect

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

    1988-01-05

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

  11. Nucleotide excision repair and chromatin remodeling.

    PubMed

    Ura, Kiyoe; Hayes, Jeffrey J

    2002-05-01

    The organization of DNA within eukaryotic cell nuclei poses special problems and opportunities for the cell. For example, assembly of DNA into chromatin is thought to be a principle mechanism by which adventitious general transcription is repressed. However, access to genomic DNA for events such as DNA repair must be facilitated by energy-intensive processes that either directly alter chromatin structure or impart post-translational modifications, leading to increased DNA accessibility. The assembly of DNA into chromatin affects both the incidence of damage to DNA and repair of that damage. Correction of most damage to DNA caused by UV irradiation occurs via the nucleotide excision repair (NER) process. NER requires extensive involvement of large multiprotein complexes with relatively large stretches of DNA. Here, we review recent evidence suggesting that at least some steps of NER require ATP-dependent chromatin remodeling activities while perhaps others do not. PMID:11985610

  12. Predictive Computational Modeling of Chromatin Folding

    NASA Astrophysics Data System (ADS)

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

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

  13. FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin

    PubMed Central

    Giresi, Paul G.; Kim, Jonghwan; McDaniell, Ryan M.; Iyer, Vishwanath R.; Lieb, Jason D.

    2007-01-01

    DNA segments that actively regulate transcription in vivo are typically characterized by eviction of nucleosomes from chromatin and are experimentally identified by their hypersensitivity to nucleases. Here we demonstrate a simple procedure for the isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements). To perform FAIRE, chromatin is crosslinked with formaldehyde in vivo, sheared by sonication, and phenol-chloroform extracted. The DNA recovered in the aqueous phase is fluorescently labeled and hybridized to a DNA microarray. FAIRE performed in human cells strongly enriches DNA coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, and active promoters. Evidence for cell-type–specific patterns of FAIRE enrichment is also presented. FAIRE has utility as a positive selection for genomic regions associated with regulatory activity, including regions traditionally detected by nuclease hypersensitivity assays. PMID:17179217

  14. 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

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

    PubMed

    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

    2014-03-01

    To maintain genome function and stability, DNA sequence and its organization into chromatin must be duplicated during cell division. Understanding how entire chromosomes are copied remains a major challenge. Here, we use nascent chromatin capture (NCC) to profile chromatin proteome dynamics during replication in human cells. NCC relies on biotin-dUTP labelling of replicating DNA, affinity purification and quantitative proteomics. Comparing nascent chromatin with mature post-replicative chromatin, we provide association dynamics for 3,995 proteins. The replication machinery and 485 chromatin factors such as CAF-1, DNMT1 and SUV39h1 are enriched in nascent chromatin, whereas 170 factors including histone H1, DNMT3, MBD1-3 and PRC1 show delayed association. This correlates with H4K5K12diAc removal and H3K9me1 accumulation, whereas H3K27me3 and H3K9me3 remain unchanged. Finally, we combine NCC enrichment 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

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

    PubMed Central

    Zhou, Jian; Troyanskaya, Olga G.

    2016-01-01

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

  17. Nucleosome repeat lengths and columnar chromatin structure.

    PubMed

    Trifonov, Edward N

    2016-06-01

    Thorough quantitative study of nucleosome repeat length (NRL) distributions, conducted in 1992 by J. Widom, resulted in a striking observation that the linker lengths between the nucleosomes are quantized. Comparison of the NRL average values with the MNase cut distances predicted from the hypothetical columnar structure of chromatin (this work) shows a close correspondence between the two. This strongly suggests that the NRL distribution, actually, reflects the dominant role of columnar chromatin structure common for all eukaryotes. PMID:26208520

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

    SciTech Connect

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

    2006-10-15

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

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

    PubMed Central

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

    2015-01-01

    It has been well established that the architecture of chromatin in cell nuclei is not random but functionally correlated. Chromatin damage caused by ionizing radiation raises complex repair machineries. This is accompanied by local chromatin rearrangements and structural changes which may for instance improve the accessibility of damaged sites for repair protein complexes. Using stably transfected HeLa cells expressing either green fluorescent protein (GFP) labelled histone H2B or yellow fluorescent protein (YFP) labelled histone H2A, we investigated the positioning of individual histone proteins in cell nuclei by means of high resolution localization microscopy (Spectral Position Determination Microscopy = SPDM). The cells were exposed to ionizing radiation of different doses and aliquots were fixed after different repair times for SPDM imaging. In addition to the repair dependent histone protein pattern, the positioning of antibodies specific for heterochromatin and euchromatin was separately recorded by SPDM. The present paper aims to provide a quantitative description of structural changes of chromatin after irradiation and during repair. It introduces a novel approach to analyse SPDM images by means of statistical physics and graph theory. The method is based on the calculation of the radial distribution functions as well as edge length distributions for graphs defined by a triangulation of the marker positions. The obtained results show that through the cell nucleus the different chromatin re-arrangements as detected by the fluorescent nucleosomal pattern average themselves. In contrast heterochromatic regions alone indicate a relaxation after radiation exposure and re-condensation during repair whereas euchromatin seemed to be unaffected or behave contrarily. SPDM in combination with the analysis techniques applied allows the systematic elucidation of chromatin re-arrangements after irradiation and during repair, if selected sub-regions of nuclei are investigated. PMID:26042422

  20. Chromatin remodeling agents for cancer therapy.

    PubMed

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

    2008-09-01

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

  1. Stable Chromosome Condensation Revealed by Chromosome Conformation Capture.

    PubMed

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

    2015-11-01

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

  2. Poly(ADP-ribosyl)ation regulates CTCF-dependent chromatin insulation.

    PubMed

    Yu, Wenqiang; Ginjala, Vasudeva; Pant, Vinod; Chernukhin, Igor; Whitehead, Joanne; Docquier, France; Farrar, Dawn; Tavoosidana, Gholamreza; Mukhopadhyay, Rituparna; Kanduri, Chandrasekhar; Oshimura, Mitsuo; Feinberg, Andrew P; Lobanenkov, Victor; Klenova, Elena; Ohlsson, Rolf

    2004-10-01

    Chromatin insulators demarcate expression domains by blocking the cis effects of enhancers or silencers in a position-dependent manner. We show that the chromatin insulator protein CTCF carries a post-translational modification: poly(ADP-ribosyl)ation. Chromatin immunoprecipitation analysis showed that a poly(ADP-ribosyl)ation mark, which exclusively segregates with the maternal allele of the insulator domain in the H19 imprinting control region, requires the bases that are essential for interaction with CTCF. Chromatin immunoprecipitation-on-chip analysis documented that the link between CTCF and poly(ADP-ribosyl)ation extended to more than 140 mouse CTCF target sites. An insulator trap assay showed that the insulator function of most of these CTCF target sites is sensitive to 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase activity. We suggest that poly(ADP-ribosyl)ation imparts chromatin insulator properties to CTCF at both imprinted and nonimprinted loci, which has implications for the regulation of expression domains and their demise in pathological lesions. PMID:15361875

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

    PubMed Central

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

    2014-01-01

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

  4. 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

  5. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2008-01-01

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

  8. 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

  9. Large-scale chromatin morpho-functional changes during mammalian oocyte growth and differentiation

    PubMed Central

    Luciano, A.M.; Lodde, V.; Franciosi, F.; Tessaro, I.; Corbani, D.; Modina, S.C.

    2012-01-01

    Mammalian oocyte development is characterized by impressive changes in chromatin structure and function within the germinal vesicle (GV). These changes are crucial to confer the oocyte with meiotic and developmental competencies. In cow, oocytes collected from early and middle antral follicles present four patterns of chromatin configuration, from GV0 to GV3, and its progressive condensation has been related to the achievement of developmental potential. During oogenesis, follicular cells are essential for the acquisition of meiotic and developmental competencies and communicate with the oocyte by paracrine and gap junction mediated mechanisms. We recently analyzed the role of gap junction communications (GJC) on chromatin remodeling process during the specific phase of folliculogenesis that coincides with the transcriptional silencing and sequential acquisition of meiotic and developmental capabilities. Our studies demonstrated that GJC between germinal and somatic compartments plays a fundamental role in the regulation of chromatin remodeling and transcription activities during the final oocyte differentiation, throughout cAMP dependent mechanism(s). PMID:23027353

  10. 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

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

    PubMed

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

    2015-11-24

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

  12. Changing Chromatin Fiber Conformation by Nucleosome Repositioning

    PubMed Central

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

    2014-01-01

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

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

    ERIC Educational Resources Information Center

    Eirin-Lopez, Jose M.

    2013-01-01

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

  14. 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

  15. The chromatin scaffold protein SAFB1 renders chromatin permissive for DNA damage signaling.

    PubMed

    Altmeyer, Matthias; Toledo, Luis; Gudjonsson, Thorkell; Grøfte, Merete; Rask, Maj-Britt; Lukas, Claudia; Akimov, Vyacheslav; Blagoev, Blagoy; Bartek, Jiri; Lukas, Jiri

    2013-10-24

    Although the general relevance of chromatin modifications for genotoxic stress signaling, cell-cycle checkpoint activation, and DNA repair is well established, how these modifications reach initial thresholds in order to trigger robust responses remains largely unexplored. Here, we identify the chromatin-associated scaffold attachment factor SAFB1 as a component of the DNA damage response and show that SAFB1 cooperates with histone acetylation to allow for efficient γH2AX spreading and genotoxic stress signaling. SAFB1 undergoes a highly dynamic exchange at damaged chromatin in a poly(ADP-ribose)-polymerase 1- and poly(ADP-ribose)-dependent manner and is required for unperturbed cell-cycle checkpoint activation and guarding cells against replicative stress. Altogether, our data reveal that transient recruitment of an architectural chromatin component is required in order to overcome physiological barriers by making chromatin permissive for DNA damage signaling, whereas the ensuing exclusion of SAFB1 may help prevent excessive signaling. PMID:24055346

  16. 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

  17. Gearing up chromatin: A role for chromatin remodeling during the transcriptional restart upon DNA damage.

    PubMed

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

    2014-01-01

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

  18. Targeting chromatin to improve radiation response.

    PubMed

    Olcina, M M; O'Dell, S; Hammond, E M

    2015-03-01

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

  19. Chromatin remodeling in cardiovascular development and physiology

    PubMed Central

    Han, Pei; Hang, Calvin T.; Yang, Jin; Chang, Ching-Pin

    2010-01-01

    Chromatin regulation provides an important means of controlling cardiac gene expression under different physiological and pathological conditions. Processes that direct the development of normal embryonic hearts and pathology of stressed adult hearts may share general mechanisms that govern cardiac gene expression by chromatin-regulating factors. These common mechanisms may provide a framework for us to investigate the interactions among diverse chromatin remodelers/modifiers and various transcription factors in the fine regulation of gene expression, essential for all aspects of cardiovascular biology. Aberrant cardiac gene expression, triggered by a variety of pathological insults, can cause heart diseases in both animals and humans. The severity of cardiomyopathy and heart failure correlates strongly with abnormal cardiac gene expression. Therefore, controlling cardiac gene expression presents a promising approach to the treatment of human cardiomyopathy. This review focuses on the roles of ATP-dependent chromatin-remodeling factors and chromatin-modifying enzymes in the control of gene expression during cardiovascular development and disease. PMID:21293009

  20. Targeting chromatin to improve radiation response

    PubMed Central

    Olcina, M M; O'Dell, S

    2015-01-01

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

  1. Chromatin Ring Formation at Plant Centromeres.

    PubMed

    Schubert, Veit; Ruban, Alevtina; Houben, Andreas

    2016-01-01

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

  2. Chromatin Ring Formation at Plant Centromeres

    PubMed Central

    Schubert, Veit; Ruban, Alevtina; Houben, Andreas

    2016-01-01

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

  3. Condensates in Jovian Atmospheres

    NASA Technical Reports Server (NTRS)

    West, R.

    1999-01-01

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

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

    SciTech Connect

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

    2010-11-11

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

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

    PubMed Central

    Tajbakhsh, Jian

    2011-01-01

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

  6. 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

  7. Nucleosome dynamics during chromatin remodeling in vivo.

    PubMed

    Ramachandran, Srinivas; Henikoff, Steven

    2016-03-01

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

  8. 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

  9. 4D chromatin dynamics in cycling cells

    PubMed Central

    Strickfaden, Hilmar; Zunhammer, Andreas; van Koningsbruggen, Silvana; Köhler, Daniela

    2010-01-01

    This live cell study of chromatin dynamics in four dimensions (space and time) in cycling human cells provides direct evidence for three hypotheses first proposed by Theodor Boveri in seminal studies of fixed blastomeres from Parascaris equorum embryos: (I) Chromosome territory (CT) arrangements are stably maintained during interphase. (II) Chromosome proximity patterns change profoundly during prometaphase. (III) Similar CT proximity patterns in pairs of daughter nuclei reflect symmetrical chromosomal movements during anaphase and telophase, but differ substantially from the arrangement in mother cell nucleus. Hypothesis I could be confirmed for the majority of interphase cells. A minority, however, showed complex, rotational movements of CT assemblies with large-scale changes of CT proximity patterns, while radial nuclear arrangements were maintained. A new model of chromatin dynamics is proposed. It suggests that long-range DNA-DNA interactions in cell nuclei may depend on a combination of rotational CT movements and locally constrained chromatin movements. PMID:21327076

  10. Autism genes keep turning up chromatin

    PubMed Central

    LaSalle, Janine M.

    2013-01-01

    Autism-spectrum disorders (ASD) are complex genetic disorders collectively characterized by impaired social interactions and language as well as repetitive and restrictive behaviors. Of the hundreds of genes implicated in ASD, those encoding proteins acting at neuronal synapses have been most characterized by candidate gene studies. However, recent unbiased genome-wide analyses have turned up a multitude of novel candidate genes encoding nuclear factors implicated in chromatin remodeling, histone demethylation, histone variants, and the recognition of DNA methylation. Furthermore, the chromatin landscape of the human genome has been shown to influence the location of de novo mutations observed in ASD as well as the landscape of DNA methylation underlying neurodevelopmental and synaptic processes. Understanding the interactions of nuclear chromatin proteins and DNA with signal transduction pathways and environmental influences in the developing brain will be critical to understanding the relevance of these ASD candidate genes and continued uncovering of the “roots” of autism etiology. PMID:24404383

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

    NASA Astrophysics Data System (ADS)

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

    2001-10-01

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

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

    PubMed

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

    2004-06-01

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

  13. Chromatin connections to pluripotency and cellular reprogramming

    PubMed Central

    Orkin, Stuart H.; Hochedlinger, Konrad

    2016-01-01

    The pluripotent state of embryonic stem (ES) cells provides a unique perspective on regulatory programs that govern self-renewal and differentiation, and somatic cell reprogramming. Here we review the highly connected protein and transcriptional networks that maintain pluripotency, and how they are intertwined with factors that affect chromatin structure and function. The complex interrelationships between pluripotency and chromatin factors are illustrated by X-chromosome inactivation, regulatory control by non-coding RNAs, and environmental influences on cell states. Recent findings suggest a model in which environmental cues and growth conditions may direct the fate of cells transitioning a “plastic state” induced during reprogramming. PMID:21663790

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

    PubMed Central

    Chen, Hsiuyi V.; Rando, Oliver J.

    2014-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

  17. Chromatin organization of gammaherpesvirus latent genomes.

    PubMed

    Tempera, Italo; Lieberman, Paul M

    2010-01-01

    The gammaherpesviruses are a subclass of the herpesvirus family that establish stable latent infections in proliferating lymphoid and epithelial cells. The latent genomes are maintained as multicopy chromatinized episomes that replicate in synchrony with the cellular genome. Importantly, most of the episomes do not integrate into the host chromosome. Therefore, it is essential that the viral "minichromosome" establish a chromatin structure that is suitable for gene expression, DNA replication, and chromosome segregation. Evidence suggests that chromatin organization is important for each of these functions and plays a regulatory role in the establishment and maintenance of latent infection. Here, we review recent studies on the chromatin organization of the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). We discuss the potential role of viral origins of DNA replication and viral encoded origin-binding proteins like EBNA1 and LANA in establishment of viral chromosome organization during latent infection. We also discuss the roles of host cell factors, like CTCF and cohesins, that contribute to higher-order chromosome structures that may be important for stable gene expression programs during latent infection in proliferating cells. PMID:19853673

  18. Chromatin regulation: How complex does it get?

    PubMed Central

    Meier, Karin; Brehm, Alexander

    2014-01-01

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

  19. Chemical biology: Chromatin chemistry goes cellular

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  20. Oncogene-mediated alterations in chromatin conformation

    PubMed Central

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

    2012-01-01

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

  1. Epigenetic chromatin silencing: bistability and front propagation

    PubMed Central

    Sedighi, Mohammad; Sengupta, Anirvan M

    2008-01-01

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

  2. Chromatin modifiers, cognitive disorders, and imprinted genes

    PubMed Central

    Cunningham, Melissa D.; Kassis, Judith A.; Pfeifer, Karl

    2010-01-01

    In this issue of Developmental Cell, Kernohan et al. link the chromatin regulatory proteins ATRX, MeCP2, CTCF, and cohesin with silencing of H19 and other imprinted genes during critical stages of postnatal brain development, perhaps suggesting a common etiology for several human diseases that exhibit defects in brain development and function. PMID:20159587

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

    PubMed Central

    Cui, Mingxue; Han, Min

    2007-01-01

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

  4. 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

  5. Inferential modeling of 3D chromatin structure

    PubMed Central

    Wang, Siyu; Xu, Jinbo; Zeng, Jianyang

    2015-01-01

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

  6. Inferential modeling of 3D chromatin structure.

    PubMed

    Wang, Siyu; Xu, Jinbo; Zeng, Jianyang

    2015-04-30

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

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

    PubMed

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

    2015-02-01

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

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

    PubMed Central

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

    2012-01-01

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

  9. 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 γH2AX 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

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

    PubMed Central

    Ellis, Kaitlyn; Friedman, Chloe; Yedvobnick, Barry

    2015-01-01

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

  11. Ensuring condensate recovery efficiency.

    PubMed

    Mayoh, Paul

    2012-09-01

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

  12. O-GlcNAc Transferase Regulates Mitotic Chromatin Dynamics*

    PubMed Central

    Sakabe, Kaoru; Hart, Gerald W.

    2010-01-01

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

  13. Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

    SciTech Connect

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

    2005-11-01

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

  14. Chromatin signature discovery via histone modification profile alignments

    PubMed Central

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

    2012-01-01

    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

  15. Hyperdynamic Plasticity of Chromatin Proteins in Pluripotent Embryonic Stem Cells

    PubMed Central

    Meshorer, Eran; Yellajoshula, Dhananjay; George, Eric; Scambler, Peter J.; Brown, David T.; Misteli, Tom

    2007-01-01

    Summary Differentiation of embryonic stem (ES) cells from a pluripotent to a committed state involves global changes in genome expression patterns. Gene activity is critically determined by chromatin structure and interactions of chromatin binding proteins. Here, we show that major architectural chromatin proteins are hyperdynamic and bind loosely to chromatin in ES cells. Upon differentiation, the hyperdynamic proteins become immobilized on chromatin. Hyperdynamic binding is a property of pluripotent cells, but not of undifferentiated cells that are already lineage committed. ES cells lacking the nucleosome assembly factor HirA exhibit elevated levels of unbound histones, and formation of embryoid bodies is accelerated. In contrast, ES cells, in which the dynamic exchange of H1 is restricted, display differentiation arrest. We suggest that hyperdynamic binding of structural chromatin proteins is a functionally important hallmark of pluripotent ES cells that contributes to the maintenance of plasticity in undifferentiated ES cells and to establishing higher-order chromatin structure. PMID:16399082

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

    PubMed

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

    2015-12-17

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

  17. Epigenetic Regulation of Chromatin Structure and Gene Function by Biotin1,2

    PubMed Central

    Hassan, Yousef I.; Zempleni, Janos

    2006-01-01

    Covalent modifications of histones are a crucial component of epigenetic events that regulate chromatin structures and gene function. Evidence has been provided that distinct lysine residues in histones are modified by covalent attachment of the vitamin biotin, catalyzed by biotinidase and holocarboxylase synthetase. Biotinylation of histones appears to be conserved across species. The following biotinylation sites have been identified by using both mass spectrometry and enzymatic biotinylation of synthetic peptides: K9, K13, K125, K127, and K129 in histone H2A, K4, K9, and K18 in histone H3, and K8 and K12 in histone H4. Evidence has been provided that biotinylated histone H4 is enriched in pericentrometric heterochromatin, and that biotinylation of histone H4 participates in gene silencing, mitotic condensation of chromatin, and the cellular response to DNA damage. Biotinylation of histones is a reversible process and depends on exogenous biotin supply, but the identities of histone debiotinylases remain uncertain. We propose that some effects of biotin deficiency can be attributed to abnormal chromatin structures. PMID:16772434

  18. A novel role for microtubules in apoptotic chromatin dynamics and cellular fragmentation

    PubMed Central

    Moss, David K.; Betin, Virginie M.; Malesinski, Soazig D.; Lane, Jon D.

    2006-01-01

    Summary Dramatic changes in cellular dynamics characterise the apoptotic execution phase, culminating in fragmentation into membrane-bound apoptotic bodies. Previous evidence suggests that actin-myosin plays dominant roles in apoptotic cellular remodelling, while all other cytoskeletal elements dismantle. We have used fixed and live-cell imaging to confirm that interphase microtubules rapidly depolymerise at the start of the execution phase. At around this time, pericentriolar components (pericentrin, ninein and γ-tubulin) are lost from the centrosomal region. Subsequently, however, extensive non-centrosomal bundles of densely packed, dynamic microtubules rapidly assemble throughout the cytoplasm in all cell-lines tested. These microtubules play important roles in the peripheral relocation of chromatin in the dying cell, because nocodazole treatment restricts the dispersal of condensed apoptotic chromatin into surface blebs, and causes the withdrawal of chromatin fragments back towards the cell centre. Importantly, nocodazole and taxol are both potent inhibitors of apoptotic fragmentation in A431 cells, implicating dynamic microtubules in apoptotic body formation. Live-cell imaging studies indicate that fragmentation is accompanied by the extension of rigid microtubule-rich spikes that project through the cortex of the dying cell. These structures enhance interactions between apoptotic cells and phagocytes in vitro, by providing additional sites for attachment to neighbouring cells. PMID:16723742

  19. Genetic damage in soybean workers exposed to pesticides: evaluation with the comet and buccal micronucleus cytome assays.

    PubMed

    Benedetti, Danieli; Nunes, Emilene; Sarmento, Merielen; Porto, Carem; Dos Santos, Carla Eliete Iochims; Dias, Johnny Ferraz; da Silva, Juliana

    2013-04-15

    Soybean cultivation is widespread in the State of Rio Grande do Sul (RS, Brazil), especially in the city of Espumoso. Soybean workers in this region are increasingly exposed to a wide combination of chemical agents present in formulations of fungicides, herbicides, and insecticides. In the present study, the comet assay in peripheral leukocytes and the buccal micronucleus (MN) cytome assay (BMCyt) in exfoliated buccal cells were used to assess the effects of exposures to pesticides in soybean farm workers from Espumoso. A total of 127 individuals, 81 exposed and 46 non-exposed controls, were evaluated. Comet assay and BMCyt (micronuclei and nuclear buds) data revealed DNA damage in soybean workers. Cell death was also observed (condensed chromatin, karyorhectic, and karyolitic cells). Inhibition of non-specific choline esterase (BchE) was not observed in the workers. The trace element contents of buccal samples were analyzed by Particle-Induced X-ray Emission (PIXE). Higher concentrations of Mg, Al, Si, P, S, and Cl were observed in cells from workers. No associations with use of personal protective equipment, gender, or mode of application of pesticides were observed. Our findings indicate the advisability of monitoring genetic toxicity in soybean farm workers exposed to pesticides. PMID:23347873

  20. Intergenic Locations of Rice Centromeric Chromatin

    PubMed Central

    Yan, Huihuang; Talbert, Paul B; Lee, Hye-Ran; Jett, Jamie; Henikoff, Steven; Chen, Feng; Jiang, Jiming

    2008-01-01

    Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. Plant and animal centromeres are typically located in megabase-sized arrays of tandem satellite repeats, making their precise mapping difficult. However, some rice centromeres are largely embedded in nonsatellite DNA, providing an excellent model to study centromere structure and evolution. We used chromatin immunoprecipitation and 454 sequencing to define the boundaries of nine of the 12 centromeres of rice. Centromere regions from chromosomes 8 and 9 were found to share synteny, most likely reflecting an ancient genome duplication. For four centromeres, we mapped discrete subdomains of binding by the centromeric histone variant CENH3. These subdomains were depleted in both intact and nonfunctional genes relative to interspersed subdomains lacking CENH3. The intergenic location of rice centromeric chromatin resembles the situation for human neocentromeres and supports a model of the evolution of centromeres from gene-poor regions. PMID:19067486

  1. Diversity in the organization of centromeric chromatin.

    PubMed

    Steiner, Florian A; Henikoff, Steven

    2015-04-01

    Centromeric chromatin is distinguished primarily by nucleosomes containing the histone variant cenH3, which organizes the kinetochore that links the chromosome to the spindle apparatus. Whereas budding yeast have simple 'point' centromeres with single cenH3 nucleosomes, and fission yeast have 'regional' centromeres without obvious sequence specificity, the centromeres of most organisms are embedded in highly repetitive 'satellite' DNA. Recent studies have revealed a remarkable diversity in centromere chromatin organization among different lineages, including some that have lost cenH3 altogether. We review recent progress in understanding point, regional and satellite centromeres, as well as less well-studied centromere types, such as holocentromeres. We also discuss the formation of neocentromeres, the role of pericentric heterochromatin, and the structure and composition of the cenH3 nucleosome. PMID:25956076

  2. Chromatin dynamics during plant sexual reproduction

    PubMed Central

    She, Wenjing; Baroux, Célia

    2014-01-01

    Plants have the remarkable ability to establish new cell fates throughout their life cycle, in contrast to most animals that define all cell lineages during embryogenesis. This ability is exemplified during sexual reproduction in flowering plants where novel cell types are generated in floral tissues of the adult plant during sporogenesis, gametogenesis, and embryogenesis. While the molecular and genetic basis of cell specification during sexual reproduction is being studied for a long time, recent works disclosed an unsuspected role of global chromatin organization and its dynamics. In this review, we describe the events of chromatin dynamics during the different phases of sexual reproduction and discuss their possible significance particularly in cell fate establishment. PMID:25104954

  3. Chromatin regulation by AML1 complex.

    PubMed

    Yoshida, Hitoshi; Kitabayashi, Issay

    2008-01-01

    The AML1 gene is the most frequent target of chromosomal translocations in acute leukemias. AML1 is essential for definitive hematopoiesis and regulates transcription of its target genes by binding to the specific DNA sequence. AML1 forms large multiprotein complexes including CBFbeta as a "core component" as well as several classes of chromatin modulators such as p300/CBP, MOZ, PML and HIPK2 as "regulatory complex". In this review, we describe the mechanisms by which AML1 complex regulates gene transcription and hematopoiesis, and its disruption by the leukemia-associated chromosomal translocations that affect genes for components of AML1 complex in view of deregulation of chromatin structure. PMID:18224409

  4. Advanced microscopy methods for visualizing chromatin structure.

    PubMed

    Lakadamyali, Melike; Cosma, Maria Pia

    2015-10-01

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

  5. The polymorphisms of the chromatin fiber

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  6. The polymorphisms of the chromatin fiber.

    PubMed

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

    2015-01-28

    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. PMID:25437138

  7. Measure Guideline: Evaporative Condensers

    SciTech Connect

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

    2012-03-01

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

  8. Cadmium induced apoptotic changes in chromatin structure and subphases of nuclear growth during the cell cycle in CHO cells.

    PubMed

    Banfalvi, G; Gacsi, M; Nagy, G; Kiss, Z B; Basnakian, A G

    2005-05-01

    CHO cells were grown in the presence of 1 mu M CdCl(2) and subjected to ATP-dependent replicative DNA synthesis after permeabilization. By decreasing the density of the cell culture replicative DNA synthesis was diminishing. At higher than 2 x 10(6) cell/ml concentration Cd had virtually no effect on the rate of DNA replication. Growth at higher cell concentrations could be suppressed by increasing Cd concentration. After Cd treatment cells were synchronized by counterflow centrifugal elutriation. Cadmium toxicity on cell growth in early and mid S phase led to the accumulation of enlarged cells in late S phase. Flow cytometry showed increased cellular and nuclear sizes after Cd treatment. As the cells progressed through the S phase, 11 subpopulations of nuclear sizes were distinguished. Apoptotic chromatin changes were visualized by fluorescent microscopy in a cell cycle dependent manner. In the control untreated cells the main transitory forms of chromatin corresponded to those we have published earlier (veil-like, supercoiled chromatin, fibrous, ribboned structures, chromatin strings, elongated prechromosomes, precondensed chromosomes). Cadmium treatment caused: (a) the absence of decondensed veil-like structures and premature chromatin condensation in the form of apoptotic bodies in early S phase (2.2-2.4 average C-value), (b) the absence of fibrous structures, the lack of supercoiled chromatin, the appearance of uncoiled ribboned chromatin and perichromatin semicircles, in early mid S phase (2.5-2.9 C), (c) the presence of perichromatin fibrils and chromatin bodies in mid S phase (2.9-3.2 C), (d) early intra-nuclear inclusions, elongated forms of premature chromosomes, the extrusion and rupture of nuclear membrane later in mid S phase (3.3-3.4 C), (e) the exclusion of chromatin bodies and the formation of clusters of large-sized perichromatin granules in late S phase (3.5-3.8 C) and (f) large extensive disruptions and holes in the nuclear membrane and the clumping of incompletely folded chromosomes (3.8-4. C). PMID:15909124

  9. Chromatin signature of widespread monoallelic expression

    PubMed Central

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

    2013-01-01

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

  10. Higher order chromatin organization in cancer

    PubMed Central

    Reddy, Karen L.; Feinberg, Andrew P.

    2013-01-01

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

  11. Default assembly of early adenovirus chromatin

    SciTech Connect

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

    2007-03-01

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

  12. 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

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

    PubMed Central

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

    PubMed Central

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

    2007-01-01

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

  15. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-01

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest. PMID:19081993

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

    NASA Astrophysics Data System (ADS)

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

    2008-04-01

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

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

    PubMed Central

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

    2015-01-01

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

  18. Formation of stress-specific p53 binding patterns is influenced by chromatin but not by modulation of p53 binding affinity to response elements.

    PubMed

    Millau, Jean-François; Bandele, Omari J; Perron, Josiann; Bastien, Nathalie; Bouchard, Eric F; Gaudreau, Luc; Bell, Douglas A; Drouin, Régen

    2011-04-01

    The p53 protein is crucial for adapting programs of gene expression in response to stress. Recently, we revealed that this occurs partly through the formation of stress-specific p53 binding patterns. However, the mechanisms that generate these binding patterns remain largely unknown. It is not established whether the selective binding of p53 is achieved through modulation of its binding affinity to certain response elements (REs) or via a chromatin-dependent mechanism. To shed light on this issue, we used a microsphere assay for protein-DNA binding to measure p53 binding patterns on naked DNA. In parallel, we measured p53 binding patterns within chromatin using chromatin immunoprecipitation and DNase I coupled to ligation-mediated polymerase chain reaction footprinting. Through this experimental approach, we revealed that UVB and Nutlin-3 doses, which lead to different cellular outcomes, induce similar p53 binding patterns on naked DNA. Conversely, the same treatments lead to stress-specific p53 binding patterns on chromatin. We show further that altering chromatin remodeling using an histone acetyltransferase inhibitor reduces p53 binding to REs. Altogether, our results reveal that the formation of p53 binding patterns is not due to the modulation of sequence-specific p53 binding affinity. Rather, we propose that chromatin and chromatin remodeling are required in this process. PMID:21177650

  19. Freeze-Tolerant Condensers

    NASA Technical Reports Server (NTRS)

    Crowley, Christopher J.; Elkouhk, Nabil

    2004-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  1. Mps1 phosphorylation of condensin II controls chromosome condensation at the onset of mitosis.

    PubMed

    Kagami, Yuya; Nihira, Keishi; Wada, Shota; Ono, Masaya; Honda, Mariko; Yoshida, Kiyotsugu

    2014-06-23

    During mitosis, genomic DNA is condensed into chromosomes to promote its equal segregation into daughter cells. Chromosome condensation occurs during cell cycle progression from G2 phase to mitosis. Failure of chromosome compaction at prophase leads to subsequent misregulation of chromosomes. However, the molecular mechanism that controls the early phase of mitotic chromosome condensation is largely unknown. Here, we show that Mps1 regulates initial chromosome condensation during mitosis. We identify condensin II as a novel Mps1-associated protein. Mps1 phosphorylates one of the condensin II subunits, CAP-H2, at Ser492 during mitosis, and this phosphorylation event is required for the proper loading of condensin II on chromatin. Depletion of Mps1 inhibits chromosomal targeting of condensin II and accurate chromosome condensation during prophase. These findings demonstrate that Mps1 governs chromosomal organization during the early stage of mitosis to facilitate proper chromosome segregation. PMID:24934155

  2. The chromatin remodeling complex NoRC targets HDAC1 to the ribosomal gene promoter and represses RNA polymeraseI transcription

    PubMed Central

    Zhou, Yonggang; Santoro, Raffaella; Grummt, Ingrid

    2002-01-01

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

  3. Direct evidence for pitavastatin induced chromatin structure change in the KLF4 gene in endothelial cells.

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

    Friedland, W; Kundrát, P

    2015-09-01

    The module that simulates the kinetics and yields of radiation-induced chromosome aberrations within the biophysical code PARTRAC is described. Radiation track structures simulated by Monte Carlo methods are overlapped with multi-scale models of DNA and chromatin to assess the resulting DNA damage. Spatial mobility of individual DNA ends from double-strand breaks is modelled simultaneously with their processing by the non-homologous end-joining enzymes. To score diverse types of chromosome aberrations, the joined ends are classified regarding their original chromosomal location, orientation and the involvement of centromeres. A comparison with experimental data on dicentrics induced by gamma and alpha particles shows that their relative dose dependence is predicted correctly, although the absolute yields are overestimated. The critical model assumptions on chromatin mobility and on the initial damage recognition and chromatin remodelling steps and their future refinements to solve this issue are discussed. PMID:25883314

  6. Dual Roles of p300 in Chromatin Assembly and Transcriptional Activation in Cooperation with Nucleosome Assembly Protein 1 In Vitro

    PubMed Central

    Asahara, Hiroshi; Tartare-Deckert, Sophie; Nakagawa, Takeya; Ikehara, Tsuyoshi; Hirose, Fumiko; Hunter, Tony; Ito, Takashi; Montminy, Marc

    2002-01-01

    In a yeast two-hybrid screen to identify proteins that bind to the KIX domain of the coactivator p300, we obtained cDNAs encoding nucleosome assembly protein 1 (NAP-1), a 60-kDa histone H2A-H2B shuttling protein that promotes histone deposition. p300 associates preferentially with the H2A-H2B-bound form of NAP-1 rather than with the unbound form of NAP-1. Formation of NAP-1-p300 complexes was found to increase during S phase, suggesting a potential role for p300 in chromatin assembly. In micrococcal nuclease and supercoiling assays, addition of p300 promoted efficient chromatin assembly in vitro in conjunction with NAP-1 and ATP-utilizing chromatin assembly and remodeling factor; this effect was dependent in part on the intrinsic histone acetyltransferase activity of p300. Surprisingly, NAP-1 potently inhibited acetylation of core histones by p300, suggesting that efficient assembly requires acetylation of either NAP-1 or p300 itself. As p300 acted cooperatively with NAP-1 in stimulating transcription from a chromatin template in vitro, our results suggest a dual role of NAP-1-p300 complexes in promoting chromatin assembly and transcriptional activation. PMID:11940655

  7. A Method to Study the Epigenetic Chromatin States of Rare Hematopoietic Stem and Progenitor Cells; MiniChIPChip

    PubMed Central

    2010-01-01

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

  8. Depletion of the chromatin looping proteins CTCF and cohesin causes chromatin compaction: insight into chromatin folding by polymer modelling.

    PubMed

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

    2014-10-01

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

  9. Luciferase assay.

    PubMed

    Smale, Stephen T

    2010-05-01

    When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. Because transfection efficiency in such assays can be low, promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. The reporter protein's activity or fluorescence within a transfected cell population is approximately proportional to the steady-state mRNA level. A commonly used reporter gene is the luciferase gene from the firefly Photinus pyralis. This gene encodes a 61-kDa enzyme that oxidizes D-luciferin in the presence of ATP, oxygen, and Mg(++), yielding a fluorescent product that can be quantified by measuring the released light. Including coenzyme A in the reaction enhances the sensitivity of the assay and provides a sustained light reaction. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a detergent-containing buffer. Cell debris is removed by microcentrifugation and luciferase activity is measured using a luminometer. Some luminometers directly inject the reagents into the cell lysate. Such automation allows the signal to be measured at a precise time following injection, which can increase the consistency of the results. For manual luminometers, the substrate solution is mixed by hand with the cell lysate, and the fluorescence is read at a defined time following mixing. The luciferase assay is extremely rapid, simple, relatively inexpensive, sensitive, and possesses a broad linear range. PMID:20439408

  10. Chromosome and genetic testing using ChIP assay.

    PubMed

    Kohzaki, Hidetsugu; Asano, Maki

    2016-01-01

    Chromatin immunoprecipitation (ChIP) assay can be used to easily visualize information about proteins, DNA, and RNA on chromosomes and is widely used for analysis of genomes, epigenomes, mRNAs, and non-coding RNAs. The ChIP assay can detect, not only DNA-binding proteins of various organisms, but also the temporal and spatial regulating mechanisms of RNA-binding proteins. Because of these features, demand for ChIP assay is expected to grow. Here, by using yeast and Drosophila as examples, we describe the superiority of the improved ChIP assay that we have developed. PMID:27100707

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

    NASA Astrophysics Data System (ADS)

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

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Bao, Yunhe

    2011-06-01

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

  14. Dynamic Chromatin Regulation from a Single Molecule Perspective.

    PubMed

    Fierz, Beat

    2016-03-18

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

  15. Methods for identifying higher-order chromatin structure.

    PubMed

    Sajan, Samin A; Hawkins, R David

    2012-01-01

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

  16. Condenser macrofouling control technologies

    SciTech Connect

    Mussalli, Y.G.

    1984-06-01

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

  17. Ghost condensate busting

    SciTech Connect

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

    2008-09-15

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

  18. Key condenser failure mechanisms

    SciTech Connect

    Buecker, B.

    2009-04-15

    Eight practical lessons highlight many of the factors that can influence condenser tube corrosion at coal-fired utilities and the effects contaminant in-leakage can have on steam generating units. 1 ref., 4 figs.

  19. 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.

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

    PubMed Central

    Singh, Purva; Schwarzbauer, Jean E.

    2014-01-01

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

  1. Histone demethylases in chromatin biology and beyond

    PubMed Central

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

    2015-01-01

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

  2. Assessment of sera for chromatin-immunoprecipitation.

    PubMed

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

    2008-01-01

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

  3. 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

  4. Assay system

    SciTech Connect

    Patzke, J.B.; Rosenberg, B.J.

    1984-02-07

    The accuracy of assays for monitoring concentrations of basic drugs in biological fluids containing a/sub 1/-acid glycoproteins, such as blood (serum or plasma), is improved by the addition of certain organic phosphate compounds to minimize the ''protein effect.'' Kits containing the elements of the invention are also disclosed.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  6. Epigenetics: Beyond Chromatin Modifications and Complex Genetic Regulation1

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-10-30

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

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

    PubMed

    Fukagawa, Tatsuo; Earnshaw, William C

    2014-09-01

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

  9. 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

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

    PubMed Central

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

    2013-01-01

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

  11. 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.

  12. 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

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

    PubMed

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

    2015-03-01

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

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

    PubMed Central

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

    2014-01-01

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

  15. c-Myb Binding Sites in Haematopoietic Chromatin Landscapes

    PubMed Central

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

    2015-01-01

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

  16. DNA-enrichment microfluidic chip for chromatin immunoprecipitation.

    PubMed

    Oh, Hyun Jik; Park, Joong Yull; Park, Sung Eun; Lee, Bo Yun; Park, Jong Sung; Kim, Suel-Kee; Yoon, Tae Joong; Lee, Sang-Hoon

    2009-04-15

    Chromatin immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting association of individual proteins with specific genomic regions; the technique requires several complex steps and is tedious. In this paper, we develop a microbead-packed microfluidic chip which eliminates most of the laborious, time-consuming, and skill-dependent processes of the ChIP procedure. A computational fluid dynamics model was established to analyze fluidic behavior in a microbead-packed microchannel. With the use of the new chip, a ChIP procedure was performed to purify the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene from human embryonic kidney cells (cell line 293). The ChIP capability of the microfluidic chip was evaluated and compared with that of a commercial assay kit; the precipitation performance of both methods was almost identical as shown by quantitative measurement of DNA. However, our chip offers the advantage of low resin volume, and the experimental time is greatly reduced. In addition, our method is less dependent on individual technical skill. Therefore, we expect that our microfluidic chip-based ChIP method will be widely used in DNA-, gene-, and protein-related research and will improve experimental efficiency. PMID:19298056

  17. Single-cell chromatin accessibility reveals principles of regulatory variation

    PubMed Central

    Buenrostro, Jason D.; Wu, Beijing; Litzenburger, Ulrike M.; Ruff, Dave; Gonzales, Michael L.; Snyder, Michael P.; Chang, Howard Y.; Greenleaf, William J.

    2015-01-01

    Cell-to-cell variation is a universal feature of life that impacts a wide range of biological phenomena, from developmental plasticity1,2 to tumor heterogeneity3. While recent advances have improved our ability to document cellular phenotypic variation4–8 the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of cellular DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells via assay for transposase-accessible chromatin using sequencing (ATAC-seq). Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single-cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provides insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type specific accessibility variance across 8 cell types. Targeted perturbations of cell cycle or transcription factor signaling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome topological domains9 de novo, linking single-cell accessibility variation to three-dimensional genome organization. All together, single-cell analysis of DNA accessibility provides new insight into cellular variation of the “regulome.” PMID:26083756

  18. Histone modification and chromatin remodeling during NER.

    PubMed

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

    2015-12-01

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

  19. Red oak condensate: its apparent lack of cytotoxic and genotoxic effects as compared with three other wood-drying condensates.

    PubMed

    Mark, H F; Naram, R; Bastan, W C; Cherkes, J K; LaMarche, P H

    1995-01-01

    A major activity of the lumber industry is the kiln-drying of wood. In order to ascertain whether wood-drying condensates pose a possible environmental hazard, the cytotoxicity and genotoxicity of these condensates in vitro, were tested using an assay validated using Chinese hamster ovary (CHO) cells and a known genotoxicant, mitomycin C. Subsequently, the assay was developed for the human peripheral blood lymphocyte (HPBL) system, as it was felt that results derived from human cells would reflect the situation more closely in vivo. Condensates from Southern yellow pine, Eastern white pine and Douglas fir trees were tested in CHO and HPBL systems and have demonstrated cytotoxic and genotoxic effects in vitro, as reported elsewhere. Red oak condensate has also been tested using the HPBL system. Thus far, results are consistent with the hypothesis that there is no difference between the cytotoxic and genotoxic effects of treated cells versus controls. This finding indicates either that the condensate of red oak poses no appreciable genetic hazard as measured by cytotoxicity and genotoxicity assays, or that the condensate has lost its potency with time and storage; both of these possibilities have important environmental implications. PMID:8829893

  20. Global chromatin fibre compaction in response to DNA damage

    SciTech Connect

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

    2011-11-04

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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    1990-01-01

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

  3. A developmentally stable chromatin structure in the human beta-globin gene cluster.

    PubMed Central

    Forrester, W C; Thompson, C; Elder, J T; Groudine, M

    1986-01-01

    The DNase I-hypersensitive sites in the human embryonic beta-globin gene region have been mapped in erythroid-enriched fractions of disaggregated fetal livers, in adult nucleated red blood cells, and in fetal brain tissue. Our analysis of a region extending 11 kilobases (kb) 5' of the epsilon-globin gene reveals many minor nuclease-hypersensitive sites and one major site located 6.1 kb upstream of the epsilon-globin gene. All of these hypersensitive sites are erythroid-specific, and the major site is stable throughout erythroid development. As assayed by nuclear runoff transcription, little or no epsilon-globin gene expression is detectable in fetal or adult erythroid cells. Thus, the presence of the major hypersensitive site 5' of the epsilon-globin gene in both fetal and adult erythroid cells demonstrates that this site is not specifically correlated with transcription of the gene or with a particular stage of development. Rather, this site may reflect an early event in erythroid differentiation. In addition, DNase I has been used to probe the overall sensitivity of epsilon-globin chromatin in fetal erythroid cells. Our findings indicate that the epsilon-globin gene as well as the other genes in the beta-globin cluster reside within the chromatin domain that is more DNase I-sensitive than "bulk" chromatin. Images PMID:3456593

  4. Estrogen induces tissue specific changes in the chromatin conformation of the vitellogenin genes in Xenopus.

    PubMed Central

    Gerber-Huber, S; Felber, B K; Weber, R; Ryffel, G U

    1981-01-01

    Nuclei from male Xenopus liver were digested extensively with DNase I and the residual amount of the four vitellogenin genes measured by hybridization with a moderate excess of vitellogenin cDNA. The saturation value was about twofold lower in chromatin isolated from liver cells of estrogen treated than from untreated males or from erythrocytes. Analyzing the disappearance of several defined restriction fragments specific for the A1 and A2 vitellogenin genes, after limited digestion with DNase I, suggested that the entire A1 and A2 vitellogenin genes are about twofold more sensitive to DNase I in chromatin of hepatocytes isolated from estrogen treated than from untreated males. Using the same assay no change in the DNase I sensitivity of the two vitellogenin genes in erythrocyte chromatin was observed. Analysis of the beta 1-globin and an albumin gene demonstrated that the DNase I sensitivity of these genes in both cell types is not altered by estrogen. All these data indicate that estrogen stimulation results in an increased DNase I sensitivity specific for the vitellogenin genes in hepatocytes. Images PMID:6269051

  5. The polyomavirus enhancer activates chromatin accessibility on integration into the HPRT gene.

    PubMed Central

    Pikaart, M; Feng, J; Villeponteau, B

    1992-01-01

    Recent studies suggest that enhancers may increase the accessibility of chromatin to transcription factors. To test the effects of a viral enhancer on chromatin accessibility, we have inserted minigenes with or without the polyomavirus enhancer into the third exon of the hypoxanthine phosphoribosyltransferase (HPRT) gene by homologous recombination and have prepared high-resolution maps of gene accessibility by using a novel polymerase chain reaction assay for DNase I sensitivity. In its native state, we find that the HPRT gene has low sensitivity to DNase I in fibrosarcoma cells. Insertion of the polyomavirus enhancer and neo reporter gene into exon 3 confers altered HPRT DNase I sensitivity for several kilobases on either side of the enhancer. The changes in DNase I sensitivity peak near the enhancer and decline with distance from the enhancer. The increase in HPRT DNase I sensitivity persisted when the tk promoter was deleted from the inserted construct but disappeared when the enhancer was deleted. These experiments identify the polyomavirus enhancer as a cis-acting initiator of chromatin accessibility. Images PMID:1333045

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2007-05-01

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

  10. Remodeling of chromatin structure within the promoter is important for bmp-2-induced fgfr3 expression

    PubMed Central

    Sun, Fenyong; Chen, Qiongyu; Yang, Songhai; Pan, Qiuhui; Ma, Ji; Wan, Yang; Chang, Chih-Hao; Hong, An

    2009-01-01

    Fibroblast growth factor receptor 3 (FGFR3) plays an important role in cartilage development. Although upregulation of FGFR3 expression in response to bone morphogenetic protein-2 (BMP-2) has been reported, the molecular mechanisms remain unknown. In this study, we used in vivo approaches to characterize BMP-2-induced alterations in the chromatin organization of the FGFR3 core promoter. Chromatin immunoprecipitation analysis demonstrated that the binding of Brg1, a component of the SWI/SNF remodeling complex, may selectively remodel a chromatin region (encompassing nucleotide –90 to +35), uncovering the transcription start site and three Sp1-binding sites, as revealed by nuclease digestion hypersensitivity assays. We then showed an increase in the association of Sp1 with the proximal promoter, followed by the recruitment of p300, resulting in a change of the histone ‘code’, such as in phosphorylation and methylation. Collectively, our study results suggest a model for BMP-2-induced FGFR3 expression in which the core promoter architecture is specifically regulated. PMID:19401440

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

    PubMed Central

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

    1977-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2015-11-20

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2013-12-10

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

  17. Mass condensation on networks

    NASA Astrophysics Data System (ADS)

    Waclaw, B.; Sopik, J.; Janke, W.; Meyer-Ortmanns, H.

    2010-09-01

    We construct classical stochastic mass transport processes for stationary states which are chosen to factorize over pairs of sites of an undirected, connected, but otherwise arbitrary graph. For the special topology of a ring we derive static properties such as the critical point of the transition between the liquid and the condensed phase, the shape of the condensate and its scaling with the system size. It turns out that the shape is not universal, but determined by the interplay of local and ultralocal interactions. In two dimensions the effect of anisotropic interactions of hopping rates can be treated analytically, since the partition function allows a dimensional reduction to an effective one-dimensional zero-range process. Here we predict the onset, shape and scaling of the condensate on a square lattice. We indicate further extensions in the outlook.

  18. First order kaon condensate

    SciTech Connect

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

    1999-08-01

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

  19. First order kaon condensate

    NASA Astrophysics Data System (ADS)

    Glendenning, Norman K.; Schaffner-Bielich, Jürgen

    1999-08-01

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

  20. Chromatin regulators of the AID induced DNA damage response

    PubMed Central

    Daniel, Jeremy A.; Nussenzweig, André

    2013-01-01

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

  1. Stimulation of transcription of mouse kidney chromatin by sulfated polysaccharides.

    PubMed Central

    Warnick, C T; Zazarus, H M

    1975-01-01

    The sulfated polysaccharides polydextran sulfate (PDS) and heparin stimulate in vitro transcription of mouse kidney chromatin by E. coli RNA polymerase by about 100 and 40 fold respectively. Heparin which has been N-desulfated and N-acetylated stimulates only 13 fold. Chondroitin sulfate B and heparitin sulfate do not stimulate transcription under similar conditions. PDS inhibits transcription of deproteinized chromatin. Therefore, the stimulation with chromatin is due to interaction with the chromatin and not the polymerase. Polydextran sulfate has no effect on the size of the RNA that is made either under conditions in which the enzyme can reinitiate or under conditions in which reinitiation is blocked. If reinitiation of the enzyme is blocked, the time required to complete the synthesis of the RNA is the same whether or not the enzyme is stimulated by PDS. These observations indicate that sulfated polysaccharides stimulate transcription by making available new RNA polymerase binding sites on the chromatin. PMID:1144063

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

    PubMed

    Donà, Mattia; Mittelsten Scheid, Ortrun

    2015-08-01

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

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

    PubMed Central

    Bühler, Marc; Gasser, Susan M

    2009-01-01

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

  4. ATP Dependent Chromatin Remodeling Enzymes in Embryonic Stem Cells

    PubMed Central

    Saladi, Srinivas Vinod

    2010-01-01

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

  5. Revisiting Higher-order and Large-scale Chromatin Organization

    PubMed Central

    Bian, Qian

    2012-01-01

    The past several years has seen increasing appreciation for plasticity of higher-level chromatin folding. Four distinct “30 nm” chromatin fiber structures have been identified, while new in situ imaging approaches have questioned the universality of 30 nm chromatin fibers as building blocks for chromosome folding in vivo. 3C-based approaches have provided a non-microscopic, genomic approach to investigating chromosome folding while uncovering a plethora of long-distance cis interactions difficult to accommodate in traditional hierarchical chromatin folding models. Recent microscopy based studies have suggested complex topologies co-existing within linear interphase chromosome structures. These results call for a reappraisal of traditional models of higher-level chromatin folding. PMID:22459407

  6. Induction of condensed determination

    SciTech Connect

    Langley, P.

    1996-12-31

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

  7. Keeping condensers clean

    SciTech Connect

    Wicker, K.

    2006-04-15

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

  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. MYST-family histone acetyltransferases: beyond chromatin.

    PubMed

    Sapountzi, Vasileia; Ct, Jacques

    2011-04-01

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

  10. Identification of alternative topological domains in chromatin

    PubMed Central

    2014-01-01

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

  11. Oligodendroglial Development: New Roles for Chromatin Accessibility.

    PubMed

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

    2015-12-01

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

  12. Angiogenesis Assays.

    PubMed

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

    2016-01-01

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

  13. 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.

  14. 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

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

    EPA Science Inventory

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

  16. 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...

  17. Condensate removal device

    DOEpatents

    Maddox, James W.; Berger, David D.

    1984-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

    Loidl, P; Gröbner, P

    1987-01-01

    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. Images PMID:3118335

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

    PubMed

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

    1984-09-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  3. Minireview: role of kinases and chromatin remodeling in progesterone signaling to chromatin.

    PubMed

    Vicent, Guillermo P; Nacht, A Silvina; Zaurín, Roser; Ballaré, Cecilia; Clausell, Jaime; Beato, Miguel

    2010-11-01

    Steroid hormones regulate gene expression by interaction of their receptors with hormone-responsive elements on DNA or with other transcription factors, but they can also activate cytoplasmic signaling cascades. Rapid activation of Erk by progestins via an interaction of the progesterone receptor (PR) with the estrogen receptor is critical for transcriptional activation of the mouse mammary tumor virus (MMTV) promoter and other progesterone target genes. Erk activation leads to the phosphorylation of PR, activation of mitogen- and stress-activated protein kinase 1, and the recruitment of a complex of the three activated proteins and of P300/CBP-associated factor (PCAF) to a single nucleosome, resulting in the phosphoacetylation of histone H3 and the displacement of heterochromatin protein 1γ. Hormone-dependent gene expression requires ATP-dependent chromatin remodeling complexes. Two switch/sucrose nonfermentable-like complexes, Brahma-related gene 1-associated factor (BAF) and polybromo-BAF are present in breast cancer cells, but only BAF is recruited to the MMTV promoter and cooperates with PCAF during activation of hormone-responsive promoters. PCAF acetylates histone H3 at K14, an epigenetic mark recognized by BAF subunits, thus anchoring the complex to chromatin. BAF catalyzes localized displacement of histones H2A and H2B, facilitating access of nuclear factor 1 and additional PR complexes to the hidden hormone-responsive elements on the MMTV promoter. The linker histone H1 is a structural component of chromatin generally regarded as a general repressor of transcription. However, it contributes to a better regulation of the MMTV promoter by favoring a more homogeneous nucleosome positioning, thus reducing basal transcription and actually enhancing hormone induced transcription. During transcriptional activation, H1 is phosphorylated and displaced from the promoter. The kinase cyclin-dependent kinase 2 is activated after progesterone treatment and could catalyze progesterone-induced phosphorylation of histone H1 by chromatin remodeling complexes. The initial steps of gene induction by progestins involve changes in the chromatin organization of target promoters that require the activation of several kinase signaling pathways initiated by membrane anchored PR. Because these pathways also respond to other external signals, they serve to integrate the hormonal response in the global context of the cellular environment. PMID:20484412

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

    PubMed Central

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

    2015-01-01

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

  5. 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

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

    PubMed Central

    Diesinger, Philipp M.; Heermann, Dieter W.

    2009-01-01

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

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

    PubMed Central

    2012-01-01

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

  8. CTCF-Mediated Functional Chromatin Interactome in Pluripotent Cells

    PubMed Central

    Handoko, Lusy; Xu, Han; Li, Guoliang; Ngan, Chew Yee; Chew, Elaine; Schnapp, Marie; Lee, Charlie Wah Heng; Ye, Chaopeng; Ping, Joanne Lim Hui; Mulawadi, Fabianus; Wong, Eleanor; Sheng, Jianpeng; Zhang, Yubo; Poh, Thompson; Chan, Chee Seng; Kunarso, Galih; Shahab, Atif; Bourque, Guillaume; Cacheux-Rataboul, Valere; Sung, Wing-Kin; Ruan, Yijun; Wei, Chia-Lin

    2011-01-01

    Mammalian genomes are viewed as functional organizations that orchestrate spatial and temporal gene regulation. CTCF, the most characterized insulator-binding protein, has been implicated as a key genome organizer. Yet, little is known about CTCF-associated higher order chromatin structures at a global scale. Here, we applied Chromatin Interaction Analysis by Paired-End-Tag sequencing to elucidate the CTCF-chromatin interactome in pluripotent cells. From this analysis, 1,480 cis and 336 trans interacting loci were identified with high reproducibility and precision. Associating these chromatin interaction loci with their underlying epigenetic states, promoter activities, enhancer binding and nuclear lamina occupancy, we uncovered five distinct chromatin domains that suggest potential new models of CTCF function in chromatin organization and transcriptional control. Specifically, CTCF interactions demarcate chromatin-nuclear membrane attachments and influence proper gene expression through extensive crosstalk between promoters and regulatory elements. This highly complex nuclear organization offers insights towards the unifying principles governing genome plasticity and function. PMID:21685913

  9. Nucleosome positioning and composition modulate in silico chromatin flexibility

    PubMed Central

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

    2015-01-01

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

  10. 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.

  11. Maintaining steam/condensate lines

    SciTech Connect

    Russum, S.A. )

    1992-03-05

    Steam and condensate systems must be maintained with the same diligence as the boiler itself. Unfortunately, they often are not. The water treatment program, critical to keeping the boiler at peak efficiency and optimizing operating life, should not stop with the boiler. The program must encompass the steam and condensate system as well. A properly maintained condensate system maximizes condensate recovery, which is a cost-free energy source. The fuel needed to turn the boiler feedwater into steam has already been provided. Returning the condensate allows a significant portion of that fuel cost to be recouped. Condensate has a high heat content. Condensate is a readily available, economical feedwater source. Properly treated, it is very pure. Condensate improves feedwater quality and reduces makeup water demand and pretreatment costs. Higher quality feedwater means more reliable boiler operation.

  12. Chromatin regulation at the frontier of synthetic biology

    PubMed Central

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

    2016-01-01

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

  13. Multivalent engagement of chromatin modifications by linked binding modules

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2002-01-01

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

  15. Stress-induced structural changes in plant chromatin.

    PubMed

    Probst, Aline V; Mittelsten Scheid, Ortrun

    2015-10-01

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

  16. Mapping regulatory factors by immunoprecipitation from native chromatin

    PubMed Central

    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. PMID:25827087

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

    PubMed

    Witkowski, Leora; Foulkes, William D

    2015-12-01

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

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

    TOXLINE Toxicology Bibliographic Information

    Khalifa T; Lymberopoulos A

    2013-12-01

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

  19. Chromatin structures of the rat tyrosine aminotransferase gene relate to the function of its cis-acting elements.

    PubMed Central

    Nitsch, D; Stewart, A F; Boshart, M; Mestril, R; Weih, F; Schütz, G

    1990-01-01

    The relationship between DNase I-hypersensitive sites (HSs) and transcriptional enhancers of the rat tyrosine aminotransferase (TAT) gene was examined by comparing HSs in and around the TAT gene with the activity of the corresponding DNA sequences in transient transfection assays. In this manner, we identified two HSs as liver-specific enhancers. Of three hepatoma cell lines examined, only one sustained TAT mRNA levels comparable to those of liver. In this cell line, both enhancers were strongly active, and strong hypersensitivity in chromatin over the enhancers was evident. The other two hepatoma cell lines had reduced levels of TAT mRNA and no or altered hypersensitivity over either the enhancers or the promoter. One of these lines carried a negative regulator of the TAT gene, the tissue specific extinguisher Tse-1. This cell line exhibited all HSs characteristic of the strongly active gene except at the promoter; however, one enhancer was inactive even though hypersensitive in chromatin. In a TAT-nonexpressing cell line, inactivity of both enhancers correlated with absence of the respective HSs. We conclude that although hypersensitivity in chromatin necessarily accompanies cell-type-specific enhancer activity, the occurrence of cell-type-specific HSs does not imply that the underlying sequences harbor enhancers active in transient transfection assays. Images PMID:1972541

  20. Feshbach-Einstein Condensates

    SciTech Connect

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

    2009-01-09

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

  1. CW laser light condensation.

    PubMed

    Zhurahov, Michael; Bekker, Alexander; Levit, Boris; Weill, Rafi; Fischer, Baruch

    2016-03-21

    We present a first experimental demonstration of classical CW laser light condensation (LC) in the frequency (mode) domain that verifies its prediction (Fischer and Weill, Opt. Express20, 26704 (2012)). LC is based on weighting the modes in a noisy environment in a loss-gain measure compared to an energy (frequency) scale in Bose-Einstein condensation (BEC). It is characterized by a sharp transition from multi- to single-mode oscillation, occurring when the spectral-filtering (loss-trap) has near the lowest-loss mode ("ground-state") a power-law dependence with an exponent smaller than 1. An important meaning of the many-mode LC system stems from its relation to lasing and photon-BEC. PMID:27136845

  2. 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 = -ρv 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 ℓ of fluid with equation of state p = +ρ, 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 kBℓMc/, instead of the Bekenstein–Hawking entropy formula, SBH = 4πkBGM2/c. Hence, unlike black holes, the new solution is thermodynamically stable and has no information paradox. PMID:15210982

  3. Inhomogeneous chiral condensates

    NASA Astrophysics Data System (ADS)

    Buballa, Michael; Carignano, Stefano

    2015-03-01

    The chiral condensate, which is constant in vacuum, may become spatially modulated at moderately high densities where in the traditional picture of the QCD phase diagram a first-order chiral phase transition occurs. We review the current status of this idea, which originally dates back to Migdal's pion condensation, but recently received new momentum through studies on the nature of the chiral critical point and by the conjecture of a quarkyonic-matter phase. We discuss how these nonuniform phases emerge in generalized Ginzburg-Landau analyses as well as in specific calculations, both within effective models and in Dyson-Schwinger or large-Nc approaches to QCD. Questions about the most favored shape of the modulations and its dimension, and about the effects of nonzero isospin chemical potential, strange quarks, color superconductivity, and external magnetic fields on these inhomogeneous phases will be addressed as well.

  4. Multilayer graphene condenser microphone

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  5. Gravity triggered neutrino condensates

    SciTech Connect

    Barenboim, Gabriela

    2010-11-01

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

  6. Bose-Einstein Condensation

    SciTech Connect

    El-Sherbini, Th.M.

    2005-03-17

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

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

    PubMed

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

    2015-08-01

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

  8. Asymmetric condensed dark matter

    NASA Astrophysics Data System (ADS)

    Aguirre, Anthony; Diez-Tejedor, Alberto

    2016-04-01

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

  9. Chromatin and extracellular vesicle associated sperm RNAs

    PubMed Central

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

    2015-01-01

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

  10. Centromeric chromatin and its dynamics in plants.

    PubMed

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

    2015-07-01

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

  11. Sperm cryopreservation: effects on chromatin structure.

    PubMed

    Paoli, Donatella; Lombardo, Francesco; Lenzi, Andrea; Gandini, Loredana

    2014-01-01

    Cryopreservation is a technique that can keep sperm alive indefinitely, enabling the conservation of male fertility. It involves the cooling of semen samples and their storage at -196°C in liquid nitrogen. At this temperature all metabolic processes are arrested. Sperm cryopreservation is of fundamental importance for patients undergoing medical or surgical treatments that could induce sterility, such as cancer patients about to undergo genotoxic chemotherapy or radiotherapy, as it offers these patients not only the hope of future fertility but also psychological support in dealing with the various stages of the treatment protocols.Despite its importance for assisted reproduction technology (ART) and its success in terms of babies born, this procedure can cause cell damage and impaired sperm function. Various studies have evaluated the impact of cryopreservation on chromatin structure, albeit with contradictory results. Some, but not all, authors found significant sperm DNA damage after cryopreservation. However, studies attempting to explain the mechanisms involved in the aetiology of cryopreservation-induced DNA damage are still limited. Some reported an increase in sperm with activated caspases after cryopreservation, while others found an increase in the percentage of oxidative DNA damage. There is still little - and contradictory - information on the mechanism of the generation of DNA fragmentation after cryopreservation. More studies are needed to establish the true importance of such damage, especially to improve the results of ART. PMID:23955677

  12. Regulation of meiotic chromatin loop size by chromosomal position.

    PubMed Central

    Heng, H H; Chamberlain, J W; Shi, X M; Spyropoulos, B; Tsui, L C; Moens, P B

    1996-01-01

    At meiotic prophase, chromatin loops around a proteinaceous core, with the sizes of these loops varying between species. Comparison of the morphology of sequence-related inserts at different sites in transgenic mice demonstrates that loop size also varies with chromosomal geography. Similarly, chromatin loop lengths differ dramatically for interstitially and terminally located hamster telomeric sequences. Sequences, telomeric or otherwise, located at chromosome termini, closely associate with the meiotic proteinaceous core, forming shorter loops than identical interstitial sequences. Thus, we present evidence that different chromatin packaging mechanisms exist for interstitial versus terminal chromosomal regions, which act separately from those operating at the level of the DNA sequence. Chromosomal position plays the dominant role in chromatin packaging. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8610120

  13. 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

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

    PubMed Central

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

    1978-01-01

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

  15. Effect of irradiation and endogenous nucleases on rat liver chromatin

    SciTech Connect

    Gelderblom, D.; Smit, B.J.; Boehm, L.

    1984-08-01

    The assessment of the consequences of irradiation on chromatin is complicated by endogenous nucleases. Isolation and prolonged storage of rat liver nuclei in buffers containing divalent metal ions activates these enzymes and promotes the degradation of chromatin. Irradiation of rat liver nuclei to dose levels of 20,000 rad under conditions in which endogenous nucleases are inhibited and analysis of the irradiated chromatin by sucrose density gradient centrifugation gave no evidence for monosomes or oligosomes. When chromatin from irradiated nuclei was digested with micrococcal nuclease, the levels of monosomes and oligosomes were identical to those of micrococcal nuclease digests of unirradiated control nuclei. These results suggest that irradiation results in neither a direct fragmentation of linkers nor the sensitization of linkers for subsequent cleavage by micrococcal nuclease.

  16. Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin.

    PubMed

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

    2016-02-11

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

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

    PubMed Central

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

    2015-01-01

    Histological subtypes of basal cell carcinoma have biological, evolutionary and distinct prognostic behavior. The analysis of characteristics of the nucleus can provide data on their cellular physiology and behavior. The authors of this study evaluated nuclear morphological parameters and textural patterns of chromatin from different subtypes of basal cell carcinoma: nodular (n=37), superficial (n=28) and sclerodermiform (n=28). The parameters were compared between neoplasms' subtypes and with unaffected adjacent basal epithelium. Nuclear area and diameter of sclerodermiform neoplasms were superior to the other subtypes. Chromatin's color intensity and fractal dimension were less intense in superficial subtypes. Nuclear roundness and chromatin's entropy presented lower values in tumors than in normal epithelium. There was significant correlation between morphological and textural variables of normal skin and tumors. Morphometric elements and textural chromatin's homogeneity of basal cell carcinomas may be related to evolutionary, biological and behavior particularities related to each histotype. PMID:26734870

  18. 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

  19. Probing Chromatin-modifying Enzymes with Chemical Tools.

    PubMed

    Fischle, Wolfgang; Schwarzer, Dirk

    2016-03-18

    Chromatin is the universal template of genetic information in all eukaryotic organisms. Chemical modifications of the DNA-packaging histone proteins and the DNA bases are crucial signaling events in directing the use and readout of eukaryotic genomes. The enzymes that install and remove these chromatin modifications as well as the proteins that bind these marks govern information that goes beyond the sequence of DNA. Therefore, these so-called epigenetic regulators are intensively studied and represent promising drug targets in modern medicine. We summarize and discuss recent advances in the field of chemical biology that have provided chromatin research with sophisticated tools for investigating the composition, activity, and target sites of chromatin modifying enzymes and reader proteins. PMID:26845102

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2016-01-01

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

  2. Nuclear envelope and chromatin, lock and key of genome integrity.

    PubMed

    Gay, Sophie; Foiani, Marco

    2015-01-01

    More than as an inert separation between the inside and outside of the nucleus, the nuclear envelope (NE) constitutes an active toll, which controls the import and export of molecules, and also a hub for a diversity of genomic processes, such as transcription, DNA repair, and chromatin dynamics. Proteins localized at the inner surface of the NE (such as lamins, nuclear pore proteins, lamin-associated proteins) interact with chromatin in a dynamic manner, contributing to the establishment of topological domains. In this review, we address the complex interplay between chromatin and NE. We discuss the divergence of this cross talk during evolution and comment both on the current established models and the most recent findings. In particular, we focus our attention on how the NE cooperates with chromatin in protecting the genome integrity. PMID:26008788

  3. Polycomb-mediated chromatin compaction weathers the STORM.

    PubMed

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

    2016-01-01

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

  4. Condenser performance monitoring and cleaning

    SciTech Connect

    Walden, J.V.

    1998-12-31

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

  5. 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

  6. Active and Repressive Chromatin-Associated Proteome after MPA Treatment and the Role of Midkine in Epithelial Monolayer Permeability

    PubMed Central

    Khan, Niamat; Lenz, Christof; Binder, Lutz; Pantakani, Dasaradha Venkata Krishna; Asif, Abdul R.

    2016-01-01

    Mycophenolic acid (MPA) is prescribed to maintain allografts in organ-transplanted patients. However, gastrointestinal (GI) complications, particularly diarrhea, are frequently observed as a side effect following MPA therapy. We recently reported that MPA altered the tight junction (TJ)-mediated barrier function in a Caco-2 cell monolayer model system. This study investigates whether MPA induces epigenetic changes which lead to GI complications, especially diarrhea. Methods: We employed a Chromatin Immunoprecipitation-O-Proteomics (ChIP-O-Proteomics) approach to identify proteins associated with active (H3K4me3) as well as repressive (H3K27me3) chromatin histone modifications in MPA-treated cells, and further characterized the role of midkine, a H3K4me3-associated protein, in the context of epithelial monolayer permeability. Results: We identified a total of 333 and 306 proteins associated with active and repressive histone modification marks, respectively. Among them, 241 proteins were common both in active and repressive chromatin, 92 proteins were associated exclusively with the active histone modification mark, while 65 proteins remained specific to repressive chromatin. Our results show that 45 proteins which bind to the active and seven proteins which bind to the repressive chromatin region exhibited significantly altered abundance in MPA-treated cells as compared to DMSO control cells. A number of novel proteins whose function is not known in bowel barrier regulation were among the identified proteins, including midkine. Our functional integrity assays on the Caco-2 cell monolayer showed that the inhibition of midkine expression prior to MPA treatment could completely block the MPA-mediated increase in barrier permeability. Conclusions: The ChIP-O-Proteomics approach delivered a number of novel proteins with potential implications in MPA toxicity. Consequently, it can be proposed that midkine inhibition could be a potent therapeutic approach to prevent the MPA-mediated increase in TJ permeability and leak flux diarrhea in organ transplant patients. PMID:27104530

  7. A Role for CARM1-Mediated Histone H3 Arginine Methylation in Protecting Histone Acetylation by Releasing Corepressors from Chromatin

    PubMed Central

    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 TIF1β 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 TIF1β with an episomal reporter and that CARM1 is required in MEF cells for LPS-induced dissociation of NuRD from a NF-κb 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

  8. HMG Nuclear Proteins: Linking Chromatin Structure to Cellular Phenotype

    PubMed Central

    Reeves, Raymond

    2009-01-01

    I. Summary Although the three families of mammalian HMG proteins (HMGA, HMGB and HMGN) participate in many of the same nuclear processes, each family plays its own unique role in modulating chromatin structure and regulating genomic function. This review focuses on the similarities and differences in the mechanisms by which the different HMG families impact chromatin structure and influence cellular phenotype. The biological implications of having three architectural transcription factor families with complementary, but partially overlapping, nuclear functions are discussed. PMID:19748605

  9. Unsupervised pattern discovery in human chromatin structure through genomic segmentation.

    PubMed

    Hoffman, Michael M; Buske, Orion J; Wang, Jie; Weng, Zhiping; Bilmes, Jeff A; Noble, William Stafford

    2012-05-01

    We trained Segway, a dynamic Bayesian network method, simultaneously on chromatin data from multiple experiments, including positions of histone modifications, transcription-factor binding and open chromatin, all derived from a human chronic myeloid leukemia cell line. In an unsupervised fashion, we identified patterns associated with transcription start sites, gene ends, enhancers, transcriptional regulator CTCF-binding regions and repressed regions. Software and genome browser tracks are at http://noble.gs.washington.edu/proj/segway/. PMID:22426492

  10. Chromosome condensation in mitosis and meiosis of rye (Secale cereale L.).

    PubMed

    Zoller, J F; Herrmann, R G; Wanner, G

    2004-01-01

    Structural investigation and morphometry of meiotic chromosomes by scanning electron microscopy (in comparison to light microscopy) of all stages of condensation of meiosis I + II show remarkable differences during chromosome condensation in mitosis and meiosis I of rye (Secale cereale) with respect to initiation, mode and degree of condensation. Mitotic chromosomes condense in a linear fashion, shorten in length and increase moderately in diameter. In contrast, in meiosis I, condensation of chromosomes in length and diameter is a sigmoidal process with a retardation in zygotene and pachytene and an acceleration from diplotene to diakinesis. The basic structural components of mitotic chromosomes of rye are "parallel fibers" and "chromomeres" which become highly compacted in metaphase. Although chromosome architecture in early prophase of meiosis seems similar to mitosis in principle, there is no equivalent stage during transition to metaphase I when chromosomes condense to a much higher degree and show a characteristic "smooth" surface. No indication was found for helical winding of chromosomes either in mitosis or in meiosis. Based on measurements, we propose a mechanism for chromosome dynamics in mitosis and meiosis, which involves three individual processes: (i) aggregation of chromatin subdomains into a chromosome filament, (ii) condensation in length, which involves a progressive increase in diameter and (iii) separation of chromatids. PMID:15218269

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-11-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    SciTech Connect

    Bradbury, E.M.

    1992-11-01

    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.

  16. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2002-08-01

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

  18. Fragmentation of chromatin with 125I radioactive disintegrations.

    PubMed Central

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

    1976-01-01

    The DNA in Chinese hamster cells was labeled first for 3 h with [3H]TdR and then for 3 h with [125I]UdR. Chromatin was extracted, frozen, and stored at -30 degrees C until 1.0 X 10(17) and 1.25 X 10(17) disintegrations/g of labeled DNA occurred for 125I and 3H respectively. Velocity sedimentation of chromatin (DNA with associated chromosomal proteins) in neutral sucrose gradients indicated that the localized energy from the 125I disintegrations, which gave about 1 double-strand break/disintegration plus an additional 1.3 single strand breaks, selectively fragmented the [125I] chromatin into pieces smaller than the [3H] chromatin. In other words, 125I disintegrations caused much more localized damage in the chromatin labeled with 125I than in the chromatin labeled with 3H, and fragments induced in DNA by 125I disintegrations were not held together by the associated chromosomal proteins. Use of this 125I technique for studying chromosomal proteins associated with different regions in the cellular DNA is discussed. For these studies, the number of disintegrations required for fragmenting DNA molecules of different sizes is illustrated. PMID:963201

  19. 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

  20. Histone Depletion Facilitates Chromatin Loops on the Kilobasepair Scale

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

    Liu, Liang; Zhao, Weiling; Zhou, Xiaobo

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. 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.

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

    PubMed

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

    2011-06-01

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

  7. 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

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

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Wong, Michael; Hada, Megumi; Wu, Honglu

    2015-01-01

    Microgravity has been shown to alter global gene expression patterns and protein levels both in cultured cells and animal models. It has been suggested that the packaging of chromatin fibers in the interphase nucleus is closely related to genome function, and the changes in transcriptional activity are tightly correlated with changes in chromatin folding. This study explores the changes of chromatin conformation and chromatin-chromatin interactions in the simulated microgravity environment, and investigates their correlation to the expression of genes located at different regions of the chromosome. To investigate the folding of chromatin in interphase under various culture conditions, human epithelial cells, fibroblasts, and lymphocytes were fixed in the G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome as separate colors. After images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multi-mega base pair scale. In order to determine the effects of microgravity on chromosome conformation and orientation, measures such as distance between homologous pairs, relative orientation of chromosome arms about a shared midpoint, and orientation of arms within individual chromosomes were all considered as potentially impacted by simulated microgravity conditions. The studies revealed non-random folding of chromatin in interphase, and suggested an association of interphase chromatin folding with radiation-induced chromosome aberration hotspots. Interestingly, the distributions of genes with expression changes over chromosome 3 in cells cultured under microgravity environment are apparently clustered on specific loci and chromosomes. This data provides important insights into how mammalian cells respond to microgravity at molecular level.

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

    PubMed

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

    2013-11-01

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

  10. Condensed Matter Nuclear Science

    NASA Astrophysics Data System (ADS)

    Biberian, Jean-Paul

    2006-02-01

    1. General. A tribute to gene Mallove - the "Genie" reactor / K. Wallace and R. Stringham. An update of LENR for ICCF-11 (short course, 10/31/04) / E. Storms. New physical effects in metal deuterides / P. L. Hagelstein ... [et al.]. Reproducibility, controllability, and optimization of LENR experiments / D. J. Nagel -- 2. Experiments. Electrochemistry. Evidence of electromagnetic radiation from Ni-H systems / S. Focardi ... [et al.]. Superwave reality / I. Dardik. Excess heat in electrolysis experiments at energetics technologies / I. Dardik ... [et al.]. "Excess heat" during electrolysis in platinum/K[symbol]CO[symbol]/nickel light water system / J. Tian ... [et al.]. Innovative procedure for the, in situ, measurement of the resistive thermal coefficient of H(D)/Pd during electrolysis; cross-comparison of new elements detected in the Th-Hg-Pd-D(H) electrolytic cells / F. Celani ... [et al.]. Emergence of a high-temperature superconductivity in hydrogen cycled Pd compounds as an evidence for superstoihiometric H/D sites / A. Lipson ... [et al.]. Plasma electrolysis. Calorimetry of energy-efficient glow discharge - apparatus design and calibration / T. B. Benson and T. O. Passell. Generation of heat and products during plasma electrolysis / T. Mizuno ... [et al.]. Glow discharge. Excess heat production in Pd/D during periodic pulse discharge current in various conditions / A. B. Karabut. Beam experiments. Accelerator experiments and theoretical models for the electron screening effect in metallic environments / A. Huke, K. Czerski, and P. Heide. Evidence for a target-material dependence of the neutron-proton branching ratio in d+d reactions for deuteron energies below 20keV / A. Huke ... [et al.]. Experiments on condensed matter nuclear events in Kobe University / T. Minari ... [et al.]. Electron screening constraints for the cold fusion / K. Czerski, P. Heide, and A. Huke. Cavitation. Low mass 1.6 MHz sonofusion reactor / R. Stringham. Particle detection. Research into characteristics of X-ray emission laser beams from solidstate cathode medium of high-current glow discharge / A. B. Karabut. Charged particles from Ti and Pd foils / L. Kowalski ... [et al.]. Cr-39 track detectors in cold fusion experiments: review and perspectives / A. S. Roussetski. Energetic particle shower in the vapor from electrolysis / R. A. Oriani and J. C. Fisher. Nuclear reactions produced in an operating electrolysis cell / R. A. Oriani and J. C. Fisher. Evidence of microscopic ball lightning in cold fusion experiments / E. H. Lewis. Neutron emission from D[symbol] gas in magnetic fields under low temperature / T. Mizuno ... [et al.]. Energetic charged particle emission from hydrogen-loaded Pd and Ti cathodes and its enhancement by He-4 implantation / A. G. Lipson ... [et al.]. H-D permeation. Observation of nuclear transmutation reactions induced by D[symbol] gas permeation through Pd complexes / Y. Iwamura ... [et al.]. Deuterium (hydrogen) flux permeating through palladium and condensed matter nuclear science / Q. M. Wei ... [et al.]. Triggering. Precursors and the fusion reactions in polarized Pd/D-D[symbol]O system: effect of an external electric field / S. Szpak, P. A. Mosier-Boss, and F. E. Gordon. Calorimetric and neutron diagnostics of liquids during laser irradiation / Yu. N. Bazhutov ... [et al.]. Anomalous neutron capture and plastic deformation of Cu and Pd cathodes during electrolysis in a weak thermalized neutron field: evidence of nuclei-lattice exchange / A. G. Lipson and G. H. Miley. H-D loading. An overview of experimental studies on H/Pd over-loading with thin Pd wires and different electrolytic solutions / A. Spallone ... [et al.] -- 3. Transmutations. Photon and particle emission, heat production, and surface transformation in Ni-H system / E. Campari ... [et al.]. Surface analysis of hydrogen-loaded nickel alloys / E. Campari ... [et al.]. Low-energy nuclear reactions and the leptonic monopole / G. Lochak and L. Urutskoev. Results of analysis of Ti foil after glow discharge with deuterium / I. B. Savvatimova and D. V. Gavritenkov. Enhancement mechanisms of low-energy nuclear reactions / F. A. Gareev, I. E. Zhidkova, and Y. L. Ratis. Co-deposition of palladium with hydrogen isotopes / J. Dash and A. Ambadkar. Variation of the concentration of isotopes copper and zinc in human plasmas of patients affected by cancer / A. Triassi. Transmutation of metal at low energy in a confined plasma in water / D. Cirillo and V. Iorio. The conditions and realization of self-similar Coulomb collapse of condensed target and low-energy laboratory nucleosynthesis / S. V. Adamenko and V. I. Vysotskii. The spatial structure of water and the problem of controlled low-energy nuclear reactions in water matrix / V. I. Vysotskii and A. A. Kornilova. Experiments on controlled decontamination of water mixture of longlived active isotopes in biological cells / V. I. Vysotskii. Assessment of the biological effects of "strange" radiation / E. A. Pryakhin ... [et al.]. Possible nuclear transmutation of nitrogen in the earth's atmosphere / M. Fukuhara. Evidences on the occurrence of LENR-type processes in alchemical transmutations / J. Pérez-Pariente. History of the discovery of transmutation at Texas A&M University / J. O.-M. Bockris -- 4. Theory. Quantum electrodynamics. Concerning the modeling of systems in terms of quantum electro dynamics: the special case of "cold fusion" / M. Abyaneh ... [et al.]. Screening. Theoretical model of the probability of fusion between deuterons within deformed lattices with microcracks at room temperature / F. Fulvio. Resonant tunnelling. Effective interaction potential in the deuterium plasma and multiple resonance scattering / T. Toimela. Multiple scattering theory and condensed matter nuclear science - "super-absorption" in a crystal latice / X. Z. Li ... [et al.]. Ion band states. Framework for understanding LENR processes, using conventional condensed matter physics / S. R. Chubb. I. Bloch ions / T. A. Chubb. II. Inhibited diffusion driven surface transmutations / T. A. Chubb. III. Bloch nuclides, Iwamura transmutations, and Oriani showers / T. A. Chubb. Bose-Einstein condensate. Theoretical study of nuclear reactions induced by Bose-Einstein condensation in Pd / K.-I. Tsuchiya and H. Okumura. Proposal for new experimental tests of the Bose-Einstein condensation mechanism for low-energy nuclear reaction and transmutation processes in deuterium loaded micro- and nano-scale cavities / Y. E. Kim ... [et al.]. Mixtures of charged bosons confined in harmonic traps and Bose-Einstein condensation mechanism for low-energy nuclear reactions and transmutation processes in condensed matters / Y. E. Kim and A. L. Zubarev. Alternative interpretation of low-energy nuclear reaction processes with deuterated metals based on the Bose-Einstein condensation mechanism / Y. E. Kim and T. O. Passell. Multi-body fusion. [symbol]He/[symbol]He production ratios by tetrahedral symmetric condensation / A. Takahashi. Phonon coupling. Phonon-exchange models: some new results / P. L. Hagelstein. Neutron clusters. Cold fusion phenomenon and solid state nuclear physics / H. Kozima. Neutrinos, magnetic monopoles. Neutrino-driven nuclear reactions of cold fusion and transmutation / V. Filimonov. Light monopoles theory: an overview of their effects in physics, chemistry, biology, and nuclear science (weak interactions) / G. Lochak. Electrons clusters and magnetic monopoles / M. Rambaut. Others. Effects of atomic electrons on nuclear stability and radioactive decay / D. V. Filippov, L. I. Urutskoev, and A. A. Rukhadze. Search for erzion nuclear catalysis chains from cosmic ray erzions stopping in organic scintillator / Yu. N. Bazhutov and E. V. Pletnikov. Low-energy nuclear reactions resulting as picometer interactions with similarity to K-shell electron capture / H. Hora ... [et al.] -- 5. Other topics. On the possible magnetic mechanism of shortening the runaway of RBMK-1000 reactor at Chernobyl Nuclear Power Plant / D. V. Filippov ... [et al.]. Cold fusion in the context of a scientific revolution in physics: history and economic ramifications / E. Lewis. The nucleovoltaic cell / D. D. Moon. Introducing the book "Cold Fusion and the Future" / J. Rothwell. Recent cold fusion claims: are they valid? / L. Kowalski. History of attempts to publish a paper / L. Kowalski.

  11. Confinement Contains Condensates

    SciTech Connect

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

    2012-03-12

    Dynamical chiral symmetry breaking and its connection to the generation of hadron masses has historically been viewed as a vacuum phenomenon. We argue that confinement makes such a position untenable. If quark-hadron duality is a reality in QCD, then condensates, those quantities that have commonly been viewed as constant empirical mass-scales that fill all spacetime, are instead wholly contained within hadrons; i.e., they are a property of hadrons themselves and expressed, e.g., in their Bethe-Salpeter or light-front wave functions. We explain that this paradigm is consistent with empirical evidence, and incidentally expose misconceptions in a recent Comment.

  12. Condensed Plasmas under Microgravity

    NASA Technical Reports Server (NTRS)

    Morfill, G. E.; Thomas, H. M.; Konopka, U.; Rothermel, H.; Zuzic, M.; Ivlev, A.; Goree, J.; Rogers, Rick (Technical Monitor)

    1999-01-01

    Experiments under microgravity conditions were carried out to study 'condensed' (liquid and crystalline) states of a colloidal plasma (ions, electrons, and charged microspheres). Systems with approximately 10(exp 6) microspheres were produced. The observed systems represent new forms of matter--quasineutral, self-organized plasmas--the properties of which are largely unexplored. In contrast to laboratory measurements, the systems under microgravity are clearly three dimensional (as expected); they exhibit stable vortex flows, sometimes adjacent to crystalline regions, and a central 'void,' free of microspheres.

  13. Allelic Imbalance in Regulation of ANRIL through Chromatin Interaction at 9p21 Endometriosis Risk Locus.

    PubMed

    Nakaoka, Hirofumi; Gurumurthy, Aishwarya; Hayano, Takahide; Ahmadloo, Somayeh; Omer, Waleed H; Yoshihara, Kosuke; Yamamoto, Akihito; Kurose, Keisuke; Enomoto, Takayuki; Akira, Shigeo; Hosomichi, Kazuyoshi; Inoue, Ituro

    2016-04-01

    Genome-wide association studies (GWASs) have discovered numerous single nucleotide polymorphisms (SNPs) associated with human complex disorders. However, functional characterization of the disease-associated SNPs remains a formidable challenge. Here we explored regulatory mechanism of a SNP on chromosome 9p21 associated with endometriosis by leveraging "allele-specific" functional genomic approaches. By re-sequencing 1.29 Mb of 9p21 region and scrutinizing DNase-seq data from the ENCODE project, we prioritized rs17761446 as a candidate functional variant that was in perfect linkage disequilibrium with the original GWAS SNP (rs10965235) and located on DNase I hypersensitive site. Chromosome conformation capture followed by high-throughput sequencing revealed that the protective G allele of rs17761446 exerted stronger chromatin interaction with ANRIL promoter. We demonstrated that the protective allele exhibited preferential binding affinities to TCF7L2 and EP300 by bioinformatics and chromatin immunoprecipitation (ChIP) analyses. ChIP assays for histone H3 lysine 27 acetylation and RNA polymerase II reinforced the enhancer activity of the SNP site. The allele specific expression analysis for eutopic endometrial tissues and endometrial carcinoma cell lines showed that rs17761446 was a cis-regulatory variant where G allele was associated with increased ANRIL expression. Our work illuminates the allelic imbalances in a series of transcriptional regulation from factor binding to gene expression mediated by chromatin interaction underlie the molecular mechanism of 9p21 endometriosis risk locus. Functional genomics on common disease will unlock functional aspect of genotype-phenotype correlations in the post-GWAS stage. PMID:27055116

  14. Allelic Imbalance in Regulation of ANRIL through Chromatin Interaction at 9p21 Endometriosis Risk Locus

    PubMed Central

    Nakaoka, Hirofumi; Gurumurthy, Aishwarya; Hayano, Takahide; Ahmadloo, Somayeh; Omer, Waleed H; Yoshihara, Kosuke; Yamamoto, Akihito; Kurose, Keisuke; Enomoto, Takayuki; Akira, Shigeo; Hosomichi, Kazuyoshi; Inoue, Ituro

    2016-01-01

    Genome-wide association studies (GWASs) have discovered numerous single nucleotide polymorphisms (SNPs) associated with human complex disorders. However, functional characterization of the disease-associated SNPs remains a formidable challenge. Here we explored regulatory mechanism of a SNP on chromosome 9p21 associated with endometriosis by leveraging “allele-specific” functional genomic approaches. By re-sequencing 1.29 Mb of 9p21 region and scrutinizing DNase-seq data from the ENCODE project, we prioritized rs17761446 as a candidate functional variant that was in perfect linkage disequilibrium with the original GWAS SNP (rs10965235) and located on DNase I hypersensitive site. Chromosome conformation capture followed by high-throughput sequencing revealed that the protective G allele of rs17761446 exerted stronger chromatin interaction with ANRIL promoter. We demonstrated that the protective allele exhibited preferential binding affinities to TCF7L2 and EP300 by bioinformatics and chromatin immunoprecipitation (ChIP) analyses. ChIP assays for histone H3 lysine 27 acetylation and RNA polymerase II reinforced the enhancer activity of the SNP site. The allele specific expression analysis for eutopic endometrial tissues and endometrial carcinoma cell lines showed that rs17761446 was a cis-regulatory variant where G allele was associated with increased ANRIL expression. Our work illuminates the allelic imbalances in a series of transcriptional regulation from factor binding to gene expression mediated by chromatin interaction underlie the molecular mechanism of 9p21 endometriosis risk locus. Functional genomics on common disease will unlock functional aspect of genotype-phenotype correlations in the post-GWAS stage. PMID:27055116

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  17. Use of Chromatin Immunoprecipitation To Clone Novel E2F Target Promoters

    PubMed Central

    Weinmann, Amy S.; Bartley, Stephanie M.; Zhang, Theresa; Zhang, Michael Q.; Farnham, Peggy J.

    2001-01-01

    We have taken a new approach to the identification of E2F-regulated promoters. After modification of a chromatin immunoprecipitation assay, we cloned nine chromatin fragments which represent both strong and weak in vivo E2F binding sites. Further characterization of three of the cloned fragments revealed that they are bound in vivo not only by E2Fs but also by members of the retinoblastoma tumor suppressor protein family and by RNA polymerase II, suggesting that these fragments represent promoters regulated by E2F transcription complexes. In fact, database analysis indicates that all three fragments correspond to genomic DNA located just upstream of start sites for previously identified mRNAs. One clone, ChET 4, corresponds to the promoter region for beclin 1, a candidate tumor suppressor protein. We demonstrate that another of the clones, ChET 8, is strongly bound by E2F family members in vivo but does not contain a consensus E2F binding site. However, this fragment functions as a promoter whose activity can be repressed by E2F1. Finally, we demonstrate that the ChET 9 promoter contains a consensus E2F binding site, can be activated by E2F1, and drives expression of an mRNA that is upregulated in colon and liver tumors. Interestingly, the characterized ChET promoters do not display regulation patterns typical of known E2F target genes in a U937 cell differentiation system. In summary, we have provided evidence that chromatin immunoprecipitation can be used to identify E2F-regulated promoters which contain both consensus and nonconsensus binding sites and have shown that not all E2F-regulated promoters show identical expression profiles. PMID:11564866

  18. 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...

  19. 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...

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

    PubMed Central

    Collepardo-Guevara, Rosana; Schlick, Tamar

    2014-01-01

    Deciphering the factors that control chromatin fiber structure is key to understanding fundamental chromosomal processes. Although details remain unknown, it is becoming clear that chromatin is polymorphic depending on internal and external factors. In particular, different lengths of the linker DNAs joining successive nucleosomes (measured in nucleosome-repeat lengths or NRLs) that characterize different cell types and cell cycle stages produce different structures. NRL is also nonuniform within single fibers, but how this diversity affects chromatin fiber structure is not clear. Here we perform Monte Carlo simulations of a coarse-grained oligonucleosome model to help interpret fiber structure subject to intrafiber NRL variations, as relevant to proliferating cells of interphase chromatin, fibers subject to remodeling factors, and regulatory DNA sequences. We find that intrafiber NRL variations have a profound impact on chromatin structure, with a wide range of different architectures emerging (highly bent narrow forms, canonical and irregular zigzag fibers, and polymorphic conformations), depending on the NRLs mixed. This stabilization of a wide range of fiber forms might allow NRL variations to regulate both fiber compaction and selective DNA exposure. The polymorphic forms spanning canonical to sharply bent structures, like hairpins and loops, arise from large NRL variations and are surprisingly more compact than uniform NRL structures. They are distinguished by tail-mediated far-nucleosome interactions, in addition to the near-nucleosome interactions of canonical 30-nm fibers. Polymorphism is consistent with chromatins diverse biological functions and heterogeneous constituents. Intrafiber NRL variations, in particular, may contribute to fiber bending and looping and thus to distant communication in associated regulatory processes. PMID:24847063

  1. Structural Fluctuations of the Chromatin Fiber within Topologically Associating Domains.

    PubMed

    Tiana, Guido; Amitai, Assaf; Pollex, Tim; Piolot, Tristan; Holcman, David; Heard, Edith; Giorgetti, Luca

    2016-03-29

    Experiments based on chromosome conformation capture have shown that mammalian genomes are partitioned into topologically associating domains (TADs), within which the chromatin fiber preferentially interacts. TADs may provide three-dimensional scaffolds allowing genes to contact their appropriate distal regulatory DNA sequences (e.g., enhancers) and thus to be properly regulated. Understanding the cell-to-cell and temporal variability of the chromatin fiber within TADs, and what determines them, is thus of great importance to better understand transcriptional regulation. We recently described an equilibrium polymer model that can accurately predict cell-to-cell variation of chromosome conformation within single TADs, from chromosome conformation capture-based data. Here we further analyze the conformational and energetic properties of our model. We show that the chromatin fiber within TADs can easily fluctuate between several conformational states, which are hierarchically organized and are not separated by important free energy barriers, and that this is facilitated by the fact that the chromatin fiber within TADs is close to the onset of the coil-globule transition. We further show that in this dynamic state the properties of the chromatin fiber, and its contact probabilities in particular, are determined in a nontrivial manner not only by site-specific interactions between strongly interacting loci along the fiber, but also by nonlocal correlations between pairs of contacts. Finally, we use live-cell experiments to measure the dynamics of the chromatin fiber in mouse embryonic stem cells, in combination with dynamical simulations, and predict that conformational changes within one TAD are likely to occur on timescales that are much shorter than the duration of one cell cycle. This suggests that genes and their regulatory elements may come together and disassociate several times during a cell cycle. These results have important implications for transcriptional regulation as they support the concept of highly dynamic interactions driven by a complex interplay between site-specific interactions and the intrinsic biophysical properties of the chromatin fiber. PMID:27028634

  2. Characterization of nucleosome unwrapping within chromatin fibers using magnetic tweezers.

    PubMed

    Chien, Fan-Tso; van der Heijden, Thijn

    2014-07-15

    Nucleosomal arrays fold into chromatin fibers and the higher-order folding of chromatin plays a strong regulatory role in all processes involving DNA access, such as transcription and replication. A fundamental understanding of such regulation requires insight into the folding properties of the chromatin fiber in molecular detail. Despite this, the structure and the mechanics of chromatin fibers remain highly disputed. Single-molecule force spectroscopy experiments have the potential to provide such insight, but interpretation of the data has been hampered by the large variations in experimental force-extension traces. Here we explore the possibility that chromatin fibers are composed of both single-turn and fully wrapped histone octamers. By characterizing the force-dependent behavior of in vitro reconstituted chromatin fibers and reanalyzing existing data, we show the unwrapping of the outer turn of nucleosomal DNA at 3 pN. We present a model composed of two freely-jointed chains, which reveals that nucleosomes within the chromatin fiber show identical force-extension behavior to mononucleosomes, indicating that nucleosome-nucleosome interactions are orders-of-magnitude smaller than previously reported and therefore can be overcome by thermal fluctuations. We demonstrate that lowering the salt concentration externally increases the wrapping energy significantly, indicative of the electrostatic interaction between the wrapped DNA and the histone octamer surface. We propose that the weak interaction between nucleosomes could allow easy access to nucleosomal DNA, while DNA unwrapping from the histone core could provide a stable yet dynamic structure during DNA maintenance. PMID:25028879

  3. Chromatin perturbations during the DNA damage response in higher eukaryotes.

    PubMed

    Bakkenist, Christopher J; Kastan, Michael B

    2015-12-01

    The DNA damage response is a widely used term that encompasses all signaling initiated at DNA lesions and damaged replication forks as it extends to orchestrate DNA repair, cell cycle checkpoints, cell death and senescence. ATM, an apical DNA damage signaling kinase, is virtually instantaneously activated following the introduction of DNA double-strand breaks (DSBs). The MRE11-RAD50-NBS1 (MRN) complex, which has a catalytic role in DNA repair, and the KAT5 (Tip60) acetyltransferase are required for maximal ATM kinase activation in cells exposed to low doses of ionizing radiation. The sensing of DNA lesions occurs within a highly complex and heterogeneous chromatin environment. Chromatin decondensation and histone eviction at DSBs may be permissive for KAT5 binding to H3K9me3 and H3K36me3, ATM kinase acetylation and activation. Furthermore, chromatin perturbation may be a prerequisite for most DNA repair. Nucleosome disassembly during DNA repair was first reported in the 1970s by Smerdon and colleagues when nucleosome rearrangement was noted during the process of nucleotide excision repair of UV-induced DNA damage in human cells. Recently, the multi-functional protein nucleolin was identified as the relevant histone chaperone required for partial nucleosome disruption at DBSs, the recruitment of repair enzymes and for DNA repair. Notably, ATM kinase is activated by chromatin perturbations induced by a variety of treatments that do not directly cause DSBs, including treatment with histone deacetylase inhibitors. Central to the mechanisms that activate ATR, the second apical DNA damage signaling kinase, outside of a stalled and collapsed replication fork in S-phase, is chromatin decondensation and histone eviction associated with DNA end resection at DSBs. Thus, a stress that is common to both ATM and ATR kinase activation is chromatin perturbations, and we argue that chromatin perturbations are both sufficient and required for induction of the DNA damage response. PMID:26391293

  4. Chromatin remodeling in the aging genome of Drosophila

    PubMed Central

    Wood, Jason G.; Hillenmeyer, Sara; Lawrence, Charles; Chang, Chengyi; Hosier, Suzanne; Lightfoot, Will; Mukherjee, Eric; Jiang, Nan; Schorl, Christoph; Brodsky, Alexander S.; Neretti, Nicola; Helfand, Stephen L.

    2010-01-01

    Summary Chromatin structure affects the accessibility of DNA to transcription, repair and replication. Changes in chromatin structure occur during development, but less is known about changes during aging. We examined the state of chromatin structure and its effect on gene expression during aging in Drosophila at the whole genome and cellular level using whole genome tiling microarrays of activation and repressive chromatin marks, whole genome transcriptional microarrays and single cell immunohistochemistry. We found dramatic reorganization of chromosomal regions with age. Mapping of H3K9me3 and HP1 signals to fly chromosomes reveals in young flies the expected high enrichment in the pericentric regions, the 4th chromosome and islands of facultative heterochromatin dispersed throughout the genome. With age there is a striking reduction in this enrichment resulting in a nearly equivalent level of H3K9me3 and HP1 in the pericentric regions, the 4th chromosome, facultative heterochromatin and euchromatin. These extensive changes in repressive chromatin marks are associated with alterations in age-related gene expression. Large-scale changes in repressive marks with age are further substantiated by single cell immunohistochemistry that show changes in nuclear distribution of H3K9me3 and HP1 marks with age. Such epigenetic changes are expected to directly or indirectly impinge upon important cellular functions such as gene expression, DNA repair and DNA replication. The combination of genome-wide approaches such as whole genome chromatin immunoprecipitation and transcriptional studies in conjunction with single cell immunohistochemistry as shown here provide a first step toward defining how changes in chromatin may contribute to the process of aging in metazoans. PMID:20961390

  5. Chromatin perturbations during the DNA damage response in higher eukaryotes

    PubMed Central

    Bakkenist, Christopher J.; Kastan, Michael B.

    2016-01-01

    The DNA damage response is a widely used term that encompasses all signaling initiated at DNA lesions and damaged replication forks as it extends to orchestrate DNA repair, cell cycle checkpoints, cell death and senescence. ATM, an apical DNA damage signaling kinase, is virtually instantaneously activated following the introduction of DNA double-strand breaks (DSBs). The MRE11-RAD50-NBS1 (MRN) complex, which has a catalytic role in DNA repair, and the KAT5 (Tip60) acetyltransferase are required for maximal ATM kinase activation in cells exposed to low doses of ionizing radiation. The sensing of DNA lesions occurs within a highly complex and heterogeneous chromatin environment. Chromatin decondensation and histone eviction at DSBs may be permissive for KAT5 binding to H3K9me3 and H3K36me3, ATM kinase acetylation and activation. Furthermore, chromatin perturbation may be a prerequisite for most DNA repair. Nucleosome disassembly during DNA repair was first reported in the 1970s by Smerdon and colleagues when nucleosome rearrangement was noted during the process of nucleotide excision repair of UV-induced DNA damage in human cells. Recently, the multi-functional protein nucleolin was identified as the relevant histone chaperone required for partial nucleosome disruption at DBSs, the recruitment of repair enzymes and for DNA repair. Notably, ATM kinase is activated by chromatin perturbations induced by a variety of treatments that do not directly cause DSBs, including treatment with histone deacetylase inhibitors. Central to the mechanisms that activate ATR, the second apical DNA damage signaling kinase, outside of a stalled and collapsed replication fork in S-phase, is chromatin decondensation and histone eviction associated with DNA end resection at DSBs. Thus, a stress that is common to both ATM and ATR kinase activation is chromatin perturbations, and we argue that chromatin perturbations are both sufficient and required for induction of the DNA damage response. PMID:26391293

  6. Condensates in relativistic scalar theories

    NASA Astrophysics Data System (ADS)

    Moore, Guy D.

    2016-03-01

    Scalar field theory with large infrared initial occupancy develops very large deep-infrared occupancy, which locally resembles a Bose-Einstein condensate. We study the structure and spatial coherence of this condensate. The O (N ) symmetric theory with N >1 is qualitatively different than N =1 . We explain the thermodynamical reason why, for N >1 , the condensate locally carries nearly maximal conserved charge density. We also show how this property impedes the condensate's decay, and we show that it prevents the condensate from ever becoming fully spatially homogeneous. For N ≤4 the condensate can carry topological defects, but these do not appear to control the large-k tail in its power spectrum, which is the same for N =8 where there are no topological defects.

  7. Cosmic curvature and condensation

    NASA Technical Reports Server (NTRS)

    Harwit, Martin

    1992-01-01

    It is shown that the universe may consist of a patchwork of domains with different Riemann curvature constants k = 0, +/-1. Features of a phase transition in which flat space breaks up in a transition 2k0 - k(-) + k(+) with initial scale factors R(-) = R(+) are postulated and explored. It is shown that such a transition is energetically permitted, has the equivalent of a Curie temperature, and can lead in a natural way to the formation of voids and galaxies. It is predicted that, if the ambient universe on average is well fitted by a purely k(-) space, with only occasional domains of k(+) containing galaxies, a density parameter of (A(z sub c + 1)) super -1 should be expected, where z sub c represents the redshift of the earliest objects to have condensed, and A takes on values ranging from about 5 to 3. Present observations of quasars would suggest a density of about 0.03 or 0.05, respectively, but it could be lower if earlier condensation took place.

  8. 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.

  9. A soybean cDNA encoding a chromatin-binding peptide inhibits mitosis of mammalian cells.

    PubMed

    Galvez, A F; de Lumen, B O

    1999-05-01

    A soybean cDNA encoding the small subunit peptide of a cotyledon-specific 2S albumin (Gm2S-1) is thought to play a role in arresting mitosis during the DNA endoreduplication and cell expansion phase of seed development. The peptide (termed lunasin) contains the cell adhesion motif Arg-Gly-Asp (RGD) followed by eight aspartic acid residues at its C-terminal end. A chimeric gene encoding the lunasin peptide tagged with green fluorescent protein (GFP) arrested cell division, caused abnormal spindle fiber elongation, chromosomal fragmentation, and cell lysis when transiently transfected into murine embryo fibroblast, murine hepatoma, and human breast cancer cells. Deletion of the polyaspartyl end abolished the antimitotic effect. Subcellular localization of lunasin and immunobinding assay using synthetic peptides revealed the preferential adherence of lunasin to chromatin. Immunofluorescence showed that kinetochore proteins were displaced from the centromere in lunasin-transfected cells. These observations suggest that lunasin binds to the chromatin, leading to disruption of kinetochore formation and inhibition of mitosis. PMID:10331812

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

    PubMed Central

    2012-01-01

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

  12. Chromatin loop organization of the junb locus in mouse dendritic cells

    PubMed Central

    Salem, Tamara; Gomard, Tiphanie; Court, Franck; Moquet-Torcy, Gabriel; Brockly, Frédérique; Forné, Thierry; Piechaczyk, Marc

    2013-01-01

    The junb gene behaves as an immediate early gene in bacterial lipopolysaccharide (LPS)-stimulated dendritic cells (DCs), where its transient transcriptional activation is necessary for the induction of inflammatory cytokines. junb is a short gene and its transcriptional activation by LPS depends on the binding of NF-κB to an enhancer located just downstream of its 3′ UTR. Here, we have addressed the mechanisms underlying the transcriptional hyper-reactivity of junb. Using transfection and pharmacological assays to complement chromatin immunoprecipitation analyses addressing the localization of histones, polymerase II, negative elongation factor (NELF)-, DRB sensitivity-inducing factor (DSIF)- and Positive Transcription Factor b complexes, we demonstrate that junb is a RNA Pol II-paused gene where Pol II is loaded in the transcription start site domain but poorly active. Moreover, High salt-Recovered Sequence, chromosome conformation capture (3C)- and gene transfer experiments show that (i) junb is organized in a nuclear chromatin loop bringing into close spatial proximity the upstream promoter region and the downstream enhancer and (ii) this configuration permits immediate Pol II release on the junb body on binding of LPS-activated NF-κB to the enhancer. Thus, our work unveils a novel topological framework underlying fast junb transcriptional response in DCs. Moreover, it also points to a novel layer of complexity in the modes of action of NF-κB. PMID:23921639

  13. SIRT7 Is Activated by DNA and Deacetylates Histone H3 in the Chromatin Context.

    PubMed

    Tong, Zhen; Wang, Yi; Zhang, Xiaoyu; Kim, David D; Sadhukhan, Sushabhan; Hao, Quan; Lin, Hening

    2016-03-18

    Mammalian sirtuins (SIRT1-7) are members of a highly conserved family of nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylases that regulate many biological processes including metabolism, genome stability, and transcription. Among the seven human sirtuins, SIRT7 is the least understood, to a large extent due to the lack of enzymatic activity in vitro. Here, we reported that SIRT7 can be activated by DNA to hydrolyze the acetyl group from lysine residues in vitro on histone peptides and histones in the chromatin context. Both N- and C- termini of SIRT7 are important for the DNA-activated deacetylase activity. The regulatory mechanism of SIRT7 is different from that of SIRT6, which also showed increased activity on chromatin substrates, but the deacetylase activity of SIRT6 on a peptide substrate cannot be activated by DNA. This finding provides an improved enzymatic activity assay of SIRT7 that will promote the development of SIRT7 modulators. Further investigation into the activation mechanism of SIRT7 by DNA could provide new insights into its biological function and help the development of sirtuin activators. PMID:26907567

  14. Chromatin redistribution of the DEK oncoprotein represses hTERT transcription in leukemias.

    PubMed

    Karam, Maroun; Thenoz, Morgan; Capraro, Valrie; Robin, Jean-Philippe; Pinatel, Christiane; Lancon, Agns; 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

  15. Exhaled breath condensate: an overview.

    PubMed

    Davis, Michael D; Montpetit, Alison; Hunt, John

    2012-08-01

    Exhaled breath condensate (EBC) is a promising source of biomarkers of lung disease. EBC may be thought of either as a body fluid or as a condensate of exhaled gas. There are 3 principal contributors to EBC: variable-sized particles or droplets that are aerosolized from the airway lining fluid, distilled water that condenses from gas phase out of the nearly water-saturated exhalate, and water-soluble volatiles that are exhaled and absorbed into the condensing breath. The nonvolatile constituents and the water-soluble volatile constituents are of particular interest. Several key issues are discussed in this article. PMID:22877615

  16. Condensation heat transfer in a microgravity environment

    NASA Technical Reports Server (NTRS)

    Chow, L. C.; Parish, R. C.

    1986-01-01

    In the present treatment of the condensation heat transfer process in a microgravity environment, two mechanisms for condensate removal are analyzed in light of two problems: (1) film condensation on a flat, porous plate, with condensate being removed by wall suction; and (2) the analytical prediction of the heat transfer coefficient of condensing annular flows, where the condensate film is driven by vapor shear. Both suction and vapor shear can effectively drain the condensate, ensuring continuous operation in microgravity.

  17. Extensive chromatin fragmentation improves enrichment of protein binding sites in chromatin immunoprecipitation experiments.

    PubMed

    Fan, Xiaochun; Lamarre-Vincent, Nathan; Wang, Qian; Struhl, Kevin

    2008-11-01

    Extensive sonication of formaldehyde-crosslinked chromatin can generate DNA fragments averaging 200 bp in length (range 75-300 bp). Fragmentation is largely random with respect to genomic region and nucleosome position. ChIP experiments employing such extensively fragmented samples show 2- to 4-fold increased enrichment of protein binding sites over control genomic regions, when compared to samples sonicated to a more conventional size range (300-500 bp). The basis of improved fold enrichments is that immunoprecipitation of protein-bound regions is unaffected by fragment size, whereas immunoprecipitation of control genomic regions decreases progressively along with reduced fragment size due to fewer nonspecific binding sites. The use of extensively sonicated samples improves mapping of protein binding sites, and it extends the dynamic range for quantitative measurements of histone density. We show that many yeast promoter regions are virtually devoid of histones. PMID:18765474

  18. Remodeling of chromatin under low intensity diffuse ultrasound

    PubMed Central

    Noriega, Sandra; Budhiraja, Gaurav; Subramanian, Anuradha

    2012-01-01

    A variety of mechanotransduction pathways mediate the response of fibroblasts or chondrocytes to ultrasound stimulation. In addition, regulatory pathways that co-ordinate stimulus-specific cellular responses are likely to exist. In this study, analysis was confined to the hypothesis that ultrasound stimulation (US) influences the chromatin structure, and that these changes may reflect a regulatory pathway that connects nuclear architecture, chromatin structure and gene expression. Murine fibroblasts seeded on tissue culture plates were stimulated with US (5.0 MHz (14 kPa), 51-s per application) and the thermal denaturation profiles of nuclei isolated from fibroblasts were assessed by dynamic scanning calorimetry (DSC). When compared to the thermal profiles obtained from the nuclei of non-stimulated cells, the nuclei obtained from stimulated cells showed a change in peak profiles and peak areas, which is indicative of chromatin remodeling. Independently, US was also observed to impact the histone (H1):chromatin association as measured indirectly by DAPI staining. Based on our work, it appears plausible that US can produce a remodeling of chromatin, thus triggering signal cascade and other intracellular mechanisms. PMID:22575092

  19. Topological constraints strongly affect chromatin reconstitution in silico

    PubMed Central

    Brackley, C.A.; Allan, J.; Keszenman-Pereyra, D.; Marenduzzo, D.

    2015-01-01

    The fundamental building block of chromatin, and of chromosomes, is the nucleosome, a composite material made up from DNA wrapped around a histone octamer. In this study we provide the first computer simulations of chromatin self-assembly, starting from DNA and histone proteins, and use these to understand the constraints which are imposed by the topology of DNA molecules on the creation of a polynucleosome chain. We take inspiration from the in vitro chromatin reconstitution protocols which are used in many experimental studies. Our simulations indicate that during self-assembly, nucleosomes can fall into a number of topological traps (or local folding defects), and this may eventually lead to the formation of disordered structures, characterised by nucleosome clustering. Remarkably though, by introducing the action of topological enzymes such as type I and II topoisomerase, most of these defects can be avoided and the result is an ordered 10-nm chromatin fibre. These findings provide new insight into the biophysics of chromatin formation, both in the context of reconstitution in vitro and in terms of the topological constraints which must be overcome during de novo nucleosome formation in vivo, e.g. following DNA replication or repair. PMID:25432958

  20. Nucleosomal chromatin in the mature sperm of Drosophila melanogaster

    PubMed Central

    Elnfati, Abdul Hakim; Iles, David; Miller, David

    2015-01-01

    During spermiogenesis in mammals and many other vertebrate classes, histone-containing nucleosomes are replaced by protamine toroids, which can repackage chromatin at a 10 to 20-fold higher density than in a typical somatic nucleus. However, recent evidence suggests that sperm of many species, including human and mouse retain a small compartment of nucleosomal chromatin, particularly near genes important for embryogenesis. As in mammals, spermiogenesis in the fruit fly, Drosophila melanogaster has also been shown to undergo a programmed substitution of nucleosomes with protamine-like proteins. Using chromatin immunoprecipitation (ChIP) and whole-genome tiling array hybridization (ChIP-chip), supported by immunocytochemical evidence, we show that in a manner analogous to nucleosomal chromatin retention in mammalian spermatozoa, distinct domains packaged by the canonical histones H2A, H2B, H3 and H4 are present in the fly sperm nucleus. We also find evidence for the retention of nucleosomes with specific histone H3 trimethylation marks characteristic of chromatin repression (H3K9me3, H3K27me3) and active transcription (H3K36me3). Raw and processed data from the experiments are available at GEO, accession GSE52165. PMID:26981400

  1. Nucleosomal chromatin in the mature sperm of Drosophila melanogaster.

    PubMed

    Elnfati, Abdul Hakim; Iles, David; Miller, David

    2016-03-01

    During spermiogenesis in mammals and many other vertebrate classes, histone-containing nucleosomes are replaced by protamine toroids, which can repackage chromatin at a 10 to 20-fold higher density than in a typical somatic nucleus. However, recent evidence suggests that sperm of many species, including human and mouse retain a small compartment of nucleosomal chromatin, particularly near genes important for embryogenesis. As in mammals, spermiogenesis in the fruit fly, Drosophila melanogaster has also been shown to undergo a programmed substitution of nucleosomes with protamine-like proteins. Using chromatin immunoprecipitation (ChIP) and whole-genome tiling array hybridization (ChIP-chip), supported by immunocytochemical evidence, we show that in a manner analogous to nucleosomal chromatin retention in mammalian spermatozoa, distinct domains packaged by the canonical histones H2A, H2B, H3 and H4 are present in the fly sperm nucleus. We also find evidence for the retention of nucleosomes with specific histone H3 trimethylation marks characteristic of chromatin repression (H3K9me3, H3K27me3) and active transcription (H3K36me3). Raw and processed data from the experiments are available at GEO, accession GSE52165. PMID:26981400

  2. Micron-scale coherence in interphase chromatin dynamics

    PubMed Central

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

    2013-01-01

    Chromatin structure and dynamics control all aspects of DNA biology yet are poorly understood, especially at large length scales. We developed an approach, displacement correlation spectroscopy based on time-resolved image correlation analysis, to map chromatin dynamics simultaneously across the whole nucleus in cultured human cells. This method revealed that chromatin movement was coherent across large regions (4–5 µm) for several seconds. Regions of coherent motion extended beyond the boundaries of single-chromosome territories, suggesting elastic coupling of motion over length scales much larger than those of genes. These large-scale, coupled motions were ATP dependent and unidirectional for several seconds, perhaps accounting for ATP-dependent directed movement of single genes. Perturbation of major nuclear ATPases such as DNA polymerase, RNA polymerase II, and topoisomerase II eliminated micron-scale coherence, while causing rapid, local movement to increase; i.e., local motions accelerated but became uncoupled from their neighbors. We observe similar trends in chromatin dynamics upon inducing a direct DNA damage; thus we hypothesize that this may be due to DNA damage responses that physically relax chromatin and block long-distance communication of forces. PMID:24019504

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

    PubMed Central

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

    2015-01-01

    The chromatin fiber undergoes significant structural changes during the cell’s life cycle to modulate DNA accessibility. Detailed mechanisms of such structural transformations of chromatin fibers as affected by various internal and external conditions such as the ionic conditions of the medium, the linker DNA length, and the presence of linker histones, constitute an open challenge. Here we utilize Monte Carlo (MC) simulations of a coarse grained model of chromatin with nonuniform linker DNA lengths as found in vivo to help explain some aspects of this challenge. We investigate the unfolding mechanisms of chromatin fibers with alternating linker lengths of 26-62 bp and 44-79 bp using a series of end-to-end stretching trajectories with and without linker histones and compare results to uniform-linker-length fibers. We find that linker histones increase overall resistance of nonuniform fibers and lead to fiber unfolding with superbeads-on-a-string cluster transitions. Chromatin fibers with nonuniform linker DNA lengths display a more complex, multi-step yet smoother process of unfolding compared to their uniform counterparts, likely due to the existence of a more continuous range of nucleosome-nucleosome interactions. This finding echoes the theme that some heterogeneity in fiber component is biologically advantageous. PMID:25564319

  4. Analysis of epigenetic alterations to chromatin during development

    PubMed Central

    Minard, Meghan E.; Jain, Abhinav K.; Barton, Michelle Craig

    2009-01-01

    Each cell within a multicellular organism has distinguishable characteristics established by its unique patterns of gene expression. This individual identity is determined by the expression of genes in a time and place-dependent manner. It is becoming increasingly clear that chromatin plays a fundamental role in the control of gene transcription in multicellular organisms. Understanding the regulation of chromatin, and how the distinct identity of a cell is passed to daughter cells during development, is paramount. Techniques with which to study chromatin have advanced rapidly over the past decade. Development of high throughput techniques and their proper applications has provided us essential tools to understand the regulation of epigenetic phenomena and its effect on gene expression. Understanding the changes that occur in chromatin during the course of development will not only contribute to our knowledge of normal gene expression, but will also add to our knowledge of how gene expression goes awry during disease. This review opens with an introduction to some of the key premises of epigenetic regulation of gene expression. A discussion of experimental techniques with which one can study epigenetic alterations to chromatin during development follows, emphasizing recent breakthroughs in this area. We then present examples of epigenetic mechanisms exploited in the control of developmental cell fate and regulation of tissue-specific gene expression. Finally, we discuss some of the frontiers and challenges in this area of research. PMID:19603511

  5. 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

  6. Extended chromatin fibers: evidence from scanning force microscopy studies

    NASA Astrophysics Data System (ADS)

    Leuba, Sanford S.; Yang, Guoliang; Robert, Charles; van Holde, Kensal; Zlatanova, Jordanka; Bustamante, Carlos J.

    1995-03-01

    Unfixed chicken erythrocyte fibers in very low salt have been imaged using the scanning force microscope (SFM) operating in the tapping mode in air at ambient humidity. These images reveal a 3D organization of the fibers. The planar 'zig-zag' conformation is rare, and extended 'beads- on-a-string' fibers are seen only in chromatin depleted of H1 and H5. Glutaraldehyde fixation reveals very similar structures. Fibers fixed in 10 mM salt appear somewhat more compacted. These results, when compared with modeling studies indicate that chromatin fibers may exist as irregular 3D arrays of nucleosomes even at low ionic strength. The basic subunit of chromatin, the nucleosome, is composed of a core particle of 146 bp of DNA wrapping 1.75 left-handed superhelical turns around an octamer of core histones and of DNA connecting consecutive core particles. The linker of lysine-rich histones (H1 family) bind the DNA entering and exiting the nucleosome core particle. We suggest that by binding the entry/exit DNA, histone H1 may fix the entry/exit DNA angle. The fixed entry/exit angle, the rigidity of the linker DNA at low ionic strength, and the natural variability of the linker DNA length determine an irregular 3D fiber of chromatin. Our results emphasize the role of H1 in determining the entry/exit DNA angle, which further helps determine the mutual disposition of adjacent nucleosomes an the packing of the chromatin fiber.

  7. Visualization and analysis of chromatin by scanning force microscopy.

    PubMed

    Bustamante, C; Zuccheri, G; Leuba, S H; Yang, G; Samori, B

    1997-05-01

    The use of the scanning force microscope (SFM) to visualize and analyze chromatin fiber structures is presented. Protocols to prepare chromatin fibers for SFM imaging of fibers in air and in buffer are first discussed. Next, the conditions for acquiring high-quality SFM images such as optimal instrumental parameters, appropriate deposition substrates, and adequate procedures of sample deposition are described. It is shown that analysis and quantitation of the SFM images support an irregular, three-dimensional arrangement of nucleosomes in the native chromatin fiber. This structure is lost in linker histone-depleted fibers, which show, instead, a beads-on-a-string structure. Molecular modeling of the chromatin fiber structures and computer simulation of the SFM imaging process indicate that the natural variability of the linker length may be the major determinant of the structural irregularity of the native chromatin fiber. Removal of linker histones (H1/H5) may change the amount of DNA wrapped around the histone octamer, which in turn may induce the transition from a three-dimensional irregular helix to an extended beads-on-a-string structure. Studies of trinucleosomes indicate that both the average successive nucleosome center-to-center distance and the average angle between two successive linkers increase upon the removal of linker histone. PMID:9169197

  8. 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

  9. Chromatin: a tunable spring at work inside chromosomes.

    PubMed

    Ben-Haïm, E; Lesne, A; Victor, J M

    2001-11-01

    This paper focuses on mechanical aspects of chromatin biological functioning. Within a basic geometric modeling of the chromatin assembly, we give a 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 wormlike 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. PMID:11735982

  10. 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

  11. Genotoxicity and DNA adduct formation of incense smoke condensates: comparison with environmental tobacco smoke condensates.

    PubMed

    Chen, C C; Lee, H

    1996-03-01

    Indoor air pollution has now been recognized as a potentially important problem for public health, since people spend most of their day in closed environments. Incense burning is possibly associated with elevated risks of leukemia and brain tumor in children from the epidemiological studies. Thus, evaluation of the genotoxicity of smoke condensates from incense burning is needed. We examined the genotoxicity of incense smoke condensates (ISC) using the Ames test in S. typhimurium strains with different mutagenic specificity and level of metabolic enzyme, the SOS chromotest in E. coli PQ37, and sister chromatid exchange assay in Chinese hamster ovary cells (SCE/CHO). The genotoxicity of environmental tobacco smoke condensates (TSC) was also evaluated by the three assays to compare with the genotoxicity of ISC. ISC showed a positive response in TA98, but not in TA100. It suggested that ISC only contained frame shift mutagens. The mutagenicity of ISC in both strains of TA98NR with deficient nitroreductase and TA98/1,8-DNP6 with deficient O-acetyltransferase was markedly decreased compared to that in TA98 strain. However, the mutagenicity was enhanced in YG1024 with overexpression of O-acetyltransferase activity. Thus, nitroarenes seemed to be responsible in part for the mutagenicity of ISC. Interestingly, all of the four ISC and two TSC samples showed a dose-dependent genotoxic response in the SOS chromotest with E. coli PQ37 but a low SCE induction of those samples were observed in CHO cells. When the genotoxicity was analyzed based on the condensates per one gram of original samples, the genotoxicity of two TSC condensates in prokaryotic cells was higher than that of four ISC samples except for the genotoxicity of TSC-2 in TA98 strain. However, the genotoxicity of certain ISC in eukaryotic cells based on the SCE/CHO assay was higher than that of TSC. To compare the covalent binding of DNA reactive intermediates of ISC and TSC to S. typhimurium TA98, the DNA adducts were evaluated by the 32P-postlabeling method with butanol extraction version. Similar diagonal radioactive zone (DRZ) was observed between ISC and CSC. However, DNA adduct levels induced by TSC were much greater than that of ISC. PMID:8600366

  12. Chromas from chromatin: sonification of the epigenome

    PubMed Central

    Cittaro, Davide; Lazarevic, Dejan; Provero, Paolo

    2016-01-01

    The epigenetic modifications are organized in patterns determining the functional properties of the underlying genome. Such patterns, typically measured by ChIP-seq assays of histone modifications, can be combined and translated into musical scores, summarizing multiple signals into a single waveform. As music is recognized as a universal way to convey meaningful information, we wanted to investigate properties of music obtained by sonification of ChIP-seq data. We show that the music produced by such quantitative signals is perceived by human listeners as more pleasant than that produced from randomized signals. Moreover, the waveform can be analyzed to predict phenotypic properties, such as differential gene expression. PMID:27019695

  13. Chromosome Condensation in the Absence of the Non-SMC Subunits of MukBEF†

    PubMed Central

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

    2006-01-01

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

  14. Condensation Processes in Geothermal Systems

    NASA Astrophysics Data System (ADS)

    Norman, D. I.; Moore, J. N.

    2005-12-01

    We model condensation processes in geothermal systems to understand how this process changes fluid chemistry. We assume two processes operate in geothermal systems: 1) condensation of a vapor phase derived by boiling an aqueous geothermal fluid into a cool near surface water and 2) condensation of a magmatic vapor by a deep circulating meteoric thermal fluid. It is assumed that the condensation process has two stages. Initially the condensing fluid is under saturated in gaseous species. Condensation of the vapor phase continues until the pressure on the fluid equals the sum of the partial pressures of water and the dissolved gaseous species. At that time bubbles flux through the condensing fluid. In time the fluid and fluxing gas phase come to equilibrium. Calculation shows that during the second stage of the condensation process the liquid phase becomes enriched in more soluble gaseous species like CO2 and H2S, and depleted in less soluble species like CH4 and N2. Stage 2 condensation processes can therefore be monitored by ratios of more and less condensable species like CO2/N2. Condensation of vapor released by boiling geothermal fluids results in liquids with high concentrations of H2S and CO2 like is seen in geothermal system steam-heated waters. Condensation of a magmatic vapor into circulating meteoric water has been proposed, but not well demonstrated. We compare to our models the Cerro Prieto, Mexico gas analysis data set collected over twelve years time by USGS personnel. It was assumed for modeling that the Cerro Prieto geothermal fluids are circulating meteoritic fluids with N2/Ar ratios about 40 to which is added a magmatic vapor with N2/Ar ratio = 400. The Cerro Prieto analyses show a strong correlation between N2/Ar and CO2/N2 as predicted by calculation. Two dimensional image plots of well N2/Ar + CO2/N2 show a bull's-eye pattern on the geothermal field. Image plots of analyses collected over a year or less time show N2/Ar and CO2/N2 hot spots. Plotting data for individual wells show a hysteresis like loops on time vs. CO2/N2 diagrams. Our analysis demonstrates that condensation of magmatic vapor into convecting meteoric waters is a viable process. Condensation explains variations in Cerro Prieto geothermal system gas chemistry and is compatible with helium isotope data. Locally condensation appears to wax and wane over a time periods of about 10 years.

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

    SciTech Connect

    Seitz, L.C.; Tang, Keliang; Cummings, W.J.; Zolan, M.E.

    1996-04-01

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

  16. Condenser for photolithography system

    DOEpatents

    Sweatt, William C.

    2004-03-02

    A condenser for a photolithography system, in which a mask image from a mask is projected onto a wafer through a camera having an entrance pupil, includes a source of propagating radiation, a first mirror illuminated by the radiation, a mirror array illuminated by the radiation reflected from said first mirror, and a second mirror illuminated by the radiation reflected from the array. The mirror array includes a plurality of micromirrors. Each of the micromirrors is selectively actuatable independently of each other. The first mirror and the second mirror are disposed such that the source is imaged onto a plane of the mask and the mirror array is imaged into the entrance pupil of the camera.

  17. Microgravity condensing heat exchanger

    NASA Technical Reports Server (NTRS)

    Thomas, Christopher M. (Inventor); Ma, Yonghui (Inventor); North, Andrew (Inventor); Weislogel, Mark M. (Inventor)

    2011-01-01

    A heat exchanger having a plurality of heat exchanging aluminum fins with hydrophilic condensing surfaces which are stacked and clamped between two cold plates. The cold plates are aligned radially along a plane extending through the axis of a cylindrical duct and hold the stacked and clamped portions of the heat exchanging fins along the axis of the cylindrical duct. The fins extend outwardly from the clamped portions along approximately radial planes. The spacing between fins is symmetric about the cold plates, and are somewhat more closely spaced as the angle they make with the cold plates approaches 90.degree.. Passageways extend through the fins between vertex spaces which provide capillary storage and communicate with passageways formed in the stacked and clamped portions of the fins, which communicate with water drains connected to a pump externally to the duct. Water with no entrained air is drawn from the capillary spaces.

  18. Intrinsic coupling of lagging-strand synthesis to chromatin assembly.

    PubMed

    Smith, Duncan J; Whitehouse, Iestyn

    2012-03-22

    Fifty per cent of the genome is discontinuously replicated on the lagging strand as Okazaki fragments. Eukaryotic Okazaki fragments remain poorly characterized and, because nucleosomes are rapidly deposited on nascent DNA, Okazaki fragment processing and nucleosome assembly potentially affect one another. Here we show that ligation-competent Okazaki fragments in Saccharomyces cerevisiae are sized according to the nucleosome repeat. Using deep sequencing, we demonstrate that ligation junctions preferentially occur near nucleosome midpoints rather than in internucleosomal linker regions. Disrupting chromatin assembly or lagging-strand polymerase processivity affects both the size and the distribution of Okazaki fragments, suggesting a role for nascent chromatin, assembled immediately after the passage of the replication fork, in the termination of Okazaki fragment synthesis. Our studies represent the first high-resolution analysis--to our knowledge--of eukaryotic Okazaki fragments in vivo, and reveal the interconnection between lagging-strand synthesis and chromatin assembly. PMID:22419157

  19. Identifying different types of chromatin using Giemsa staining.

    PubMed

    Stockert, Juan C; Blázquez-Castro, Alfonso; Horobin, Richard W

    2014-01-01

    Mixtures of polychrome methylene blue-eosin Y (i.e., Giemsa stain) are widely used in biological staining. They induce a striking purple coloration of chromatin DNA (the Romanowsky-Giemsa effect), which contrasts with the blue-stained RNA-containing cytoplasm and nucleoli. After specific prestaining treatments that induce chromatin disorganization (giving banded or harlequin chromosomes), Giemsa staining produces a differential coloration, with C- and G-bands appearing in purple whereas remaining chromosome regions are blue. Unsubstituted (TT) and bromo-substituted (BT) DNAs also appear purple and blue, respectively. The same occurs in the case of BT and BB chromatids.In addition to discussing the use of Giemsa stain as a suitable method to reveal specific features of chromosome structure, some molecular processes and models are also described to explain Giemsa staining mechanisms of chromatin. PMID:24162977

  20. Absence of canonical active chromatin marks in developmentally regulated genes

    PubMed Central

    Ruiz-Romero, Marina; Corominas, Montserrat; Guigó, Roderic

    2015-01-01

    The interplay of active and repressive histone modifications is assumed to play a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated to stable production of RNA, while unmarked chromatin would permit rapid gene activation and de-activation during development. In this case, regulation by transcription factors would play a comparatively more important regulatory role. PMID:26280901