WorldWideScience

Sample records for chromatin

  1. Reprogramming chromatin

    DEFF Research Database (Denmark)

    Ehrensberger, Andreas Hasso; Svejstrup, Jesper Qualmann

    2012-01-01

    attributed to high kinetic barriers that affect all cells equally and can only be overcome by rare stochastic events. The barriers to reprogramming are likely to involve transformations of chromatin state because (i) inhibitors of chromatin-modifying enzymes can enhance the efficiency of reprogramming...... and (ii) knockdown or knock-out of chromatin-modifying enzymes can lower the efficiency of reprogramming. Here, we review the relationship between chromatin state transformations (chromatin reprogramming) and cellular reprogramming, with an emphasis on transcription factors, chromatin remodeling factors...

  2. Chromatin Immunoprecipitation.

    Science.gov (United States)

    Wiehle, Laura; Breiling, Achim

    2016-01-01

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

  3. Chromatin is wonderful stuff.

    NARCIS (Netherlands)

    R. van Driel

    2007-01-01

    Chromatin molecules have properties that set them aside from all other biomacromolecules in the cell. (i) Chromosomes, which are single chromatin molecules, are the largest macromolecules in eukaryotic cells. (ii) Chromatin molecules carry the cell's genetic and epigenetic information and all contro

  4. Chromatin Structure and Function

    CERN Document Server

    Wolffe, Alan P

    1999-01-01

    The Third Edition of Chromatin: Structure and Function brings the reader up-to-date with the remarkable progress in chromatin research over the past three years. It has been extensively rewritten to cover new material on chromatin remodeling, histone modification, nuclear compartmentalization, DNA methylation, and transcriptional co-activators and co-repressors. The book is written in a clear and concise fashion, with 60 new illustrations. Chromatin: Structure and Function provides the reader with a concise and coherent account of the nature, structure, and assembly of chromatin and its active

  5. Where splicing joins chromatin

    OpenAIRE

    Hnilicová, Jarmila; Staněk, David

    2011-01-01

    There are numerous data suggesting that two key steps in gene expression—transcription and splicing influence each other closely. For a long time it was known that chromatin modifications regulate transcription, but only recently it was shown that chromatin and histone modifications play a significant role in pre-mRNA splicing. Here we summarize interactions between splicing machinery and chromatin and discuss their potential functional significance. We focus mainly on histone acetylation and...

  6. Prenucleosomes and Active Chromatin

    Science.gov (United States)

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

    2016-01-01

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

  7. Reprogramming the chromatin landscape

    DEFF Research Database (Denmark)

    Miranda, Tina B; Voss, Ty C; Sung, Myong-Hee;

    2013-01-01

    , mechanistic details defining the cellular interactions between ER and GR are poorly understood. We investigated genome-wide binding profiles for ER and GR upon coactivation and characterized the status of the chromatin landscape. We describe a novel mechanism dictating the molecular interplay between ER...... and GR. Upon induction, GR modulates access of ER to specific sites in the genome by reorganization of the chromatin configuration for these elements. Binding to these newly accessible sites occurs either by direct recognition of ER response elements or indirectly through interactions with other factors...

  8. Chromatin replication and epigenome maintenance

    DEFF Research Database (Denmark)

    Alabert, Constance; Groth, Anja

    2012-01-01

    initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA...

  9. Chromatin chemistry goes cellular.

    OpenAIRE

    W. Fischle; D. Schwarzer; Mootz, H.

    2015-01-01

    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.

  10. Chromatin assembly using Drosophila systems.

    Science.gov (United States)

    Fyodorov, Dmitry V; Levenstein, Mark E

    2002-05-01

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

  11. Proteomic interrogation of human chromatin.

    Directory of Open Access Journals (Sweden)

    Mariana P Torrente

    Full Text Available Chromatin proteins provide a scaffold for DNA packaging and a basis for epigenetic regulation and genomic maintenance. Despite understanding its functional roles, mapping the chromatin proteome (i.e. the "Chromatome" is still a continuing process. Here, we assess the biological specificity and proteomic extent of three distinct chromatin preparations by identifying proteins in selected chromatin-enriched fractions using mass spectrometry-based proteomics. These experiments allowed us to produce a chromatin catalog, including several proteins ranging from highly abundant histone proteins to less abundant members of different chromatin machinery complexes. Using a Normalized Spectral Abundance Factor approach, we quantified relative abundances of the proteins across the chromatin enriched fractions giving a glimpse into their chromosomal abundance. The large-scale data sets also allowed for the discovery of a variety of novel post-translational modifications on the identified chromatin proteins. With these comparisons, we find one of the probed methods to be qualitatively superior in specificity for chromatin proteins, but inferior in proteomic extent, evidencing a compromise that must be made between biological specificity and broadness of characterization. Additionally, we attempt to identify proteins in eu- and heterochromatin, verifying the enrichments by characterizing the post-translational modifications detected on histone proteins from these chromatin regions. In summary, our results provide insights into the value of different methods to extract chromatin-associated proteins and provide starting points to study the factors that may be involved in directing gene expression and other chromatin-related processes.

  12. Effects of fast neutrons on chromatin: dependence on chromatin structure

    Energy Technology Data Exchange (ETDEWEB)

    Radu, L. [Dept. of Molecular Genetics, V. Babes National Inst., Bd. Timisoara, Bucharest (Romania); Constantinescu, B. [Dept. of Cyclotron, H. Hulubei National Inst., Bucharest (Romania); Gazdaru, D. [Dept. of Biophysics, Physics Faculty, Univ. of Bucharest (Romania)

    2002-07-01

    The effects of fast neutrons (10-100 Gy) on chromatin extracted from normal (liver of Wistar rats) and tumor (Walker carcinosarcoma maintained on Wistar rats) tissues were compared. The spectroscopic assays used were (i) chromatin intrinsic fluorescence, (ii) time-resolved fluorescence of chromatin-proflavine complexes, and (iii) fluorescence resonance energy transfer (FRET) between dansyl chloride and acridine orange coupled to chromatin. For both normal and tumor chromatin, the intensity of intrinsic fluorescence specific for acidic and basic proteins decreased with increasing dose. The relative contributions of the excited-state lifetime of proflavine bound to chromatin were reduced upon fast-neutron irradiation, indicating a decrease in the proportion of chromatin DNA available for ligand binding. The Forster energy transfer efficiencies were also modified by irradiation. These effects were larger for chromatin from tumor tissue. In the range 0-100 Gy, fast neutrons induced alterations in DNA and acidic and basic proteins, as well as in global chromatin structure. The radiosensitivity of chromatin extracted from tumor tissue seems to be higher than that of chromatin extracted from normal tissue, probably because of its higher euchromatin (loose)-heterochromatin (compact) ratio. (author)

  13. Cas9 Functionally Opens Chromatin

    OpenAIRE

    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.

  14. Cas9 Functionally Opens Chromatin

    OpenAIRE

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

    2015-01-01

    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.

  15. Cas9 Functionally Opens Chromatin.

    Directory of Open Access Journals (Sweden)

    Amira A Barkal

    Full Text Available 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.

  16. Chromatin Flavors: Chromatin composition and domain organization in Drosophila melanogaster

    NARCIS (Netherlands)

    J.G. van Bemmel (Joke)

    2012-01-01

    textabstractChromatin was originally identified by W. Flemming in 1882 as not much more than the stainable substance of the cell nucleus. Flemming named this substance according to the Greek word “chroma”, meaning color. In 1911 chromatin was characterized as proteins, named histones, that were atta

  17. Epigenetics & chromatin: Interactions and processes

    NARCIS (Netherlands)

    S. Henikoff (Steven); F.G. Grosveld (Frank)

    2013-01-01

    textabstractOn 11 to 13 March 2013, BioMed Central will be hosting its inaugural conference, Epigenetics & Chromatin: Interactions and Processes, at Harvard Medical School, Cambridge, MA, USA. Epigenetics & Chromatin has now launched a special article series based on the general themes of the confer

  18. Chromatin, epigenetics and stem cells.

    Science.gov (United States)

    Roloff, Tim C; Nuber, Ulrike A

    2005-03-01

    Epigenetics is a term that has changed its meaning with the increasing biological knowledge on developmental processes. However, its current application to stem cell biology is often imprecise and is conceptually problematic. This article addresses two different subjects, the definition of epigenetics and chromatin states of stem and differentiated cells. We describe mechanisms that regulate chromatin changes and provide an overview of chromatin states of stem and differentiated cells. Moreover, a modification of the current epigenetics definition is proposed that is not restricted by the heritability of gene expression throughout cell divisions and excludes translational gene expression control. PMID:15819395

  19. Vernalization-mediated chromatin changes.

    Science.gov (United States)

    Zografos, Brett R; Sung, Sibum

    2012-07-01

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

  20. Brain Function and Chromatin Plasticity

    OpenAIRE

    Dulac, Catherine

    2010-01-01

    The characteristics of epigenetic control, including the potential for long-lasting, stable effects on gene expression that outlive an initial transient signal, could be of singular importance for post-mitotic neurons, which are subject to changes with short- to long-lasting influence on their activity and connectivity. Persistent changes in chromatin structure are thought to contribute to mechanisms of epigenetic inheritance. Recent advances in chromatin biology offer new avenues to investig...

  1. Single Molecule Studies of Chromatin

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-02-06

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

  2. Chromatin remodelers and their roles in chromatin organization

    OpenAIRE

    Strålfors, Annelie

    2012-01-01

    The DNA in the eukaryotic nucleus is organized into a complex DNA-protein structure called chromatin. The basic repeating unit of chromatin is the nucleosome, which consists of 147 bp of DNA wrapped around a histone protein octamer. The nucleosomes form a “beads on a string” structure, which can be folded into higherorder structures that allow an extensive degree of DNA compaction. This compaction is so effective that 2 meters of DNA can fit into the human cell nucleus with a ...

  3. Guarding against Collateral Damage during Chromatin Transactions

    DEFF Research Database (Denmark)

    Altmeyer, Matthias; Lukas, Jiri

    2013-01-01

    Signal amplifications are vital for chromatin function, yet they also bear the risk of transforming into unrestrained, self-escalating, and potentially harmful responses. Examples of inbuilt limitations are emerging, revealing how chromatin transactions are confined within physiological boundaries....

  4. Chromatin structure regulates gene conversion.

    Directory of Open Access Journals (Sweden)

    W Jason Cummings

    2007-10-01

    Full Text Available Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors. We found that the immunoglobulin Vlambda pseudogene array is characterized by histone modifications associated with active chromatin. We directly demonstrated the importance of chromatin structure for gene conversion, using a regulatable experimental system in which the heterochromatin protein HP1 (Drosophila melanogaster Su[var]205, expressed as a fusion to Escherichia coli lactose repressor, is tethered to polymerized lactose operators integrated within the pseudo-Vlambda donor array. Tethered HP1 diminished histone acetylation within the pseudo-Vlambda array, and altered the outcome of Vlambda diversification, so that nontemplated mutations rather than templated mutations predominated. Thus, chromatin structure regulates homology-directed repair. These results suggest that histone modifications may contribute to maintaining genomic stability by preventing recombination between repetitive sequences.

  5. Predicting chromatin organization using histone marks

    OpenAIRE

    Huang, Jialiang; Marco, Eugenio; Pinello, Luca; Yuan, Guo-Cheng

    2015-01-01

    Genome-wide mapping of three dimensional chromatin organization is an important yet technically challenging task. To aid experimental effort and to understand the determinants of long-range chromatin interactions, we have developed a computational model integrating Hi-C and histone mark ChIP-seq data to predict two important features of chromatin organization: chromatin interaction hubs and topologically associated domain (TAD) boundaries. Our model accurately and robustly predicts these feat...

  6. Impact of Chromatin on HIV Replication

    OpenAIRE

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

    2015-01-01

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

  7. Chromatin Dynamics of Circadian Transcription

    OpenAIRE

    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. Intrigui...

  8. Proteomics of a fuzzy organelle: interphase chromatin

    Science.gov (United States)

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

    2014-01-01

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

  9. Chromatin Modification and Remodeling in Heart Development

    Directory of Open Access Journals (Sweden)

    Paul Delgado-Olguín

    2006-01-01

    Full Text Available In organogenesis, cell types are specified from determined precursors as morphogenetic patterning takes place. These events are largely controlled by tissue-specific transcription factors. These transcription factors must function within the context of chromatin to activate or repress target genes. Recent evidence suggests that chromatin-remodeling and -modifying factors may have tissue-specific function. Here we review the potential roles for chromatin-remodeling and -modifying proteins in the development of the mammalian heart.

  10. Transcriptional networks and chromatin remodeling controlling adipogenesis

    DEFF Research Database (Denmark)

    Siersbæk, Rasmus; Nielsen, Ronni; Mandrup, Susanne

    2012-01-01

    Adipocyte differentiation is tightly controlled by a transcriptional cascade, which directs the extensive reprogramming of gene expression required to convert fibroblast-like precursor cells into mature lipid-laden adipocytes. Recent global analyses of transcription factor binding and chromatin...... remodeling have revealed 'snapshots' of this cascade and the chromatin landscape at specific time-points of differentiation. These studies demonstrate that multiple adipogenic transcription factors co-occupy hotspots characterized by an open chromatin structure and specific epigenetic modifications....... Such transcription factor hotspots are likely to represent key signaling nodes which integrate multiple adipogenic signals at specific chromatin sites, thereby facilitating coordinated action on gene expression....

  11. The architects of crenarchaeal chromatin : A biophysical characterization of chromatin proteins from Sulfolobus solfataricus

    NARCIS (Netherlands)

    Driessen, Rosalie Paula Catharina

    2014-01-01

    Understanding of chromatin organization and compaction in Archaea is currently limited. The genome of several megabasepairs long is folded by a set of small chromatin proteins to fit into the micron-sized cell. A first step in understanding archaeal chromatin organization is to study the action of i

  12. Computational strategies to address chromatin structure problems.

    Science.gov (United States)

    Perišić, Ognjen; Schlick, Tamar

    2016-01-01

    While the genetic information is contained in double helical DNA, gene expression is a complex multilevel process that involves various functional units, from nucleosomes to fully formed chromatin fibers accompanied by a host of various chromatin binding enzymes. The chromatin fiber is a polymer composed of histone protein complexes upon which DNA wraps, like yarn upon many spools. The nature of chromatin structure has been an open question since the beginning of modern molecular biology. Many experiments have shown that the chromatin fiber is a highly dynamic entity with pronounced structural diversity that includes properties of idealized zig-zag and solenoid models, as well as other motifs. This diversity can produce a high packing ratio and thus inhibit access to a majority of the wound DNA. Despite much research, chromatin's dynamic structure has not yet been fully described. Long stretches of chromatin fibers exhibit puzzling dynamic behavior that requires interpretation in the light of gene expression patterns in various tissue and organisms. The properties of chromatin fiber can be investigated with experimental techniques, like in vitro biochemistry, in vivo imagining, and high-throughput chromosome capture technology. Those techniques provide useful insights into the fiber's structure and dynamics, but they are limited in resolution and scope, especially regarding compact fibers and chromosomes in the cellular milieu. Complementary but specialized modeling techniques are needed to handle large floppy polymers such as the chromatin fiber. In this review, we discuss current approaches in the chromatin structure field with an emphasis on modeling, such as molecular dynamics and coarse-grained computational approaches. Combinations of these computational techniques complement experiments and address many relevant biological problems, as we will illustrate with special focus on epigenetic modulation of chromatin structure. PMID:27345617

  13. A microscopic analysis of Arabidopsis chromatin

    NARCIS (Netherlands)

    Willemse, J.J.

    2007-01-01

    Genetic information of eukaryotic organisms is stored as DNA in the nuclei of their cells. Nuclear DNA is associated with several proteins, which together form chromatin. The most abundant chromatin proteins arehistones,they arrange the initial packaging step of the DNA. DNA

  14. Expression-dependent folding of interphase chromatin.

    Directory of Open Access Journals (Sweden)

    Hansjoerg Jerabek

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

  15. Chromatin dynamics resolved with force spectroscopy

    NARCIS (Netherlands)

    Chien, Fan-Tso

    2011-01-01

    In eukaryotic cells, genomic DNA is organized in chromatin fibers composed of nucleosomes as structural units. A nucleosome contains 1.7 turns of DNA wrapped around a histone octamer and is connected to the adjacent nucleosomes with linker DNA. The folding of chromatin fibers effectively increases t

  16. Chromatin challenges during DNA replication and repair

    DEFF Research Database (Denmark)

    Groth, Anja; Rocha, Walter; Verreault, Alain;

    2007-01-01

    the challenge of maintenance, cells have evolved efficient nucleosome-assembly pathways and chromatin-maturation mechanisms that reproduce chromatin organization in the wake of DNA replication and repair. The aim of this Review is to describe how these pathways operate and to highlight how the epigenetic...

  17. Chromatin Remodelers: From Function to Dysfunction

    Directory of Open Access Journals (Sweden)

    Gernot Längst

    2015-06-01

    Full Text Available 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.

  18. Chromatin-modifying proteins in cancer

    DEFF Research Database (Denmark)

    Fog, Cathrine K; Jensen, Klaus T; Lund, Anders Henrik

    2007-01-01

    -despite the fact that all cells in the organism contain the same genetic information. A large amount of data gathered over the last decades has demonstrated that deregulation of chromatin-modifying proteins is etiologically involved in the development and progression of cancer. Here we discuss how epigenetic...... alterations influence cancer development and review known cancer-associated alterations in chromatin-modifying proteins....

  19. A Long-Distance Chromatin Affair

    NARCIS (Netherlands)

    Denker, Annette; de Laat, Wouter

    2015-01-01

    Changes in transcription factor binding sequences result in correlated changes in chromatin composition locally and at sites hundreds of kilobases away. New studies demonstrate that this concordance is mediated via spatial chromatin interactions that constitute regulatory modules of the human genome

  20. Chromatin domain boundaries: insulators and beyond

    Institute of Scientific and Technical Information of China (English)

    Gong Hong WEI; De Pei LIU; Chih Chuan LIANG

    2005-01-01

    The eukaryotic genome is organized into functionally and structurally distinct domains, representing regulatory units for gene expression and chromosome behavior. DNA sequences that mark the border between adjacent domains are the insulators or boundary elements, which are required in maintenance of the function of different domains. Some insulators need others enable to play insulation activity. Chromatin domains are defined by distinct sets of post-translationally modified histones. Recent studies show that these histone modifications are also involved in establishment of sharp chromatin boundaries in order to prevent the spreading of distinct domains. Additionally, in some loci, the high-order chromatin structures for long-range looping interactions also have boundary activities, suggesting a correlation between insulators and chromatin loop domains. In this review, we will discuss recent progress in the field of chromatin domain boundaries.

  1. Computational strategies to address chromatin structure problems

    Science.gov (United States)

    Perišić, Ognjen; Schlick, Tamar

    2016-06-01

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

  2. Extensive Variation in Chromatin States Across Humans

    KAUST Repository

    Kasowski, M.

    2013-10-17

    The majority of disease-associated variants lie outside protein-coding regions, suggesting a link between variation in regulatory regions and disease predisposition. We studied differences in chromatin states using five histone modifications, cohesin, and CTCF in lymphoblastoid lines from 19 individuals of diverse ancestry. We found extensive signal variation in regulatory regions, which often switch between active and repressed states across individuals. Enhancer activity is particularly diverse among individuals, whereas gene expression remains relatively stable. Chromatin variability shows genetic inheritance in trios, correlates with genetic variation and population divergence, and is associated with disruptions of transcription factor binding motifs. Overall, our results provide insights into chromatin variation among humans.

  3. Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

    KAUST Repository

    Bigeard, Jean

    2014-07-10

    In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

  4. Chromatin Domains: The Unit of Chromosome Organization.

    Science.gov (United States)

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

    2016-06-01

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

  5. Chromatin proteins and modifications as drug targets

    DEFF Research Database (Denmark)

    Helin, Kristian; Dhanak, Dashyant

    2013-01-01

    A plethora of groundbreaking studies have demonstrated the importance of chromatin-associated proteins and post-translational modifications of histones, proteins and DNA (so-called epigenetic modifications) for transcriptional control and normal development. Disruption of epigenetic control...

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

    Science.gov (United States)

    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

  7. The Chromatin Fiber: Multiscale Problems and Approaches

    OpenAIRE

    Ozer, Gungor; Luque, Antoni; Schlick, Tamar

    2015-01-01

    The structure of chromatin, affected by many factors from DNA linker lengths to posttranslational modifications, is crucial to the regulation of eukaryotic cells. Combined experimental and computational methods have led to new insights into its structural and dynamical features, from interactions due to the flexible core histone tails of the nucleosomes to the physical mechanism driving the formation of chromosomal domains. Here we present a perspective of recent advances in chromatin modelin...

  8. Etiology and Evaluation of Sperm Chromatin Anomalies

    Directory of Open Access Journals (Sweden)

    Marziyeh Tavalaee

    2008-01-01

    Full Text Available Evidence suggests that human sperm chromatin anomalies adversely affect reproductive outcomesand infertile men possess substantially amount of sperm with chromatin anomalies than fertilemen.Routine semen analysis evaluates parameters such as sperm motility and morphology, but doesnot examine the nuclear DNA integrity of spermatozoa. It has been suggested that altered nuclearchromatin structure or damaged DNA in spermatozoa could modify the special cellular functionsof human spermatozoa, and thereby affect the fertility potential. Intra-cytoplasmic sperm injection(ICSI bypass the barriers to fertilization for such a sperm, then the effect of chromatin anomalies onthe development remains a concern. Therefore, it is essential to develop and use accurate diagnostictests, which may provide better prognostic capabilities than the standard sperm assessments. Thisreview discusses our current understanding of the structure and organization of sperm DNA,the different procedures for assessment of sperm chromatin anomalies including comet assay,Chromomycin A3 (CMA3, sperm chromatin structure assay (SCSA, acridine orange test (AOT,terminal TdT-mediated dUTP-nick-end labelling (TUNEL assay, aniline blue and sperm chromatindispersion (SCD test and the impact of chromatin anomalies on reproductive outcome.

  9. New mitotic regulators released from chromatin

    Directory of Open Access Journals (Sweden)

    Hideki eYokoyama

    2013-12-01

    Full Text Available Faithful action of the mitotic spindle segregates duplicated chromosomes into daughter cells. Perturbations of this process result in chromosome mis-segregation, leading to chromosomal instability and cancer development. Chromosomes are not simply passengers segregated by spindle microtubules but rather play a major active role in spindle assembly. The GTP bound form of the Ran GTPase (RanGTP, produced around chromosomes, locally activates spindle assembly factors. Recent studies have uncovered that chromosomes organize mitosis beyond spindle formation. They distinctly regulate other mitotic events, such as spindle maintenance in anaphase, which is essential for chromosome segregation. Furthermore, the direct function of chromosomes is not only to produce RanGTP but, in addition, to release key mitotic regulators from chromatin. Chromatin-remodeling factors and nuclear pore complex proteins, which have established functions on chromatin in interphase, dissociate from mitotic chromatin and function in spindle assembly or maintenance. Thus, chromosomes actively organize their own segregation using chromatin-releasing mitotic regulators as well as RanGTP.

  10. Chromatin associations in Arabidopsis interphase nuclei

    Directory of Open Access Journals (Sweden)

    Veit eSchubert

    2014-11-01

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

  11. Neutron-scattering studies of chromatin

    International Nuclear Information System (INIS)

    It is clear that a knowledge of the basic molecular structure of chromatin is a prerequisite for any progress toward an understanding of chromosome organization. With a two-component system, protein and nucleic acid, neutrons have a particularly powerful application to studies of the spatial arrangements of these components because of the ability, by contrast matching with H2O-D2O mixtures, to obtain neutron-scattering data on the individual components. With this approach it has been shown that the neutron diffraction of chromatin is consistent with a ''beads on a string'' model in which the bead consists of a protein core with DNA coiled on the outside. However, because chromatin is a gel and gives limited structural data, confirmation of such a model requires extension of the neutron studies by deuteration of specific chromatin components and the isolation of chromatin subunits. Although these studies are not complete, the neutron results so far obtained support the subunit model described above

  12. Chromatin ring formation at plant centromeres

    Directory of Open Access Journals (Sweden)

    Veit eSchubert

    2016-02-01

    Full Text Available 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.

  13. Nucleosome dynamics during chromatin remodeling in vivo.

    Science.gov (United States)

    Ramachandran, Srinivas; Henikoff, Steven

    2016-01-01

    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

  14. Functions of the Proteasome on Chromatin

    Science.gov (United States)

    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

  15. Functions of the Proteasome on Chromatin

    Directory of Open Access Journals (Sweden)

    Tyler S. McCann

    2014-11-01

    Full Text Available 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.

  16. CHD chromatin remodelers and the transcription cycle.

    Science.gov (United States)

    Murawska, Magdalena; Brehm, Alexander

    2011-01-01

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

  17. Local Nucleosome Dynamics Facilitate Chromatin Accessibility in Living Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Saera Hihara

    2012-12-01

    Full Text Available Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (∼50 nm movement/30 ms, which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.

  18. CTCF Binding Polarity Determines Chromatin Looping

    NARCIS (Netherlands)

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

    2015-01-01

    CCCTC-binding factor (CTCF) is an architectural protein involved in the three-dimensional (3D) organization of chromatin. In this study, we assayed the 3D genomic contact profiles of a large number of CTCF binding sites with high-resolution 4C-seq. As recently reported, our data also suggest that ch

  19. Unraveling the mechanisms of chromatin fibril packaging.

    Science.gov (United States)

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

    2016-05-01

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

  20. Chromatin and epigenetics in all their states

    NARCIS (Netherlands)

    Bey, Till; Jamge, Suraj; Klemme, Sonja; Komar, Dorota Natalia; Gall, Le Sabine; Mikulski, Pawel; Schmidt, Martin; Zicola, Johan; Berr, Alexandre

    2016-01-01

    In January 2016, the first Epigenetic and Chromatin Regulation of Plant Traits conference was held in Strasbourg, France. An all-star lineup of speakers, a packed audience of 130 participants from over 20 countries, and a friendly scientific atmosphere contributed to make this conference a meetin

  1. Single Chromatin Fibre Assembly Using Optical Tweezers

    NARCIS (Netherlands)

    Bennink, M.L.; Pope, L.H.; Leuba, S.H.; Grooth, de B.G.; Greve, J.

    2001-01-01

    Here we observe the formation of a single chromatin fibre using optical tweezers. A single -DNA molecule was suspended between two micron-sized beads, one held by a micropipette and the other in an optical trap. The constrained DNA molecule was incubated with Xenopus laevis egg extract in order to r

  2. Research Discovers Frequent Mutations of Chromatin

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    With the support of National Natural Science Foundation of China, BGI, the largest genomics organization in the world, and Peking University Shenzhen Hospital, published online in Nature Geneticsics that the study on frequent mutations of chromatin remodeling genes in transitional cell carcinoma (TCC) of thebladder on August 8th, 2011. Their study provides a valuable genetic basis for future studies on TCC,

  3. Epigenetic chromatin silencing: bistability and front propagation

    Science.gov (United States)

    Sedighi, Mohammad; Sengupta, Anirvan M.

    2007-12-01

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

  4. Impact of chromatin structure on PR signaling

    DEFF Research Database (Denmark)

    Grøntved, Lars; Hager, Gordon L

    2012-01-01

    but also to the glucocorticoid receptor (GR), as these receptors share many similarities regarding interaction with, and remodeling of, chromatin. Both receptors can bind nucleosomal DNA and have accordingly been described as pioneering factors. However recent genomic approaches (ChIP-seq and DHS-seq) show...

  5. Histone variants: key players of chromatin.

    Science.gov (United States)

    Biterge, Burcu; Schneider, Robert

    2014-06-01

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

  6. The great repression: chromatin and cryptic transcription.

    Science.gov (United States)

    Hennig, Bianca P; Fischer, Tamás

    2013-01-01

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

  7. The Chromatin Scaffold Protein SAFB1 Renders Chromatin Permissive for DNA Damage Signaling

    DEFF Research Database (Denmark)

    Altmeyer, Matthias; Toledo Lazaro, Luis Ignacio; Gudjonsson, Thorkell;

    2013-01-01

    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...

  8. Chromatin Dynamics of the mouse β-globin locus

    NARCIS (Netherlands)

    M.P.C. van de Corput (Mariëtte); E. de Boer (Ernie); T.A. Knoch (Tobias); W.A. van Cappellen (Gert); M. Lesnussa (Michael); H.J.F.M.M. Eussen (Bert)

    2010-01-01

    textabstractLately it has become more clear that (subtle) changes in 3D organization of chromatin can either trigger transcription or silence genes or gene clusters. It has also been postulated that due to changes in chromatin structure, a change in chromatin accessibility of transcription factors

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2014-04-01

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

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

    Science.gov (United States)

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

    2016-06-30

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

  12. Replicating chromatin: a tale of histones

    DEFF Research Database (Denmark)

    Groth, Anja

    2009-01-01

    Chromatin serves structural and functional roles crucial for genome stability and correct gene expression. This organization must be reproduced on daughter strands during replication to maintain proper overlay of epigenetic fabric onto genetic sequence. Nucleosomes constitute the structural...... reassembly on nascent DNA strands. The aim of this review is to discuss how histones - new and old - are handled at the replication fork, highlighting new mechanistic insights and revisiting old paradigms....

  13. Keystone Symposia on Epigenomics and Chromatin Dynamics

    DEFF Research Database (Denmark)

    Ravnskjær, Kim

    2012-01-01

    Keystone Symposia kicked off the start of 2012 with two joint meetings on Epigenomics and Chromatin Dynamics and a star-studded list of speakers. Held in Keystone, CO, January 17-22, and organized by Steven Jacobsen and Steven Henikoff and by Bradley Cairns and Geneviève Almouzni, respectively, t......, there was plenty happening in these sessions that it did not seem to matter that the ski-slope conditions were not ideal....

  14. Identification of alternative topological domains in chromatin

    OpenAIRE

    Filippova, Darya; Patro, Rob; Duggal, Geet; Kingsford, Carl

    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 r...

  15. Multiscale Identification of Topological Domains in Chromatin

    OpenAIRE

    Filippova, Darya; Patro, Rob; Duggal, Geet; Kingsford, Carl

    2013-01-01

    Recent 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 va...

  16. Chromatin regulation in drug addiction and depression

    OpenAIRE

    Renthal, William; Nestler, Eric J.

    2009-01-01

    Alterations in gene expression are implicated in the pathogenesis of several neuropsychiatrie disorders, including drug addiction and depression, increasing evidence indicates that changes in gene expression in neurons, in the context of animal models of addiction and depression, are mediated in part by epigenetic mechanisms that alter chromatin structure on specific gene promoters. This review discusses recent findings from behavioral, molecular, and bioinformatic approaches that are being u...

  17. Titration and hysteresis in epigenetic chromatin silencing

    International Nuclear Information System (INIS)

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

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

    KAUST Repository

    Jégu, Teddy

    2015-10-12

    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.

  19. Spectroscopic study of fast-neutron-irradiated chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Radu, L. [V. Babes National Inst., Dept. of Molecular Genetics, Bucharest (Romania)]. E-mail: serbanradu@pcnet.ro; Gazdaru, D. [Bucharest Univ., Dept. of Biophysics, Physics Faculty, Bucharest (Romania); Constantinescu, B. [H. Hulubei National Inst., Dept. of Cyclotron, Bucharest (Romania)

    2004-02-01

    The effects produced by fast neutrons (0-100 Gy) on chromatin structure were analyzed by (i) [{sup 1}H]-NMR spectroscopy, (ii) time resolved spectroscopy, and (iii) fluorescence resonance energy transfer (FRET). Two types of chromatin were tested: (i) a chromatin from a normal tissue (liver of Wistar rats) and (ii) a chromatin from a tumoral tissue (Guerin limphotrope epithelioma, a rat solid tumor). The fast-neutron action on chromatin determines greater values of the [{sup 1}H]-NMR transverse relaxation time, indicating a more injured structure. Time-resolved fluorescence measurements show that the relative contribution of the excited state lifetime of bound ethidium bromide to chromatin DNA diminishes with increasing irradiation doses. This reflects the damage that occurs in DNA structure: production of single- and double-strand breaks due to sugar and base modifications. By the FRET method, the distance between dansyl chloride and acridine orange coupled at chromatin was determined. This distance increases upon fast-neutron action. The radiosensitivity of the tumor tissue chromatin seems higher than that of the normal tissue chromatin, probably because of its higher (loose) euchromatin/(compact) heterochromatin ratio. As the values of the physical parameters analyzed are specific for a determined dose, the establishment of these parameters may constitute a criterion for the microdosimetry of chromatin radiolesions produced by fast neutrons. (author)

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

    Directory of Open Access Journals (Sweden)

    Justin W Walley

    2008-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Mariliis Tark-Dame

    2014-10-01

    Full Text Available 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.

  2. Prevalence of X-chromatin in Jordanian women

    International Nuclear Information System (INIS)

    This study was conducted to evaluate the distribution of X-chromatin among Jordanian women at different age groups. Results will be compared with other studies for possible racial and environmental effects on X-chromatin distribution. Blood samples were drawn from all women subjected to this study by finger prick and stained with Wright's stain. X-chromatin positive polymorphonuclear cells were counted and corrected for percentage. Samples were taken during the late 2002 and early 2003 from healthy women attending routine checkup in health centers in Northern Jordan. The number of X-chromatin was highest in the 50 and above years age group. The number of X-chromatin was 14-18% in other age groups. These results were in accordance with other studies. It seems that racial and environmental factors are ineffective on distribution of X-chromatin in Jordanian women. These data could be used as as reference for further studies. (author)

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

    Science.gov (United States)

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

    2016-02-01

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

  4. Inverstigation of chromatin folding patterns by atomic force microscopy

    Institute of Scientific and Technical Information of China (English)

    ZHANGYi; OUYANGZhenqian; 等

    1999-01-01

    The chromatin folding patterns in air and liquid were studied by atomic force microscopy(AFM),A gentle water-air interface method was adopted to spread chromatin from interphase nucleus of chicken erythrocyte.The chromatin was absorbed on APS-mica surface and studied with AFM,Beads-on a-string were observed and many higher-order structrues such as superbeads with dimensions 40-60nm in diameter and 4-7nm in height were found to string together to make chromation fibers.When sample spreading and absorbing time were shortened.higher-order chromatin fibers with 60-120nm in width were observed in air as well as under water environment.These chromatin structures may reflect chromatin folding patterns in the living cells.

  5. Long Noncoding RNAs, Chromatin, and Development

    Directory of Open Access Journals (Sweden)

    Daniel P. Caley

    2010-01-01

    Full Text Available The way in which the genome of a multicellular organism can orchestrate the differentiation of trillions of cells and many organs, all from a single fertilized egg, is the subject of intense study. Different cell types can be defined by the networks of genes they express. This differential expression is regulated at the epigenetic level by chromatin modifications, such as DNA and histone methylation, which interact with structural and enzymatic proteins, resulting in the activation or silencing of any given gene. While detailed mechanisms are emerging on the role of different chromatin modifications and how these functions are effected at the molecular level, it is still unclear how their deposition across the epigenomic landscape is regulated in different cells. A raft of recent evidence is accumulating that implicates long noncoding RNAs (lncRNAs in these processes. Most genomes studied to date undergo widespread transcription, the majority of which is not translated into proteins. In this review, we will describe recent work suggesting that lncRNAs are more than transcriptional "noise", but instead play a functional role by acting as tethers and guides to bind proteins responsible for modifying chromatin and mediating their deposition at specific genomic locations. We suggest that lncRNAs are at the heart of developmental regulation, determining the epigenetic status and transcriptional network in any given cell type, and that they provide a means to integrate external differentiation cues with dynamic nuclear responses through the regulation of a metastable epigenome. Better characterization of the lncRNA-protein "interactome" may eventually lead to a new molecular toolkit, allowing researchers and clinicians to modulate the genome at the epigenetic level to treat conditions such as cancer.

  6. Chromatin remodeling regulated by steroid and nuclear receptors

    Institute of Scientific and Technical Information of China (English)

    1997-01-01

    Coactivators and corepressors regulate transcription by controlling interactions between sequence-specific transcription factors,the basal transcriptional machinery and the chromatin environment,This review consider the access of nuclear and steroid receptors to chromatin,their use of corepressors and coactivators to modify chromatin structure and the implications for transcriptional control.The assembly of specific nucleoprotein architectures and targeted histone modification emerge as central controlling elements for gene expression.

  7. Combinatorial epigenetic patterns as quantitative predictors of chromatin biology

    OpenAIRE

    Cieślik, Marcin; Bekiranov, Stefan

    2014-01-01

    Background Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is the most widely used method for characterizing the epigenetic states of chromatin on a genomic scale. With the recent availability of large genome-wide data sets, often comprising several epigenetic marks, novel approaches are required to explore functionally relevant interactions between histone modifications. Computational discovery of "chromatin states" defined by such combinatorial interactions enabled desc...

  8. Hydrogen peroxide mediates higher order chromatin degradation.

    Science.gov (United States)

    Bai, H; Konat, G W

    2003-01-01

    Although a large body of evidence supports a causative link between oxidative stress and neurodegeneration, the mechanisms are still elusive. We have recently demonstrated that hydrogen peroxide (H(2)O(2)), the major mediator of oxidative stress triggers higher order chromatin degradation (HOCD), i.e. excision of chromatin loops at the matrix attachment regions (MARs). The present study was designed to determine the specificity of H(2)O(2) in respect to HOCD induction. Rat glioma C6 cells were exposed to H(2)O(2) and other oxidants, and the fragmentation of genomic DNA was assessed by field inversion gel electrophoresis (FIGE). S1 digestion before FIGE was used to detect single strand fragmentation. The exposure of C6 cells to H(2)O(2) induced a rapid and extensive HOCD. Thus, within 30 min, total chromatin was single strandedly digested into 50 kb fragments. Evident HOCD was elicited by H(2)O(2) at concentrations as low as 5 micro M. HOCD was mostly reversible during 4-8h following the removal of H(2)O(2) from the medium indicating an efficient relegation of the chromatin fragments. No HOCD was induced by H(2)O(2) in isolated nuclei indicating that HOCD-endonuclease is activated indirectly by cytoplasmic signal pathways triggered by H(2)O(2). The exposure of cells to a synthetic peroxide, i.e. tert-butyrylhydroperoxide (tBH) also induced HOCD, but to a lesser extent than H(2)O(2). Contrary to the peroxides, the exposure of cells to equitoxic concentration of hypochlorite and spermine NONOate, a nitric oxide generator, failed to induce rapid HOCD. These results indicate that rapid HOCD is not a result of oxidative stress per se, but is rather triggered by signaling cascades initiated specifically by H(2)O(2). Furthermore, the rapid and extensive HOCD was observed in several rat and human cell lines challenged with H(2)O(2), indicating that the process is not restricted to glial cells, but rather represents a general response of cells to H(2)O(2). PMID:12421592

  9. Genome-wide Association of Yorkie with Chromatin and Chromatin-Remodeling Complexes

    Directory of Open Access Journals (Sweden)

    Hyangyee Oh

    2013-02-01

    Full Text Available The Hippo pathway regulates growth through the transcriptional coactivator 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. Coimmunoprecipitation and mass spectrometry identify GAGA factor (GAF, the Brahma complex, and the 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 messenger RNA 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.

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

    Science.gov (United States)

    Bawa-Khalfe, Tasneem

    2016-01-01

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

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

    Science.gov (United States)

    Jafari, Mahvash; Nateghi, M; Rabbani, A

    2010-01-01

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

  12. Organization of higher-level chromatin structures (chromomere, chromonema and chromatin block) examined using visible light-induced chromatin photo-stabilization.

    Science.gov (United States)

    Sheval, E V; Prusov, A N; Kireev, I I; Fais, D; Polyakov, V Yu

    2002-01-01

    The method of chromatin photo-stabilization by the action of visible light in the presence of ethidium bromide was used for investigation of higher-level chromatin structures in isolated nuclei. As a model we used rat hepatocyte nuclei isolated in buffers which stabilized or destabilized nuclear matrix. Several higher-level chromatin structures were visualized: 100nm globules-chromomeres, chains of chromomeres-chromonemata, aggregates of chromomeres-blocks of condensed chromatin. All these structures were completely destroyed by 2M NaCl extraction independent of the matrix state, and DNA was extruded from the residual nuclei (nuclear matrices) into a halo. These results show that nuclear matrix proteins do not play the main role in the maintenance of higher-level chromatin structures. Preliminary irradiation led to the reduction of the halo width in the dose-dependent manner. In regions of condensed chromatin of irradiated nucleoids there were discrete complexes consisting of DNA fibers radiating from an electron-dense core and resembling the decondensed chromomeres or the rosette-like structures. As shown by the analysis of proteins bound to irradiated nuclei upon high-salt extraction, irradiation presumably stabilized the non-histone proteins. These results suggest that in interphase nuclei loop domains are folded into discrete higher-level chromatin complexes (chromomeres). These complexes are possibly maintained by putative non-histone proteins, which are extracted with high-salt buffers from non-irradiated nuclei. PMID:12127937

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    to insulate genes from regulatory elements or to take part in long-distance interactions. Using a high-resolution chromatin conformation capture (H3C) method, we show that the Drosophila gypsy insulator behaves as a conformational chromatin border that is able to prohibit contacts between a Polycomb response...... element (PRE) and a distal promoter. On the other hand, two spaced gypsy elements form a chromatin loop that is able to bring an upstream PRE in contact with a downstream gene to mediate its repression. Chromatin immunoprecipitation (ChIP) profiles of the Polycomb protein and its associated H3K27me3...

  14. Lessons from Anaplasma phagocytophilum: Chromatin Remodeling by Bacterial Effectors

    OpenAIRE

    Rennoll-Bankert, Kristen E.; Dumler, J. Stephen

    2012-01-01

    Bacterial pathogens can alter global host gene expression via histone modifications and chromatin remodeling in order to subvert host responses, including those involved with innate immunity, allowing for bacterial survival. Shigella flexneri, Listeria monocytogenes, Chlamydia trachomatis, and Anaplasma phagocytophilum express effector proteins that modify host histones and chromatin structure. A. phagocytophilum modulates granulocyte respiratory burst in part by dampening transcription of se...

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

    Science.gov (United States)

    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 (Partificial insemination. PMID:27175169

  16. Rapid genome-scale mapping of chromatin accessibility in tissue

    DEFF Research Database (Denmark)

    Grøntved, Lars; Bandle, Russell; John, Sam;

    2012-01-01

    BACKGROUND: The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on la...

  17. A Broad Set of Chromatin Factors Influences Splicing

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-07-01

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

  19. Chromatin architecture and gene expression in Escherichia coli

    DEFF Research Database (Denmark)

    Willenbrock, Hanni; Ussery, David

    2004-01-01

    Two recent genome-scale analyses underscore the importance of DNA topology and chromatin structure in regulating transcription in Escherichia coli.......Two recent genome-scale analyses underscore the importance of DNA topology and chromatin structure in regulating transcription in Escherichia coli....

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

    Science.gov (United States)

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

    2003-10-24

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

  1. Genome maintenance in the context of 4D chromatin condensation.

    Science.gov (United States)

    Yu, Sonia; Yang, Fan; Shen, Wen H

    2016-08-01

    The eukaryotic genome is packaged in the three-dimensional nuclear space by forming loops, domains, and compartments in a hierarchical manner. However, when duplicated genomes prepare for segregation, mitotic cells eliminate topologically associating domains and abandon the compartmentalized structure. Alongside chromatin architecture reorganization during the transition from interphase to mitosis, cells halt most DNA-templated processes such as transcription and repair. The intrinsically condensed chromatin serves as a sophisticated signaling module subjected to selective relaxation for programmed genomic activities. To understand the elaborate genome-epigenome interplay during cell cycle progression, the steady three-dimensional genome requires a time scale to form a dynamic four-dimensional and a more comprehensive portrait. In this review, we will dissect the functions of critical chromatin architectural components in constructing and maintaining an orderly packaged chromatin environment. We will also highlight the importance of the spatially and temporally conscious orchestration of chromatin remodeling to ensure high-fidelity genetic transmission. PMID:27098512

  2. The AID-induced DNA damage response in chromatin

    DEFF Research Database (Denmark)

    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 in which AID-induced DNA damage is targeted and repaired. Outstanding questions remain regarding the direct roles...

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

    Science.gov (United States)

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

    2016-09-01

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

  4. On the mechanochemical machinery underlying chromatin remodeling

    Science.gov (United States)

    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.

  5. Chromatin structure near transcriptionally active genes

    International Nuclear Information System (INIS)

    Hypersensitive domains are the most prominent features of transcriptionally active chromatin. In the case of the β/sup A/-globin gene, it seems likely that two or more protein factors are capable of binding to the DNA so tightly that the nucleosome is prevented from binding. We have shown that nucleosomes, once bound in the assembly process in vitro, cannot be displaced. The interaction of the 5S gene transcription factor TFIIIA with its target DNA also is blocked by histones, and it has been suggested that the activation of the gene must occur during replication, before histones are reassembled on the DNA. We suppose that a similar mechanism may govern the binding of the hypersensitivity factors. It should be noted that nucleosomes are excluded not only from the sites to which the factors bind, but also from the regions between the two domains and at either side. 12 refs., 6 figs

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

    DEFF Research Database (Denmark)

    Alabert, Constance; Bukowski-Wills, Jimi-Carlo; Lee, Sung-Po;

    2014-01-01

    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...

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

    Science.gov (United States)

    Hirakawa, Takeshi; Matsunaga, Sachihiro

    2016-01-01

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

  8. Characteristics of thymine dimer excision from xeroderma pigmentosum chromatin

    International Nuclear Information System (INIS)

    We investigated thymine dimer excision from xeroderma pigmentosum (XP) chromatin in the cell-free reconstruction system. The normal-cell extract performed specific dimer excision from native chromatin and DNA isolated from 100 J/m2-irradiated cells. Such an excision in vitro was rapid and required high concentrations of extract. The extracts of XP group A, C and G cells were unable to excise from their own native-chromatin, but capable of excising from chromatin deprived of loosely bound nonhistone proteins with 0.35 M NaCl, as were from purified DNA. Thus, group A, C and G cells are most likely to be defective in the specific XP factors facilitating the excising activity under multicomponent regulation at the chromatin level. Further, either of group A, C and G extracts successfully complemented the native chromatin of the alternative groups. Uniquely, the XP group D extract excised dimers from native chromatin in the normal fashion under the condition. These results suggest that XP group A, C, D and G cells examined may not be defective in the dimer specific endonuclease and exonuclease per se. 19 references, 3 figures, 2 tables

  9. PREDICTION OF CHROMATIN STATES USING DNA SEQUENCE PROPERTIES

    KAUST Repository

    Bahabri, Rihab R.

    2013-06-01

    Activities of DNA are to a great extent controlled epigenetically through the internal struc- ture of chromatin. This structure is dynamic and is influenced by different modifications of histone proteins. Various combinations of epigenetic modification of histones pinpoint to different functional regions of the DNA determining the so-called chromatin states. How- ever, the characterization of chromatin states by the DNA sequence properties remains largely unknown. In this study we aim to explore whether DNA sequence patterns in the human genome can characterize different chromatin states. Using DNA sequence motifs we built binary classifiers for each chromatic state to eval- uate whether a given genomic sequence is a good candidate for belonging to a particular chromatin state. Of four classification algorithms (C4.5, Naive Bayes, Random Forest, and SVM) used for this purpose, the decision tree based classifiers (C4.5 and Random Forest) yielded best results among those we evaluated. Our results suggest that in general these models lack sufficient predictive power, although for four chromatin states (insulators, het- erochromatin, and two types of copy number variation) we found that presence of certain motifs in DNA sequences does imply an increased probability that such a sequence is one of these chromatin states.

  10. Anti-chromatin antibodies in juvenile rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    V. Gerloni

    2011-09-01

    Full Text Available Objective: to evaluate the prevalence and clinical significance of anti-chromatin antibodies (Abs in juvenile rheumatoid arthritis (JRA. Methods: IgG anti-chromatin Abs were detected by an enzyme-linked immunosorbent assay (ELISA, in sera of 94 children with JRA (10 children with systemic, 38 with polyarticular and 46 with oligoarticular disease onset. As control group, 33 age- and-sex-matched healthy children (HC were also examined. Results: Abs to chromatin were detected in 24/94 (25,5% of children suffering from JRA. Particularly, the higher prevalence of anti-chromatin Abs has been found in children with oligoarticular (30,4% and polyarticular (23,7% onset JRA. In these groups Abs titers were significantly higher compared to systemic JRA and HC (p=0.003. Anti-chromatin Abs were observed more frequently in patients with oligoarticular disease and chronic uveitis (21,7%. Furthermore, higher levels of anti-chromatin Abs has been found in all the patients treated with anti-TNFα therapy (p<0.0001. Conclusions: our results confirm previous data about the prevalence of anti-chromatin Abs in JRA. These Abs were significantly higher in the group of patients with oligoarticular onset with past or present hystory of ocular involvement and in the group with polyarticular JRA treated with biologic therapy. A long-term follow-up study could be useful to evaluate the potential utility of these autoantibodies.

  11. Fractal Characterization of Chromatin Decompaction in Live Cells.

    Science.gov (United States)

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

    2015-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Timsy Uppal

    2015-01-01

    Full Text Available 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Uppal, Timsy [Department of Microbiology and Immunology, School of Medicine, University of Nevada, 1664 N Virginia Street, MS 320, Reno, NV 89557 (United States); Jha, Hem C. [Department of Microbiology and the Tumor Virology Program of the Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, 201E Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104 (United States); Verma, Subhash C. [Department of Microbiology and Immunology, School of Medicine, University of Nevada, 1664 N Virginia Street, MS 320, Reno, NV 89557 (United States); Robertson, Erle S., E-mail: erle@mail.med.upenn.edu [Department of Microbiology and the Tumor Virology Program of the Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, 201E Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104 (United States)

    2015-01-14

    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.

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

    International Nuclear Information System (INIS)

    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

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

    Directory of Open Access Journals (Sweden)

    Hinde Elizabeth

    2012-12-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

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

    2015-02-18

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

  17. Interaction and conformational changes of chromatin with divalent ions.

    OpenAIRE

    Borochov, N; Ausio, J; Eisenberg, H

    1984-01-01

    We have investigated the interaction of divalent ions with chromatin towards a closer understanding of the role of metal ions in the cell nucleus. The first row transition metal ion chlorides MnCl2, CoCl2, NiCl2 and CuCl2 lead to precipitation of chicken erythrocyte chromatin at a significantly lower concentration than the alkali earth metal chlorides MgCl2, CaCl2 and BaCl2. A similar distinction can be made for the compaction of chromatin to the "30 nm" solenoid higher order structure which ...

  18. SUMO-2 Orchestrates Chromatin Modifiers in Response to DNA Damage

    DEFF Research Database (Denmark)

    Hendriks, Ivo A; Treffers, Louise W; Verlaan-de Vries, Matty;

    2015-01-01

    dynamically SUMOylated interaction networks of chromatin modifiers, transcription factors, DNA repair factors, and nuclear body components. SUMOylated chromatin modifiers include JARID1B/KDM5B, JARID1C/KDM5C, p300, CBP, PARP1, SetDB1, and MBD1. Whereas SUMOylated JARID1B was ubiquitylated by the SUMO......-targeted ubiquitin ligase RNF4 and degraded by the proteasome in response to DNA damage, JARID1C was SUMOylated and recruited to the chromatin to demethylate histone H3K4....

  19. Sperm chromatin structure and male fertility: biological and clinical aspects

    Institute of Scientific and Technical Information of China (English)

    J. Erenpreiss; M. Spano; J. Erenpreisa; M. Bungum; A. Giwercman

    2006-01-01

    Aim: Sperm chromatin/DNA integrity is essential for the accurate transmission of paternal genetic information, and normal sperm chromatin structure is important for sperm fertilizing ability. The routine examination of semen, which includes sperm concentration, motility and morphology, does not identify defects in sperm chromatin structure. The origin of sperm DNA damage and a variety of methods for its assessment are described. Evaluation of sperm DNA damage appears to be a useful tool for assessing male fertility potential both in vivo and in vitro. The possible impact of sperm DNA defects on the offspring is also discussed.

  20. Lamin C and chromatin organization in Drosophila

    Indian Academy of Sciences (India)

    B. V. Gurudatta; L. S. Shashidhara; Veena K. Parnaik

    2010-04-01

    Drosophila lamin C (LamC) is a developmentally regulated component of the nuclear lamina. The lamC gene is situated in the fifth intron of the essential gene tout velu (ttv). We carried out genetic analysis of lamC during development. Phenotypic analyses of RNAi-mediated downregulation of lamC expression as well as targeted misexpression of lamin C suggest a role for lamC in cell survival. Of particular interest in the context of laminopathies is the caspase-dependent apoptosis induced by the overexpression of lamin C. Interestingly, misexpression of lamin C in the central nervous system, where it is not normally expressed, did not affect organization of the nuclear lamina. lamC mutant alleles suppressed position effect variegation normally displayed at near-centromeric and telomeric regions. Further, both downregulation and misexpression of lamin C affected the distribution of heterochromatin protein 1. Our results suggest that Drosophila lamC has a tissue-specific role during development and is required for chromatin organization.

  1. Control of chromatin structure by long noncoding RNA

    Science.gov (United States)

    Böhmdorfer, Gudrun; Wierzbicki, Andrzej T.

    2015-01-01

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

  2. R-loop: an emerging regulator of chromatin dynamics

    Institute of Scientific and Technical Information of China (English)

    Qais Al-Hadid; Yanzhong Yang

    2016-01-01

    The dynamic structure of chromatin,which exists in two conformational states:heterochromatin and euchromatin,alters the accessibility of the DNA to regulatory factors during transcription,replication,recombination,and DNA damage repair.Chemical modifications of histones and DNA,as well as adenosine triphospahate-dependent nucleosome remodeling,have been the major focus of research on chromatin dynamics over the past two decades.However,recent studies using a DNA-RNA hybrid-specific antibody and next-generation seque,ncing approaches have revealed that the formation of R-loops,one of the most common non-canonical DNA structures,is an emerging regulator of chromatin states.This review focuses on recent insights into the interplay between R-loop formation and the epigenetic modifications of chromatin in normal and disease states.

  3. In vitro binding of nitracrine to DNA in chromatin.

    Science.gov (United States)

    Wilmańska, D; Szmigiero, L; Gniazdowski, M

    1989-01-01

    In the presence of sulfhydryl compounds nitracrine, an anticancer drug, binds covalently to DNA. The accessibility of DNA in chromatin both to nitracrine and to 8-methoxypsoralen, which was used as a reference compound in this study, when assayed in NaCl concentrations from 0 to 2 M show similar characteristics. The initial decrease reaches a minimum at 0.15 M NaCl above which dissociation of non-histone proteins and histones at higher ionic strengths is demonstrated by an increase in accessible sites. The relative accessibility of DNA in chromatin to nitracrine is, however, lower than that found for 8-methoxypsoralen. Partial dissociation of chromatin with 0.7 M NaCl increases the accessibility of DNA in chromatin when assayed in the absence of NaCl but has no apparent influence when estimated at ionic strength close to physiological conditions. PMID:2742691

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

    Science.gov (United States)

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

    2016-01-01

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

  5. Probing Chromatin-modifying Enzymes with Chemical Tools

    KAUST Repository

    Fischle, Wolfgang

    2016-02-04

    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.

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

    Science.gov (United States)

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

    2012-07-01

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

  7. Insights into Chromatin Structure and Dynamics in Plants

    Directory of Open Access Journals (Sweden)

    Stefanie Rosa

    2013-11-01

    Full Text Available 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.

  8. Does seminal fluid viscosity influence sperm chromatin integrity?

    Science.gov (United States)

    Gopalkrishnan, K; Padwal, V; Balaiah, D

    2000-01-01

    A retrospective study was undertaken to investigate whether viscosity alters sperm chromatin integrity. Semen samples were obtained from 269 men attending the infertility clinic. The viscosity was measured quantitatively by needle and syringe method and the viscosity ratio was calculated against distilled water. The chromatin integrity was evaluated by in vitro decondensation test using 1% SDS and 6 mM EDTA. According to the viscosity ratios the samples were divided into 2 groups: I, normal (ratio 9, n = 30) viscosity. Chromatin integrity was significantly lower in the group with higher viscosity. Significant decrease in sperm count and motility were seen in group II as compared to group I. Thus, hyperviscosity of seminal fluid alters the sperm chromatin integrity. PMID:11028927

  9. Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin.

    Science.gov (United States)

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

    2016-02-11

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

  10. Neutron scattering studies on chromatin higher-order structure

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-12-31

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

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

    DEFF Research Database (Denmark)

    Birch, Joanna L; Tan, Bertrand C-M; Panov, Kostya I;

    2009-01-01

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

  12. Chromatin structure modulates DNA repair by photolyase in vivo.

    OpenAIRE

    Suter, B.; Livingstone-Zatchej, M; Thoma, F

    1997-01-01

    Yeast and many other organisms use nucleotide excision repair (NER) and photolyase in the presence of light (photoreactivation) to repair cyclobutane pyrimidine dimers (CPDs), a major class of DNA lesions generated by UV light. To study the role of photoreactivation at the chromatin level in vivo, we used yeast strains which contained minichromosomes (YRpTRURAP, YRpCS1) with well-characterized chromatin structures. The strains were either proficient (RAD1) or deficient (rad1 delta) in NER. In...

  13. Higher order chromatin structure: bridging physics and biology

    OpenAIRE

    Fudenberg, Geoffrey; Mirny, Leonid A.

    2012-01-01

    Recent advances in microscopy and genomic techniques have provided new insight into spatial chromatin organization inside of the nucleus. In particular, chromosome conformation capture data has highlighted the relevance of polymer physics for high-order chromatin organization. In this context, we review basic polymer states, discuss how an appropriate polymer model can be determined from experimental data, and examine the success and limitations of various polymer models of high-order interph...

  14. Ectopically tethered CP190 induces large-scale chromatin decondensation

    Science.gov (United States)

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

    2014-01-01

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

  15. Minor groove binder distamycin remodels chromatin but inhibits transcription.

    Directory of Open Access Journals (Sweden)

    Parijat Majumder

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

  16. ATP-Dependent Chromatin Remodeling Factors and Their Roles in Affecting Nucleosome Fiber Composition

    Directory of Open Access Journals (Sweden)

    Alexandra Lusser

    2011-10-01

    Full Text Available ATP-dependent chromatin remodeling factors of the SNF2 family are key components of the cellular machineries that shape and regulate chromatin structure and function. Members of this group of proteins have broad and heterogeneous functions ranging from controlling gene activity, facilitating DNA damage repair, promoting homologous recombination to maintaining genomic stability. Several chromatin remodeling factors are critical components of nucleosome assembly processes, and recent reports have identified specific functions of distinct chromatin remodeling factors in the assembly of variant histones into chromatin. In this review we will discuss the specific roles of ATP-dependent chromatin remodeling factors in determining nucleosome composition and, thus, chromatin fiber properties.

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

    Science.gov (United States)

    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.

  18. Structural Fluctuations of the Chromatin Fiber within Topologically Associating Domains.

    Science.gov (United States)

    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

  19. Chromatin perturbations during the DNA damage response in higher eukaryotes.

    Science.gov (United States)

    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

  20. The dynamics of individual nucleosomes controls the chromatin condensation pathway: direct AFM visualization of variant chromatin

    CERN Document Server

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

    2009-01-01

    Chromatin organization and dynamics is studied in this work at scales ranging from single nucleosome to nucleosomal array by using a unique combination of biochemical assays, single molecule imaging technique and numerical modeling. We demonstrate that a subtle modification in the nucleosome structure induced by the histone variant H2A.Bbd drastically modifies the higher order organization of the nucleosomal arrays. Importantly, as directly visualized by AFM, conventional H2A nucleosomal arrays exhibit specific local organization, in contrast to H2A.Bbd arrays, which show ?beads on a string? structure. The combination of systematic image analysis and theoretical modeling allows a quantitative description relating the observed gross structural changes of the arrays to their local organization. Our results strongly suggest that higher-order organization of H1-free nucleosomal arrays is mainly determined by the fluctuation properties of individual nucleosomes. Moreover, numerical simulations suggest the existenc...

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

    Science.gov (United States)

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

    2015-01-01

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

  2. CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  4. Circadian rhythms and memory formation: regulation by chromatin remodeling.

    Science.gov (United States)

    Sahar, Saurabh; Sassone-Corsi, Paolo

    2012-01-01

    Epigenetic changes, such as DNA methylation or histone modification, can remodel the chromatin and regulate gene expression. Remodeling of chromatin provides an efficient mechanism of transducing signals, such as light or nutrient availability, to regulate gene expression. CLOCK:BMAL1 mediated activation of clock-controlled genes (CCGs) is coupled to circadian changes in histone modification at their promoters. Several chromatin modifiers, such as the deacetylases SIRT1 and HDAC3 or methyltransferase MLL1, have been shown to be recruited to the promoters of the CCGs in a circadian manner. Interestingly, the central element of the core clock machinery, the transcription factor CLOCK, also possesses histone acetyltransferase activity. Rhythmic expression of the CCGs is abolished in the absence of these chromatin modifiers. Recent research has demonstrated that chromatin remodeling is at the cross-roads of circadian rhythms and regulation of metabolism and aging. It would be of interest to identify if similar pathways exist in the epigenetic regulation of memory formation. PMID:22470318

  5. Circadian Rhythms and Memory Formation: Regulation by Chromatin Remodeling

    Directory of Open Access Journals (Sweden)

    Saurabh eSahar

    2012-03-01

    Full Text Available Epigenetic changes, such as DNA methylation or histone modification, can remodel the chromatin and regulate gene expression. Remodeling of chromatin provides an efficient mechanism of transducing signals, such as light or nutrient availability, to regulate gene expression. CLOCK:BMAL1 mediated activation of clock-controlled genes (CCGs is coupled to circadian changes in histone modification at their promoters. Several chromatin modifiers, such as the deacetylases SIRT1 and HDAC3 or methyltransferase MLL1, have been shown to be recruited to the promoters of the CCGs in a circadian manner. Interestingly, the central element of the core clock machinery, the transcription factor CLOCK, also possesses histone acetyltransferase activity. Rhythmic expression of the CCGs is abolished in the absence of these chromatin modifiers. Recent research has demonstrated that chromatin remodeling is at the cross-roads of circadian rhythms and regulation of metabolism and aging. It would be of interest to identify if similar pathways exist in the epigenetic regulation of memory formation.

  6. Deciphering Noncoding RNA and Chromatin Interactions: Multiplex Chromatin Interaction Analysis by Paired-End Tag Sequencing (mChIA-PET).

    Science.gov (United States)

    Choy, Jocelyn; Fullwood, Melissa J

    2017-01-01

    Genomic DNA is dynamically associated with protein factors and folded to form chromatin fibers. The 3-dimensional (3D) configuration of the chromatin will enable the distal genetic elements to come into close proximity, allowing transcriptional regulation. Noncoding RNA can mediate the 3D structure of chromatin. Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a valuable and powerful technique in molecular biology which allows the study of unbiased, genome-wide de novo chromatin interactions with paired-end tags. Here, we describe the standard version of ChIA-PET and a Multiplex ChIA-PET version. PMID:27662871

  7. Spatial organization of chromatin domains and compartments in single chromosomes.

    Science.gov (United States)

    Wang, Siyuan; Su, Jun-Han; Beliveau, Brian J; Bintu, Bogdan; Moffitt, Jeffrey R; Wu, Chao-ting; Zhuang, Xiaowei

    2016-08-01

    The spatial organization of chromatin critically affects genome function. Recent chromosome-conformation-capture studies have revealed topologically associating domains (TADs) as a conserved feature of chromatin organization, but how TADs are spatially organized in individual chromosomes remains unknown. Here, we developed an imaging method for mapping the spatial positions of numerous genomic regions along individual chromosomes and traced the positions of TADs in human interphase autosomes and X chromosomes. We observed that chromosome folding deviates from the ideal fractal-globule model at large length scales and that TADs are largely organized into two compartments spatially arranged in a polarized manner in individual chromosomes. Active and inactive X chromosomes adopt different folding and compartmentalization configurations. These results suggest that the spatial organization of chromatin domains can change in response to regulation. PMID:27445307

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

    CERN Document Server

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

    2006-01-01

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

  9. H4K44 Acetylation Facilitates Chromatin Accessibility during Meiosis

    Directory of Open Access Journals (Sweden)

    Jialei Hu

    2015-12-01

    Full Text Available Meiotic recombination hotspots are associated with histone post-translational modifications and open chromatin. However, it remains unclear how histone modifications and chromatin structure regulate meiotic recombination. Here, we identify acetylation of histone H4 at Lys44 (H4K44ac occurring on the nucleosomal lateral surface. We show that H4K44 is acetylated at pre-meiosis and meiosis and displays genome-wide enrichment at recombination hotspots in meiosis. Acetylation at H4K44 is required for normal meiotic recombination, normal levels of double-strand breaks (DSBs during meiosis, and optimal sporulation. Non-modifiable H4K44R results in increased nucleosomal occupancy around DSB hotspots. Our results indicate that H4K44ac functions to facilitate chromatin accessibility favorable for normal DSB formation and meiotic recombination.

  10. Oxidative stress signaling to chromatin in health and disease

    KAUST Repository

    Kreuz, Sarah

    2016-06-20

    Oxidative stress has a significant impact on the development and progression of common human pathologies, including cancer, diabetes, hypertension and neurodegenerative diseases. Increasing evidence suggests that oxidative stress globally influences chromatin structure, DNA methylation, enzymatic and non-enzymatic post-translational modifications of histones and DNA-binding proteins. The effects of oxidative stress on these chromatin alterations mediate a number of cellular changes, including modulation of gene expression, cell death, cell survival and mutagenesis, which are disease-driving mechanisms in human pathologies. Targeting oxidative stress-dependent pathways is thus a promising strategy for the prevention and treatment of these diseases. We summarize recent research developments connecting oxidative stress and chromatin regulation.

  11. Human pescadillo induces large-scale chromatin unfolding

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hao; FANG Yan; HUANG Cuifen; YANG Xiao; YE Qinong

    2005-01-01

    The human pescadillo gene encodes a protein with a BRCT domain. Pescadillo plays an important role in DNA synthesis, cell proliferation and transformation. Since BRCT domains have been shown to induce chromatin large-scale unfolding, we tested the role of Pescadillo in regulation of large-scale chromatin unfolding. To this end, we isolated the coding region of Pescadillo from human mammary MCF10A cells. Compared with the reported sequence, the isolated Pescadillo contains in-frame deletion from amino acid 580 to 582. Targeting the Pescadillo to an amplified, lac operator-containing chromosome region in the mammalian genome results in large-scale chromatin decondensation. This unfolding activity maps to the BRCT domain of Pescadillo. These data provide a new clue to understanding the vital role of Pescadillo.

  12. Sliding and peeling of histone during chromatin remodelling

    CERN Document Server

    Garai, Ashok; Chowdhury, Debashish

    2011-01-01

    ATP-dependent chromatin remodeling enzymes (CRE) are bio-molecular motors in eukaryotic cells. These are driven by a chemical fuel, namely, adenosine triphosphate (ATP). CREs actively participate in many cellular processes that require accessibility of specific stretches of DNA which are packaged as chromatin. The basic unit of chromatin is a nucleosome where 146 bp $\\sim$ 50 nm of a double stranded DNA (dsDNA) is wrapped around a spool formed by histone proteins. We investigate the mechanism of peeling of the histone spool, and its complete detachment, from the dsDNA by a CRE. Our two-state model of a CRE captures effectively two distinct chemical (or conformational) states in the mechano-chemical cycle of each ATP-dependent CRE. We calculate the mean times for histone detachment. Our predictions on the ATP-dependence of the measurable quantities can be tested by carrying out {\\it in-vitro} experiments.

  13. Absence of canonical active chromatin marks in developmentally regulated genes

    Science.gov (United States)

    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

  14. TALE proteins bind to both active and inactive chromatin.

    Science.gov (United States)

    Scott, James N F; Kupinski, Adam P; Kirkham, Christopher M; Tuma, Roman; Boyes, Joan

    2014-02-15

    TALE (transcription activator-like effector) proteins can be tailored to bind to any DNA sequence of choice and thus are of immense utility for genome editing and the specific delivery of transcription activators. However, to perform these functions, they need to occupy their sites in chromatin. In the present study, we have systematically assessed TALE binding to chromatin substrates and find that in vitro TALEs bind to their target site on nucleosomes at the more accessible entry/exit sites, but not at the nucleosome dyad. We show further that in vivo TALEs bind to transcriptionally repressed chromatin and that transcription increases binding by only 2-fold. These data therefore imply that TALEs are likely to bind to their target in vivo even at inactive loci.

  15. Rapid genome-scale mapping of chromatin accessibility in tissue

    Science.gov (United States)

    2012-01-01

    Background The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on large amounts of purified nuclei as starting material. This complicates analysis of trace clinical tissue samples that are often stored frozen. We have developed an alternative nuclease based procedure to bypass nuclear preparation to interrogate nuclease accessible regions in frozen tissue samples. Results Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh). The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergence between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases and accordingly accessibility of CpG islands is probed more efficiently using TACh. Conclusion The TACh methodology identifies accessible chromatin derived from frozen tissue samples. We propose that this simple, robust approach can be applied across a broad range of

  16. Rapid genome-scale mapping of chromatin accessibility in tissue

    Directory of Open Access Journals (Sweden)

    Grøntved Lars

    2012-06-01

    Full Text Available Abstract Background The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on large amounts of purified nuclei as starting material. This complicates analysis of trace clinical tissue samples that are often stored frozen. We have developed an alternative nuclease based procedure to bypass nuclear preparation to interrogate nuclease accessible regions in frozen tissue samples. Results Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh. The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergence between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases and accordingly accessibility of CpG islands is probed more efficiently using TACh. Conclusion The TACh methodology identifies accessible chromatin derived from frozen tissue samples. We propose that this simple, robust approach can be applied

  17. Dicer is associated with ribosomal DNA chromatin in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Lasse Sinkkonen

    Full Text Available BACKGROUND: RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer(-/- ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines. CONCLUSION/SIGNIFICANCE: We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity. However, localization of Dicer to the transcribed region suggests that transcription may contribute to the Dicer deposition at rDNA chromatin. We hypothesize that Dicer functions in maintaining integrity of rDNA arrays.

  18. Single-epitope recognition imaging of native chromatin

    Directory of Open Access Journals (Sweden)

    Wang Hongda

    2008-12-01

    Full Text Available Abstract Background Direct visualization of chromatin has the potential to provide important insights into epigenetic processes. In particular, atomic force microscopy (AFM can visualize single nucleosomes under physiological ionic conditions. However, AFM has mostly been applied to chromatin that has been reconstituted in vitro, and its potential as a tool for the dissection of native nucleosomes has not been explored. Recently we applied AFM to native Drosophila chromatin containing the centromere-specific histone 3 (CenH3, showing that it is greatly enriched in smaller particles. Taken together with biochemical analyses of CenH3 nucleosomes, we propose that centromeric nucleosomes are hemisomes, with one turn of DNA wrapped around a particle consisting of one molecule each of centromere-specific CenH3, H4, H2A and H2B. Results Here we apply a recognition mode of AFM imaging to directly identify CenH3 within histone core particles released from native centromeric chromatin. More than 90% of these particles were found to be tetrameric in height. The specificity of recognition was confirmed by blocking with a CenH3 peptide, and the strength of the interaction was quantified by force measurements. These results imply that the particles imaged by AFM are indeed mature CenH3-containing hemisomes. Conclusion Efficient and highly specific recognition of CenH3 in histone core particles isolated from native centromeric chromatin demonstrates that tetramers are the predominant form of centromeric nucleosomes in mature tetramers. Our findings provide proof of principle that this approach can yield insights into chromatin biology using direct and rapid detection of native nucleosomes in physiological salt concentrations.

  19. Chromatin Repressive Complexes in Stem Cells, Development, and Cancer

    DEFF Research Database (Denmark)

    Laugesen, Anne; Helin, Kristian

    2014-01-01

    of the polycomb repressive complexes, PRC1 and PRC2, and the HDAC1- and HDAC2-containing complexes, NuRD, Sin3, and CoREST, in stem cells, development, and cancer, as well as the ongoing efforts to develop therapies targeting these complexes in human cancer. Furthermore, we discuss the role of repressive......The chromatin environment is essential for the correct specification and preservation of cell identity through modulation and maintenance of transcription patterns. Many chromatin regulators are required for development, stem cell maintenance, and differentiation. Here, we review the roles...... complexes in modulating thresholds for gene activation and their importance for specification and maintenance of cell fate....

  20. The human chromosome. Electron microscopic observations on chromatin fiber organization.

    Science.gov (United States)

    Abuelo, J G; Moore, D E

    1969-04-01

    Human lymphocytes were grown in short-term tissue culture and were arrested in metaphase with Colcemid. Their chromosomes were prepared by the Langmuir trough-critical point drying technique and were examined under the electron microscope. In addition, some chromosomes were digested with trypsin, Pronase, or DNase. The chromosomes consist entirely of tightly packed, 240 +/- 50-A chromatin fibers. Trypsin and Pronase treatments induce relaxation of fiber packing and reveal certain underlying fiber arrangements. Furthermore, trypsin treatment demonstrates that the chromatin fiber has a 25-50 A trypsin-resistant core surrounded by a trypsin-sensitive sheath. DNase digestion suggests that this core contains DNA.

  1. Chromatin versus pathogens: the function of epigenetics in plant immunity

    Directory of Open Access Journals (Sweden)

    Bo eDing

    2015-09-01

    Full Text Available To defend against pathogens, plants have developed a sophisticated innate immunity that includes effector recognition, signal transduction, and rapid defense responses. Recent evidence has demonstrated that plants utilize the epigenetic control of gene expression to fine-tune their defense when challenged by pathogens. In this review, we highlight the current understanding of the molecular mechanisms of histone modifications (i.e., methylation, acetylation, and ubiquitination and chromatin remodeling that contribute to plant immunity against pathogens. Functions of key histone-modifying and chromatin remodeling enzymes are discussed.

  2. Retention of the Native Epigenome in Purified Mammalian Chromatin.

    Directory of Open Access Journals (Sweden)

    Andreas H Ehrensberger

    Full Text Available A protocol is presented for the isolation of native mammalian chromatin as fibers of 25-250 nucleosomes under conditions that preserve the natural epigenetic signature. The material is composed almost exclusively of histones and DNA and conforms to the structure expected by electron microscopy. All sequences probed for were retained, indicating that the material is representative of the majority of the genome. DNA methylation marks and histone marks resembled the patterns observed in vivo. Importantly, nucleosome positions also remained largely unchanged, except on CpG islands, where nucleosomes were found to be unstable. The technical challenges of reconstituting biochemical reactions with native mammalian chromatin are discussed.

  3. Genomic and chromatin signals underlying transcription start-site selection

    DEFF Research Database (Denmark)

    Valen, Eivind; Sandelin, Albin Gustav

    2011-01-01

    ; the field is now faced with the daunting challenge of translating these descriptive maps into quantitative and predictive models describing the underlying biology. We review here the genomic and chromatin features that underlie TSS selection and usage, focusing on the differences between the major classes....... In recent years substantial progress has been made towards this goal, spurred by the possibility of applying genome-wide, sequencing-based analysis. We now have a large collection of high-resolution datasets identifying locations of TSSs, protein-DNA interactions, and chromatin features over whole genomes...

  4. Chromatin structure and evolution in the human genome

    Directory of Open Access Journals (Sweden)

    Dunlop Malcolm G

    2007-05-01

    Full Text Available Abstract Background Evolutionary rates are not constant across the human genome but genes in close proximity have been shown to experience similar levels of divergence and selection. The higher-order organisation of chromosomes has often been invoked to explain such phenomena but previously there has been insufficient data on chromosome structure to investigate this rigorously. Using the results of a recent genome-wide analysis of open and closed human chromatin structures we have investigated the global association between divergence, selection and chromatin structure for the first time. Results In this study we have shown that, paradoxically, synonymous site divergence (dS at non-CpG sites is highest in regions of open chromatin, primarily as a result of an increased number of transitions, while the rates of other traditional measures of mutation (intergenic, intronic and ancient repeat divergence as well as SNP density are highest in closed regions of the genome. Analysis of human-chimpanzee divergence across intron-exon boundaries indicates that although genes in relatively open chromatin generally display little selection at their synonymous sites, those in closed regions show markedly lower divergence at their fourfold degenerate sites than in neighbouring introns and intergenic regions. Exclusion of known Exonic Splice Enhancer hexamers has little affect on the divergence observed at fourfold degenerate sites across chromatin categories; however, we show that closed chromatin is enriched with certain classes of ncRNA genes whose RNA secondary structure may be particularly important. Conclusion We conclude that, overall, non-CpG mutation rates are lowest in open regions of the genome and that regions of the genome with a closed chromatin structure have the highest background mutation rate. This might reflect lower rates of DNA damage or enhanced DNA repair processes in regions of open chromatin. Our results also indicate that dS is a poor

  5. The importance of topoisomerases for chromatin regulated genes

    DEFF Research Database (Denmark)

    Fredsøe, Jacob Christian; Pedersen, Jakob Madsen; Rødgaard, Morten Terpager;

    2013-01-01

    DNA topoisomerases are enzymes, which function to relieve torsional stress in the DNA helix by introducing transient breaks into the DNA molecule. By use of Saccharomyces cerevisiae and microarray technology we have previously shown that topoisomerases are required for the activation of chromatin...... topoisomerases for optimal activation, but in contrast to the PHO5 gene, topoisomerases are not required for chromatin remodeling of the GAL1/10 promoter region, indicating a different role of the enzymes. We are currently performing a detailed investigation of the GAL genes to elucidate the precise role...

  6. CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

    OpenAIRE

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

    2015-01-01

    Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, ...

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

    Science.gov (United States)

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

    2015-01-01

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

  8. ATP independent and ATP dependent chromatin remodeling in wheat

    International Nuclear Information System (INIS)

    Unraveling the biochemistry of chromatin dynamics during DNA replication, repair, recombination as well as transcription is the current challenge in biology. The nucleosomes containing histone octamer are the crucial elements responsible for winding and unwinding eukaryotic DNA. During DNA centric events, these nucleosomes translocate along the DNA with concomitant covalent modifications of histones. We explored these mechanisms in wheat seedlings after irradiation with survivable dose of 60Co-γ radiations. The histones isolated from irradiated seedlings showed that global acetylation of H3 decreased and H4 increased in dose depend manner till 100 grays. Time course of individual modifications showed that for H3K4 and H3K9 acetylation decreased, whereas H3S10, phosphorylation increased. There were fluctuations in acetylation of H4K5, H4K12 and H4K16, whereas H4K8 showed hyperacetylation. We found ATP-dependent chromatin remodeling activity as trans-transfer of the nucleosomes from wheat native donor chromatin on a labeled nucleosome positioning sequence and cis-transfer of the mononucleosomes in vitro. However, there was no significant change in this activity in extracts obtained from irradiated wheat seedlings. This is the first report on, demonstration of ATP-dependent chromatin remodeling activity and site specific H3 and H4 modifications in response to exposure to ionizing radiation in case of plants. (author)

  9. Chromatin Structure in Cell Differentiation, Aging and Cancer

    NARCIS (Netherlands)

    S. Kheradmand Kia (Sima)

    2009-01-01

    textabstractChromatin is the structure that the eukaryotic genome is packaged into, allowing over a metre of DNA to fit into the small volume of the nucleus. It is composed of DNA and proteins, most of which are histones. This DNA-protein complex is the template for a number of essential cell proces

  10. Chromatin remodelers in the DNA double strand break response

    NARCIS (Netherlands)

    Smeenk, Godelieve

    2012-01-01

    During my PhD project, I studied the role of several chromatin remodelers in the DNA double strand break (DSB) response. We discovered that both CHD4 and SMARCA5 are required for ubiquitin signaling through the E3 ubiquitin ligases RNF8 and RNF168, which is a central signaling event in the response

  11. Generation of bivalent chromatin domains during cell fate decisions

    Directory of Open Access Journals (Sweden)

    De Gobbi Marco

    2011-06-01

    Full Text Available Abstract Background In self-renewing, pluripotent cells, bivalent chromatin modification is thought to silence (H3K27me3 lineage control genes while 'poising' (H3K4me3 them for subsequent activation during differentiation, implying an important role for epigenetic modification in directing cell fate decisions. However, rather than representing an equivalently balanced epigenetic mark, the patterns and levels of histone modifications at bivalent genes can vary widely and the criteria for identifying this chromatin signature are poorly defined. Results Here, we initially show how chromatin status alters during lineage commitment and differentiation at a single well characterised bivalent locus. In addition we have determined how chromatin modifications at this locus change with gene expression in both ensemble and single cell analyses. We also show, on a global scale, how mRNA expression may be reflected in the ratio of H3K4me3/H3K27me3. Conclusions While truly 'poised' bivalently modified genes may exist, the original hypothesis that all bivalent genes are epigenetically premarked for subsequent expression might be oversimplistic. In fact, from the data presented in the present work, it is equally possible that many genes that appear to be bivalent in pluripotent and multipotent cells may simply be stochastically expressed at low levels in the process of multilineage priming. Although both situations could be considered to be forms of 'poising', the underlying mechanisms and the associated implications are clearly different.

  12. Regulation of chromatin structure by poly(ADP-ribosylation

    Directory of Open Access Journals (Sweden)

    Sascha eBeneke

    2012-09-01

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

  13. Is chromatin remodeling required to build sister-chromatid cohesion?

    NARCIS (Netherlands)

    Riedel, Christian G; Gregan, Juraj; Gruber, Stephan; Nasmyth, Kim

    2004-01-01

    Chromosome segregation during mitosis and meiosis depends on the linkage of sister DNA molecules after replication. These links, known as sister-chromatid cohesion, are provided by a multi-subunit complex called cohesin. Recent papers suggest that chromatin-remodeling complexes also have a role in t

  14. Functional Insights into Chromatin Remodelling from Studies on CHARGE Syndrome

    NARCIS (Netherlands)

    Basson, M. Albert; van Ravenswaaij-Arts, Conny

    2015-01-01

    CHARGE syndrome is a rare genetic syndrome characterised by a unique combination of multiple organ anomalies. Dominant loss-of-function mutations in the gene encoding chromodomain helicase DNA binding protein 7 (CHD7), which is an ATP-dependent chromatin remodeller, have been identified as the cause

  15. Trichostatin A induced histone acetylation causes decondensation of interphase chromatin.

    NARCIS (Netherlands)

    T.A. Knoch (Tobias); M. Wachsmuth (Malte); M. Frank-Stöhr (Monika); M. Stöhr (Michael); C.P. Bacher (Christian); K. Rippe (Karsten)

    2004-01-01

    textabstractThe effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a rev

  16. Control of the Transition to Flowering by Chromatin Modifications

    Institute of Scientific and Technical Information of China (English)

    Yuehui He

    2009-01-01

    The timing of floral transition is critical to reproductive success in angiosperms and is genetically controlled by a network of flowering genes.In Arabidopsis,expression of certain flowering genes is regulated by various chromatin modifications,among which are two central regulators of flowering,namely FLOWERING LOCUS C(FLC) and FLOWERING LOCUS T(FT).Recent studies have revealed that a number of chromatin-modifying components are involved in activation or repression of FLC expression.Activation of FLC expression is associated with various 'active' chromatin modifications including acetylation of core histone tails,histone H3 lysine-4 (H3K4) methylation,H2B monoubiquitination,H3 lysine-36 (H3K36) di- and tri-methylation and deposition of the histone variant H2A.Z,whereas various 'repressive' histone modifications are associated with FLC repression,including histone deacetylation,H3K4 demethylation,histone H3 lysine-9(H3Kg) and H3 lysine-27 (H3K27) methylation,and histone arginine methylation.In addition,recent studies have revealed that Polycomb group gene-mediated transcriptional-silencing mechanism not only represses FLC expression,but also directly represses FT expression.Regulation of FLC expression provides a paradigm for control of the expression of other developmental genes in plants through chromatin mechanisms.

  17. The epigenetic regulation of cell cycle and chromatin dynamic by sirtuins

    OpenAIRE

    Martínez Redondo, Paloma

    2014-01-01

    Tesi realitzada a l'Institut d'Investigació Biomèdica de Bellvitge (IDIBELL) The chromatin consists of a hierarchical and dynamical structure that is modulated during the different cell cycle stages in order to maintain genome integrity and preserve the genetic information coded in the DNA. The dynamic structure of the chromatin depends on the coordination of the different chromatin remodeling processes: histone modifications, chromatin remodeling enzymes/complexes, DNA methylation and chr...

  18. Transcriptional repression of the yeast CHA1 gene requires the chromatin-remodeling complex RSC

    DEFF Research Database (Denmark)

    Moreira, José Manuel Alfonso; Holmberg, S

    1999-01-01

    In eukaryotes, DNA is packaged into chromatin, a compact structure that must be disrupted when genes are transcribed by RNA polymerase II. For transcription to take place, chromatin is remodeled via nucleosome disruption or displacement, a fundamental transcriptional regulatory mechanism in eukar......In eukaryotes, DNA is packaged into chromatin, a compact structure that must be disrupted when genes are transcribed by RNA polymerase II. For transcription to take place, chromatin is remodeled via nucleosome disruption or displacement, a fundamental transcriptional regulatory mechanism...

  19. Citrullination regulates pluripotency and histone H1 binding to chromatin

    Science.gov (United States)

    Christophorou, Maria A.; Castelo-Branco, Gonçalo; Halley-Stott, Richard P.; Oliveira, Clara Slade; Loos, Remco; Radzisheuskaya, Aliaksandra; Mowen, Kerri A.; Bertone, Paul; Silva, José C. R.; Zernicka-Goetz, Magdalena; Nielsen, Michael L.; Gurdon, John B.; Kouzarides, Tony

    2014-03-01

    Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.

  20. Dynamic chromatin: the regulatory domain organization of eukaryotic gene loci.

    Science.gov (United States)

    Bonifer, C; Hecht, A; Saueressig, H; Winter, D M; Sippel, A E

    1991-10-01

    It is hypothesized that nuclear DNA is organized in topologically constrained loop domains defining basic units of higher order chromatin structure. Our studies are performed in order to investigate the functional relevance of this structural subdivision of eukaryotic chromatin for the control of gene expression. We used the chicken lysozyme gene locus as a model to examine the relation between chromatin structure and gene function. Several structural features of the lysozyme locus are known: the extension of the region of general DNAasel sensitivity of the active gene, the location of DNA-sequences with high affinity for the nuclear matrix in vitro, and the position of DNAasel hypersensitive chromatin sites (DHSs). The pattern of DHSs changes depending on the transcriptional status of the gene. Functional studies demonstrated that DHSs mark the position of cis-acting regulatory elements. Additionally, we discovered a novel cis-activity of the border regions of the DNAasel sensitive domain (A-elements). By eliminating the position effect on gene expression usually observed when genes are randomly integrated into the genome after transfection, A-elements possibly serve as punctuation marks for a regulatory chromatin domain. Experiments using transgenic mice confirmed that the complete structurally defined lysozyme gene domain behaves as an independent regulatory unit, expressing the gene in a tissue specific and position independent manner. These expression features were lost in transgenic mice carrying a construct, in which the A-elements as well as an upstream enhancer region were deleted, indicating the lack of a locus activation function on this construct. Experiments are designed in order to uncover possible hierarchical relationships between the different cis-acting regulatory elements for stepwise gene activation during cell differentiation. We are aiming at the definition of the basic structural and functional requirements for position independent and high

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

    Science.gov (United States)

    Walker, P R; LeBlanc, J; Sikorska, M

    1989-05-01

    The reactivity of aluminum and several other divalent and trivalent metallic cations toward chromatin from rat brain and liver has been investigated. Two criteria are used to determine the relative reactivity of these cations toward chromatin. The first involves the ability of the ions to compact the chromatin fibers to the point where chromatin precipitates. The second criterion measures the ability of cations to interfere with the accessibility of exogenous structural probes (nucleases) to chromatin. Of the divalent cations tested, nickel, cobalt, zinc, cadmium, and mercury were the most reactive toward chromatin, on the basis of their ability to induce precipitation of chromatin in the micromolar concentration range. The divalent cations magnesium, calcium, copper, strontium, and barium were much less effective, although all cations precipitate chromatin if their concentration is increased. Of the trivalent cations tested, aluminum, indium, and gallium were very effective precipitants, whereas iron and scandium were without effect at the concentrations tested. Of all the cations tested, aluminum was the most reactive. Aluminum's ability to alter the structure of chromatin was investigated further by testing its ability to interfere with nuclease accessibility. This test confirmed that aluminum does induce considerable changes in chromatin structure at micromolar concentrations. Furthermore, chromatin from cortical areas of the brain was much more sensitive to aluminum than chromatin from liver. These results are discussed in light of the known toxicity of these cations, with particular emphasis on the possible role of aluminum in Alzheimer's disease. PMID:2752000

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

    Directory of Open Access Journals (Sweden)

    Wioletta Czaja

    2012-09-01

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

  3. Retroviruses hijack chromatin loops to drive oncogene expression and highlight the chromatin architecture around proto-oncogenic loci.

    Directory of Open Access Journals (Sweden)

    Jillian M Pattison

    Full Text Available The majority of the genome consists of intergenic and non-coding DNA sequences shown to play a major role in different gene regulatory networks. However, the specific potency of these distal elements as well as how these regions exert function across large genomic distances remains unclear. To address these unresolved issues, we closely examined the chromatin architecture around proto-oncogenic loci in the mouse and human genomes to demonstrate a functional role for chromatin looping in distal gene regulation. Using cell culture models, we show that tumorigenic retroviral integration sites within the mouse genome occur near existing large chromatin loops and that this chromatin architecture is maintained within the human genome as well. Significantly, as mutagenesis screens are not feasible in humans, we demonstrate a way to leverage existing screens in mice to identify disease relevant human enhancers and expose novel disease mechanisms. For instance, we characterize the epigenetic landscape upstream of the human Cyclin D1 locus to find multiple distal interactions that contribute to the complex cis-regulation of this cell cycle gene. Furthermore, we characterize a novel distal interaction upstream of the Cyclin D1 gene which provides mechanistic evidence for the abundant overexpression of Cyclin D1 occurring in multiple myeloma cells harboring a pathogenic translocation event. Through use of mapped retroviral integrations and translocation breakpoints, our studies highlight the importance of chromatin looping in oncogene expression, elucidate the epigenetic mechanisms crucial for distal cis-regulation, and in one particular instance, explain how a translocation event drives tumorigenesis through upregulation of a proto-oncogene.

  4. Retroviruses Hijack Chromatin Loops to Drive Oncogene Expression and Highlight the Chromatin Architecture around Proto-Oncogenic Loci

    Science.gov (United States)

    Pattison, Jillian M.; Wright, Jason B.; Cole, Michael D.

    2015-01-01

    The majority of the genome consists of intergenic and non-coding DNA sequences shown to play a major role in different gene regulatory networks. However, the specific potency of these distal elements as well as how these regions exert function across large genomic distances remains unclear. To address these unresolved issues, we closely examined the chromatin architecture around proto-oncogenic loci in the mouse and human genomes to demonstrate a functional role for chromatin looping in distal gene regulation. Using cell culture models, we show that tumorigenic retroviral integration sites within the mouse genome occur near existing large chromatin loops and that this chromatin architecture is maintained within the human genome as well. Significantly, as mutagenesis screens are not feasible in humans, we demonstrate a way to leverage existing screens in mice to identify disease relevant human enhancers and expose novel disease mechanisms. For instance, we characterize the epigenetic landscape upstream of the human Cyclin D1 locus to find multiple distal interactions that contribute to the complex cis-regulation of this cell cycle gene. Furthermore, we characterize a novel distal interaction upstream of the Cyclin D1 gene which provides mechanistic evidence for the abundant overexpression of Cyclin D1 occurring in multiple myeloma cells harboring a pathogenic translocation event. Through use of mapped retroviral integrations and translocation breakpoints, our studies highlight the importance of chromatin looping in oncogene expression, elucidate the epigenetic mechanisms crucial for distal cis-regulation, and in one particular instance, explain how a translocation event drives tumorigenesis through upregulation of a proto-oncogene. PMID:25799187

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

    Science.gov (United States)

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

    2015-06-01

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

  6. Proteomics and the genetics of sperm chromatin condensation

    Institute of Scientific and Technical Information of China (English)

    Rafael Oliva; Judit Castillo

    2011-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  8. Cellular Fractionation and Isolation of Chromatin-Associated RNA.

    Science.gov (United States)

    Conrad, Thomas; Ørom, Ulf Andersson

    2017-01-01

    In eukaryotic cells, the synthesis, processing, and functions of RNA molecules are confined to distinct subcellular compartments. Biochemical fractionation of cells prior to RNA isolation thus enables the analysis of distinct steps in the lifetime of individual RNA molecules that would be masked in bulk RNA preparations from whole cells. Here, we describe a simple two-step differential centrifugation protocol for the isolation of cytoplasmic, nucleoplasmic, and chromatin-associated RNA that can be used in downstream applications such as qPCR or deep sequencing. We discuss various aspects of this fractionation protocol, which can be readily applied to many mammalian cell types. For the study of long noncoding RNAs and enhancer RNAs in regulation of transcription especially the preparation of chromatin-associated RNA can contribute significantly to further developments.

  9. Cellular Fractionation and Isolation of Chromatin-Associated RNA.

    Science.gov (United States)

    Conrad, Thomas; Ørom, Ulf Andersson

    2017-01-01

    In eukaryotic cells, the synthesis, processing, and functions of RNA molecules are confined to distinct subcellular compartments. Biochemical fractionation of cells prior to RNA isolation thus enables the analysis of distinct steps in the lifetime of individual RNA molecules that would be masked in bulk RNA preparations from whole cells. Here, we describe a simple two-step differential centrifugation protocol for the isolation of cytoplasmic, nucleoplasmic, and chromatin-associated RNA that can be used in downstream applications such as qPCR or deep sequencing. We discuss various aspects of this fractionation protocol, which can be readily applied to many mammalian cell types. For the study of long noncoding RNAs and enhancer RNAs in regulation of transcription especially the preparation of chromatin-associated RNA can contribute significantly to further developments. PMID:27662865

  10. Changes in chromatin state in donors subjected to physical stress

    OpenAIRE

    Shckorbatov, Yuriy; Samokhvalov, Valeriy; Bevziuk, Dariya; Kovaliov, Maxim

    2009-01-01

    The purpose of the present study is to evaluate changes in chromatin of human buccal epithelium under the influence of stressing factor - dosed physical activity. Investigations were performed in a group of students (13 men) of age 19-23. Cells were stained on a slide by a 2% orcein solution in 45% acetic acid during 1 h. The following physiological indexes were determined: arterial blood pressure, pulse frequency, and frequency of breathing. The physical stress produced by the dosed physical...

  11. Effect of saffron on rat sperm chromatin integrity

    OpenAIRE

    Mohammad Mardani; Ahmad Vaez; Shahnaz Razavi

    2014-01-01

    Background: Currently, relation between reactive oxygen species (ROS) ROS concentration and semen quality was indicated. Saffron has traditionally been not only considered as a food additive but also as a medicinal herb, which has a good antioxidant properties. Objective: The aim of this study was to evaluate the protection potency of saffron and vitamin E on sperm chromatin integrity. Materials and Methods: Thirty adult male Wistar rats divided equally into saffron (100 mg/kg), vitamin E (10...

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

    Science.gov (United States)

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

    1978-05-01

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

  13. Quality of histone modification antibodies undermines chromatin biology research

    OpenAIRE

    Goran Kungulovski; Albert Jeltsch

    2015-01-01

    Histone post-translational modification (PTM) antibodies are essential research reagents in chromatin biology. However, they suffer from variable properties and insufficient documentation of quality. Antibody manufacturers and vendors should provide detailed lot-specific documentation of quality, rendering further quality checks by end-customers unnecessary. A shift from polyclonal antibodies towards sustainable reagents like monoclonal or recombinant antibodies or histone binding domains wou...

  14. Spermine-induced aggregation of DNA, nucleosome, and chromatin.

    OpenAIRE

    Raspaud, E.; Chaperon, I; Leforestier, A; Livolant, F

    1999-01-01

    We have analyzed the conditions of aggregation or precipitation of DNA in four different states: double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), mononucleosome core particles (NCP), and H1-depleted chromatin fragments (ChF) in the presence of the multivalent cation spermine (4+). In an intermediate regime of DNA concentration, these conditions are identical for the four states. This result demonstrates that the mechanism involved is general from flexible chains to rigid rods and qua...

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

    Science.gov (United States)

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

    1998-04-01

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

  16. Repression and activation by multiprotein complexes that alter chromatin structure.

    Science.gov (United States)

    Kingston, R E; Bunker, C A; Imbalzano, A N

    1996-04-15

    Recent studies have provided strong evidence that macromolecular complexes are used in the cell to remodel chromatin structure during activation and to create an inaccessible structure during repression, Although there is not yet any rigorous demonstration that modification of chromatin structure plays a direct, causal role in either activation or repression, there is sufficient smoke to indicate the presence of a blazing inferno nearby. It is clear that complexes that remodel chromatin are tractable in vitro; hopefully this will allow the establishment of systems that provide a direct analysis of the role that remodeling might play in activation. These studies indicate that establishment of functional systems to corroborate the elegant genetic studies on repression might also be tractable. As the mechanistic effects of these complexes are sorted out, it will become important to understand how the complexes are regulated. In many of the instances discussed above, the genes whose products make up these complexes were identified in genetic screens for effects on developmental processes. This implies a regulation of the activity of these complexes in response to developmental cues and further implies that the work to fully understand these complexes will occupy a generation of scientists.

  17. Biphasic Chromatin Structure and FISH Signals Reflect Intranuclear Order

    Directory of Open Access Journals (Sweden)

    Jyoti P. Chaudhuri

    2005-01-01

    Full Text Available Background and Aim: One of the two parental allelic genes may selectively be expressed, regulated by imprinting, X-inactivation or by other less known mechanisms. This study aims to reflect on such genetic mechanisms. Materials and Methods: Slides from short term cultures or direct smears of blood, bone marrow and amniotic fluids were hybridized with FISH probes singly, combined or sequentially. Two to three hundred cells were examined from each preparation. Results and Aignificance: A small number of cells (up to about 5%, more frequent in leukemia cases, showed the twin features: (1 nuclei with biphasic chromatin, one part decondensed and the other condensed; and (2 homologous FISH signals distributed equitably in those two regions. The biphasic chromatin structure with equitable distribution of the homologous FISH signals may correspond to the two sets of chromosomes, supporting observations on ploidywise intranuclear order. The decondensed chromatin may relate to enhanced transcriptions or advanced replications. Conclusions: Transcriptions of only one of the two parental genomes cause allelic exclusion. Genomes may switch with alternating monoallelic expression of biallelic genes as an efficient genetic mechanism. If genomes fail to switch, allelic exclusion may lead to malignancy. Similarly, a genome-wide monoallelic replication may tilt the balance of heterozygosity resulting in aneusomy, initiating early events in malignant transformation and in predicting cancer mortality.

  18. High sperm chromatin stability in semen with high viscosity.

    Science.gov (United States)

    Gonzales, G F; Sánchez, A

    1994-01-01

    This study was designed to determine the effects of high semen viscosity on sperm chromatin stability. Semen samples obtained from men with normal and high viscosity were studied. Sperm chromatin stability was tested by exposure to sodium dodecyl sulfate (SDS) only and SDS together with a zinc-chelating agent, disodium ethylene diamine tetraacetate (SDS+EDTA). After SDS incubation, stable sperm was 61.36 +/- 3.0 and 54.71 +/- 3.42% for normal and high semen viscosity, respectively (P:NS), and after SDS+EDTA, it was further reduced to 12.48 +/- 0.99% in semen samples with normal consistency and in a less magnitude in semen samples with high viscosity (25.6 +/- 5.2). Comparing values obtained in SDS+EDTA, a high sperm stability was observed in samples with hyperviscosity (p hyperviscosity is associated with a high sperm chromatin stability in situations when a zinc-chelating agent is present. PMID:8122934

  19. Senataxin controls meiotic silencing through ATR activation and chromatin remodeling.

    Science.gov (United States)

    Yeo, Abrey J; Becherel, Olivier J; Luff, John E; Graham, Mark E; Richard, Derek; Lavin, Martin F

    2015-01-01

    Senataxin, defective in ataxia oculomotor apraxia type 2, protects the genome by facilitating the resolution of RNA-DNA hybrids (R-loops) and other aspects of RNA processing. Disruption of this gene in mice causes failure of meiotic recombination and defective meiotic sex chromosome inactivation, leading to male infertility. Here we provide evidence that the disruption of Setx leads to reduced SUMOylation and disruption of protein localization across the XY body during meiosis. We demonstrate that senataxin and other DNA damage repair proteins, including ataxia telangiectasia and Rad3-related protein-interacting partner, are SUMOylated, and a marked downregulation of both ataxia telangiectasia and Rad3-related protein-interacting partner and TopBP1 leading to defective activation and signaling through ataxia telangiectasia and Rad3-related protein occurs in the absence of senataxin. Furthermore, chromodomain helicase DNA-binding protein 4, a component of the nucleosome remodeling and deacetylase chromatin remodeler that interacts with both ataxia telangiectasia and Rad3-related protein and senataxin was not recruited efficiently to the XY body, triggering altered histone acetylation and chromatin conformation in Setx (-/-) pachytene-staged spermatocytes. These results demonstrate that senataxin has a critical role in ataxia telangiectasia and Rad3-related protein- and chromodomain helicase DNA-binding protein 4-mediated transcriptional silencing and chromatin remodeling during meiosis providing greater insight into its critical role in gene regulation to protect against neurodegeneration. PMID:27462424

  20. Histone chaperones link histone nuclear import and chromatin assembly.

    Science.gov (United States)

    Keck, Kristin M; Pemberton, Lucy F

    2013-01-01

    Histone chaperones are proteins that shield histones from nonspecific interactions until they are assembled into chromatin. After their synthesis in the cytoplasm, histones are bound by different histone chaperones, subjected to a series of posttranslational modifications and imported into the nucleus. These evolutionarily conserved modifications, including acetylation and methylation, can occur in the cytoplasm, but their role in regulating import is not well understood. As part of histone import complexes, histone chaperones may serve to protect the histones during transport, or they may be using histones to promote their own nuclear localization. In addition, there is evidence that histone chaperones can play an active role in the import of histones. Histone chaperones have also been shown to regulate the localization of important chromatin modifying enzymes. This review is focused on the role histone chaperones play in the early biogenesis of histones, the distinct cytoplasmic subcomplexes in which histone chaperones have been found in both yeast and mammalian cells and the importins/karyopherins and nuclear localization signals that mediate the nuclear import of histones. We also address the role that histone chaperone localization plays in human disease. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.

  1. The shades of gray of the chromatin fiber: recent literature provides new insights into the structure of chromatin.

    Science.gov (United States)

    Ausió, Juan

    2015-01-01

    The chromatin fiber consists of a string of nucleosomes connected by linker DNA regions. The hierarchy of folding of this fiber within the cell has long been controversial, and the existence of an originally described 30 nm fiber has been debated and reviewed extensively. This review contextualizes two recent papers on this topic that suggest the 30 nm fiber to be an over-simplification. The idealized model from the first study provides good insight into the constraints and histone participation in the maintenance of the fiber structure. The second paper provides a theoretical description of a more realistic view of the highly heterogeneous and dynamic chromatin organization in the in vivo setting. It is now time to abandon the highly regular "one start" solenoidal 30 nm structure and replace it with a more realistic highly dynamic, polymorphic fiber.

  2. The protein encoded by the proto-oncogene DEK changes the topology of chromatin and reduces the efficiency of DNA replication in a chromatin-specific manner

    DEFF Research Database (Denmark)

    Alexiadis, V; Waldmann, T; Andersen, Jens S.;

    2000-01-01

    The structure of chromatin regulates the genetic activity of the underlying DNA sequence. We report here that the protein encoded by the proto-oncogene DEK, which is involved in acute myelogenous leukemia, induces alterations of the superhelical density of DNA in chromatin. The change in topology...

  3. RNA is an integral component of chromatin that contributes to its structural organization.

    Directory of Open Access Journals (Sweden)

    Antonio Rodríguez-Campos

    Full Text Available Chromatin structure is influenced by multiples factors, such as pH, temperature, nature and concentration of counterions, post-translational modifications of histones and binding of structural non-histone proteins. RNA is also known to contribute to the regulation of chromatin structure as chromatin-induced gene silencing was shown to depend on the RNAi machinery in S. pombe, plants and Drosophila. Moreover, both in Drosophila and mammals, dosage compensation requires the contribution of specific non-coding RNAs. However, whether RNA itself plays a direct structural role in chromatin is not known. Here, we report results that indicate a general structural role for RNA in eukaryotic chromatin. RNA is found associated to purified chromatin prepared from chicken liver, or cultured Drosophila S2 cells, and treatment with RNase A alters the structural properties of chromatin. Our results indicate that chromatin-associated RNAs, which account for 2%-5% of total chromatin-associated nucleic acids, are polyA(- and show a size similar to that of the DNA contained in the corresponding chromatin fragments. Chromatin-associated RNA(s are not likely to correspond to nascent transcripts as they are also found bound to chromatin when cells are treated with alpha-amanitin. After treatment with RNase A, chromatin fragments of molecular weight >3.000 bp of DNA showed reduced sedimentation through sucrose gradients and increased sensitivity to micrococcal nuclease digestion. This structural transition, which is observed both at euchromatic and heterochromatic regions, proceeds without loss of histone H1 or any significant change in core-histone composition and integrity.

  4. Temporal profiling of the chromatin proteome reveals system-wide responses to replication inhibition

    DEFF Research Database (Denmark)

    Khoudoli, Guennadi A; Gillespie, Peter J; Stewart, Graeme;

    2008-01-01

    Although the replication, expression, and maintenance of DNA are well-studied processes, the way that they are coordinated is poorly understood. Here, we report an analysis of the changing association of proteins with chromatin (the chromatin proteome) during progression through interphase...... of the cell cycle. Sperm nuclei were incubated in Xenopus egg extracts, and chromatin-associated proteins were analyzed by mass spectrometry at different times. Approximately 75% of the proteins varied in abundance on chromatin by more than 15%, suggesting that the chromatin proteome is highly dynamic....... Proteins were then assigned to one of 12 different clusters on the basis of their pattern of chromatin association. Each cluster contained functional groups of proteins involved in different nuclear processes related to progression through interphase. We also blocked DNA replication by inhibiting either...

  5. Contribution of Topological Domains and Loop Formation to 3D Chromatin Organization

    Directory of Open Access Journals (Sweden)

    Vuthy Ea

    2015-07-01

    Full Text Available Recent investigations on 3D chromatin folding revealed that the eukaryote genomes are both highly compartmentalized and extremely dynamic. This review presents the most recent advances in topological domains’ organization of the eukaryote genomes and discusses the relationship to chromatin loop formation. CTCF protein appears as a central factor of these two organization levels having either a strong insulating role at TAD borders, or a weaker architectural role in chromatin loop formation. TAD borders directly impact on chromatin dynamics by restricting contacts within specific genomic portions thus confining chromatin loop formation within TADs. We discuss how sub-TAD chromatin dynamics, constrained into a recently described statistical helix conformation, can produce functional interactions by contact stabilization.

  6. Analysis of Myc-induced histone modifications on target chromatin.

    Directory of Open Access Journals (Sweden)

    Francesca Martinato

    Full Text Available The c-myc proto-oncogene is induced by mitogens and is a central regulator of cell growth and differentiation. The c-myc product, Myc, is a transcription factor that binds a multitude of genomic sites, estimated to be over 10-15% of all promoter regions. Target promoters generally pre-exist in an active or poised chromatin state that is further modified by Myc, contributing to fine transcriptional regulation (activation or repression of the afferent gene. Among other mechanisms, Myc recruits histone acetyl-transferases to target chromatin and locally promotes hyper-acetylation of multiple lysines on histones H3 and H4, although the identity and combination of the modified lysines is unknown. Whether Myc dynamically regulates other histone modifications (or marks at its binding sites also remains to be addressed. Here, we used quantitative chromatin immunoprecipitation (qChIP to profile a total of 24 lysine-acetylation and -methylation marks modulated by Myc at target promoters in a human B-cell line with a regulatable c-myc transgene. Myc binding promoted acetylation of multiple lysines, primarily of H3K9, H3K14, H3K18, H4K5 and H4K12, but significantly also of H4K8, H4K91 and H2AK5. Dimethylation of H3K79 was also selectively induced at target promoters. A majority of target promoters showed co-induction of multiple marks - in various combinations - correlating with recruitment of the two HATs tested (Tip60 and HBO1, incorporation of the histone variant H2A.Z and transcriptional activation. Based on this and previous findings, we surmise that Myc recruits the Tip60/p400 complex to achieve a coordinated histone acetylation/exchange reaction at activated promoters. Our data are also consistent with the additive and redundant role of multiple acetylation events in transcriptional activation.

  7. A unique chromatin signature uncovers early developmental enhancers in humans.

    Science.gov (United States)

    Rada-Iglesias, Alvaro; Bajpai, Ruchi; Swigut, Tomek; Brugmann, Samantha A; Flynn, Ryan A; Wysocka, Joanna

    2011-02-10

    Cell-fate transitions involve the integration of genomic information encoded by regulatory elements, such as enhancers, with the cellular environment. However, identification of genomic sequences that control human embryonic development represents a formidable challenge. Here we show that in human embryonic stem cells (hESCs), unique chromatin signatures identify two distinct classes of genomic elements, both of which are marked by the presence of chromatin regulators p300 and BRG1, monomethylation of histone H3 at lysine 4 (H3K4me1), and low nucleosomal density. In addition, elements of the first class are distinguished by the acetylation of histone H3 at lysine 27 (H3K27ac), overlap with previously characterized hESC enhancers, and are located proximally to genes expressed in hESCs and the epiblast. In contrast, elements of the second class, which we term 'poised enhancers', are distinguished by the absence of H3K27ac, enrichment of histone H3 lysine 27 trimethylation (H3K27me3), and are linked to genes inactive in hESCs and instead are involved in orchestrating early steps in embryogenesis, such as gastrulation, mesoderm formation and neurulation. Consistent with the poised identity, during differentiation of hESCs to neuroepithelium, a neuroectoderm-specific subset of poised enhancers acquires a chromatin signature associated with active enhancers. When assayed in zebrafish embryos, poised enhancers are able to direct cell-type and stage-specific expression characteristic of their proximal developmental gene, even in the absence of sequence conservation in the fish genome. Our data demonstrate that early developmental enhancers are epigenetically pre-marked in hESCs and indicate an unappreciated role of H3K27me3 at distal regulatory elements. Moreover, the wealth of new regulatory sequences identified here provides an invaluable resource for studies and isolation of transient, rare cell populations representing early stages of human embryogenesis.

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

    OpenAIRE

    Roopa, T.; Shivashankar, G. V.

    2006-01-01

    Chromatin assembly is condensed by histone tail-tail interactions and other nuclear proteins into a highly compact structure. Using an optical trap modulation force spectroscopy, we probe the effect of tail interactions on local chromatin fluidity. Chromatin fibers, purified from mammalian cells, are tethered between a microscope coverslip and a glass micropipette. Mechanical unzipping of tail interactions, using the micropipette, lead to the enhancement of local fluidity. This is measured us...

  9. Biochemical and structural characterization of Cren7, a novel chromatin protein conserved among Crenarchaea

    OpenAIRE

    Guo, Li; Feng, Yingang; Zhang, Zhenfeng; Yao, Hongwei; Luo, Yuanming; Wang, Jinfeng; Huang, Li

    2007-01-01

    Archaea contain a variety of chromatin proteins consistent with the evolution of different genome packaging mechanisms. Among the two main kingdoms in the Archaea, Euryarchaeota synthesize histone homologs, whereas Crenarchaeota have not been shown to possess a chromatin protein conserved at the kingdom level. We report the identification of Cren7, a novel family of chromatin proteins highly conserved in the Crenarchaeota. A small, basic, methylated and abundant protein, Cren7 displays a high...

  10. Study on Resistance of Human Sperm Chromatin to Heparin Decondensation

    Institute of Scientific and Technical Information of China (English)

    褚劲松; 李建国; 薛同一; 王一飞

    1995-01-01

    Resistance of human sperm chromatin to heparin deeondensatinn was investigated by image analysis. The level of DNA deeondensation was determined by measuring the α, [red fluorescence/(red + green) fluoreseence] of sperm. The optimal experimental conditions were incubating sperms with 1000 IU/ml of heparin at 37℃ for 13 minutes and analysing the sperms with excitation F488, red fluoreseenee F630, green fluoreseence F530. The result showed that 72.93±14.73 percent of 20 fertile human sperms resist heparin deeondensa tion.

  11. Evaluation of sperm chromatin structure in boar semen

    Directory of Open Access Journals (Sweden)

    Banaszewska Dorota

    2015-06-01

    Full Text Available This study was an attempt to evaluate sperm chromatin structure in the semen of insemination boars. Preparations of semen were stained with acridine orange, aniline blue, and chromomycin A3. Abnormal protamination occurred more frequently in young individuals whose sexual development was not yet complete, but may also be an individual trait. This possibility is important to factor into the decision regarding further exploitation of insemination boars. Thus a precise assessment of abnormalities in the protamination process would seem to be expedient as a tool supplementing morphological and molecular evaluation of semen. Disruptions in nucleoprotein structure can be treated as indicators of the biological value of sperm cells.

  12. Hydrodynamic evidence in support of spacer regions in chromatin.

    Science.gov (United States)

    Schmitz, K S; Shaw, B R

    1977-08-12

    Quasi-elastic light scattering and sedimentation velocity methods were used to study the hydrodynamic properties of purified dimer subunits obtained from partial digestion of chicken erythrocyte chromatin with staphylococcal nuclease. The experimental value of 1.87 +/- 0.08 X 10(-7) gram per second for the friction factor of these dimer subunits in low ionic strength buffer cannot be reasonably interpreted in terms of a contiguous sphere model. Analysis by means of an equivalent dimer method suggests that the spacer region accounts for a maximum of 19 percent of the friction properties of the dimer.

  13. Effects of nuclear isolation on psoralen affinity for chromatin

    International Nuclear Information System (INIS)

    We have tested the effects of nuclear isolation on intercalation of TMP (a psoralen) at specific sequences and in total DNA of cultured human cells. DNA in nuclei photobound about 20% more TMP than in cells and about 10% as much as purified DNA. In contrast, a transcribed ras gene and a randomly selected polymorphic sequence each bound about 20% more TMP than total DNA in cells. However, in nuclei, as in purified DNA, both sequences were just as sensitive as total DNA. Apparently, chromatin in cells exists within diverse TMP-binding environments and some of this diversity was lost upon nuclear isolation

  14. Nucleosome conformational flexibility in experiments with single chromatin fibers

    Directory of Open Access Journals (Sweden)

    Sivolob A. V.

    2010-09-01

    Full Text Available Studies on the chromatin nucleosome organization play an ever increasing role in our comprehension of mechanisms of the gene activity regulation. This minireview describes the results on the nucleosome conformational flexibility, which were obtained using magnetic tweezers to apply torsion to oligonucleosome fibers reconstituted on single DNA molecules. Such an approach revealed a new structural form of the nucleosome, the reversome, in which DNA is wrapped in a right-handed superhelix around a distorted histone octamer. Molecular mechanisms of the nucleosome structural flexibility and its biological relevance are discussed.

  15. Analysis of histone posttranslational modifications from nucleolus-associated chromatin by mass spectrometry.

    Science.gov (United States)

    Dillinger, Stefan; Garea, Ana Villar; Deutzmann, Rainer; Németh, Attila

    2014-01-01

    Chromatin is unevenly distributed within the eukaryote nucleus and it contributes to the formation of morphologically and functionally distinct substructures, called chromatin domains and nuclear bodies. Here we describe an approach to assess specific chromatin features, the histone posttranslational modifications (PTMs), of the largest nuclear sub-compartment, the nucleolus. In this chapter, methods for the isolation of nucleolus-associated chromatin from native or formaldehyde-fixed cells and the effect of experimental procedures on the outcome of mass spectrometry analysis of histone PTMs are compared.

  16. MRN1 implicates chromatin remodeling complexes and architectural factors in mRNA maturation

    DEFF Research Database (Denmark)

    Düring, Louis; Thorsen, Michael; Petersen, Darima;

    2012-01-01

    A functional relationship between chromatin structure and mRNA processing events has been suggested, however, so far only a few involved factors have been characterized. Here we show that rsc nhp6¿¿ mutants, deficient for the function of the chromatin remodeling factor RSC and the chromatin....... Genetic interactions are observed between 2 µm-MRN1 and the splicing deficient mutants snt309¿, prp3, prp4, and prp22, and additional genetic analyses link MRN1, SNT309, NHP6A/B, SWI/SNF, and RSC supporting the notion of a role of chromatin structure in mRNA processing....

  17. Restoring chromatin after replication: How new and old histone marks come together

    DEFF Research Database (Denmark)

    Jasencakova, Zusana; Groth, Anja

    2010-01-01

    replication and chromatin assembly processes in time and space. Dynamic recycling and de novo deposition of histones are fundamental for chromatin restoration. Histone post-translational modifications (PTMs) are thought to have a causal role in establishing distinct chromatin structures. Here we discuss PTMs...... present on new and parental histones and how they influence genome stability and restoration of epigenetically defined domains. Newly deposited histones must change their signature in the process of chromatin restoration, this may occur in a step-wise fashion involving replication-coupled processes...... and information from recycled parental histones....

  18. Identification of potential nuclear reprogramming and differentiation factors by a novel selection method for cloning chromatin-binding proteins

    Institute of Scientific and Technical Information of China (English)

    LiuWang; AihuaZheng; LingYi; ChongrenXu; MingxiaoDing; HongkuiDeng

    2005-01-01

    Nuclear reprogramming is critical for animal cloning and stem cell creation through nuclear transfer, which requires extensive remodeling of chromosomal architecture involving dramatic changes in chromatin-binding proteins. To understand the mechanism of nuclear reprogramming, it is critical to identify chromatin-binding factors specify the reprogramming process. In this report, we have developed a high-throughput selection method, based on T7 phage display and chromatin immunoprecipitation, to isolate chromatin-binding factors expressed in mouse embryonic stem cells using primary mouse embryonic fibroblast chromatin. Seven chromatin-binding proteins have been isolated by this method. We have also isolated several chromatin-binding proteins involved in hepatocyte differentiation. Our method provides a powerful tool to rapidly and selectively identify chromatin-binding proteins. The method can be used to study epigenetic modification of chromatin during nuclear reprogramming, cell differentiation, and transdifferentiation.

  19. Chromatin factors affecting DNA repair in mammalian cell nuclei

    International Nuclear Information System (INIS)

    We are investigating chromatin factors that participate in the incision step of DNA repair in eukaryotic cells. Localization of repair activity within nuclei, the stability and extractability of activity, the specificity for recognizing damage in chromatin or purified DNA as substrates are of interest in this investigation of human cells, CHO cells, and their radiation sensitive mutants. We have developed procedures that provide nuclei in which their DNA behaves as a collection of circular molecules. The integrity of the DNA in human nuclei can be maintained during incubation in appropriate buffers for as long as 60 minutes. When cells or nuclei are exposed to uv light prior to incubation, incisions presumably associated with DNA repair can be demonstrated. Incision activity is stable to prior extraction of nuclei with 0.6 M NaCl, which removes many nonhistone proteins. Our studies are consistent with an hypothesis that factors responsible for initiating DNA repair are localized in the nuclear matrix. 18 references, 3 figures

  20. Chromatin Assembly at Kinetochores Is Uncoupled from DNA Replication

    Science.gov (United States)

    Shelby, Richard D.; Monier, Karine; Sullivan, Kevin F.

    2000-01-01

    The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric “state” on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [3H]thymidine we demonstrate that CENP-A–associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation. PMID:11086012

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

    Science.gov (United States)

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

    2014-11-13

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

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

    Science.gov (United States)

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

    2014-11-13

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

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

    Directory of Open Access Journals (Sweden)

    Mora Xavier

    2007-05-01

    Full Text Available Abstract Background Histone H1 is involved in the formation and maintenance of chromatin higher order structure. H1 has multiple isoforms; the subtypes differ in timing of expression, extent of phosphorylation and turnover rate. In vertebrates, the amino acid substitution rates differ among subtypes by almost one order of magnitude, suggesting that each subtype might have acquired a unique function. We have devised a competitive assay to estimate the relative binding affinities of histone H1 mammalian somatic subtypes H1a-e and H1° for long chromatin fragments (30–35 nucleosomes in physiological salt (0.14 M NaCl at constant stoichiometry. Results The H1 complement of native chromatin was perturbed by adding an additional amount of one of the subtypes. A certain amount of SAR (scaffold-associated region DNA was present in the mixture to avoid precipitation of chromatin by excess H1. SAR DNA also provided a set of reference relative affinities, which were needed to estimate the relative affinities of the subtypes for chromatin from the distribution of the subtypes between the SAR and the chromatin. The amounts of chromatin, SAR and additional H1 were adjusted so as to keep the stoichiometry of perturbed chromatin similar to that of native chromatin. H1 molecules freely exchanged between the chromatin and SAR binding sites. In conditions of free exchange, H1a was the subtype of lowest affinity, H1b and H1c had intermediate affinities and H1d, H1e and H1° the highest affinities. Subtype affinities for chromatin differed by up to 19-fold. The relative affinities of the subtypes for chromatin were equivalent to those estimated for a SAR DNA fragment and a pUC19 fragment of similar length. Avian H5 had an affinity ~12-fold higher than H1e for both DNA and chromatin. Conclusion H1 subtypes freely exchange in vitro between chromatin binding sites in physiological salt (0.14 M NaCl. The large differences in relative affinity of the H1 subtypes for

  4. Chromatin and epigenetics in all their states: Meeting report of the first conference on Epigenetic and Chromatin Regulation of Plant Traits - January 14 - 15, 2016 - Strasbourg, France.

    Science.gov (United States)

    Bey, Till; Jamge, Suraj; Klemme, Sonja; Komar, Dorota Natalia; Le Gall, Sabine; Mikulski, Pawel; Schmidt, Martin; Zicola, Johan; Berr, Alexandre

    2016-08-01

    In January 2016, the first Epigenetic and Chromatin Regulation of Plant Traits conference was held in Strasbourg, France. An all-star lineup of speakers, a packed audience of 130 participants from over 20 countries, and a friendly scientific atmosphere contributed to make this conference a meeting to remember. In this article we summarize some of the new insights into chromatin, epigenetics, and epigenomics research and highlight nascent ideas and emerging concepts in this exciting area of research. PMID:27184433

  5. A Testis-Specific Chaperone and the Chromatin Remodeler ISWI Mediate Repackaging of the Paternal Genome

    Directory of Open Access Journals (Sweden)

    Cécile M. Doyen

    2015-11-01

    Full Text Available During spermatogenesis, the paternal genome is repackaged into a non-nucleosomal, highly compacted chromatin structure. Bioinformatic analysis revealed that Drosophila sperm chromatin proteins are characterized by a motif related to the high-mobility group (HMG box, which we termed male-specific transcript (MST-HMG box. MST77F is a MST-HMG-box protein that forms an essential component of sperm chromatin. The deposition of MST77F onto the paternal genome requires the chaperone function of tNAP, a testis-specific NAP protein. MST77F, in turn, enables the stable incorporation of MST35Ba and MST35Bb into sperm chromatin. Following MST-HMG-box protein deposition, the ATP-dependent chromatin remodeler ISWI mediates the appropriate organization of sperm chromatin. Conversely, at fertilization, maternal ISWI targets the paternal genome and drives its repackaging into de-condensed nucleosomal chromatin. Failure of this transition in ISWI mutant embryos is followed by mitotic defects, aneuploidy, and haploid embryonic divisions. Thus, ISWI enables bi-directional transitions between two fundamentally different forms of chromatin.

  6. Phytochrome B and histone deacetylase 6 control light-induced chromatin compaction in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Federico Tessadori

    2009-09-01

    Full Text Available Natural genetic variation in Arabidopsis thaliana exists for many traits and often reflects acclimation to local environments. Studying natural variation has proven valuable in the characterization of phenotypic traits and, in particular, in identifying genetic factors controlling these traits. It has been previously shown that chromatin compaction changes during development and biotic stress. To gain more insight into the genetic control of chromatin compaction, we investigated the nuclear phenotype of 21 selected Arabidopsis accessions from different geographic origins and habitats. We show natural variation in chromatin compaction and demonstrate a positive correlation with latitude of geographic origin. The level of compaction appeared to be dependent on light intensity. A novel approach, combining Quantitative Trait Locus (QTL mapping and microscopic examination, pointed at PHYTOCHROME-B (PHYB and HISTONE DEACETYLASE-6 (HDA6 as positive regulators of light-controlled chromatin compaction. Indeed, mutant analyses demonstrate that both factors affect global chromatin organization. HDA6, in addition, strongly promotes the light-mediated compaction of the Nucleolar Organizing Regions (NORs. The accession Cape Verde Islands-0 (Cvi-0, which shows sequence polymorphism in the PHYB gene and in the HDA6 promotor, resembles the hda6 mutant in having reduced chromatin compaction and decreased methylation levels of DNA and histone H3K9 at the NORs. We provide evidence that chromatin organization is controlled by light intensity. We propose that chromatin plasticity is associated with acclimation of Arabidopsis to its environment. The polymorphic alleles such as PHYB and HDA6 control this process.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-05-01

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

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

    Science.gov (United States)

    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.

  9. Assembly of Two Transgenes in an Artificial Chromatin Domain Gives Highly Coordinated Expression in Tobacco

    NARCIS (Netherlands)

    Mlynárová, Ludmila; Loonen, Annelies; Mietkiewska, Elzbieta; Jansen, Ritsert C.; Nap, Jan-Peter

    2002-01-01

    The chromatin loop model predicts that genes within the same chromatin domain exhibit coordinated regulation. We here present the first direct experimental support for this model in plants. Two reporter genes, the E. coli β-glucuronidase gene and the firefly luciferase gene, driven by different prom

  10. Relationship between chromatin structure and sensitivity to molecularly targeted auger electron radiation therapy.

    NARCIS (Netherlands)

    Terry, S.Y.A.; Vallis, K.A.

    2012-01-01

    PURPOSE: The open structure of euchromatin renders it susceptible to DNA damage by ionizing radiation (IR) compared with compact heterochromatin. The effect of chromatin configuration on the efficacy of Auger electron radiotherapy was investigated. METHODS AND MATERIALS: Chromatin structure was alte

  11. Prediction of highly expressed genes in microbes based on chromatin accessibility

    DEFF Research Database (Denmark)

    Willenbrock, Hanni; Ussery, David

    2007-01-01

    BACKGROUND: It is well known that gene expression is dependent on chromatin structure in eukaryotes and it is likely that chromatin can play a role in bacterial gene expression as well. Here, we use a nucleosomal position preference measure of anisotropic DNA flexibility to predict highly expressed...

  12. Requirements for chromatin reassembly during transcriptional downregulation of a heat shock gene in S. cerevisiae

    DEFF Research Database (Denmark)

    Jensen, Mette Moesgaard; Christensen, Marianne Skovgaard; Bonven, Bjarne Juul;

    2008-01-01

    , but not Asf1p, reassociation of H3 with DNA is compromised. However, despite a lasting open chromatin structure, transcription ceases normally in the spt6 mutant. Thus, transcriptional downregulation can be uncoupled from histone redepositioning and ongoing transcription is not required to prevent chromatin...

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

    International Nuclear Information System (INIS)

    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. (perspective)

  14. Herpes simplex virus 1 induces egress channels through marginalized host chromatin.

    Science.gov (United States)

    Myllys, Markko; Ruokolainen, Visa; Aho, Vesa; Smith, Elizabeth A; Hakanen, Satu; Peri, Piritta; Salvetti, Anna; Timonen, Jussi; Hukkanen, Veijo; Larabell, Carolyn A; Vihinen-Ranta, Maija

    2016-01-01

    Lytic infection with herpes simplex virus type 1 (HSV-1) induces profound modification of the cell nucleus including formation of a viral replication compartment and chromatin marginalization into the nuclear periphery. We used three-dimensional soft X-ray tomography, combined with cryogenic fluorescence, confocal and electron microscopy, to analyse the transformation of peripheral chromatin during HSV-1 infection. Our data showed an increased presence of low-density gaps in the marginalized chromatin at late infection. Advanced data analysis indicated the formation of virus-nucleocapsid-sized (or wider) channels extending through the compacted chromatin of the host. Importantly, confocal and electron microscopy analysis showed that these gaps frequently contained viral nucleocapsids. These results demonstrated that HSV-1 infection induces the formation of channels penetrating the compacted layer of cellular chromatin and allowing for the passage of progeny viruses to the nuclear envelope, their site of nuclear egress. PMID:27349677

  15. High-Frequency Promoter Firing Links THO Complex Function to Heavy Chromatin Formation

    DEFF Research Database (Denmark)

    Mouaikel, John; Causse, Sébastien Z; Rougemaille, Mathieu;

    2013-01-01

    The THO complex is involved in transcription, genome stability, and messenger ribonucleoprotein (mRNP) formation, but its precise molecular function remains enigmatic. Under heat shock conditions, THO mutants accumulate large protein-DNA complexes that alter the chromatin density of target genes...... (heavy chromatin), defining a specific biochemical facet of THO function and a powerful tool of analysis. Here, we show that heavy chromatin distribution is dictated by gene boundaries and that the gene promoter is necessary and sufficient to convey THO sensitivity in these conditions. Single......-molecule fluorescence insitu hybridization measurements show that heavy chromatin formation correlates with an unusually high firing pace of the promoter with more than 20 transcription events per minute. Heavy chromatin formation closely follows the modulation of promoter firing and strongly correlates with polymerase...

  16. Assembly of telomeric chromatin to create ALTernative endings.

    Science.gov (United States)

    O'Sullivan, Roderick J; Almouzni, Genevieve

    2014-11-01

    Circumvention of the telomere length-dependent mechanisms that control the upper boundaries of cellular proliferation is necessary for the unlimited growth of cancer. Most cancer cells achieve cellular immortality by up-regulating the expression of telomerase to extend and maintain their telomere length. However, a small but significant number of cancers do so via the exchange of telomeric DNA between chromosomes in a pathway termed alternative lengthening of telomeres, or ALT. Although it remains to be clarified why a cell chooses the ALT pathway and how ALT is initiated, recently identified mutations in factors that shape the chromatin and epigenetic landscape of ALT telomeres are shedding light on these mechanisms. In this review, we examine these recent findings and integrate them into the current models of the ALT mechanism. PMID:25172551

  17. Quantitative Immunofluorescence Analysis of Nucleolus-Associated Chromatin.

    Science.gov (United States)

    Dillinger, Stefan; Németh, Attila

    2016-01-01

    The nuclear distribution of eu- and heterochromatin is nonrandom, heterogeneous, and dynamic, which is mirrored by specific spatiotemporal arrangements of histone posttranslational modifications (PTMs). Here we describe a semiautomated method for the analysis of histone PTM localization patterns within the mammalian nucleus using confocal laser scanning microscope images of fixed, immunofluorescence stained cells as data source. The ImageJ-based process includes the segmentation of the nucleus, furthermore measurements of total fluorescence intensities, the heterogeneity of the staining, and the frequency of the brightest pixels in the region of interest (ROI). In the presented image analysis pipeline, the perinucleolar chromatin is selected as primary ROI, and the nuclear periphery as secondary ROI. PMID:27576710

  18. Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase

    DEFF Research Database (Denmark)

    Maiolica, Alessio; de Medina-Redondo, Maria; Schoof, Erwin;

    2014-01-01

    Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr3 of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date...... protein- protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg2 and Lys4 adjacent to the Haspin phosphorylated threonine......, histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin- associated proteins and identified a Haspin...

  19. Impact of sperm DNA chromatin in the clinic.

    Science.gov (United States)

    Ioannou, Dimitrios; Miller, David; Griffin, Darren K; Tempest, Helen G

    2016-02-01

    The paternal contribution to fertilization and embryogenesis is frequently overlooked as the spermatozoon is often considered to be a silent vessel whose only function is to safely deliver the paternal genome to the maternal oocyte. In this article, we hope to demonstrate that this perception is far from the truth. Typically, infertile men have been unable to conceive naturally (or through regular IVF), and therefore, a perturbation of the genetic integrity of sperm heads in infertile males has been under-considered. The advent of intracytoplasmic sperm injection (ICSI) however has led to very successful treatment of male factor infertility and subsequent widespread use in IVF clinics worldwide. Until recently, little concern has been raised about the genetic quality of sperm in ICSI patients or the impact genetic aberrations could have on fertility and embryogenesis. This review highlights the importance of chromatin packaging in the sperm nucleus as essential for the establishment and maintenance of a viable pregnancy. PMID:26678492

  20. Interaction of maize chromatin-associated HMG proteins with mononucleosomes

    DEFF Research Database (Denmark)

    Lichota, J.; Grasser, Klaus D.

    2003-01-01

    maize HMGA and five different HMGB proteins with mononucleosomes (containing approx. 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin. The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA...... in the presence of H1 differed from that observed in the absence of H1. HMGA and the HMGB proteins bound H1-containing nucleosome particles with similar affinity. The plant HMG proteins could also bind nucleosomes that were briefly treated with trypsin (removing the N-terminal domains of the core histones......), suggesting that the histone N-termini are dispensable for HMG protein binding. In the presence of untreated nucleosomes and trypsinised nucleosomes, HMGB1 could be chemically crosslinked with a core histone, which indicates that the trypsin-resistant part of the histones within the nucleosome is the main...

  1. Chromatin Remodeling in Stem Cell Maintenance in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Lin Xu; Wen-Hui Shen

    2009-01-01

    Pluripotent stem cells are able to both self-renew and generate undifferentiated cells for the formation of new tissues and organs.In higher plants,stem cells found in the shoot apical meristem (SAM) and the root apical meristem (RAM) are origins of organogenesis occurring post-embryonically.It is important to understand how the regulation of stem cell fate is coordinated to enable the meristem to constantly generate different types of lateral organs.Much knowledge has accumulated on specific transcription factors controlling SAM and RAM activity.Here,we review recent evidences for a role of chromatin remodeling in the maintenance of stable expression states of transcription factor genes and the control of stem cell activity in Arabidopsis.

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

    Science.gov (United States)

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

    2011-07-01

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

  3. Chromatin Dynamics in Vivo: A Game of Musical Chairs

    Directory of Open Access Journals (Sweden)

    Daniël P. Melters

    2015-08-01

    Full Text Available Histones are a major component of chromatin, the nucleoprotein complex fundamental to regulating transcription, facilitating cell division, and maintaining genome integrity in almost all eukaryotes. In addition to canonical, replication-dependent histones, replication-independent histone variants exist in most eukaryotes. In recent years, steady progress has been made in understanding how histone variants assemble, their involvement in development, mitosis, transcription, and genome repair. In this review, we will focus on the localization of the major histone variants H3.3, CENP-A, H2A.Z, and macroH2A, as well as how these variants have evolved, their structural differences, and their functional significance in vivo.

  4. Snake-like chromatin in conjunctival cells of a population aged 30-60 years from Copenhagen City

    DEFF Research Database (Denmark)

    Bjerrum, Kirsten Birgitte

    1998-01-01

    ophthalmology, keratoconjunctivitis sicca, Sjögrens Syndrome, epidemiology, imprint biopsy, snake-like chromatin......ophthalmology, keratoconjunctivitis sicca, Sjögrens Syndrome, epidemiology, imprint biopsy, snake-like chromatin...

  5. C/EBP maintains chromatin accessibility in liver and facilitates glucocorticoid receptor recruitment to steroid response elements

    DEFF Research Database (Denmark)

    Grøntved, Lars; John, Sam; Baek, Songjoon;

    2013-01-01

    Mechanisms regulating transcription factor interaction with chromatin in intact mammalian tissues are poorly understood. Exploiting an adrenalectomized mouse model with depleted endogenous glucocorticoids, we monitor changes of the chromatin landscape in intact liver tissue following glucocortico...

  6. Genomic aberrations frequently alter chromatin regulatory genes in chordoma.

    Science.gov (United States)

    Wang, Lu; Zehir, Ahmet; Nafa, Khedoudja; Zhou, Nengyi; Berger, Michael F; Casanova, Jacklyn; Sadowska, Justyna; Lu, Chao; Allis, C David; Gounder, Mrinal; Chandhanayingyong, Chandhanarat; Ladanyi, Marc; Boland, Patrick J; Hameed, Meera

    2016-07-01

    Chordoma is a rare primary bone neoplasm that is resistant to standard chemotherapies. Despite aggressive surgical management, local recurrence and metastasis is not uncommon. To identify the specific genetic aberrations that play key roles in chordoma pathogenesis, we utilized a genome-wide high-resolution SNP-array and next generation sequencing (NGS)-based molecular profiling platform to study 24 patient samples with typical histopathologic features of chordoma. Matching normal tissues were available for 16 samples. SNP-array analysis revealed nonrandom copy number losses across the genome, frequently involving 3, 9p, 1p, 14, 10, and 13. In contrast, copy number gain is uncommon in chordomas. Two minimum deleted regions were observed on 3p within a ∼8 Mb segment at 3p21.1-p21.31, which overlaps SETD2, BAP1 and PBRM1. The minimum deleted region on 9p was mapped to CDKN2A locus at 9p21.3, and homozygous deletion of CDKN2A was detected in 5/22 chordomas (∼23%). NGS-based molecular profiling demonstrated an extremely low level of mutation rate in chordomas, with an average of 0.5 mutations per sample for the 16 cases with matched normal. When the mutated genes were grouped based on molecular functions, many of the mutation events (∼40%) were found in chromatin regulatory genes. The combined copy number and mutation profiling revealed that SETD2 is the single gene affected most frequently in chordomas, either by deletion or by mutations. Our study demonstrated that chordoma belongs to the C-class (copy number changes) tumors whose oncogenic signature is non-random multiple copy number losses across the genome and genomic aberrations frequently alter chromatin regulatory genes. © 2016 Wiley Periodicals, Inc.

  7. Genomic aberrations frequently alter chromatin regulatory genes in chordoma.

    Science.gov (United States)

    Wang, Lu; Zehir, Ahmet; Nafa, Khedoudja; Zhou, Nengyi; Berger, Michael F; Casanova, Jacklyn; Sadowska, Justyna; Lu, Chao; Allis, C David; Gounder, Mrinal; Chandhanayingyong, Chandhanarat; Ladanyi, Marc; Boland, Patrick J; Hameed, Meera

    2016-07-01

    Chordoma is a rare primary bone neoplasm that is resistant to standard chemotherapies. Despite aggressive surgical management, local recurrence and metastasis is not uncommon. To identify the specific genetic aberrations that play key roles in chordoma pathogenesis, we utilized a genome-wide high-resolution SNP-array and next generation sequencing (NGS)-based molecular profiling platform to study 24 patient samples with typical histopathologic features of chordoma. Matching normal tissues were available for 16 samples. SNP-array analysis revealed nonrandom copy number losses across the genome, frequently involving 3, 9p, 1p, 14, 10, and 13. In contrast, copy number gain is uncommon in chordomas. Two minimum deleted regions were observed on 3p within a ∼8 Mb segment at 3p21.1-p21.31, which overlaps SETD2, BAP1 and PBRM1. The minimum deleted region on 9p was mapped to CDKN2A locus at 9p21.3, and homozygous deletion of CDKN2A was detected in 5/22 chordomas (∼23%). NGS-based molecular profiling demonstrated an extremely low level of mutation rate in chordomas, with an average of 0.5 mutations per sample for the 16 cases with matched normal. When the mutated genes were grouped based on molecular functions, many of the mutation events (∼40%) were found in chromatin regulatory genes. The combined copy number and mutation profiling revealed that SETD2 is the single gene affected most frequently in chordomas, either by deletion or by mutations. Our study demonstrated that chordoma belongs to the C-class (copy number changes) tumors whose oncogenic signature is non-random multiple copy number losses across the genome and genomic aberrations frequently alter chromatin regulatory genes. © 2016 Wiley Periodicals, Inc. PMID:27072194

  8. Genomic Aberrations Frequently Alter Chromatin Regulatory Genes in Chordoma

    Science.gov (United States)

    Wang, Lu; Zehir, Ahmet; Nafa, Khedoudja; Zhou, Nengyi; Berger, Michael F.; Casanova, Jacklyn; Sadowska, Justyna; Lu, Chao; Allis, C. David; Gounder, Mrinal; Chandhanayingyong, Chandhanarat; Ladanyi, Marc; Boland, Patrick J; Hameed, Meera

    2016-01-01

    Chordoma is a rare primary bone neoplasm that is resistant to standard chemotherapies. Despite aggressive surgical management, local recurrence and metastasis is not uncommon. To identify the specific genetic aberrations that play key roles in chordoma pathogenesis, we utilized a genome-wide high-resolution SNP-array and next generation sequencing (NGS)-based molecular profiling platform to study 24 patient samples with typical histopathologic features of chordoma. Matching normal tissues were available for 16 samples. SNP-array analysis revealed nonrandom copy number losses across the genome, frequently involving 3, 9p, 1p, 14, 10, and 13. In contrast, copy number gain is uncommon in chordomas. Two minimum deleted regions were observed on 3p within a ~8 Mb segment at 3p21.1–p21.31, which overlaps SETD2, BAP1 and PBRM1. The minimum deleted region on 9p was mapped to CDKN2A locus at 9p21.3, and homozygous deletion of CDKN2A was detected in 5/22 chordomas (~23%). NGS-based molecular profiling demonstrated an extremely low level of mutation rate in chordomas, with an average of 0.5 mutations per sample for the 16 cases with matched normal. When the mutated genes were grouped based on molecular functions, many of the mutation events (~40%) were found in chromatin regulatory genes. The combined copy number and mutation profiling revealed that SETD2 is the single gene affected most frequently in chordomas, either by deletion or by mutations. Our study demonstrated that chordoma belongs to the C-class (copy number changes) tumors whose oncogenic signature is non-random multiple copy number losses across the genome and genomic aberrations frequently alter chromatin regulatory genes. PMID:27072194

  9. Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    Directory of Open Access Journals (Sweden)

    Sha Ky

    2010-08-01

    Full Text Available Abstract Background Tissue differentiation is accompanied by genome-wide changes in the underlying chromatin structure and dynamics, or epigenome. By controlling when, where, and what regulatory factors have access to the underlying genomic DNA, the epigenome influences the cell's transcriptome and ultimately its function. Existing genomic methods for analyzing cell-type-specific changes in chromatin generally involve two elements: (i a source for purified cells (or nuclei of distinct types, and (ii a specific treatment that partitions or degrades chromatin by activity or structural features. For many cell types of great interest, such assays are limited by our inability to isolate the relevant cell populations in an organism or complex tissue containing an intertwined mixture of other cells. This limitation has confined available knowledge of chromatin dynamics to a narrow range of biological systems (cell types that can be sorted/separated/dissected in large numbers and tissue culture models or to amalgamations of diverse cell types (tissue chunks, whole organisms. Results Transgene-driven expression of DNA/chromatin modifying enzymes provides one opportunity to query chromatin structures in expression-defined cell subsets. In this work we combine in vivo expression of a bacterial DNA adenine methyltransferase (DAM with high throughput sequencing to sample tissue-specific chromatin accessibility on a genome-wide scale. We have applied the method (DALEC: Direct Asymmetric Ligation End Capture towards mapping a cell-type-specific view of genome accessibility as a function of differentiated state. Taking advantage of C. elegans strains expressing the DAM enzyme in diverse tissues (body wall muscle, gut, and hypodermis, our efforts yield a genome-wide dataset measuring chromatin accessibility at each of 538,000 DAM target sites in the C. elegans (diploid genome. Conclusions Validating the DALEC mapping results, we observe a strong association

  10. Plasticity of Fission Yeast CENP-A Chromatin Driven by Relative Levels of Histone H3 and H4

    OpenAIRE

    Castillo, Araceli G.; Mellone, Barbara G; Partridge, Janet F; William Richardson; Hamilton, Georgina L.; Allshire, Robin C.; Pidoux, Alison L.

    2007-01-01

    The histone H3 variant CENP-A assembles into chromatin exclusively at centromeres. The process of CENP-A chromatin assembly is epigenetically regulated. Fission yeast centromeres are composed of a central kinetochore domain on which CENP-A chromatin is assembled, and this is flanked by heterochromatin. Marker genes are silenced when placed within kinetochore or heterochromatin domains. It is not known if fission yeast CENP-A(Cnp1) chromatin is confined to specific sequences or whether histone...

  11. Snake-like chromatin in conjunctival cells of normal elderly persons and of patients with primary Sjögren's syndrome and other connective tissue diseases

    DEFF Research Database (Denmark)

    Bjerrum, Kirsten Birgitte

    1995-01-01

    Ophthalmology, snake-like chromatin, cytoplasm ratio, keratoconjunctivitis sicca, nucleus, goblet cell......Ophthalmology, snake-like chromatin, cytoplasm ratio, keratoconjunctivitis sicca, nucleus, goblet cell...

  12. Organophosphorous pesticide exposure alters sperm chromatin structure in Mexican agricultural workers

    International Nuclear Information System (INIS)

    Our objective was to evaluate alterations in sperm chromatin structure in men occupationally exposed to a mixture of organophosphorus pesticides (OP) because these alterations have been proposed to compromise male fertility and offspring development. Chromatin susceptibility to in situ acid-induced denaturation structure was assessed by the sperm chromatin structure assay (SCSA). Urinary levels of alkylphosphates (DAP) were used to assess exposure. Diethylthiophosphate (DETP) was the most frequent OP metabolite found in urine samples indicating that compounds derived from thiophosphoric acid were mainly used. Chromatin structure was altered in most samples. About 75% of semen samples were classified as having poor fertility potential (>30% of Percentage of DNA Fragmentation Index [DFI%]), whereas individuals without OP occupational exposure showed average DFI% values of 9.9%. Most parameters of conventional semen analysis were within normality except for the presence of immature cells (IGC) in which 82% of the samples were above reference values. There were significant direct associations between urinary DETP concentrations and mean DFI and SD-DFI but marginally (P = 0.079) with DFI%, after adjustment for potential confounders, including IGC. This suggests that OP exposure alters sperm chromatin condensation, which could be reflected in an increased number of cells with greater susceptibility to DNA denaturation. This study showed that human sperm chromatin is a sensitive target to OP exposure and may contribute to adverse reproductive outcomes. Further studies on the relevance of protein phosphorylation as a possible mechanism by which OP alter sperm chromatin are required

  13. SIRT6 recruits SNF2H to DNA break sites, preventing genomic instability through chromatin remodeling.

    Science.gov (United States)

    Toiber, Debra; Erdel, Fabian; Bouazoune, Karim; Silberman, Dafne M; Zhong, Lei; Mulligan, Peter; Sebastian, Carlos; Cosentino, Claudia; Martinez-Pastor, Barbara; Giacosa, Sofia; D'Urso, Agustina; Näär, Anders M; Kingston, Robert; Rippe, Karsten; Mostoslavsky, Raul

    2013-08-22

    DNA damage is linked to multiple human diseases, such as cancer, neurodegeneration, and aging. Little is known about the role of chromatin accessibility in DNA repair. Here, we find that the deacetylase sirtuin 6 (SIRT6) is one of the earliest factors recruited to double-strand breaks (DSBs). SIRT6 recruits the chromatin remodeler SNF2H to DSBs and focally deacetylates histone H3K56. Lack of SIRT6 and SNF2H impairs chromatin remodeling, increasing sensitivity to genotoxic damage and recruitment of downstream factors such as 53BP1 and breast cancer 1 (BRCA1). Remarkably, SIRT6-deficient mice exhibit lower levels of chromatin-associated SNF2H in specific tissues, a phenotype accompanied by DNA damage. We demonstrate that SIRT6 is critical for recruitment of a chromatin remodeler as an early step in the DNA damage response, indicating that proper unfolding of chromatin plays a rate-limiting role. We present a unique crosstalk between a histone modifier and a chromatin remodeler, regulating a coordinated response to prevent DNA damage.

  14. Chromatin Targeting of de Novo DNA Methyltransferases by the PWWP Domain

    Institute of Scientific and Technical Information of China (English)

    Ying-ZiGe; Min-TiePu; HumairaGowher; Hai-PingWu; Jian-PingDing; AlbertJeltsch; Guo-LiangXu

    2005-01-01

    DNA methylation patterns of mammalian genomes are generated in gametogenesis and early embryonic development. Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the process. Both en-zymes contain a long N-terminal regulatory region linked to a conserved C-terminal domain responsible forthe catalytic activity. Although a PWWP domain in the N-terminal region has been shown to bind DNA in vitro, it is unclear how the DNA methyltransferases access their substrate in chromatin in vivo. We show here that the two proteins are associated with chromatin including mitotic chromosomes in mammalian cells, and the PWWP domain is essential for the chromatin targeting of the enzymes. The functional significance of PWWPmediated chromatin targeting is suggested by the fact that a missense mutation in this domain of human DNMT3B causes immunodeficiency, centromeric heterochromatin instability, facial anomalies (ICF) syndrome, which is characterized by loss of methylation insatellite DNA, pericentromeric instability, and immunodeficiency. We demonstrate that the mutant protein completely loses its chromatin targeting capacity. Our data establish the PWWP domain as a novel chromatin/chromosome-targeting module and suggest that the PWWP-mediated chromatin association is essential for the function of the de novo methyltransferases during development.

  15. Non coding RNA: sequence-specific guide for chromatin modification and DNA damage signaling

    Directory of Open Access Journals (Sweden)

    Sofia eFrancia

    2015-11-01

    Full Text Available Chromatin conformation shapes the environment in which our genome is transcribed into RNA. Transcription is a source of DNA damage, thus it often occurs concomitantly to DNA damage signaling. Growing amounts of evidence suggest that different types of RNAs can, independently from their protein-coding properties, directly affect chromatin conformation, transcription and splicing, as well as promote the activation of the DNA damage response (DDR and DNA repair. Therefore, transcription paradoxically functions to both threaten and safeguard genome integrity. On the other hand, DNA damage signaling is known to modulate chromatin to suppress transcription of the surrounding genetic unit. It is thus intriguing to understand how transcription can modulate DDR signaling while, in turn, DDR signaling represses transcription of chromatin around the DNA lesion. An unexpected player in this field is the RNA interference (RNAi machinery, which play roles in transcription, splicing and chromatin modulation in several organisms. Non-coding RNAs (ncRNAs and several protein factors involved in the RNAi pathway are well known master regulators of chromatin while only recent reports suggest that ncRNAs are involved in DDR signaling and homology-mediated DNA repair. Here, we discuss the experimental evidence supporting the idea that ncRNAs act at the genomic loci from which they are transcribed to modulate chromatin, DDR signaling and DNA repair.

  16. Interaction of the Arabidopsis UV-B-Specific Signaling Component UVR8 with Chromatin

    Institute of Scientific and Technical Information of China (English)

    Catherine Cloix; Gareth I.Jenkins

    2008-01-01

    Arabidopsis UV RESISTANCE LOCUS8 (UVR8) is a UV-B-specific signaling component that regulates expression of a range of genes concerned with UV protection. Here, we investigate the interaction of UVR8 with chromatin. Using antibodies specific to UVR8 in chromatin immunoprecipitation (CHIP) assays with wild-type plants, we show that native UVR8 binds to chromatin in vivo. Similar experiments using an anti-GFP antibody with plants expressing a GFP-UVR8 fusion show that UVR8 associates with a relatively small region of chromatin containing the HY5 gene. UVR8 interacts with chromatin containing the promoter regions of other genes, but not with all the genes it regulates. UV-B is not required for the interaction of UVR8 with chromatin because association with several gene loci is observed in the absence of UV-B. Pulldown assays demonstrate that UVR8 associates with histones in vivo and competition experiments indicate that the interaction is preferentially with histone H2B. ChIP experiments using antibodies that recognize specific histone modifications indicate that the UV-B-stimulated transcription of some genes may be correlated with histone modification. In particular, the ELIP1 promoter showed a significant enrichment of diacetyl histone H3 (K9/K14) following UV-B exposure.These findings increase understanding of the interaction of the key UV-B-specific regulator UVR8 with chromatin.

  17. Sense and antisense transcription are associated with distinct chromatin architectures across genes.

    Science.gov (United States)

    Murray, Struan C; Haenni, Simon; Howe, Françoise S; Fischl, Harry; Chocian, Karolina; Nair, Anitha; Mellor, Jane

    2015-09-18

    Genes from yeast to mammals are frequently subject to non-coding transcription of their antisense strand; however the genome-wide role for antisense transcription remains elusive. As transcription influences chromatin structure, we took a genome-wide approach to assess which chromatin features are associated with nascent antisense transcription, and contrast these with features associated with nascent sense transcription. We describe a distinct chromatin architecture at the promoter and gene body specifically associated with antisense transcription, marked by reduced H2B ubiquitination, H3K36 and H3K79 trimethylation and increased levels of H3 acetylation, chromatin remodelling enzymes, histone chaperones and histone turnover. The difference in sense transcription between genes with high or low levels of antisense transcription is slight; thus the antisense transcription-associated chromatin state is not simply analogous to a repressed state. Using mutants in which the level of antisense transcription is reduced at GAL1, or altered genome-wide, we show that non-coding transcription is associated with high H3 acetylation and H3 levels across the gene, while reducing H3K36me3. Set1 is required for these antisense transcription-associated chromatin changes in the gene body. We propose that nascent antisense and sense transcription have fundamentally distinct relationships with chromatin, and that both should be considered canonical features of eukaryotic genes.

  18. Non-Coding RNA: Sequence-Specific Guide for Chromatin Modification and DNA Damage Signaling.

    Science.gov (United States)

    Francia, Sofia

    2015-01-01

    Chromatin conformation shapes the environment in which our genome is transcribed into RNA. Transcription is a source of DNA damage, thus it often occurs concomitantly to DNA damage signaling. Growing amounts of evidence suggest that different types of RNAs can, independently from their protein-coding properties, directly affect chromatin conformation, transcription and splicing, as well as promote the activation of the DNA damage response (DDR) and DNA repair. Therefore, transcription paradoxically functions to both threaten and safeguard genome integrity. On the other hand, DNA damage signaling is known to modulate chromatin to suppress transcription of the surrounding genetic unit. It is thus intriguing to understand how transcription can modulate DDR signaling while, in turn, DDR signaling represses transcription of chromatin around the DNA lesion. An unexpected player in this field is the RNA interference (RNAi) machinery, which play roles in transcription, splicing and chromatin modulation in several organisms. Non-coding RNAs (ncRNAs) and several protein factors involved in the RNAi pathway are well known master regulators of chromatin while only recent reports show their involvement in DDR. Here, we discuss the experimental evidence supporting the idea that ncRNAs act at the genomic loci from which they are transcribed to modulate chromatin, DDR signaling and DNA repair.

  19. High-resolution mapping reveals links of HP1 with active and inactive chromatin components.

    Directory of Open Access Journals (Sweden)

    Elzo de Wit

    2007-03-01

    Full Text Available Heterochromatin protein 1 (HP1 is commonly seen as a key factor of repressive heterochromatin, even though a few genes are known to require HP1-chromatin for their expression. To obtain insight into the targeting of HP1 and its interplay with other chromatin components, we have mapped HP1-binding sites on Chromosomes 2 and 4 in Drosophila Kc cells using high-density oligonucleotide arrays and the DNA adenine methyltransferase identification (DamID technique. The resulting high-resolution maps show that HP1 forms large domains in pericentric regions, but is targeted to single genes on chromosome arms. Intriguingly, HP1 shows a striking preference for exon-dense genes on chromosome arms. Furthermore, HP1 binds along entire transcription units, except for 5' regions. Comparison with expression data shows that most of these genes are actively transcribed. HP1 target genes are also marked by the histone variant H3.3 and dimethylated histone 3 lysine 4 (H3K4me2, which are both typical of active chromatin. Interestingly, H3.3 deposition, which is usually observed along entire transcription units, is limited to the 5' ends of HP1-bound genes. Thus, H3.3 and HP1 are mutually exclusive marks on active chromatin. Additionally, we observed that HP1-chromatin and Polycomb-chromatin are nonoverlapping, but often closely juxtaposed, suggesting an interplay between both types of chromatin. These results demonstrate that HP1-chromatin is transcriptionally active and has extensive links with several other chromatin components.

  20. Structural hierarchy of chromatin in chicken erythrocyte nuclei based on small-angle neutron scattering: Fractal nature of the large-scale chromatin organization

    International Nuclear Information System (INIS)

    The chromatin organization in chicken erythrocyte nuclei was studied by small-angle neutron scattering in the scattering-vector range from 1.5 x 10-1 to 10-4 A-1 with the use of the contrast-variation technique. This scattering-vector range corresponds to linear dimensions from 4 nm to 6 μm and covers the whole hierarchy of chromatin structures, from the nucleosomal structure to the entire nucleus. The results of the present study allowed the following conclusions to be drawn: (1) both the chromatin-protein structure and the structure of the nucleic acid component in chicken erythrocyte nuclei have mass-fractal properties, (2) the structure of the protein component of chromatin exhibits a fractal behavior on scales extending over two orders of magnitude, from the nucleosomal size to the size of an entire nucleus, and (3) the structure of the nucleic acid component of chromatin in chicken erythrocyte nuclei is likewise of a fractal nature and has two levels of organization or two phases with the crossover point at about 300-400 nm

  1. To spread or not to spread - chromatin modifications in response to DNA damage

    DEFF Research Database (Denmark)

    Altmeyer, M.; Lukas, J.

    2013-01-01

    Chromatin modifications in response to DNA damage are vital for genome integrity. Multiple proteins and pathways required to generate specialized chromatin domains around DNA lesions have been identified and the increasing amount of information calls for unifying concepts that would allow us...... to grasp the ever-increasing complexity. This review aims at contributing to this trend by focusing on feed-forward and feedback mechanisms, which in mammalian cells determine the extent of chromatin modifications after DNA damage. We highlight the emerging notion that the nodal points of these highly...

  2. Effective chromosome pairing requires chromatin remodeling at the onset of meiosis

    Science.gov (United States)

    Colas, Isabelle; Shaw, Peter; Prieto, Pilar; Wanous, Michael; Spielmeyer, Wolfgang; Mago, Rohit; Moore, Graham

    2008-01-01

    During meiosis, homologous chromosomes (homologues) recognize each other and then intimately associate. Studies exploiting species with large chromosomes reveal that chromatin is remodeled at the onset of meiosis before this intimate association. However, little is known about the effect the remodeling has on pairing. We show here in wheat that chromatin remodeling of homologues can only occur if they are identical or nearly identical. Moreover, a failure to undergo remodeling results in reduced pairing between the homologues. Thus, chromatin remodeling at the onset of meiosis enables the chromosomes to become competent to pair and recombine efficiently. PMID:18417451

  3. Genome-Wide Chromatin Immunoprecipitation in Candida albicans and Other Yeasts

    Science.gov (United States)

    Lohse, Matthew B.; Kongsomboonvech, Pisiwat; Madrigal, Maria; Hernday, Aaron D.; Nobile, Clarissa J.

    2016-01-01

    Chromatin immunoprecipitation experiments are critical to investigating the interactions between DNA and a wide range of nuclear proteins within a cell or biological sample. In this chapter we outline an optimized protocol for genome-wide chromatin immunoprecipitation that has been used successfully for several distinct morphological forms of numerous yeast species, and include an optimized method for amplification of chromatin immunoprecipitated DNA samples and hybridization to a high-density oligonucleotide tiling microarray. We also provide detailed suggestions on how to analyze the complex data obtained from these experiments. PMID:26483022

  4. Nucleosomal organization of chromatin in sperm nuclei of the bivalve mollusc Aulacomya ater.

    Science.gov (United States)

    Olivares, C; Ruiz, S

    1991-03-13

    The sperm nuclei of Aulacomya ater, family Mitylidae, contain three proteins (X, Aa5 and Aa6) which are specific to this cell type coexisting with a set of five somatic-type histones. Information about the chromatin structure resulting from this kind of association is scarce. Therefore, we have probed the structure of this sperm chromatin through digestion with micrococcal nuclease in combination with salt fractionation. The data obtained have allowed us to propose a nucleosomal arrangement for this chromatin. However, two types of nucleosomes would be present in agreement with their protein components. PMID:1861676

  5. Data on force-dependent structural changes of chromatin fibers measured with magnetic tweezers

    Directory of Open Access Journals (Sweden)

    Fan-Tso Chien

    2014-12-01

    Full Text Available The compaction of chromatin fibers regulates the accessibility of embedded DNA, highly associated with transcriptional activities [1]. Single molecule force spectroscopy has revealed the great details of the structural changes of chromatin fibers in the presence of external exerted force [2–7]. However, most of the studies focus on a specific force regime [2,3,8,9]. The data here show force-extension (FE traces of chromatin fibers as measured with magnetic tweezers, covering the force regime from 0 pN to 27 pN. Those traces provide information for further studies at varied force regimes.

  6. Tagging of MADS domain proteins for chromatin immunoprecipitation

    Directory of Open Access Journals (Sweden)

    van Zuijlen Lisette GC

    2007-09-01

    Full Text Available Abstract Background Most transcription factors fulfill their role in complexes and regulate their target genes upon binding to DNA motifs located in upstream regions or introns. To date, knowledge about transcription factor target genes and their corresponding transcription factor binding sites are still very limited. Two related methods that allow in vivo identification of transcription factor binding sites are chromatin immunoprecipitation (ChIP and chromatin affinity purification (ChAP. For ChAP, the protein of interest is tagged with a peptide or protein, which can be used for affinity purification of the protein-DNA complex and hence, the identification of the target gene. Results Here, we present the results of experiments aiming at the development of a generic tagging approach for the Arabidopsis MADS domain proteins AGAMOUS, SEPALLATA3, and FRUITFULL. For this, Arabidopsis wild type plants were transformed with constructs containing a MADS-box gene fused to either a double Strep-tag® II-FLAG-tag, a triple HA-tag, or an eGFP-tag, all under the control of the constitutive double 35S Cauliflower Mosaic Virus (CaMV promoter. Strikingly, in all cases, the number of transformants with loss-of-function phenotypes was much larger than those with an overexpression phenotype. Using endogenous promoters in stead of the 35S CaMV resulted in a dramatic reduction in the frequency of loss-of-function phenotypes. Furthermore, pleiotropic defects occasionally caused by an overexpression strategy can be overcome by using the native promoter of the gene. Finally, a ChAP result is presented using GFP antibody on plants carrying a genomic fragment of a MADS-box gene fused to GFP. Conclusion This study revealed that MADS-box proteins are very sensitive to fusions with small peptide tags and GFP tags. Furthermore, for the expression of chimeric versions of MADS-box genes it is favorable to use the entire genomic region in frame to the tag of choice

  7. Circulating chromatin-anti-chromatin antibody complexes bind with high affinity to dermo-epidermal structures in murine and human lupus nephritis

    DEFF Research Database (Denmark)

    Fismen, S; Hedberg, A; Fenton, K A;

    2009-01-01

    Murine and human lupus nephritis are characterized by glomerular deposits of electron-dense structures (EDS). Dominant components of EDS are chromatin fragments and IgG antibodies. Whether glomerular EDS predispose for similar deposits in skin is unknown. We analysed (i) whether dermo...... (NZBxNZW)F1 and MRL-lpr/lpr mice and from five patients with lupus nephritis were analysed by immunofluorescence, immune electron microscopy (IEM) and co-localization TUNEL IEM. Affinity of chromatin fragments for membrane structures was determined by surface plasmon resonance. Results demonstrated (i...... were present in capillary lumina in glomeruli and skin of all nephritic individuals. Thus, chromatin-IgG complexes accounting for lupus nephritis seem to reach skin through circulation, but other undetermined factors are required for these complexes to deposit within skin membranes....

  8. Chromatin and Cell Wall Staining of Schizosaccharomyces pombe.

    Science.gov (United States)

    Hagan, Iain M

    2016-01-01

    Fission yeasts grow by tip extension, maintaining a constant width until they reach a critical size threshold and divide. Division by medial fission-which gives these yeast their name-generates a new end that arises from the site of cytokinesis. The old end, which was produced during the previous cell cycle, initiates progression of the new cell cycle, and in G2, the new end is activated in a process termed new-end takeoff (NETO). In this protocol, the fluorescent stains calcofluor and 4',6-diamidino-2-phenylindole (DAPI) are used to give a rapid and informative assessment of morphogenesis and cell-cycle progression in the fission yeast Schizosaccharomyces pombe Calcofluor reveals the timing of NETO because it stains the birth scars that are generated at new ends by cytokinesis less efficiently than the rest of the cell wall. Intense calcofluor staining of the septum and measurement of cell length are also widely used to identify dividing cells and to gauge the timing of mitotic commitment. Staining nuclei with DAPI identifies mono- and binucleated cells and complements the calcofluor staining procedure to evaluate the stages of the cell cycle and identify mitotic errors. Equally simple DAPI staining procedures reveal chromatin structure in higher resolution, facilitating more accurate staging of mitotic progression and characterization of mitotic errors. PMID:27250942

  9. Cracking the chromatin code: Precise rule of nucleosome positioning

    Science.gov (United States)

    Trifonov, Edward N.

    2011-03-01

    Various aspects of packaging DNA in eukaryotic cells are outlined in physical rather than biological terms. The informational and physical nature of packaging instructions encoded in DNA sequences is discussed with the emphasis on signal processing difficulties - very low signal-to-noise ratio and high degeneracy of the nucleosome positioning signal. As the author has been contributing to the field from its very onset in 1980, the review is mostly focused at the works of the author and his colleagues. The leading concept of the overview is the role of deformational properties of DNA in the nucleosome positioning. The target of the studies is to derive the DNA bendability matrix describing where along the DNA various dinucleotide elements should be positioned, to facilitate its bending in the nucleosome. Three different approaches are described leading to derivation of the DNA deformability sequence pattern, which is a simplified linear presentation of the bendability matrix. All three approaches converge to the same unique sequence motif CGRAAATTTYCG or, in binary form, YRRRRRYYYYYR, both representing the chromatin code.

  10. HJURP is involved in the expansion of centromeric chromatin.

    Science.gov (United States)

    Perpelescu, Marinela; Hori, Tetsuya; Toyoda, Atsushi; Misu, Sadahiko; Monma, Norikazu; Ikeo, Kazuho; Obuse, Chikashi; Fujiyama, Asao; Fukagawa, Tatsuo

    2015-08-01

    The CENP-A-specific chaperone HJURP mediates CENP-A deposition at centromeres. The N-terminal region of HJURP is responsible for binding to soluble CENP-A. However, it is unclear whether other regions of HJURP have additional functions for centromere formation and maintenance. In this study, we generated chicken DT40 knockout cell lines and gene replacement constructs for HJURP to assess the additional functions of HJURP in vivo. Our analysis revealed that the middle region of HJURP associates with the Mis18 complex protein M18BP1/KNL2 and that the HJURP-M18BP1 association is required for HJURP function. In addition, on the basis of the analysis of artificial centromeres induced by ectopic HJURP localization, we demonstrate that HJURP exhibits a centromere expansion activity that is separable from its CENP-A-binding activity. We also observed centromere expansion surrounding natural centromeres after HJURP overexpression. We propose that this centromere expansion activity reflects the functional properties of HJURP, which uses this activity to contribute to the plastic establishment of a centromeric chromatin structure. PMID:26063729

  11. Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

    International Nuclear Information System (INIS)

    TIP48 is a highly conserved eukaryotic AAA+ 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

  12. First Exon Length Controls Active Chromatin Signatures and Transcription

    Directory of Open Access Journals (Sweden)

    Nicole I. Bieberstein

    2012-07-01

    Full Text Available Here, we explore the role of splicing in transcription, employing both genome-wide analysis of human ChIP-seq data and experimental manipulation of exon-intron organization in transgenic cell lines. We show that the activating histone modifications H3K4me3 and H3K9ac map specifically to first exon-intron boundaries. This is surprising, because these marks help recruit general transcription factors (GTFs to promoters. In genes with long first exons, promoter-proximal levels of H3K4me3 and H3K9ac are greatly reduced; consequently, GTFs and RNA polymerase II are low at transcription start sites (TSSs and exhibit a second, promoter-distal peak from which transcription also initiates. In contrast, short first exons lead to increased H3K4me3 and H3K9ac at promoters, higher expression levels, accuracy in TSS usage, and a lower frequency of antisense transcription. Therefore, first exon length is predictive for gene activity. Finally, splicing inhibition and intron deletion reduce H3K4me3 levels and transcriptional output. Thus, gene architecture and splicing determines transcription quantity and quality as well as chromatin signatures.

  13. Dual Chromatin and Cytoskeletal Remodeling by SETD2.

    Science.gov (United States)

    Park, In Young; Powell, Reid T; Tripathi, Durga Nand; Dere, Ruhee; Ho, Thai H; Blasius, T Lynne; Chiang, Yun-Chen; Davis, Ian J; Fahey, Catherine C; Hacker, Kathryn E; Verhey, Kristen J; Bedford, Mark T; Jonasch, Eric; Rathmell, W Kimryn; Walker, Cheryl Lyn

    2016-08-11

    Posttranslational modifications (PTMs) of tubulin specify microtubules for specialized cellular functions and comprise what is termed a "tubulin code." PTMs of histones comprise an analogous "histone code," although the "readers, writers, and erasers" of the cytoskeleton and epigenome have heretofore been distinct. We show that methylation is a PTM of dynamic microtubules and that the histone methyltransferase SET-domain-containing 2 (SETD2), which is responsible for H3 lysine 36 trimethylation (H3K36me3) of histones, also methylates α-tubulin at lysine 40, the same lysine that is marked by acetylation on microtubules. Methylation of microtubules occurs during mitosis and cytokinesis and can be ablated by SETD2 deletion, which causes mitotic spindle and cytokinesis defects, micronuclei, and polyploidy. These data now identify SETD2 as a dual-function methyltransferase for both chromatin and the cytoskeleton and show a requirement for methylation in maintenance of genomic stability and the integrity of both the tubulin and histone codes. PMID:27518565

  14. Chromatin remodeling and stem cell theory of relativity.

    Science.gov (United States)

    Cerny, Jan; Quesenberry, Peter J

    2004-10-01

    The field of stem cell biology is currently being redefined. Stem cell (hematopoietic and non-hematopoietic) differentiation has been considered hierarchical in nature, but recent data suggest that there is no progenitor/stem cell hierarchy, but rather a reversible continuum. The stem cell (hematopoietic and non-hematopoietic) phenotype, the total differentiation capacity (hematopoietic and non-hematopoietic), gene expression as well as other stem cell functional characteristics (homing, receptor and adhesion molecule expression) vary throughout a cell-cycle transit widely. This seems to be dependent on shifting chromatin and gene expression with cell-cycle transit. The published data on DNA methylation, histone acetylation, and also RNAi, the major regulators of gene expression, conjoins very well and provides an explanation for the major issues of stem cell biology. Those features of stem cells mentioned above can be rather difficult to apprehend when a classical hierarchy biology view is applied, but they become clear and easier to understand once they are correlated with the underlining epigenetic changes. We are entering a new era of stem cell biology the era of "chromatinomics." We are one step closer to the practical use of cellular therapy for degenerative diseases.

  15. Shedding Light on Large-Scale Chromatin Reorganization in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Martijn van Zanten; Federico Tessadori; Anton J.M. Peeters; Paul Fransz

    2012-01-01

    Plants need to respond quickly and appropriately to various types of light signals from the environment to optimize growth and development.The immediate response to shading,reduced photon flux (low light),and changes in spectral quality involves changes in gene regulation.In the case of more persistent shade,the plant shows a dramatic change in the organization of chromatin.Both plant responses are controlled via photoreceptor signaling proteins.Recently,several studies have revealed similar features of chromatin reorganization in response to various abiotic and biotic signals,while others have unveiled intricate molecular networks of light signaling towards gene regulation.This opinion paper briefly describes the chromatin (de)compaction response from a light-signaling perspective to provide a link between chromatin and the molecular network of photoreceptors and E3 ubiquitin ligase complexes.

  16. Protocol: fine-tuning of a Chromatin Immunoprecipitation (ChIP protocol in tomato

    Directory of Open Access Journals (Sweden)

    Iusem Norberto D

    2010-04-01

    Full Text Available Abstract Background Searching thoroughly for plant cis-elements corresponding to transcription factors is worthwhile to reveal novel gene activation cascades. At the same time, a great deal of research is currently focused on epigenetic events in plants. A widely used method serving both purposes is chromatin immunoprecipitation, which was developed for Arabidopsis and other plants but is not yet operational for tomato (Solanum lycopersicum, a model plant species for a group of economically important crops. Results We developed a chromatin immunoprecipitation protocol suitable for tomato by adjusting the parameters to optimise in vivo crosslinking, purification of nuclei, chromatin extraction, DNA shearing and precipitate analysis using real-time PCR. Results were obtained with two different antibodies, five control loci and two normalisation criteria. Conclusion Here we provide a chromatin immunoprecipitation procedure for tomato leaves that could be combined with high-throughput sequencing to generate a detailed map of epigenetic modifications or genome-wide nucleosome positioning data.

  17. Discovery and Characterization of Chromatin States for Systematic Annotation of the Human Genome

    Science.gov (United States)

    Ernst, Jason; Kellis, Manolis

    A plethora of epigenetic modifications have been described in the human genome and shown to play diverse roles in gene regulation, cellular differentiation and the onset of disease. Although individual modifications have been linked to the activity levels of various genetic functional elements, their combinatorial patterns are still unresolved and their potential for systematic de novo genome annotation remains untapped. Here, we use a multivariate Hidden Markov Model to reveal chromatin states in human T cells, based on recurrent and spatially coherent combinations of chromatin marks.We define 51 distinct chromatin states, including promoter-associated, transcription-associated, active intergenic, largescale repressed and repeat-associated states. Each chromatin state shows specific enrichments in functional annotations, sequence motifs and specific experimentally observed characteristics, suggesting distinct biological roles. This approach provides a complementary functional annotation of the human genome that reveals the genome-wide locations of diverse classes of epigenetic function.

  18. Identification of noncoding transcripts from within CENP-A chromatin at fission yeast centromeres.

    Science.gov (United States)

    Choi, Eun Shik; Strålfors, Annelie; Castillo, Araceli G; Durand-Dubief, Mickaël; Ekwall, Karl; Allshire, Robin C

    2011-07-01

    The histone H3 variant CENP-A is the most favored candidate for an epigenetic mark that specifies the centromere. In fission yeast, adjacent heterochromatin can direct CENP-A(Cnp1) chromatin establishment, but the underlying features governing where CENP-A(Cnp1) chromatin assembles are unknown. We show that, in addition to centromeric regions, a low level of CENP-A(Cnp1) associates with gene promoters where histone H3 is depleted by the activity of the Hrp1(Chd1) chromatin-remodeling factor. Moreover, we demonstrate that noncoding RNAs are transcribed by RNA polymerase II (RNAPII) from CENP-A(Cnp1) chromatin at centromeres. These analyses reveal a similarity between centromeres and a subset of RNAPII genes and suggest a role for remodeling at RNAPII promoters within centromeres that influences the replacement of histone H3 with CENP-A(Cnp1). PMID:21531710

  19. Physiological and Pathological Aging Affects Chromatin Dynamics, Structure and Function at the Nuclear Edge.

    Science.gov (United States)

    Robin, Jérôme D; Magdinier, Frédérique

    2016-01-01

    Lamins are intermediate filaments that form a complex meshwork at the inner nuclear membrane. Mammalian cells express two types of Lamins, Lamins A/C and Lamins B, encoded by three different genes, LMNA, LMNB1, and LMNB2. Mutations in the LMNA gene are associated with a group of phenotypically diverse diseases referred to as laminopathies. Lamins interact with a large number of binding partners including proteins of the nuclear envelope but also chromatin-associated factors. Lamins not only constitute a scaffold for nuclear shape, rigidity and resistance to stress but also contribute to the organization of chromatin and chromosomal domains. We will discuss here the impact of A-type Lamins loss on alterations of chromatin organization and formation of chromatin domains and how disorganization of the lamina contributes to the patho-physiology of premature aging syndromes. PMID:27602048

  20. The RSC and INO80 chromatin-remodeling complexes in DNA double-strand break repair.

    Science.gov (United States)

    Chambers, Anna L; Downs, Jessica A

    2012-01-01

    In eukaryotes, DNA is packaged into chromatin and is therefore relatively inaccessible to DNA repair enzymes. In order to perform efficient DNA repair, ATP-dependent chromatin-remodeling enzymes are required to alter the chromatin structure near the site of damage to facilitate processing and allow access to repair enzymes. Two of the best-studied remodeling complexes involved in repair are RSC (Remodels the Structure of Chromatin) and INO80 from Saccharomyces cerevisiae, which are both conserved in higher eukaryotes. RSC is very rapidly recruited to breaks and mobilizes nucleosomes to promote phosphorylation of H2A S129 and resection. INO80 enrichment at a break occurs later and is dependent on phospho-S129 H2A. INO80 activity at the break site also facilitates resection. Consequently, both homologous recombination and nonhomologous end-joining are defective in rsc mutants, while subsets of these repair pathways are affected in ino80 mutants.

  1. Till disassembly do us part: a happy marriage of nuclear envelope and chromatin.

    Science.gov (United States)

    Tsuchiya, Yuichi

    2008-02-01

    A characteristic feature of eukaryotic cells is the presence of nuclear envelope (NE) which separates genomic DNA from cytoplasm. NE is composed of inner nuclear membrane (INM), which interacts with chromatin, and outer nuclear membrane, which is connected to endoplasmic reticulum. Nuclear pore complexes are inserted into NE to form transport channels between nucleus and cytoplasm. In metazoan cells, an intermediate filament-based meshwork called as nuclear lamina exists between INM and chromatin. Sophisticated collaboration of these molecular machineries is necessary for the structure and functions of NE. Recent research advances have revealed that NE dynamically communicates with chromatin and cytoskeleton to control multiple nuclear functions. In this mini review, I briefly summarize the basic concepts and current topics of functional relationships between NE and chromatin.

  2. Active remodeling of chromatin and implications for in-vivo folding

    CERN Document Server

    Ramakrishnan, N; Kuttippurathu, Lakshmi; Kumar, P B Sunil; Rao, Madan

    2015-01-01

    Recent high resolution experiments have provided a quantitative description of the statistical properties of interphase chromatin at large scales. These findings have stimulated a search for generic physical interactions that give rise to such specific statistical conformations. Here, we show that an active chromatin model of in-vivo folding, based on the interplay between polymer elasticity, confinement, topological constraints and active stresses arising from the (un)binding of ATP-dependent chromatin-remodeling proteins gives rise to steady state conformations consistent with these experiments. Our results lead us to conjecture that the chromatin conformation resulting from this active folding optimizes information storage by co-locating gene loci which share transcription resources.

  3. Absence of canonical marks of active chromatin in developmentally regulated genes.

    Science.gov (United States)

    Pérez-Lluch, Sílvia; Blanco, Enrique; Tilgner, Hagen; Curado, Joao; Ruiz-Romero, Marina; Corominas, Montserrat; Guigó, Roderic

    2015-10-01

    The interplay of active and repressive histone modifications is assumed to have a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that the 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 with the stable production of RNA, whereas unmarked chromatin would permit rapid gene activation and deactivation during development. In the latter case, regulation by transcription factors would have a comparatively more important regulatory role than chromatin marks.

  4. Micro- and nanoscale devices for the investigation of epigenetics and chromatin dynamics

    Science.gov (United States)

    Aguilar, Carlos A.; Craighead, Harold G.

    2013-10-01

    Deoxyribonucleic acid (DNA) is the blueprint on which life is based and transmitted, but the way in which chromatin -- a dynamic complex of nucleic acids and proteins -- is packaged and behaves in the cellular nucleus has only begun to be investigated. Epigenetic modifications sit 'on top of' the genome and affect how DNA is compacted into chromatin and transcribed into ribonucleic acid (RNA). The packaging and modifications around the genome have been shown to exert significant influence on cellular behaviour and, in turn, human development and disease. However, conventional techniques for studying epigenetic or conformational modifications of chromosomes have inherent limitations and, therefore, new methods based on micro- and nanoscale devices have been sought. Here, we review the development of these devices and explore their use in the study of DNA modifications, chromatin modifications and higher-order chromatin structures.

  5. Sequential chromatin immunoprecipitation protocol for global analysis through massive parallel sequencing (reChIP-seq)

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Marco Antonio Mendoza-Parra, Shankaranarayanan Pattabhiraman & Hinrich Gronemeyer ### Abstract Chromatin immunoprecipitation combined with massive parallel sequencing (ChIP-seq) is increasingly used to study protein-chromatin interactions or local epigenetic modifications at genome-wide scale. ChIP-seq can be performed directly with several ng of immunoprecipitated DNA, which is generally obtained from a several million cells, depending on the quality of the antibody. ChI...

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-09-17

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

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

    International Nuclear Information System (INIS)

    Research highlights: → An in vitro reconstitution system was established with isolated nuclei and cytoplasm. → Chromatin fluidities were measured in the system using FRAP. → Chromatin fluidities were higher in the cytoplasm of earlier-stage embryos. → Chromatin fluidities were higher in the earlier-stage nuclei with egg-extract. → 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.

  9. Chromatin remodeling of human subtelomeres and TERRA promoters upon cellular senescence: commonalities and differences between chromosomes.

    Science.gov (United States)

    Thijssen, Peter E; Tobi, Elmar W; Balog, Judit; Schouten, Suzanne G; Kremer, Dennis; El Bouazzaoui, Fatiha; Henneman, Peter; Putter, Hein; Eline Slagboom, P; Heijmans, Bastiaan T; van der Maarel, Silvère M

    2013-05-01

    Subtelomeres are patchworks of evolutionary conserved sequence blocks and harbor the transcriptional start sites for telomere repeat containing RNAs (TERRA). Recent studies suggest that the interplay between telomeres and subtelomeric chromatin is required for maintaining telomere function. To further characterize chromatin remodeling of subtelomeres in relation to telomere shortening and cellular senescence, we systematically quantified histone modifications and DNA methylation at the subtelomeres of chromosomes 7q and 11q in primary human WI-38 fibroblasts. Upon senescence, both subtelomeres were characterized by a decrease in markers of constitutive heterochromatin, suggesting relative chromatin relaxation. However, we did not find increased levels of markers of euchromatin or derepression of the 7q VIPR2 gene. The repressed state of the subtelomeres was maintained upon senescence, which could be attributed to a rise in levels of facultative heterochromatin markers at both subtelomeres. While senescence-induced subtelomeric chromatin remodeling was similar for both chromosomes, chromatin remodeling at TERRA promoters displayed chromosome-specific patterns. At the 7q TERRA promoter, chromatin structure was co-regulated with the more proximal subtelomere. In contrast, the 11q TERRA promoter, which was previously shown to be bound by CCCTC-binding factor CTCF, displayed lower levels of markers of constitutive heterochromatin that did not change upon senescence, whereas levels of markers of facultative heterochromatin decreased upon senescence. In line with the chromatin state data, transcription of 11q TERRA but not 7q TERRA was detected. Our study provides a detailed description of human subtelomeric chromatin dynamics and shows distinct regulation of the TERRA promoters of 7q and 11q upon cellular senescence.

  10. Protocol: methodology for chromatin immunoprecipitation (ChIP) in Chlamydomonas reinhardtii

    OpenAIRE

    Strenkert Daniela; Schmollinger Stefan; Schroda Michael

    2011-01-01

    Abstract We report on a detailed chromatin immunoprecipitation (ChIP) protocol for the unicellular green alga Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example f...

  11. Chromatin Adaptor Brd4 Modulates E2 Transcription Activity and Protein Stability*

    OpenAIRE

    Lee, A-Young; Chiang, Cheng-Ming

    2009-01-01

    Brd4 is a chromatin adaptor containing tandem bromodomains binding to acetylated histone H3 and H4. Although Brd4 has been implicated in the transcriptional control of papillomavirus-encoded E2 protein, it is unclear how Brd4 regulates E2 function and whether the involvement of Brd4 in transactivation and transrepression is common to different types of E2 proteins. Using DNase I footprinting performed with in vitro reconstituted human papillomavirus (HPV) chromatin and...

  12. Effect of Seminal Vesicles and Dithiotritol (Dtt on Stability of Sperm Chromatin

    Directory of Open Access Journals (Sweden)

    MH Nasr-Esfahani

    2005-04-01

    Full Text Available Introduction: Different studies have shown that there is no relation between sperm chromatin stability and fertilization rate in both IVF and ICSI patients. However, the relation between SDS tests, as a detergent, along with DTT as reducer of disulphide bridges has not been studied so far in ICSI patients. Since different concentrations of DTT can induce different degrees of sperm chromatin decondensation, the aim of this study was to evaluate the effect of different concentrations of DTT on sperm chromatin decondensation in IVF and ICSI cases. Methods: During this study, 85 patients were divided into two groups according to their treatment procedure (IVF or ICSI.Semen samples of each patient was evaluated for sperm chromatin tests including SDS, SDS+EDTA & SDS+DTT for assessment of free thiole groups level (-SH, amount of non covalent bond between Zn and thioles(-SH Zn SH- and levels of disulfide bond (-S-S- in sperm chromatin, respectively. In this study, seminal fructose concentration, corrected seminal fructose level and true corrected fructose level as indicators of seminal vesicle function on sperm chromatin stability were assessed. Results: No correlation was observed between any of the above tests and rate of fertilization, both in IVF and ICSI cases. However, in IVF patients, a significant correlation was observed between SDS, SDS+DTT test and seminal fructose level, while in ICSI patients, only a significant correlation was observed between SDS+DTT and corrected or true fructose concentration. Conclusion: Since no correlation was observed between sperm chromatin test and fertilization rate, it is suggested that the chromatin status of these samples are adequate for fertilization to take place and extent of disulphide bridges has no effect on fertilization rate. However, the amount of disulphide bound present in sperms of ICSI and IVF patients are different, and this difference is related to seminal vesicle performance in these patients.

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

    OpenAIRE

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

    2014-01-01

    International audience Proper telomeric chromatin configuration is thought to be essential for telomere homeostasis and stability. Previous studies in mouse suggested that loss of heterochromatin marks at telomeres might favor onset of Alternative Lengthening of Telomeres (ALT) pathway, by promoting homologous recombination. However, analysis of chromatin status at human ALT telomeres has never been reported. Here, using isogenic human cell lines and cellular hybrids, which rely either on ...

  14. Structure of chromatin, protein transitions, and post-translational histone modifications in several sperm models

    OpenAIRE

    Kurtz, Katryn Lucille

    2008-01-01

    [eng] The study of chromatin structure in several simple sperm models of increasing complexity was performed. Species demonstrating different types of sperm nuclear protein transitions and structural changes in spermatic chromatin during spermiogenesis were selected as models for comparison: "H" (non-histone proteins are removed), "H->P" (protamine displaces histones), and "H->Pp->P" (precursor protamine displaces histones, and subsequently is converted into the mature protamine). This study ...

  15. CTCF-cohesin complex: architect of chromatin structure regulates V(D)J rearrangement

    Institute of Scientific and Technical Information of China (English)

    Ann J Feeney; Jiyoti Verma-Gaur

    2012-01-01

    The CTCF/cohesin complex regulates higher order chromatin structure by creating long-range chromatin loops and by insulating neighboring genes from each other.The lymphocyte antigen receptor loci have large numbers of CTCF/cohesin binding sites,and recent studies demonstrate that the CTCF/cohesin complex plays several important roles in regulating the process of V(D)J recombination at these megabase-sized receptor loci.

  16. Nuclear Fractionation Reveals Thousands of Chromatin-Tethered Noncoding RNAs Adjacent to Active Genes

    Directory of Open Access Journals (Sweden)

    Michael S. Werner

    2015-08-01

    Full Text Available A number of long noncoding RNAs (lncRNAs have been reported to regulate transcription via recruitment of chromatin modifiers or bridging distal enhancer elements to gene promoters. However, the generality of these modes of regulation and the mechanisms of chromatin attachment for thousands of unstudied human lncRNAs remain unclear. To address these questions, we performed stringent nuclear fractionation coupled to RNA sequencing. We provide genome-wide identification of human chromatin-associated lncRNAs and demonstrate tethering of RNA to chromatin by RNAPII is a pervasive mechanism of attachment. We also uncovered thousands of chromatin-enriched RNAs (cheRNAs that share molecular properties with known lncRNAs. Although distinct from eRNAs derived from active prototypical enhancers, the production of cheRNAs is strongly correlated with the expression of neighboring protein-coding genes. This work provides an updated framework for nuclear RNA organization that includes a large chromatin-associated transcript population correlated with active genes and may prove useful in de novo enhancer annotation.

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

    Science.gov (United States)

    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.

  18. Advance chromatin extraction improves capture performance of protein A affinity chromatography.

    Science.gov (United States)

    Nian, Rui; Zhang, Wei; Tan, Lihan; Lee, Jeremy; Bi, Xeuzhi; Yang, Yuansheng; Gan, Hui Theng; Gagnon, Pete

    2016-01-29

    Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%. PMID:26774119

  19. Visualization of chromatin folding patterns in chicken erythrocytes by atomic force microscopy (AFM)

    Institute of Scientific and Technical Information of China (English)

    QIANRUOLAN; ZHENGXIALIU; 等

    1997-01-01

    The organization of the higher order structure of chromatin in chicken erythrocytes has been examined with tapping-mode scanning force microscopy under conditions close to their native envirinment.Reproducible highresolution AFM images of chromatin compaction at several levels can be demonstrated.An extended beads-on-astring (width of - 15-20nm,height of - 2-3nm for each individual nucleosome) can be consistently observed.Furthermore,superbeade (width of - 40nm,height of - 7nm) are demonstrated.Visualization of the solenoid conformation at the level of 30nm chromatin fiber is attained either by using AFM or by using electron microscopy.In addition,tightly coiled chromatin fibers (- 50-60nm and - 90-110nm) can be revealed.Our data suggest that the chromatin in the interphase nucleus of chicken erythrocyte represents a high-order conformation and AFM provides useful high-resolution structural information concerning the folding pattern of interphase chromatin fibers.

  20. ISWI regulates higher-order chromatin structure and histone H1 assembly in vivo.

    Directory of Open Access Journals (Sweden)

    Davide F V Corona

    2007-09-01

    Full Text Available Imitation SWI (ISWI and other ATP-dependent chromatin-remodeling factors play key roles in transcription and other processes by altering the structure and positioning of nucleosomes. Recent studies have also implicated ISWI in the regulation of higher-order chromatin structure, but its role in this process remains poorly understood. To clarify the role of ISWI in vivo, we examined defects in chromosome structure and gene expression resulting from the loss of Iswi function in Drosophila. Consistent with a broad role in transcriptional regulation, the expression of a large number of genes is altered in Iswi mutant larvae. The expression of a dominant-negative form of ISWI leads to dramatic alterations in higher-order chromatin structure, including the apparent decondensation of both mitotic and polytene chromosomes. The loss of ISWI function does not cause obvious defects in nucleosome assembly, but results in a significant reduction in the level of histone H1 associated with chromatin in vivo. These findings suggest that ISWI plays a global role in chromatin compaction in vivo by promoting the association of the linker histone H1 with chromatin.

  1. Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

    Institute of Scientific and Technical Information of China (English)

    Sung-Bau Lee; Li-Jung Juan; Chung-Fan Lee; Derick S-C Ou; Kalpana Dulal; Liang-Hao Chang; Chen-Han Ma; Chien-Fu Huang; Hua Zhu; Young-Sun Lin

    2011-01-01

    Chromatin assembly factor 1 (CAF1) consisting of p150, p60 and p48 is known to assemble histones onto newly synthesized DNA and thus maintain the chromatin structure. Here, we show that CAF1 expression was induced in human cytomegalovirus (HCMV)-infected cells, concomitantly with global chromatin decondensation. This apparent conflict was thought to result, in part, from CAF1 mislocalization to compartments of HCMV DNA synthesis through binding of its largest subunit p150 to viral immediate-early protein 2 (IE2). p150 interaction with p60 and IE2 facilitated HCMV DNA synthesis. The IE2Q548R mutation, previously reported to result in impaired HCMV growth with unknown mechanism, disrupted IE2/p150 and IE2/histones association in our study. Moreover, IE2 interaction with histones partly depends on p150, and the HCMV-induced chromatin decondensation was reduced in cells ectopically expressing the p150 mutant defective in IE2 binding. These results not only indicate that CAF1 was hijacked by IE2 to facilitate the replication of the HCMV genome, suggesting chromatin assembly plays an important role in herpesviral DNA synthesis, but also provide a model of the virus-induced chromatin instability through CAF1.

  2. Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation.

    Science.gov (United States)

    Strickfaden, Hilmar; McDonald, Darin; Kruhlak, Michael J; Haince, Jean-Francois; Th'ng, John P H; Rouleau, Michele; Ishibashi, Toytaka; Corry, Gareth N; Ausio, Juan; Underhill, D Alan; Poirier, Guy G; Hendzel, Michael J

    2016-01-22

    Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.

  3. Prediction of transposable element derived enhancers using chromatin modification profiles.

    Directory of Open Access Journals (Sweden)

    Ahsan Huda

    Full Text Available Experimentally characterized enhancer regions have previously been shown to display specific patterns of enrichment for several different histone modifications. We modelled these enhancer chromatin profiles in the human genome and used them to guide the search for novel enhancers derived from transposable element (TE sequences. To do this, a computational approach was taken to analyze the genome-wide histone modification landscape characterized by the ENCODE project in two human hematopoietic cell types, GM12878 and K562. We predicted the locations of 2,107 and 1,448 TE-derived enhancers in the GM12878 and K562 cell lines respectively. A vast majority of these putative enhancers are unique to each cell line; only 3.5% of the TE-derived enhancers are shared between the two. We evaluated the functional effect of TE-derived enhancers by associating them with the cell-type specific expression of nearby genes, and found that the number of TE-derived enhancers is strongly positively correlated with the expression of nearby genes in each cell line. Furthermore, genes that are differentially expressed between the two cell lines also possess a divergent number of TE-derived enhancers in their vicinity. As such, genes that are up-regulated in the GM12878 cell line and down-regulated in K562 have significantly more TE-derived enhancers in their vicinity in the GM12878 cell line and vice versa. These data indicate that human TE-derived sequences are likely to be involved in regulating cell-type specific gene expression on a broad scale and suggest that the enhancer activity of TE-derived sequences is mediated by epigenetic regulatory mechanisms.

  4. Independent chromatin binding of ARGONAUTE4 and SPT5L/KTF1 mediates transcriptional gene silencing.

    Directory of Open Access Journals (Sweden)

    M Jordan Rowley

    2011-06-01

    Full Text Available Eukaryotic genomes contain significant amounts of transposons and repetitive DNA elements, which, if transcribed, can be detrimental to the organism. Expression of these elements is suppressed by establishment of repressive chromatin modifications. In Arabidopsis thaliana, they are silenced by the siRNA-mediated transcriptional gene silencing pathway where long non-coding RNAs (lncRNAs produced by RNA Polymerase V (Pol V guide ARGONAUTE4 (AGO4 to chromatin and attract enzymes that establish repressive chromatin modifications. It is unknown how chromatin modifying enzymes are recruited to chromatin. We show through chromatin immunoprecipitation (ChIP that SPT5L/KTF1, a silencing factor and a homolog of SPT5 elongation factors, binds chromatin at loci subject to transcriptional silencing. Chromatin binding of SPT5L/KTF1 occurs downstream of RNA Polymerase V, but independently from the presence of 24-nt siRNA. We also show that SPT5L/KTF1 and AGO4 are recruited to chromatin in parallel and independently of each other. As shown using methylation-sensitive restriction enzymes, binding of both AGO4 and SPT5L/KTF1 is required for DNA methylation and repressive histone modifications of several loci. We propose that the coordinate binding of SPT5L and AGO4 creates a platform for direct or indirect recruitment of chromatin modifying enzymes.

  5. Systematic dissection of roles for chromatin regulators in a yeast stress response.

    Directory of Open Access Journals (Sweden)

    Assaf Weiner

    Full Text Available Packaging of eukaryotic genomes into chromatin has wide-ranging effects on gene transcription. Curiously, it is commonly observed that deletion of a global chromatin regulator affects expression of only a limited subset of genes bound to or modified by the regulator in question. However, in many single-gene studies it has become clear that chromatin regulators often do not affect steady-state transcription, but instead are required for normal transcriptional reprogramming by environmental cues. We therefore have systematically investigated the effects of 83 histone mutants, and 119 gene deletion mutants, on induction/repression dynamics of 170 transcripts in response to diamide stress in yeast. Importantly, we find that chromatin regulators play far more pronounced roles during gene induction/repression than they do in steady-state expression. Furthermore, by jointly analyzing the substrates (histone mutants and enzymes (chromatin modifier deletions we identify specific interactions between histone modifications and their regulators. Combining these functional results with genome-wide mapping of several histone marks in the same time course, we systematically investigated the correspondence between histone modification occurrence and function. We followed up on one pathway, finding that Set1-dependent H3K4 methylation primarily acts as a gene repressor during multiple stresses, specifically at genes involved in ribosome biosynthesis. Set1-dependent repression of ribosomal genes occurs via distinct pathways for ribosomal protein genes and ribosomal biogenesis genes, which can be separated based on genetic requirements for repression and based on chromatin changes during gene repression. Together, our dynamic studies provide a rich resource for investigating chromatin regulation, and identify a significant role for the "activating" mark H3K4me3 in gene repression.

  6. FGF signalling regulates chromatin organisation during neural differentiation via mechanisms that can be uncoupled from transcription.

    Directory of Open Access Journals (Sweden)

    Nishal S Patel

    Full Text Available Changes in higher order chromatin organisation have been linked to transcriptional regulation; however, little is known about how such organisation alters during embryonic development or how it is regulated by extrinsic signals. Here we analyse changes in chromatin organisation as neural differentiation progresses, exploiting the clear spatial separation of the temporal events of differentiation along the elongating body axis of the mouse embryo. Combining fluorescence in situ hybridisation with super-resolution structured illumination microscopy, we show that chromatin around key differentiation gene loci Pax6 and Irx3 undergoes both decompaction and displacement towards the nuclear centre coincident with transcriptional onset. Conversely, down-regulation of Fgf8 as neural differentiation commences correlates with a more peripheral nuclear position of this locus. During normal neural differentiation, fibroblast growth factor (FGF signalling is repressed by retinoic acid, and this vitamin A derivative is further required for transcription of neural genes. We show here that exposure to retinoic acid or inhibition of FGF signalling promotes precocious decompaction and central nuclear positioning of differentiation gene loci. Using the Raldh2 mutant as a model for retinoid deficiency, we further find that such changes in higher order chromatin organisation are dependent on retinoid signalling. In this retinoid deficient condition, FGF signalling persists ectopically in the elongating body, and importantly, we find that inhibiting FGF receptor (FGFR signalling in Raldh2-/- embryos does not rescue differentiation gene transcription, but does elicit both chromatin decompaction and nuclear position change. These findings demonstrate that regulation of higher order chromatin organisation during differentiation in the embryo can be uncoupled from the machinery that promotes transcription and, for the first time, identify FGF as an extrinsic signal that

  7. Chromatin boundary elements organize genomic architecture and developmental gene regulation in Drosophila Hox clusters

    Science.gov (United States)

    Ma, Zhibo; Li, Mo; Roy, Sharmila; Liu, Kevin J; Romine, Matthew L; Lane, Derrick C; Patel, Sapna K; Cai, Haini N

    2016-01-01

    The three-dimensional (3D) organization of the eukaryotic genome is critical for its proper function. Evidence suggests that extensive chromatin loops form the building blocks of the genomic architecture, separating genes and gene clusters into distinct functional domains. These loops are anchored in part by a special type of DNA elements called chromatin boundary elements (CBEs). CBEs were originally found to insulate neighboring genes by blocking influences of transcriptional enhancers or the spread of silent chromatin. However, recent results show that chromatin loops can also play a positive role in gene regulation by looping out intervening DNA and “delivering” remote enhancers to gene promoters. In addition, studies from human and model organisms indicate that the configuration of chromatin loops, many of which are tethered by CBEs, is dynamically regulated during cell differentiation. In particular, a recent work by Li et al has shown that the SF1 boundary, located in the Drosophila Hox cluster, regulates local genes by tethering different subsets of chromatin loops: One subset enclose a neighboring gene ftz, limiting its access by the surrounding Scr enhancers and restrict the spread of repressive histones during early embryogenesis; and the other loops subdivide the Scr regulatory region into independent domains of enhancer accessibility. The enhancer-blocking activity of these CBE elements varies greatly in strength and tissue distribution. Further, tandem pairing of SF1 and SF2 facilitate the bypass of distal enhancers in transgenic flies, providing a mechanism for endogenous enhancers to circumvent genomic interruptions resulting from chromosomal rearrangement. This study demonstrates how a network of chromatin boundaries, centrally organized by SF1, can remodel the 3D genome to facilitate gene regulation during development.

  8. Chromatin boundary elements organize genomic architecture and developmental gene regulation in Drosophila Hox clusters.

    Science.gov (United States)

    Ma, Zhibo; Li, Mo; Roy, Sharmila; Liu, Kevin J; Romine, Matthew L; Lane, Derrick C; Patel, Sapna K; Cai, Haini N

    2016-08-26

    The three-dimensional (3D) organization of the eukaryotic genome is critical for its proper function. Evidence suggests that extensive chromatin loops form the building blocks of the genomic architecture, separating genes and gene clusters into distinct functional domains. These loops are anchored in part by a special type of DNA elements called chromatin boundary elements (CBEs). CBEs were originally found to insulate neighboring genes by blocking influences of transcriptional enhancers or the spread of silent chromatin. However, recent results show that chromatin loops can also play a positive role in gene regulation by looping out intervening DNA and "delivering" remote enhancers to gene promoters. In addition, studies from human and model organisms indicate that the configuration of chromatin loops, many of which are tethered by CBEs, is dynamically regulated during cell differentiation. In particular, a recent work by Li et al has shown that the SF1 boundary, located in the Drosophila Hox cluster, regulates local genes by tethering different subsets of chromatin loops: One subset enclose a neighboring gene ftz, limiting its access by the surrounding Scr enhancers and restrict the spread of repressive histones during early embryogenesis; and the other loops subdivide the Scr regulatory region into independent domains of enhancer accessibility. The enhancer-blocking activity of these CBE elements varies greatly in strength and tissue distribution. Further, tandem pairing of SF1 and SF2 facilitate the bypass of distal enhancers in transgenic flies, providing a mechanism for endogenous enhancers to circumvent genomic interruptions resulting from chromosomal rearrangement. This study demonstrates how a network of chromatin boundaries, centrally organized by SF1, can remodel the 3D genome to facilitate gene regulation during development. PMID:27621770

  9. ATP-dependent chromatin remodeling facilitates nucleotide excision repair of UV-induced DNA lesions in synthetic dinucleosomes

    OpenAIRE

    Ura, Kiyoe; Araki, Marito; Saeki, Hideaki; Masutani, Chikahide; Ito, Takashi; Iwai, Shigenori; Mizukoshi, Toshimi; Kaneda, Yasufumi; Hanaoka, Fumio

    2001-01-01

    To investigate the relationship between chromatin dynamics and nucleotide excision repair (NER), we have examined the effect of chromatin structure on the formation of two major classes of UV-induced DNA lesions in reconstituted dinucleosomes. Furthermore, we have developed a model chromatin-NER system consisting of purified human NER factors and dinucleosome substrates that contain pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) either at the center of the nucleosome or in the linker DNA....

  10. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles.

    Science.gov (United States)

    Cejas, Paloma; Li, Lewyn; O'Neill, Nicholas K; Duarte, Melissa; Rao, Prakash; Bowden, Michaela; Zhou, Chensheng W; Mendiola, Marta; Burgos, Emilio; Feliu, Jaime; Moreno-Rubio, Juan; Guadalajara, Héctor; Moreno, Víctor; García-Olmo, Damián; Bellmunt, Joaquim; Mullane, Stephanie; Hirsch, Michelle; Sweeney, Christopher J; Richardson, Andrea; Liu, X Shirley; Brown, Myles; Shivdasani, Ramesh A; Long, Henry W

    2016-06-01

    Extensive cross-linking introduced during routine tissue fixation of clinical pathology specimens severely hampers chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) analysis from archived tissue samples. This limits the ability to study the epigenomes of valuable, clinically annotated tissue resources. Here we describe fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq), a method that enables reliable extraction of soluble chromatin from formalin-fixed paraffin-embedded (FFPE) tissue samples for accurate detection of histone marks. We demonstrate that FiT-seq data from FFPE specimens are concordant with ChIP-seq data from fresh-frozen samples of the same tumors. By using multiple histone marks, we generate chromatin-state maps and identify cis-regulatory elements in clinical samples from various tumor types that can readily allow us to distinguish between cancers by the tissue of origin. Tumor-specific enhancers and superenhancers that are elucidated by FiT-seq analysis correlate with known oncogenic drivers in different tissues and can assist in the understanding of how chromatin states affect gene regulation.

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

    Science.gov (United States)

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

    2013-11-21

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

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

    Science.gov (United States)

    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. Analysis of topological organization of chromatin during spermatogenesis in mouse testis

    Directory of Open Access Journals (Sweden)

    Narayan Gopeshwar

    2004-01-01

    Full Text Available Eukaryotic chromatin is organized as radial DNA loops with periodical attachments to an underlying nucleoskeleton known as nuclear matrix. This higher order chromatin organization is revealed upon high salt extraction of cells. To understand the sequential change in the functional organization of chromatin during spermatogenesis, we have analysed the higher order organization of chromatin in different testicular cell types and the epididymal sperm of laboratory mouse. The expansion and contraction of the nucleoid DNA following 2 M NaCl extraction was measured in a fluorescence microscope using ethidium bromide (2.5-200 mg/mL as an intercalating dye to induce DNA positive supercoils. While the halo size varied among cell types (pachytene DNA most extended, round spermatid least, 5 mg/mL ethidium bromide (EtBr removed maximum negative supercoils in all the cell types. At higher EtBr concentrations, maximum positive supercoiling occured in pachytene DNA loops. Consistent with this, the pachytene looped domains were maximally sensitive to DNase I, while the elongated spermatids and sperms were highly resistant. Our data suggest that pachytene DNA is in the most open chromatin conformation of all testicular cell types, while round spermatids show the most compact conformation in terms of EtBr intercalation.

  14. Tagged Chromosomal Insertion Site System: A Method to Study Lamina-Associated Chromatin.

    Science.gov (United States)

    Harr, Jennifer C; Reddy, Karen L

    2016-01-01

    The three-dimensional (3D) organization of the genome is important for chromatin regulation. This organization is nonrandom and appears to be tightly correlated with or regulated by chromatin state and scaffolding proteins. To understand how specific DNA and chromatin elements contribute to the functional organization of the genome, we developed a new tool-the tagged chromosomal insertion site (TCIS) system-to identify and study minimal DNA sequences that drive nuclear compartmentalization and applied this system to specifically study the role of cis elements in targeting DNA to the nuclear lamina. The TCIS system allows Cre-recombinase-mediated site-directed integration of any DNA fragment into a locus tagged with lacO arrays, thus enabling both functional molecular studies and positional analysis of the altered locus. This system can be used to study the minimal DNA sequences that target the nuclear periphery (or other nuclear compartments), allowing researchers to understand how genome-wide results obtained, for example, by DNA adenine methyltransferase identification, chromosome conformation capture (HiC), or related methods, connect to the actual organization of DNA and chromosomes at the single-cell level. Finally, TCIS allows one to test roles for specific proteins in chromatin reorganization and to determine how changes in nuclear environment affect chromatin state and gene regulation at a single locus.

  15. Salt and divalent cations affect the flexible nature of the natural beaded chromatin structure

    DEFF Research Database (Denmark)

    Christiansen, Gunna; Griffith, J

    1977-01-01

    A natural chromatin containing simian virus 40 (SV40) DNA and histone has been used to examine changes in chromatin structure caused by various physical and chemical treatments. We find that histone H1 depleted chromatin is more compact in solutions of 0.15M NaCl or 2 mM MgCl2 than in 0.01 M Na......Cl or 0.6M NaCL, and is compact in 0.01 M NaCl solutions if histone H 1 is present. Even high concentrations of urea did not alter the fundamental beaded structure, consisting of 110A beads of 200 base pair content, each joined by thin DNA bridges of 50 base pairs. The physical bead observed by EM...... therefore contains more DNA than the 140 base pair "core particle". The natural variation in the bridge length is consistent with the broad bands observed after nuclease digestion of chromatin. Chromatin prepared for EM without fixation containing long 20A to 30A fibers possibly complexed with protein....

  16. Global genome nucleotide excision repair is organized into domains that promote efficient DNA repair in chromatin

    Science.gov (United States)

    Yu, Shirong; Evans, Katie; Bennett, Mark; Webster, Richard M.; Leadbitter, Matthew; Teng, Yumin; Waters, Raymond

    2016-01-01

    The rates at which lesions are removed by DNA repair can vary widely throughout the genome, with important implications for genomic stability. To study this, we measured the distribution of nucleotide excision repair (NER) rates for UV-induced lesions throughout the budding yeast genome. By plotting these repair rates in relation to genes and their associated flanking sequences, we reveal that, in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organized within the genome. Furthermore, by comparing genome-wide DNA repair rates in wild-type cells and cells defective in the global genome–NER (GG-NER) subpathway, we establish how this alters the distribution of NER rates throughout the genome. We also examined the genomic locations of GG-NER factor binding to chromatin before and after UV irradiation, revealing that GG-NER is organized and initiated from specific genomic locations. At these sites, chromatin occupancy of the histone acetyl-transferase Gcn5 is controlled by the GG-NER complex, which regulates histone H3 acetylation and chromatin structure, thereby promoting efficient DNA repair of UV-induced lesions. Chromatin remodeling during the GG-NER process is therefore organized into these genomic domains. Importantly, loss of Gcn5 significantly alters the genomic distribution of NER rates; this has implications for the effects of chromatin modifiers on the distribution of mutations that arise throughout the genome. PMID:27470111

  17. Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene

    International Nuclear Information System (INIS)

    Inhibition of herpes simplex virus (HSV) thymidine kinase (TK) gene transcription (pHSV-106, pML-BPV-TK4) by DNA methylation is an indirect effect, which occurs with a latency period of ∼ 8 hr microinjection of the DNA into TK- rat 2 and mouse LTK- cells. The authors have strong evidence that chromatin formation is critical for the transition of the injected DNA from methylation insensitivity to methylation sensitivity. Chromatin was reconstituted in vitro by using methylated and mock-methylated HSV TK DNA and purified chicken histone octamers. After microinjection, the methylated chromatin was always biologically inactive, as tested by autoradiography of the cells after incubation with [3H]thymidine and by RNA dot blot analysis. However, in transformed cell lines, reactivation of the methylated chromatic occurred after treatment with 5-azacytidine. Furthermore, integration of the TK chromatin into the host genome is not required to block expression of the methylated TK gene. Mouse cells that contained the pML-BPV-TK4 chromatin permanently in an episomal state also did not support TK gene expression as long as the TK DNA remained methylated

  18. Evaluation of chromatin integrity of motile bovine spermatozoa capacitated in vitro.

    Science.gov (United States)

    Reckova, Z; Machatkova, M; Rybar, R; Horakova, J; Hulinska, P; Machal, L

    2008-08-01

    The efficiency of in vitro embryo production is highly variable amongst individual sires in cattle. To eliminate that this variability is not caused by sperm chromatin damage caused by separation or capacitacion, chromatin integrity was evaluated. Seventeen of AI bulls with good NRRs but variable embryo production efficiency were used. For each bull, motile spermatozoa were separated on a Percoll gradient, resuspended in IVF-TALP medium and capacitated with or incubated without heparin for 6 h. Samples before and after separation and after 3-h and 6-h capacitacion or incubation were evaluated by the Sperm Chromatin Structure Assay (SCSA) and the proportion of sperm with intact chromatin structure was calculated. Based on changes in the non-DFI-sperm proportion, the sires were categorized as DNA-unstable (DNA-us), DNA-stable (DNA-s) and DNA-most stable (DNA-ms) bulls (n=3, n=5 and n=9, respectively). In DNA-us bulls, separation produced a significant increase of the mean non-DFI-sperm proportion (p Capacitacion produced a significant decrease in the mean non-DFI-sperm proportion in H+ sperm (p capacitacion, the mean non-DFI-sperm proportion remained almost unchanged. In DNA-ms bulls, neither separation nor capacitacion had any effect on the mean non-DFI-sperm proportion. It can be concluded that, although separation and capacitacion may produce some changes in sperm chromatin integrity, these are not associated with different in vitro fertility of the bulls involved. PMID:18578952

  19. A Chromatin-Focused siRNA Screen for Regulators of p53-Dependent Transcription.

    Science.gov (United States)

    Sammons, Morgan A; Zhu, Jiajun; Berger, Shelley L

    2016-01-01

    The protein product of the Homo sapiens TP53 gene is a transcription factor (p53) that regulates the expression of genes critical for the response to DNA damage and tumor suppression, including genes involved in cell cycle arrest, apoptosis, DNA repair, metabolism, and a number of other tumorigenesis-related pathways. Differential transcriptional regulation of these genes is believed to alter the balance between two p53-dependent cell fates: cell cycle arrest or apoptosis. A number of previously identified p53 cofactors covalently modify and alter the function of both the p53 protein and histone proteins. Both gain- and loss-of-function mutations in chromatin modifiers have been strongly implicated in cancer development; thus, we sought to identify novel chromatin regulatory proteins that affect p53-dependent transcription and the balance between the expression of pro-cell cycle arrest and proapoptotic genes. We utilized an siRNA library designed against predicted chromatin regulatory proteins, and identified known and novel chromatin-related factors that affect both global p53-dependent transcription and gene-specific regulators of p53 transcriptional activation. The results from this screen will serve as a comprehensive resource for those interested in further characterizing chromatin and epigenetic factors that regulate p53 transcription. PMID:27334938

  20. Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

    Science.gov (United States)

    Zee, Barry M.; Alekseyenko, Artyom A.; McElroy, Kyle A.; Kuroda, Mitzi I.

    2016-01-01

    Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. We recently introduced a cross-linked affinity technique, BioTAP-XL, to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. Here we advance BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore we demonstrate that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. We also integrate hydrophilic interaction chromatography to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, we greatly extend the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment. PMID:26831069

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

    Science.gov (United States)

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

    1997-01-01

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

  2. Standardizing chromatin research: a simple and universal method for ChIP-seq.

    Science.gov (United States)

    Arrigoni, Laura; Richter, Andreas S; Betancourt, Emily; Bruder, Kerstin; Diehl, Sarah; Manke, Thomas; Bönisch, Ulrike

    2016-04-20

    Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a key technique in chromatin research. Although heavily applied, existing ChIP-seq protocols are often highly fine-tuned workflows, optimized for specific experimental requirements. Especially the initial steps of ChIP-seq, particularly chromatin shearing, are deemed to be exceedingly cell-type-specific, thus impeding any protocol standardization efforts. Here we demonstrate that harmonization of ChIP-seq workflows across cell types and conditions is possible when obtaining chromatin from properly isolated nuclei. We established an ultrasound-based nuclei extraction method (NEXSON: Nuclei EXtraction by SONication) that is highly effective across various organisms, cell types and cell numbers. The described method has the potential to replace complex cell-type-specific, but largely ineffective, nuclei isolation protocols. By including NEXSON in ChIP-seq workflows, we completely eliminate the need for extensive optimization and sample-dependent adjustments. Apart from this significant simplification, our approach also provides the basis for a fully standardized ChIP-seq and yields highly reproducible transcription factor and histone modifications maps for a wide range of different cell types. Even small cell numbers (∼10,000 cells per ChIP) can be easily processed without application of modified chromatin or library preparation protocols.

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

    Science.gov (United States)

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

    2003-11-01

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

  4. Age-related reduction of chromatin fractal dimension in toluidine blue - stained hepatocytes.

    Science.gov (United States)

    Pantic, Igor; Petrovic, Danica; Paunovic, Jovana; Vucevic, Danijela; Radosavljevic, Tatjana; Pantic, Senka

    2016-07-01

    In this study, we proposed a hypothesis that chromatin of mouse hepatocytes exhibits age-related reduction of fractal dimension. This hypothesis was based on previously published works demonstrating that complexity of biological systems such as tissues, decreases during the process of physiological aging. Liver tissue was obtained from 24 male mice divided into 3 age groups: 10-days-old (young, juvenile), 210-days-old (adult) and 390-days-old. The tissue was stained using a modification of toluidine blue (nucleic acid - specific) staining method. A total of 480 chromatin structures (20 for each animal) were analyzed. For each structure, the values of fractal dimension, lacunarity, textural angular second moment and inverse difference moment were calculated using ImageJ software and its plugins. The results indicated the age-related reduction in fractal dimension and increase in lacunarity (p<0.01). Fractal dimension is a potentially good indicator of age associated changes in chromatin structure. To our knowledge, this is the first study to show that fractal complexity of hepatocyte chromatin decreases during the process of physiological aging. Fractal analysis as a method could be useful in detection of small age-related changes in chromatin distribution not otherwise visible with naked eye on conventional tissue micrographs. PMID:27412950

  5. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles.

    Science.gov (United States)

    Cejas, Paloma; Li, Lewyn; O'Neill, Nicholas K; Duarte, Melissa; Rao, Prakash; Bowden, Michaela; Zhou, Chensheng W; Mendiola, Marta; Burgos, Emilio; Feliu, Jaime; Moreno-Rubio, Juan; Guadalajara, Héctor; Moreno, Víctor; García-Olmo, Damián; Bellmunt, Joaquim; Mullane, Stephanie; Hirsch, Michelle; Sweeney, Christopher J; Richardson, Andrea; Liu, X Shirley; Brown, Myles; Shivdasani, Ramesh A; Long, Henry W

    2016-06-01

    Extensive cross-linking introduced during routine tissue fixation of clinical pathology specimens severely hampers chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) analysis from archived tissue samples. This limits the ability to study the epigenomes of valuable, clinically annotated tissue resources. Here we describe fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq), a method that enables reliable extraction of soluble chromatin from formalin-fixed paraffin-embedded (FFPE) tissue samples for accurate detection of histone marks. We demonstrate that FiT-seq data from FFPE specimens are concordant with ChIP-seq data from fresh-frozen samples of the same tumors. By using multiple histone marks, we generate chromatin-state maps and identify cis-regulatory elements in clinical samples from various tumor types that can readily allow us to distinguish between cancers by the tissue of origin. Tumor-specific enhancers and superenhancers that are elucidated by FiT-seq analysis correlate with known oncogenic drivers in different tissues and can assist in the understanding of how chromatin states affect gene regulation. PMID:27111282

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

    Directory of Open Access Journals (Sweden)

    Monica Soldi

    2013-03-01

    Full Text Available 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.

  7. ATP-dependent chromatin remodeling in the DNA-damage response

    Directory of Open Access Journals (Sweden)

    Lans Hannes

    2012-01-01

    Full Text Available Abstract The integrity of DNA is continuously challenged by metabolism-derived and environmental genotoxic agents that cause a variety of DNA lesions, including base alterations and breaks. DNA damage interferes with vital processes such as transcription and replication, and if not repaired properly, can ultimately lead to premature aging and cancer. Multiple DNA pathways signaling for DNA repair and DNA damage collectively safeguard the integrity of DNA. Chromatin plays a pivotal role in regulating DNA-associated processes, and is itself subject to regulation by the DNA-damage response. Chromatin influences access to DNA, and often serves as a docking or signaling site for repair and signaling proteins. Its structure can be adapted by post-translational histone modifications and nucleosome remodeling, catalyzed by the activity of ATP-dependent chromatin-remodeling complexes. In recent years, accumulating evidence has suggested that ATP-dependent chromatin-remodeling complexes play important, although poorly characterized, roles in facilitating the effectiveness of the DNA-damage response. In this review, we summarize the current knowledge on the involvement of ATP-dependent chromatin remodeling in three major DNA repair pathways: nucleotide excision repair, homologous recombination, and non-homologous end-joining. This shows that a surprisingly large number of different remodeling complexes display pleiotropic functions during different stages of the DNA-damage response. Moreover, several complexes seem to have multiple functions, and are implicated in various mechanistically distinct repair pathways.

  8. [Cytophotometric analysis of the chromatin structural conformity in interphase nuclei detected in UV light and by gallocyanine staining].

    Science.gov (United States)

    Zhukotskiĭ, A V; Shchegolev, A I; Butusova, N N; Nemirovskiĭ, L E; Kogan, E M

    1985-06-01

    Geometric and optical parameters of chromatin of hepatocyte nuclei have been examined before (UV, lambda = 265 nm) and after gallocyanine staining. Quantitative parameters of the chromatin structure in the same nuclei measured in situ by a scanning microscope-photometer (step size 0.125 micron) before and after staining were equal. Tinctorial properties of chromatin granules (condensed part of the nuclear material) and its diffuse part were different. It is suggested that the difference between granules and the nongranular part of chromatin is not only of optical but also of chemical nature. PMID:2410060

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    de la Chapelle, A

    1986-10-10

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

  11. Undifferentiated embryonic cell transcription factor 1 regulates ESC chromatin organization and gene expression

    DEFF Research Database (Denmark)

    Kooistra, Susanne M; van den Boom, Vincent; Thummer, Rajkumar P;

    2010-01-01

    Previous reports showed that embryonic stem (ES) cells contain hyperdynamic and globally transcribed chromatin-properties that are important for ES cell pluripotency and differentiation. Here, we demonstrate a role for undifferentiated embryonic cell transcription factor 1 (UTF1) in regulating ES...... cell chromatin structure. Using chromatin immunoprecipitation-on-chip analysis, we identified >1,700 UTF1 target genes that significantly overlap with previously identified Nanog, Oct4, Klf-4, c-Myc, and Rex1 targets. Gene expression profiling showed that UTF1 knock down results in increased expression...... of a large set of genes, including a significant number of UTF1 targets. UTF1 knock down (KD) ES cells are, irrespective of the increased expression of several self-renewal genes, Leukemia inhibitory factor (LIF) dependent. However, UTF1 KD ES cells are perturbed in their differentiation in response...

  12. A RSC/nucleosome complex determines chromatin architecture and facilitates activator binding.

    Science.gov (United States)

    Floer, Monique; Wang, Xin; Prabhu, Vidya; Berrozpe, Georgina; Narayan, Santosh; Spagna, Dan; Alvarez, David; Kendall, Jude; Krasnitz, Alexander; Stepansky, Asya; Hicks, James; Bryant, Gene O; Ptashne, Mark

    2010-04-30

    How is chromatin architecture established and what role does it play in transcription? We show that the yeast regulatory locus UASg bears, in addition to binding sites for the activator Gal4, sites bound by the RSC complex. RSC positions a nucleosome, evidently partially unwound, in a structure that facilitates Gal4 binding to its sites. The complex comprises a barrier that imposes characteristic features of chromatin architecture. In the absence of RSC, ordinary nucleosomes encroach over the UASg and compete with Gal4 for binding. Taken with our previous work, the results show that both prior to and following induction, specific DNA-binding proteins are the predominant determinants of chromatin architecture at the GAL1/10 genes. RSC/nucleosome complexes are also found scattered around the yeast genome. Higher eukaryotic RSC lacks the specific DNA-binding determinants found on yeast RSC, and evidently Gal4 works in those organisms despite whatever obstacle broadly positioned nucleosomes present.

  13. Chromatin changes in response to drought, salinity, heat, and cold stresses in plants

    Directory of Open Access Journals (Sweden)

    Jong-Myong eKim

    2015-03-01

    Full Text Available Chromatin regulation is essential to regulate genes and genome activities. In plants, the alteration of histone modification and DNA methylation are coordinated with changes in the expression of stress-responsive genes to adapt to environmental changes. Several chromatin regulators have been shown to be involved in the regulation of stress-responsive gene networks under abiotic stress conditions. Specific histone modification sites and the histone modifiers that regulate key stress-responsive genes have been identified by genetic and biochemical approaches, revealing the importance of chromatin regulation in plant stress responses. Recent studies have also suggested that histone modification plays an important role in plant stress memory. In this review, we summarize recent progress on the regulation and alteration of histone modification (acetylation, methylation, phosphorylation, and SUMOylation in response to the abiotic stresses, drought, high-salinity, heat, and cold in plants.

  14. Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo.

    Science.gov (United States)

    Ricci, Maria Aurelia; Manzo, Carlo; García-Parajo, María Filomena; Lakadamyali, Melike; Cosma, Maria Pia

    2015-03-12

    Nucleosomes help structure chromosomes by compacting DNA into fibers. To gain insight into how nucleosomes are arranged in vivo, we combined quantitative super-resolution nanoscopy with computer simulations to visualize and count nucleosomes along the chromatin fiber in single nuclei. Nucleosomes assembled in heterogeneous groups of varying sizes, here termed "clutches," and these were interspersed with nucleosome-depleted regions. The median number of nucleosomes inside clutches and their compaction defined as nucleosome density were cell-type-specific. Ground-state pluripotent stem cells had, on average, less dense clutches containing fewer nucleosomes and clutch size strongly correlated with the pluripotency potential of induced pluripotent stem cells. RNA polymerase II preferentially associated with the smallest clutches while linker histone H1 and heterochromatin were enriched in the largest ones. Our results reveal how the chromatin fiber is formed at nanoscale level and link chromatin fiber architecture to stem cell state.

  15. Introducing enteral feeding induces intestinal subclinical inflammation and respective chromatin changes in preterm pigs

    DEFF Research Database (Denmark)

    Willems, Rhea; Krych, Lukasz; Rybicki, Verena;

    2015-01-01

    AIM: To analyze how enteral food introduction affects intestinal gene regulation and chromatin structure in preterm pigs. MATERIALS & METHODS: Preterm pigs were fed parenteral nutrition plus/minus slowly increasing volumes of enteral nutrition. Intestinal gene-expression and chromatin structure...... were analyzed 5 days after birth. RESULTS: Enteral feeding led to differential upregulation of inflammatory and pattern recognition receptor genes, including IL8 (median: 5.8, 95% CI: 3.9-7.8 for formula; median: 2.2, 95% CI: 1.3-3.3 for colostrum) and TLR4 (median: 3.7, 95% CI: 2.6-4.8 for formula...... stimulation with lipopolysaccharide (median: 7.0; interquartile range: 5.63-8.85) compared with naive cells (median 4.2; interquartile range: 2.45-6.33; p = 0.03). CONCLUSION: Enteral feeding, particular with formula, induces subclinical inflammation in the premature intestine and more open chromatin...

  16. Statistical-mechanical lattice models for protein-DNA binding in chromatin

    CERN Document Server

    Teif, Vladimir B

    2010-01-01

    Statistical-mechanical lattice models for protein-DNA binding are well established as a method to describe complex ligand binding equilibriums measured in vitro with purified DNA and protein components. Recently, a new field of applications has opened up for this approach since it has become possible to experimentally quantify genome-wide protein occupancies in relation to the DNA sequence. In particular, the organization of the eukaryotic genome by histone proteins into a nucleoprotein complex termed chromatin has been recognized as a key parameter that controls the access of transcription factors to the DNA sequence. New approaches have to be developed to derive statistical mechanical lattice descriptions of chromatin-associated protein-DNA interactions. Here, we present the theoretical framework for lattice models of histone-DNA interactions in chromatin and investigate the (competitive) DNA binding of other chromosomal proteins and transcription factors. The results have a number of applications for quant...

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

    Science.gov (United States)

    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

  18. Repression of germline RNAi pathways in somatic cells by retinoblastoma pathway chromatin complexes.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Wu

    Full Text Available The retinoblastoma (Rb tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.

  19. Radiation-induced XRCC4 association with chromatin DNA analyzed by biochemical fractionation

    International Nuclear Information System (INIS)

    XRCC4, in association with DNA ligase IV, is thought to play a critical role in the ligation of two DNA ends in DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ) pathway. In the present study, we captured radiation-induced chromatin-recruitment of XRCC4 by biochemical fractionation using detergent Nonidet P-40. A subpopulation of XRCC4 changed into a form that is resistant to the extraction with 0.5% Nonidet P-40-containing buffer after irradiation. This form of XRCC4 was liberated by micrococcal nuclease treatment, indicating that it had been tethered to chromatin DNA. This chromatin-recruitment of XRCC4 could be seen immediately (<0.1 hr) after irradiation and remained up to 4 hr after 20 Gy irradiation. It was seen even after irradiation of small doses, id est (i.e.), 2 Gy, but the residence of XRCC4 on chromatin was very transient after 2 Gy irradiation, returning to near normal level in 0.2-0.5 hr after irradiation. The chromatin-bound XRCC4 represented only -1% of total XRCC4 molecules even after 20 Gy irradiation and the quantitative analysis using purified protein as the reference suggested that only a few XRCC4-DNA ligase IV complexes were recruited to each DNA end. We further show that the chromatin-recruitment of XRCC4 was not attenuated by wortmannin, an inhibitor of DNA-PK, or siRNA-mediated knockdown of the DNA-PK catalytic subunit (DNA-PKcs), indicating that this process does not require DNA-PKcs. These results would provide us with useful experimental tools and important insights to understand the DNA repair process through NHEJ pathway. (author)

  20. Diffusion-driven looping provides a consistent framework for chromatin organization.

    Directory of Open Access Journals (Sweden)

    Manfred Bohn

    Full Text Available Chromatin folding inside the interphase nucleus of eukaryotic cells is done on multiple scales of length and time. Despite recent progress in understanding the folding motifs of chromatin, the higher-order structure still remains elusive. Various experimental studies reveal a tight connection between genome folding and function. Chromosomes fold into a confined subspace of the nucleus and form distinct territories. Chromatin looping seems to play a dominant role both in transcriptional regulation as well as in chromatin organization and has been assumed to be mediated by long-range interactions in many polymer models. However, it remains a crucial question which mechanisms are necessary to make two chromatin regions become co-located, i.e. have them in spatial proximity. We demonstrate that the formation of loops can be accomplished solely on the basis of diffusional motion. The probabilistic nature of temporary contacts mimics the effects of proteins, e.g. transcription factors, in the solvent. We establish testable quantitative predictions by deriving scale-independent measures for comparison to experimental data. In this Dynamic Loop (DL model, the co-localization probability of distant elements is strongly increased compared to linear non-looping chains. The model correctly describes folding into a confined space as well as the experimentally observed cell-to-cell variation. Most importantly, at biological densities, model chromosomes occupy distinct territories showing less inter-chromosomal contacts than linear chains. Thus, dynamic diffusion-based looping, i.e. gene co-localization, provides a consistent framework for chromatin organization in eukaryotic interphase nuclei.

  1. Proteins of the origin recognition complex (ORC and DNA topoisomerases on mammalian chromatin

    Directory of Open Access Journals (Sweden)

    Baack Martina

    2009-04-01

    Full Text Available Abstract Background The process of DNA replication requires the separation of complementary DNA strands. In this process, the unwinding of circularly closed or long DNA duplices leads to torsional tensions which must be released by topoisomerases. So topoisomerases play an important role in DNA replication. In order to provide more information about topoisomerases in the initiation of mammalian replication, we investigated whether topoisomerases occur close to ORC in the chromatin of cultured human HeLa cells. Results We have used different cell fractionation procedures, namely salt and nuclease treatment of isolated nuclei as well as formaldehyde-mediated cross-linking of chromatin, to investigate the distribution of topoisomerases and proteins of the origin recognition complex (ORC in the chromatin of human HeLa cells. First we obtained no evidence for a physical interaction of either topoisomerase I or topoisomerase II with ORC. Then we found, however, that (Orc1-5 and topo II occurred together on chromatin fragments of 600 and more bp lengths. At last we showed that both topo II and Orc2 protein are enriched near the origin at the human MCM4 gene, and at least some of the topo II at the origin is active in proliferating HeLa cells. So taken together, topoisomerase II, but not topoisomerase I, is located close to ORC on chromatin. Conclusion Topoisomerase II is more highly expressed than ORC proteins in mammalian cells, so only a small fraction of total chromatin-bound topoisomerase II was found in the vicinity of ORC. The precise position of topo II relative to ORC may differ among origins.

  2. Putative molecular mechanism underlying sperm chromatin remodelling is regulated by reproductive hormones

    Directory of Open Access Journals (Sweden)

    Gill-Sharma Manjeet Kaur

    2012-12-01

    Full Text Available Abstract Background The putative regulatory role of the male reproductive hormones in the molecular mechanism underlying chromatin condensation remains poorly understood. In the past decade, we developed two adult male rat models wherein functional deficits of testosterone or FSH, produced after treatments with 20 mg/Kg/d of cyproterone acetate (CPA per os, for a period of 15 days or 3 mg/Kg/d of fluphenazine decanoate (FD subcutaneously, for a period of 60 days, respectively, affected the rate of sperm chromatin decondensation in vitro. These rat models have been used in the current study in order to delineate the putative roles of testosterone and FSH in the molecular mechanism underlying remodelling of sperm chromatin. Results We report that deficits of both testosterone and FSH affected the turnover of polyubiquitylated histones and led to their accumulation in the testis. Functional deficits of testosterone reduced expression of MIWI, the 5-methyl cap binding RNA-binding protein (PIWIlike murine homologue of the Drosophila protein PIWI/P-element induced wimpy testis containing a PAZ/Piwi-Argonaut-Zwille domain and levels of histone deacetylase1 (HDAC1, ubiquitin ligating enzyme (URE-B1/E3, 20S proteasome α1 concomitant with reduced expression of ubiquitin activating enzyme (ube1, conjugating enzyme (ube2d2, chromodomain Y like protein (cdyl, bromodomain testis specific protein (brdt, hdac6 (histone deacetylase6, androgen-dependent homeobox placentae embryonic protein (pem/RhoX5, histones h2b and th3 (testis-specific h3. Functional deficits of FSH reduced the expression of cdyl and brdt genes in the testis, affected turnover of ubiquitylated histones, stalled the physiological DNA repair mechanism and culminated in spermiation of DNA damaged sperm. Conclusions We aver that deficits of both testosterone and FSH differentially affected the process of sperm chromatin remodelling through subtle changes in the ‘chromatin condensation

  3. The epigenetics of tumour initiation: cancer stem cells and their chromatin.

    Science.gov (United States)

    Avgustinova, Alexandra; Benitah, Salvador Aznar

    2016-02-01

    Cancer stem cells (CSCs) have been identified in various tumours and are defined by their potential to initiate tumours upon transplantation, self-renew and reconstitute tumour heterogeneity. Modifications of the epigenome can favour tumour initiation by affecting genome integrity, DNA repair and tumour cell plasticity. Importantly, an in-depth understanding of the epigenomic alterations underlying neoplastic transformation may open new avenues for chromatin-targeted cancer treatment, as these epigenetic changes could be inherently more amenable to inhibition and reversal than hard-wired genomic alterations. Here we discuss how CSC function is affected by chromatin state and epigenomic instability. PMID:26874045

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

    CERN Document Server

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

    2008-01-01

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

  5. Insights into p53 transcriptional function via genome-wide chromatin occupancy and gene expression analysis

    OpenAIRE

    F Nikulenkov; Spinnler, C; Li, H.; Tonelli, C; Shi, Y; Turunen, M.; Kivioja, T; Ignatiev, I.; Kel, A; Taipale, J; Selivanova, G

    2012-01-01

    The tumor-suppressor p53 can induce various biological responses. Yet, it is not clear whether it is p53 in vivo promoter selectivity that triggers different transcription programs leading to different outcomes. Our analysis of genome-wide chromatin occupancy by p53 using chromatin immunoprecipitation (ChIP)-seq revealed ‘p53 default program', that is, the pattern of major p53-bound sites that is similar upon p53 activation by nutlin3a, reactivation of p53 and induction of tumor cell apoptosi...

  6. Selective removal of promoter nucleosomes by the RSC chromatin-remodeling complex.

    Science.gov (United States)

    Lorch, Yahli; Griesenbeck, Joachim; Boeger, Hinrich; Maier-Davis, Barbara; Kornberg, Roger D

    2011-08-01

    Purified chromatin rings, excised from the PHO5 locus of Saccharomyces cerevisiae in transcriptionally repressed and activated states, were remodeled with RSC and ATP. Nucleosomes were translocated, and those originating on the promoter of repressed rings were removed, whereas those originating on the open reading frame (ORF) were retained. Treatment of the repressed rings with histone deacetylase diminished the removal of promoter nucleosomes. These findings point to a principle of promoter chromatin remodeling for transcription, namely that promoter specificity resides primarily in the nucleosomes rather than in the remodeling complex that acts upon them.

  7. Protocol: methodology for chromatin immunoprecipitation (ChIP in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Strenkert Daniela

    2011-11-01

    Full Text Available Abstract We report on a detailed chromatin immunoprecipitation (ChIP protocol for the unicellular green alga Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example for the normalization of ChIP results as determined by real-time PCR.

  8. Diverse functions of ATP-dependent chromatin remodeling complexes in development and cancer

    Institute of Scientific and Technical Information of China (English)

    Jiang I. Wu

    2012-01-01

    Mammalian SWI/SNF like Brg1/Brm associated factors (BAF) chromatin-remodeling complexes are able to use energy derived from adenosine triphosphate (ATP) hydrolysis to change chromatin structures and regulate nuclear processes such as transcription.BAF complexes contain multiple subunits and the diverse subunit compositions provide functional specificities to BAF complexes.In this review,we summarize the functions of BAF subunits during mammalian development and in progression of various cancers.The mechanisms underlying the functional diversity and specificities of BAF complexes will be discussed.

  9. Remodelers organize cellular chromatin by counteracting intrinsic histone-DNA sequence preferences in a class-specific manner

    NARCIS (Netherlands)

    Y.M. Moshkin (Yuri); G.E. Chalkley (Gillian); T.W. Kan (Tsung Wai); B.A. Reddy (Ashok); Z. Özgür (Zeliha); W.F.J. van IJcken (Wilfred); D.H. Dekkers (Dick); J.A.A. Demmers (Jeroen); A.A. Travers (Andrew); C.P. Verrijzer (Peter)

    2012-01-01

    textabstractThe nucleosome is the fundamental repeating unit of eukaryotic chromatin. Here, we assessed the interplay between DNA sequence and ATP-dependent chromatin-remodeling factors (remodelers) in the nucleosomal organization of a eukaryotic genome. We compared the genome-wide distribution of D

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

    Science.gov (United States)

    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.

  11. High-Resolution Profiling of Drosophila Replication Start Sites Reveals a DNA Shape and Chromatin Signature of Metazoan Origins

    Directory of Open Access Journals (Sweden)

    Federico Comoglio

    2015-05-01

    Full Text Available At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, the genetic and chromatin features defining metazoan replication start sites remain largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution. We address the role of origin-proximal G-quadruplexes and suggest that they transiently stall replication forks in vivo. We dissect the chromatin configuration of replication origins and identify a rich spatial organization of chromatin features at initiation sites. DNA shape and chromatin configurations, not strict sequence motifs, mark and predict origins in higher eukaryotes. We further examine the link between transcription and origin firing and reveal that modulation of origin activity across cell types is intimately linked to cell-type-specific transcriptional programs. Our study unravels conserved origin features and provides unique insights into the relationship among DNA topology, chromatin, transcription, and replication initiation across metazoa.

  12. Targeted Histone Peptides: Insights into the Spatial Regulation of the Methyltransferase PRC2 by using a Surrogate of Heterotypic Chromatin.

    Science.gov (United States)

    Brown, Zachary Z; Müller, Manuel M; Kong, Ha Eun; Lewis, Peter W; Muir, Tom W

    2015-05-26

    Eukaryotic genomes are dynamically regulated through a host of epigenetic stimuli. The substrate for these epigenetic transactions, chromatin, is a polymer of nucleosome building blocks. In native chromatin, each nucleosome can differ from its neighbors as a result of covalent modifications to both the DNA and the histone packaging proteins. The heterotypic nature of chromatin presents a formidable obstacle to biochemical studies seeking to understand the role of context on epigenetic regulation. A chemical approach to the production of heterotypic chromatin that can be used in such studies is introduced. This method involves the attachment of a user-defined modified histone peptide to a designated nucleosome within the polymer by using a peptide nucleic acid (PNA) targeting compound. This strategy was applied to dissect the effect of chromatin context on the activity of the histone methyltransferase PRC2. The results show that PRC2 can be stimulated to produce histone H3 methylation from a defined nucleation site.

  13. Novel RNA-binding properties of the MTG chromatin regulatory proteins

    NARCIS (Netherlands)

    S. Rossetti (Stefano); L. van Unen (Leontine); N. Sacchi; A.T. Hoogeveen (Andre)

    2008-01-01

    textabstractBackground: The myeloid translocation gene (MTG) proteins are non-DNA-binding transcriptional regulators capable of interacting with chromatin modifying proteins. As a consequence of leukemia-associated chromosomal translocations, two of the MTG proteins, MTG8 and MTG16, are fused to the

  14. Chromatin-based epigenetics of adult subventricular zone neural stem cells

    Directory of Open Access Journals (Sweden)

    Gabriel eGonzales-Roybal

    2013-10-01

    Full Text Available In specific regions of the adult mammalian brain, neural stem cells (NSCs generate new neurons throughout life. Emerging evidence indicate that chromatin-based transcriptional regulation is a key epigenetic mechanism for the life-long function of adult NSCs. In the adult mouse brain, NSCs in the subventricular zone (SVZ retain the ability to produce both neurons and glia for the life of the animal. In this review, we discuss the origin and function of SVZ NSCs as they relate to key epigenetic concepts of development and potential underlying mechanism of chromatin-based transcriptional regulation. A central point of discussion is how SVZ NSCs – which possess many characteristics of mature, non-neurogenic astrocytes – maintain a youthful ability to produce both neuronal and glial lineages. In addition to reviewing data regarding the function of chromatin-modifying factors in SVZ neurogenesis, we incorporate our growing understanding that long noncoding RNAs (lncRNAs serve as an important element to chromatin-based transcriptional regulation, including that of SVZ NSCs. Discoveries regarding the epigenetic mechanisms of adult SVZ NSCs may provide key insights into fundamental principles of adult stem cell biology as well as the more complex and dynamic developmental environment of the embryonic brain.

  15. The Nuclear Oncogene SET Controls DNA Repair by KAP1 and HP1 Retention to Chromatin

    Directory of Open Access Journals (Sweden)

    Alkmini Kalousi

    2015-04-01

    Full Text Available Cells experience damage from exogenous and endogenous sources that endanger genome stability. Several cellular pathways have evolved to detect DNA damage and mediate its repair. Although many proteins have been implicated in these processes, only recent studies have revealed how they operate in the context of high-ordered chromatin structure. Here, we identify the nuclear oncogene SET (I2PP2A as a modulator of DNA damage response (DDR and repair in chromatin surrounding double-strand breaks (DSBs. We demonstrate that depletion of SET increases DDR and survival in the presence of radiomimetic drugs, while overexpression of SET impairs DDR and homologous recombination (HR-mediated DNA repair. SET interacts with the Kruppel-associated box (KRAB-associated co-repressor KAP1, and its overexpression results in the sustained retention of KAP1 and Heterochromatin protein 1 (HP1 on chromatin. Our results are consistent with a model in which SET-mediated chromatin compaction triggers an inhibition of DNA end resection and HR.

  16. 3-D Nuclear chromatin texture analysis using confocal laser scanning microscopy

    NARCIS (Netherlands)

    Huisman, André

    2007-01-01

    The transformation of a normal cell into a malignant cell is associated with genetic alterations that often result in abnormal chromosome sets (“aneuploidy”) and changes in the distribution of chromatin inside the nucleus. These changes are often subtle and are mostly referred to as “malignancy asso

  17. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling

    DEFF Research Database (Denmark)

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook;

    2015-01-01

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and...

  18. The transcriptional coactivator SAYP is a trithorax group signature subunit of the PBAP chromatin remodeling complex

    NARCIS (Netherlands)

    G.E. Chalkley (Gillian); Y.M. Moshkin (Yuri); K. Langenberg (Karin); K. Bezstarosti (Karel); A. Blastyak (Andras); H. Gyurkovics (Henrik); J.A.A. Demmers (Jeroen); C.P. Verrijzer (Peter)

    2008-01-01

    textabstractSWI/SNF ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in eukaryotic gene expression control. BAP and PBAP are the fly representatives of the two evolutionarily conserved major subclasses of SWI/SNF remodelers. Both complexes share seven core subunit

  19. Transcriptional Coactivator and Chromatin Protein PC4 Is Involved in Hippocampal Neurogenesis and Spatial Memory Extinction.

    Science.gov (United States)

    Swaminathan, Amrutha; Delage, Hélène; Chatterjee, Snehajyoti; Belgarbi-Dutron, Laurence; Cassel, Raphaelle; Martinez, Nicole; Cosquer, Brigitte; Kumari, Sujata; Mongelard, Fabien; Lannes, Béatrice; Cassel, Jean-Christophe; Boutillier, Anne-Laurence; Bouvet, Philippe; Kundu, Tapas K

    2016-09-23

    Although the elaborate combination of histone and non-histone protein complexes defines chromatin organization and hence regulates numerous nuclear processes, the role of chromatin organizing proteins remains unexplored at the organismal level. The highly abundant, multifunctional, chromatin-associated protein and transcriptional coactivator positive coactivator 4 (PC4/Sub1) is absolutely critical for life, because its absence leads to embryonic lethality. Here, we report results obtained with conditional PC4 knock-out (PC4(f/f) Nestin-Cre) mice where PC4 is knocked out specifically in the brain. Compared with the control (PC4(+/+) Nestin-Cre) mice, PC4(f/f) Nestin-Cre mice are smaller with decreased nocturnal activity but are fertile and show no motor dysfunction. Neurons in different areas of the brains of these mice show sensitivity to hypoxia/anoxia, and decreased adult neurogenesis was observed in the dentate gyrus. Interestingly, PC4(f/f) Nestin-Cre mice exhibit a severe deficit in spatial memory extinction, whereas acquisition and long term retention were unaffected. Gene expression analysis of the dorsal hippocampus of PC4(f/f) Nestin-Cre mice revealed dysregulated expression of several neural function-associated genes, and PC4 was consistently found to localize on the promoters of these genes, indicating that PC4 regulates their expression. These observations indicate that non-histone chromatin-associated proteins like PC4 play a significant role in neuronal plasticity.

  20. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters

    Science.gov (United States)

    Lavender, Christopher A.; Hoffman, Jackson A.; Trotter, Kevin W.; Gilchrist, Daniel A.; Bennett, Brian D.; Burkholder, Adam B.; Fargo, David C.; Archer, Trevor K.

    2016-01-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  1. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters.

    Science.gov (United States)

    Lavender, Christopher A; Cannady, Kimberly R; Hoffman, Jackson A; Trotter, Kevin W; Gilchrist, Daniel A; Bennett, Brian D; Burkholder, Adam B; Burd, Craig J; Fargo, David C; Archer, Trevor K

    2016-08-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  2. Recognition of chromatin by the plant alkaloid, ellipticine as a dual binder

    International Nuclear Information System (INIS)

    Recognition of core histone components of chromatin along with chromosomal DNA by a class of small molecule modulators is worth examining to evaluate their intracellular mode of action. A plant alkaloid ellipticine (ELP) which is a putative anticancer agent has so far been reported to function via DNA intercalation, association with topoisomerase II and binding to telomere region. However, its effect upon the potential intracellular target, chromatin is hitherto unreported. Here we have characterized the biomolecular recognition between ELP and different hierarchical levels of chromatin. The significant result is that in addition to DNA, it binds to core histone(s) and can be categorized as a ‘dual binder’. As a sequel to binding with histone(s) and core octamer, it alters post-translational histone acetylation marks. We have further demonstrated that it has the potential to modulate gene expression thereby regulating several key biological processes such as nuclear organization, transcription, translation and histone modifications. - Highlights: • Ellipticine acts a dual binder binding to both DNA and core histone(s). • It induces structural perturbations in chromatin, chromatosome and histone octamer. • It alters histones acetylation and affects global gene expression

  3. Changes in chromatin-associated proteins of virus-infected tobacco leaves

    NARCIS (Netherlands)

    Telgen, van H.J.

    1985-01-01

    Symptoms of viral infections in plants often resemble disturbances in growth and development. Therefore, symptoms appear to result from an interference of the virus with the regulation of growth and development of the host plant. Particularly the non-histone chromatin- associated proteins are consid

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

    Science.gov (United States)

    Daban, Joan-Ramon

    2015-10-08

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

  5. Genome-wide chromatin remodeling identified at GC-rich long nucleosome-free regions.

    Directory of Open Access Journals (Sweden)

    Karin Schwarzbauer

    Full Text Available To gain deeper insights into principles of cell biology, it is essential to understand how cells reorganize their genomes by chromatin remodeling. We analyzed chromatin remodeling on next generation sequencing data from resting and activated T cells to determine a whole-genome chromatin remodeling landscape. We consider chromatin remodeling in terms of nucleosome repositioning which can be observed most robustly in long nucleosome-free regions (LNFRs that are occupied by nucleosomes in another cell state. We found that LNFR sequences are either AT-rich or GC-rich, where nucleosome repositioning was observed much more prominently in GC-rich LNFRs - a considerable proportion of them outside promoter regions. Using support vector machines with string kernels, we identified a GC-rich DNA sequence pattern indicating loci of nucleosome repositioning in resting T cells. This pattern appears to be also typical for CpG islands. We found out that nucleosome repositioning in GC-rich LNFRs is indeed associated with CpG islands and with binding sites of the CpG-island-binding ZF-CXXC proteins KDM2A and CFP1. That this association occurs prominently inside and also prominently outside of promoter regions hints at a mechanism governing nucleosome repositioning that acts on a whole-genome scale.

  6. The Correlation of Sperm Chromatin Decondensation Following In Vitro Exposure to Heparin and Sperm Penetration Rates

    OpenAIRE

    Carrell, Douglas T.; Emery, Benjamin R.; Peterson, C. Matthew

    1998-01-01

    Purpose:The aim of this study was to evaluate the possible correlation of low-dose heparin-induced decondensation of sperm chromatin with sperm concentration, motility, morphology, membrane hypoosmotic response, ejaculate volume, and the ability of sperm to penetrate zona-free hamster oocytes.

  7. Put your 3D glasses on: plant chromatin is on show

    KAUST Repository

    Rodriguez-Granados, Natalia Y.

    2016-04-30

    The three-dimensional organization of the eukaryotic nucleus and its chromosomal conformation have emerged as important features in the complex network of mechanisms behind gene activity and genome connectivity dynamics, which can be evidenced in the regionalized chromosomal spatial distribution and the clustering of diverse genomic regions with similar expression patterns. The development of chromatin conformation capture (3C) techniques has permitted the elucidation of commonalities between the eukaryotic phyla, as well as important differences among them. The growing number of studies in the field performed in plants has shed light on the structural and regulatory features of these organisms. For instance, it has been proposed that plant chromatin can be arranged into different conformations such as Rabl, Rosette-like, and Bouquet, and that both short- and long-range chromatin interactions occur in Arabidopsis. In this review, we compile the current knowledge about chromosome architecture characteristics in plants, as well as the molecular events and elements (including long non-coding RNAs, histone and DNA modifications, chromatin remodeling complexes, and transcription factors) shaping the genome three-dimensional conformation. Furthermore, we discuss the developmental outputs of genome topology-mediated gene expression regulation. It is becoming increasingly clear that new tools and techniques with higher resolution need to be developed and implemented in Arabidopsis and other model plants in order to better understand chromosome architecture dynamics, from an integrative perspective with other fields of plant biology such as development, stress biology, and finally agriculture. © 2016 The Author 2016.

  8. Noncoding transcription by alternative rna polymerases dynamically regulates an auxin-driven chromatin loop

    KAUST Repository

    Ariel, Federico D.

    2014-08-01

    The eukaryotic epigenome is shaped by the genome topology in three-dimensional space. Dynamic reversible variations in this epigenome structure directly influence the transcriptional responses to developmental cues. Here, we show that the Arabidopsis long intergenic noncoding RNA (lincRNA) APOLO is transcribed by RNA polymerases II and V in response to auxin, a phytohormone controlling numerous facets of plant development. This dual APOLO transcription regulates the formation of a chromatin loop encompassing the promoter of its neighboring gene PID, a key regulator of polar auxin transport. Altering APOLO expression affects chromatin loop formation, whereas RNA-dependent DNA methylation, active DNA demethylation, and Polycomb complexes control loop dynamics. This dynamic chromatin topology determines PID expression patterns. Hence, the dual transcription of a lincRNA influences local chromatin topology and directs dynamic auxin-controlled developmental outputs on neighboring genes. This mechanism likely underscores the adaptive success of plants in diverse environments and may be widespread in eukaryotes. © 2014 Elsevier Inc.

  9. CHROMATIN TEXTURE OF MELANOCYTIC NUCLEI - CORRELATION BETWEEN LIGHT AND ELECTRON-MICROSCOPY

    NARCIS (Netherlands)

    ABMAYR, W; STOLZ, W; KORHERR, S; WILD, W; SCHMOECKEL, C

    1987-01-01

    Cells of a benign pigmented mole and a malignant melanoma were used to compare electron microscopy (EM) and light microscopy (LM) with high-resolution TV-scanning and multivariate analysis methods. Special emphasis was placed on different kinds of chromatin texture features and their discriminating

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Martina Belli

    2014-01-01

    Full Text Available 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.

  12. At the intersection of non-coding transcription, DNA repair, chromatin structure, and cellular senescence

    Directory of Open Access Journals (Sweden)

    Ryosuke eOhsawa

    2013-07-01

    Full Text Available It is well accepted that non-coding RNAs play a critical role in regulating gene expression. Recent paradigm-setting studies are now revealing that non-coding RNAs, other than microRNAs, also play intriguing roles in the maintenance of chromatin structure, in the DNA damage response, and in adult human stem cell aging. In this review, we will discuss the complex inter-dependent relationships among non-coding RNA transcription, maintenance of genomic stability, chromatin structure and adult stem cell senescence. DNA damage-induced non-coding RNAs transcribed in the vicinity of the DNA break regulate recruitment of the DNA damage machinery and DNA repair efficiency. We will discuss the correlation between non-coding RNAs and DNA damage repair efficiency and the potential role of changing chromatin structures around double-strand break sites. On the other hand, induction of non-coding RNA transcription from the repetitive Alu elements occurs during human stem cell aging and hinders efficient DNA repair causing entry into senescence. We will discuss how this fine balance between transcription and genomic instability may be regulated by the dramatic changes to chromatin structure that accompany cellular senescence.

  13. A chromatin immunoprecipitation (ChIP) protocol for use in whole human adipose tissue.

    Science.gov (United States)

    Haim, Yulia; Tarnovscki, Tanya; Bashari, Dana; Rudich, Assaf

    2013-11-01

    Chromatin immunoprecipitation (ChIP) has become a central method when studying in vivo protein-DNA interactions, with the major challenge being the hope to capture "authentic" interactions. While ChIP protocols have been optimized for use with specific cell types and tissues including adipose tissue-derived cells, a working ChIP protocol addressing the challenges imposed by fresh whole human adipose tissue has not been described. Utilizing human paired omental and subcutaneous adipose tissue obtained during elective abdominal surgeries, we have carefully identified and optimized individual steps in the ChIP protocol employed directly on fresh tissue fragments. We describe a complete working protocol for using ChIP on whole adipose tissue fragments. Specific steps required adaptation of the ChIP protocol to human whole adipose tissue. In particular, a cross-linking step was performed directly on fresh small tissue fragments. Nuclei were isolated before releasing chromatin, allowing better management of fat content; a sonication protocol to obtain fragmented chromatin was optimized. We also demonstrate the high sensitivity of immunoprecipitated chromatin from adipose tissue to freezing. In conclusion, we describe the development of a ChIP protocol optimized for use in studying whole human adipose tissue, providing solutions for the unique challenges imposed by this tissue. Unraveling protein-DNA interaction in whole human adipose tissue will likely contribute to elucidating molecular pathways contributing to common human diseases such as obesity and type 2 diabetes.

  14. A Development of Nucleic Chromatin Measurements as a New Prognostic Marker for Severe Chronic Heart Failure.

    Directory of Open Access Journals (Sweden)

    Machiko Kanzaki

    Full Text Available Accurate prediction of both mortality and morbidity is of significant importance, but it is challenging in patients with severe heart failure. It is especially difficult to detect the optimal time for implanting mechanical circulatory support devices in such patients. We aimed to analyze the morphometric ultrastructure of nuclear chromatin in cardiomyocytes by developing an original clinical histopathological method. Using this method, we developed a biomarker to predict poor outcome in patients with dilated cardiomyopathy (DCM.As a part of their diagnostic evaluation, 171 patients underwent endomyocardial biopsy (EMB. Of these, 63 patients diagnosed with DCM were included in this study. We used electron microscopic imaging of cardiomyocyte nuclei and an automated image analysis software program to assess whether it was possible to detect discontinuity of the nuclear periphery. Twelve months after EMB, all patients with a discontinuous nuclear periphery (Group A, n = 11 died from heart failure or underwent left ventricular assist device (VAD implantation. In contrast, in patients with a continuous nuclear periphery (Group N, n = 52 only 7 patients (13% underwent VAD implantation and there were no deaths (p<0.01. We then evaluated chromatin particle density (Nuc-CS and chromatin thickness in the nuclear periphery (Per-CS in Group N patients; these new parameters were able to identify patients with poor prognosis.We developed novel morphometric methods based on cardiomyocyte nuclear chromatin that may provide pivotal information for early prediction of poor prognosis in patients with DCM.

  15. Influence of oncogenic transcription factors on chromatin conformation and implications in prostate cancer

    Directory of Open Access Journals (Sweden)

    Yang YA

    2014-05-01

    Full Text Available Yeqing Angela Yang,1 Jung Kim,1 Jindan Yu1,21Division of Hematology/Oncology, Department of Medicine, 2Robert H Lurie Comprehensive Cancer Center, Northwestern University, Feinberg School of Medicine, Chicago, IL, USAAbstract: In recent years, facilitated by rapid technological advances, we are becoming more adept at probing the molecular processes, which take place in the nucleus, that are crucial for the hierarchical regulation and organization of chromatin architecture. With an unprecedented level of resolution, a detailed atlas of chromosomal structures (histone displacement, variants, modifications, chromosome territories, and DNA looping and mechanisms underlying their establishment, provides invaluable insight into physiological as well as pathological phenomena. In this review, we will focus on prostate cancer, a prevalent malignancy in men worldwide, and for which a curative treatment strategy is yet to be attained. We aim to catalog the most frequently observed oncogenic alterations associated with chromatin conformation, while emphasizing the TMPRSS2-ERG fusion, which is found in more than one-half of prostate cancer patients and its functions in compromising the chromatin landscape in prostate cancer.Keywords: chromatin conformation, ERG, prostate cancer

  16. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells

    Science.gov (United States)

    de Dieuleveult, Maud; Yen, Kuangyu; Hmitou, Isabelle; Depaux, Arnaud; Boussouar, Fayçal; Dargham, Daria Bou; Jounier, Sylvie; Humbertclaude, Hélène; Ribierre, Florence; Baulard, Céline; Farrell, Nina P.; Park, Bongsoo; Keime, Céline; Carrière, Lucie; Berlivet, Soizick; Gut, Marta; Gut, Ivo; Werner, Michel; Deleuze, Jean-François; Olaso, Robert; Aude, Jean-Christophe; Chantalat, Sophie; Pugh, B. Franklin; Gérard, Matthieu

    2015-01-01

    Summary ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers1–3 target specific nucleosomes to regulate transcription is unclear. Here, we present genome-wide remodeller-nucleosome interaction profiles for Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank MNase-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites (TSSs) are nevertheless chromatinized with non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and modifications (H3K4me3 and H3K27ac). RNA polymerase (pol) II therefore navigates hundreds of bp of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3′ end of the NFR. Transcriptome analysis upon remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers play either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs. PMID:26814966

  17. A polycomb group protein is retained at specific sites on chromatin in mitosis.

    Directory of Open Access Journals (Sweden)

    Nicole E Follmer

    Full Text Available Epigenetic regulation of gene expression, including by Polycomb Group (PcG proteins, may depend on heritable chromatin states, but how these states can be propagated through mitosis is unclear. Using immunofluorescence and biochemical fractionation, we find PcG proteins associated with mitotic chromosomes in Drosophila S2 cells. Genome-wide sequencing of chromatin immunoprecipitations (ChIP-SEQ from mitotic cells indicates that Posterior Sex Combs (PSC is not present at well-characterized PcG targets including Hox genes in mitosis, but does remain at a subset of interphase sites. Many of these persistent sites overlap with chromatin domain borders described by Sexton et al. (2012, which are genomic regions characterized by low levels of long range contacts. Persistent PSC binding sites flank both Hox gene clusters. We hypothesize that disruption of long-range chromatin contacts in mitosis contributes to PcG protein release from most sites, while persistent binding at sites with minimal long-range contacts may nucleate re-establishment of PcG binding and chromosome organization after mitosis.

  18. Remodeling of chromatin structure in senescent cells and its potential impact on tumor suppression and aging

    OpenAIRE

    Adams, Peter D

    2007-01-01

    Cellular senescence is an important tumor suppression process, and a possible contributor to tissue aging. Senescence is accompanied extensive changes in chromatin structure. In particular, many senescent cells accumulate specialized domains of facultative heterochromatin, called Senescence Associated Heterochromatin Foci (SAHF), which are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. This article reviews ou...

  19. New insights into protamine-like component organization in Mytilus galloprovincialis' sperm chromatin.

    Science.gov (United States)

    Vassalli, Quirino Attilio; Caccavale, Filomena; Avagnano, Stefano; Murolo, Alessandra; Guerriero, Giulia; Fucci, Laura; Ausió, Juan; Piscopo, Marina

    2015-03-01

    We have analyzed Mytilus galloprovincialis' sperm chromatin, which consists of three protamine-like proteins, PL-II, PL-III, and PL-IV, in addition to a residual amount of the four core histones. We have probed the structure of this sperm chromatin through digestion with micrococcal nuclease (MNase) in combination with salt fractionation. Furthermore, we used the electrophoretic mobility shift assay to define DNA-binding mode of PL-II and PL-III and turbidimetric assays to determine their self-association ability in the presence of sodium phosphate. Although in literature it is reported that M. galloprovincialis' sperm chromatin lacks nucleosomal organization, our results obtained by MNase digestion suggest the existence of a likely unusual organization, in which there would be a more accessible location of PL-II/PL-IV when compared with PL-III and core histones. So, we hypothesize that in M. galloprovincialis' sperm chromatin organization DNA is wrapped around a PL-III protein core and core histones and PL-II and PL-IV are bound to the flanking DNA regions (similarly to somatic histone H1). Furthermore, we propose that PL's K/R ratio affects their DNA-binding mode and self-association ability as reported previously for somatic and sperm H1 histones.

  20. SWI/SNF-like chromatin remodeling factor Fun30 supports point centromere function in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Mickaël Durand-Dubief

    2012-09-01

    Full Text Available Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture.

  1. Phosphorylation of both nucleoplasmin domains is required for activation of its chromatin decondensation activity

    DEFF Research Database (Denmark)

    Bañuelos, Sonia; Omaetxebarria, Miren J; Ramos, Isbaal;

    2007-01-01

    Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified...

  2. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells.

    Science.gov (United States)

    de Dieuleveult, Maud; Yen, Kuangyu; Hmitou, Isabelle; Depaux, Arnaud; Boussouar, Fayçal; Bou Dargham, Daria; Jounier, Sylvie; Humbertclaude, Hélène; Ribierre, Florence; Baulard, Céline; Farrell, Nina P; Park, Bongsoo; Keime, Céline; Carrière, Lucie; Berlivet, Soizick; Gut, Marta; Gut, Ivo; Werner, Michel; Deleuze, Jean-François; Olaso, Robert; Aude, Jean-Christophe; Chantalat, Sophie; Pugh, B Franklin; Gérard, Matthieu

    2016-02-01

    ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers target specific nucleosomes to regulate transcription is unclear. Here we present genome-wide remodeller-nucleosome interaction profiles for the chromatin remodellers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank micrococcal nuclease (MNase)-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites are nevertheless bound by non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and marked by H3K4me3 and H3K27ac modifications. RNA polymerase II therefore navigates hundreds of base pairs of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3' end of the NFR. Transcriptome analysis after remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers have either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs.

  3. Cotranscriptional Chromatin Remodeling by Small RNA Species: An HTLV-1 Perspective

    Directory of Open Access Journals (Sweden)

    Nishat Aliya

    2012-01-01

    Full Text Available Cell type specificity of human T cell leukemia virus 1 has been proposed as a possible reason for differential viral outcome in primary target cells versus secondary. Through chromatin remodeling, the HTLV-1 transactivator protein Tax interacts with cellular factors at the chromosomally integrated viral promoter to activate downstream genes and control viral transcription. RNA interference is the host innate defense mechanism mediated by short RNA species (siRNA or miRNA that regulate gene expression. There exists a close collaborative functioning of cellular transcription factors with miRNA in order to regulate the expression of a number of eukaryotic genes including those involved in suppression of cell growth, induction of apoptosis, as well as repressing viral replication and propagation. In addition, it has been suggested that retroviral latency is influenced by chromatin alterations brought about by miRNA. Since Tax requires the assembly of transcriptional cofactors to carry out viral gene expression, there might be a close association between miRNA influencing chromatin alterations and Tax-mediated LTR activation. Herein we explore the possible interplay between HTLV-1 infection and miRNA pathways resulting in chromatin reorganization as one of the mechanisms determining HTLV-1 cell specificity and viral fate in different cell types.

  4. Comparing genome-wide chromatin profiles using ChIP-chip or ChIP-seq

    NARCIS (Netherlands)

    Johannes, Frank; Wardenaar, Rene; Colomé Tatché, Maria; Mousson, Florence; de Graaf, Petra; Mokry, Michal; Guryev, Victor; Timmers, H. Th. Marc; Cuppen, Edwin; Jansen, Ritsert C.; Bateman, Alex

    2010-01-01

    Motivation: ChIP-chip and ChIP-seq technologies provide genomewide measurements of various types of chromatin marks at an unprecedented resolution. With ChIP samples collected from different tissue types and/ or individuals, we can now begin to characterize stochastic or systematic changes in epigen

  5. Comparing genome-wide chromatin profiles using ChIP-chip or ChIP-seq

    NARCIS (Netherlands)

    Johannes, F.; Wardenaar, R.; Colome-Tatche, M.; Mousson, F.; de Graaf, P.; Mokry, M.; Guryev, V.; Timmers, H.T.; Cuppen, E.; Jansen, R.

    2010-01-01

    MOTIVATION: ChIP-chip and ChIP-seq technologies provide genome-wide measurements of various types of chromatin marks at an unprecedented resolution. With ChIP samples collected from different tissue types and/or individuals, we can now begin to characterize stochastic or systematic changes in epigen

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

    Science.gov (United States)

    Jenkins, Jill A.

    2013-01-01

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

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

    NARCIS (Netherlands)

    Babaei, S.; Mahfouz, A.M.E.T.A.; Hulsman, M.; Lelieveldt, B.P.F.; De Ridder, J.; Reinders, M.J.T.

    2015-01-01

    The three dimensional conformation of the genome in the cell nucleus influences important biological processes such as gene expression regulation. Recent studies have shown a strong correlation between chromatin interactions and gene co-expression. However, predicting gene co-expression from frequen

  8. Effects of tamoxifen citrate on gene expression during nuclear chromatin condensation in male rats

    Institute of Scientific and Technical Information of China (English)

    Mukhtar Aleem; Varsha Padwal; Jyoti Choudhari; Nafisa Balasinor; Priyanka Parte; Manjeet Gill-Sharma

    2005-01-01

    Aim: To evaluate the effects of tamoxifen citrate on gene expression during nuclear chromatin condensation in male decondensation, acridine orange (AO) dye uptake, concentration of thiol-groups, levels and/or expression of transition proteins 1, 2 (TP1, TP2), protamine 1 (P1), cyclic AMP response element modulator-τ (CREMτ), androgenbinding protein (ABP) and cyclic adenosine 3', 5' monophosphate (cAMP) were evaluated after 60 days of exposure in adult male rats. Controls received the vehicle. Results: Tamoxifen citrate enhanced the rates of chromatin decondensation, increased AO dye uptake and reduced free thiols in caput epididymal sperms and reduced the levels of TP1, TP2, P1, and CREMτ in the testis, while cAMP was unaffected. P1 deposition was absent in the sperm. The transcripts of TP1, TP2 were increased, of P1 and ABP decreased, while those of CREMτ unaffected in the testis.Conclusion: Tamoxifen citrate reduced caput epididymal sperm chromatin compaction by reducing the testicular levels of proteins TP1, TP2 and P1 and the CREMτ involved in chromatin condensation during spermiogenesis.Tamoxifen citrate affects the expression of these genes at both the transcriptional and post-transcriptional levels.

  9. Steroid Receptors Reprogram FoxA1 Occupancy through Dynamic Chromatin Transitions

    DEFF Research Database (Denmark)

    Swinstead, Erin E; Miranda, Tina B; Paakinaho, Ville;

    2016-01-01

    between ER, GR, and FoxA1 requires further investigation. Here we show that ER and GR both have the ability to alter the genomic distribution of the FoxA1 pioneer factor. Single-molecule tracking experiments in live cells reveal a highly dynamic interaction of FoxA1 with chromatin in vivo. Furthermore...

  10. Incorporating double copies of a chromatin insulator into lentiviral vectors results in less viral integrants

    DEFF Research Database (Denmark)

    Nielsen, Troels T; Jakobsson, Johan; Rosenqvist, Nina;

    2009-01-01

    BACKGROUND: Lentiviral vectors hold great promise as gene transfer vectors in gene therapeutic settings. However, problems related to the risk of insertional mutagenesis, transgene silencing and positional effects have stalled the use of such vectors in the clinic. Chromatin insulators are boundary...

  11. Recognition of chromatin by the plant alkaloid, ellipticine as a dual binder

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Amrita; Sanyal, Sulagna; Majumder, Parijat [Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics, Block-AF, Sector-1, Bidhan Nagar, Kolkata 700064, West Bengal (India); Chakraborty, Payal [Bionivid Technology Pvt Ltd, Kasturi Nagar, Bangalore 560043 (India); Jana, Kuladip [Division of Molecular Medicine, Centre for Translational Animal Research, Bose Institute, P-1/12 C.I.T. Scheme VIIM, Kolkata 700054, West Bengal (India); Das, Chandrima, E-mail: chandrima.das@saha.ac.in [Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics, Block-AF, Sector-1, Bidhan Nagar, Kolkata 700064, West Bengal (India); Dasgupta, Dipak, E-mail: dipak.dasgupta@saha.ac.in [Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics, Block-AF, Sector-1, Bidhan Nagar, Kolkata 700064, West Bengal (India)

    2015-07-10

    Recognition of core histone components of chromatin along with chromosomal DNA by a class of small molecule modulators is worth examining to evaluate their intracellular mode of action. A plant alkaloid ellipticine (ELP) which is a putative anticancer agent has so far been reported to function via DNA intercalation, association with topoisomerase II and binding to telomere region. However, its effect upon the potential intracellular target, chromatin is hitherto unreported. Here we have characterized the biomolecular recognition between ELP and different hierarchical levels of chromatin. The significant result is that in addition to DNA, it binds to core histone(s) and can be categorized as a ‘dual binder’. As a sequel to binding with histone(s) and core octamer, it alters post-translational histone acetylation marks. We have further demonstrated that it has the potential to modulate gene expression thereby regulating several key biological processes such as nuclear organization, transcription, translation and histone modifications. - Highlights: • Ellipticine acts a dual binder binding to both DNA and core histone(s). • It induces structural perturbations in chromatin, chromatosome and histone octamer. • It alters histones acetylation and affects global gene expression.

  12. Early aberrations in chromatin dynamics in embryos produced under In vitro conditions

    DEFF Research Database (Denmark)

    Deshmukh, Rahul Shahaji; Østrup, Olga; Strejcek, Frantisek;

    2012-01-01

    In vitro production of porcine embryos by means of in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) is limited by great inefficienciy. The present study investigated chromatin and nucleolar dynamics in porcine embryos developed in vivo (IV) and compared this physiological...

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

    Directory of Open Access Journals (Sweden)

    Yu Jingjing

    2011-11-01

    Full Text Available Abstract 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

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

    Science.gov (United States)

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

    2016-01-01

    In female mammals, activation of Xist (X-inactive specific transcript) is essential for establishment of X chromosome inactivation. During early embryonic development in mice, paternal Xist is preferentially expressed whereas maternal Xist (Xm-Xist) is silenced. Unlike autosomal imprinted genes, Xist imprinting for Xm-Xist silencing was erased in cloned or parthenogenetic but not fertilized embryos. However, the molecular mechanism underlying the variable nature of Xm-Xist imprinting is poorly understood. Here, we revealed that Xm-Xist silencing depends on chromatin condensation states at the Xist/Tsix genomic region and on Rnf12 expression levels. In early preimplantation, chromatin decondensation via H3K9me3 loss and histone acetylation gain caused Xm-Xist derepression irrespective of embryo type. Although the presence of the paternal genome during pronuclear formation impeded Xm-Xist derepression, Xm-Xist was robustly derepressed when the maternal genome was decondensed before fertilization. Once Xm-Xist was derepressed by chromatin alterations, the derepression was stably maintained and rescued XmXpΔ lethality, indicating that loss of Xm-Xist imprinting was irreversible. In late preimplantation, Oct4 served as a chromatin opener to create transcriptional permissive states at Xm-Xist/Tsix genomic loci. In parthenogenetic embryos, Rnf12 overdose caused Xm-Xist derepression via Xm-Tsix repression; physiological Rnf12 levels were essential for Xm-Xist silencing maintenance in fertilized embryos. Thus, chromatin condensation and fine-tuning of Rnf12 dosage were crucial for Xist imprint maintenance by silencing Xm-Xist. PMID:27788132

  15. Histone crosstalk directed by H2B ubiquitination is required for chromatin boundary integrity.

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    Meiji Kit-Wan Ma

    2011-07-01

    Full Text Available Genomic maps of chromatin modifications have provided evidence for the partitioning of genomes into domains of distinct chromatin states, which assist coordinated gene regulation. The maintenance of chromatin domain integrity can require the setting of boundaries. The HS4 insulator element marks the 3' boundary of a heterochromatin region located upstream of the chicken β-globin gene cluster. Here we show that HS4 recruits the E3 ligase RNF20/BRE1A to mediate H2B mono-ubiquitination (H2Bub1 at this insulator. Knockdown experiments show that RNF20 is required for H2Bub1 and processive H3K4 methylation. Depletion of RNF20 results in a collapse of the active histone modification signature at the HS4 chromatin boundary, where H2Bub1, H3K4 methylation, and hyperacetylation of H3, H4, and H2A.Z are rapidly lost. A remarkably similar set of events occurs at the HSA/HSB regulatory elements of the FOLR1 gene, which mark the 5' boundary of the same heterochromatin region. We find that persistent H2Bub1 at the HSA/HSB and HS4 elements is required for chromatin boundary integrity. The loss of boundary function leads to the sequential spreading of H3K9me2, H3K9me3, and H4K20me3 over the entire 50 kb FOLR1 and β-globin region and silencing of FOLR1 expression. These findings show that the HSA/HSB and HS4 boundary elements direct a cascade of active histone modifications that defend the FOLR1 and β-globin gene loci from the pervasive encroachment of an adjacent heterochromatin domain. We propose that many gene loci employ H2Bub1-dependent boundaries to prevent heterochromatin spreading.

  16. Genetic variants in chromatin-remodeling pathway associated with lung cancer risk in a Chinese population.

    Science.gov (United States)

    Geng, Liguo; Zhu, Meng; Wang, Yuzhuo; Cheng, Yang; Liu, Jia; Shen, Wei; Li, Zhihua; Zhang, Jiahui; Wang, Cheng; Jin, Guangfu; Ma, Hongxia; Shen, Hongbing; Hu, Zhibin; Dai, Juncheng

    2016-08-10

    Chromatin remodeling complexes utilize the energy of ATP hydrolysis to remodel nucleosomes and have essential roles in transcriptional modulation. Increasing evidences indicate that these complexes directly interact with numerous proteins and regulate the formation of cancer. However, few studies reported the association of polymorphisms in chromatin remodeling genes and lung cancer. We hypothesized that variants in critical genes of chromatin remodeling pathway might contribute to the susceptibility of lung cancer. To validate this hypothesis, we systematically screened 40 polymorphisms in six key chromatin remodeling genes (SMARCA5, SMARCC2, SMARCD2, ARID1A, NR3C1 and SATB1) and evaluated them with a case-control study including 1341 cases and 1982 controls. Logistic regression revealed that four variants in NR3C1 and SATB1 were significantly associated with lung cancer risk after false discovery rate (FDR) correction [For NR3C1, rs9324921: odds ratio (OR)=1.23, P for FDR=0.029; rs12521436: OR=0.85, P for FDR=0.040; rs4912913: OR=1.17, P for FDR=0.040; For SATB1, rs6808523: OR=1.33, P for FDR=0.040]. Combing analysis presented a significant allele-dosage tendency for the number of risk alleles and lung cancer risk (Ptrendlung tumor and adjacent normal tissues in the database of The Cancer Genome Atlas (TCGA) (P=0.009 for rs6808523). These findings suggested that genetic variants in key chromatin remodeling genes may contribute to lung cancer risk in Chinese population. Further large and well-designed studies are warranted to validate our results. PMID:27179949

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

    Science.gov (United States)

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

    2016-01-01

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

  18. Androgen receptor serine 81 phosphorylation mediates chromatin binding and transcriptional activation.

    Science.gov (United States)

    Chen, Shaoyong; Gulla, Sarah; Cai, Changmeng; Balk, Steven P

    2012-03-01

    Our previous findings indicated that androgen receptor (AR) phosphorylation at serine 81 is stimulated by the mitotic cyclin-dependent kinase 1 (CDK1). In this report, we extended our previous study and confirmed that Ser-81 phosphorylation increases during mitosis, coincident with CDK1 activation. We further showed blocking cell cycle at G(1) or S phase did not disrupt androgen-induced Ser-81 phosphorylation and AR-dependent transcription, consistent with a recent report that AR was phosphorylated at Ser-81 and activated by the transcriptional CDK9. To assess the function of Ser-81 phosphorylation in prostate cancer (PCa) cells expressing endogenous AR, we developed a ligand switch strategy using a ligand-binding domain mutation (W741C) that renders AR responsive to the antagonist bicalutamide. An S81A/W741C double mutant AR stably expressed in PCa cells failed to transactivate the endogenous AR-regulated PSA or TMPRSS2 genes. ChIP showed that the S81A mutation prevented ligand-induced AR recruitment to these genes, and cellular fractionation revealed that the S81A mutation globally abrogated chromatin binding. Conversely, the AR fraction rapidly recruited to chromatin after androgen stimulation was highly enriched for Ser-81 phosphorylation. Finally, inhibition of CDK1 and CDK9 decreased AR Ser-81 phosphorylation, chromatin binding, and transcriptional activity. These findings indicate that Ser-81 phosphorylation by CDK9 stabilizes AR chromatin binding for transcription and suggest that CDK1-mediated Ser-81 phosphorylation during mitosis provides a pool of Ser-81 phosphorylation AR that can be readily recruited to chromatin for gene reactivation and may enhance AR activity in PCa.

  19. Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context.

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    Artyom A Alekseyenko

    Full Text Available The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at "entry sites" that contain a consensus sequence motif ("MSL recognition element" or MRE. However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex and female Kc cells (which lack the complex, we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.

  20. Solubilization of the chromatin-bound estrogen receptor from chicken liver and fractionation on hydroxylapatite.

    Science.gov (United States)

    Gschwendt, M

    1976-08-16

    1. High-affinity estrogen-binding sites can be solubilized from the liver chromatin of estrogenized chickens by treatment of the chromatin with 2 M KCL/5 M urea and fractionation on hydroxylapatite. Two estrogen-binding proteins are eluted from hydroxylapatite columns by 20mM phosphate (binding protein I) and 200mMphosphate (binding protein II), respectively. 2. The binding protein I is part of a non-histone protein fraction containing acid-soluble and insoluble proteins, whereas the binding protein II elutes together with high molecular weight nonhistone proteins containing acid insoluble proteins only. Both binding proteins exhibit the smae affinity for estradiol (Kd approximately 10(-9) M). 3. From chromatin of untreated chickens very small amounts of binding protein I (0.1 pmol/mg protein compared to 1.9 pmol/mg protein from estrogenized chickens) with the smae affinity for estradiol as that from estrogenized animals can be solubilized. Binding protein II is not detectable. 4. The "soluble nuclear estrogen receptor" extracted from crude liver nucleir of estrogenized chickens by 0.5 M KCL behaves on hydroxylapatite very similarly to salt/urea-dissociated chromatin with respect to the binding protein I. No binding protein II, however, can be demonstrated. 5. Chromatography of various preparations on Bio-Gel A-1.5 m indicates that the binding protein II is a residual chromatin fragment containing an unseparated binding protein-DNA complex, whereas the binding protein I represents the solubilized nucleic-acid-free chromosomal estrogen receptor. The "soluble nuclear receptor" and the binding protein I, however, are not identical with respect to their chromatographic behaviour on Bio-Gel A-1.5m, even though their estrogen binding entity remaining after trypsin treatment seems to be very similar.

  1. Differential Response of Human Hepatocyte Chromatin to HDAC Inhibitors as a Function of Microenvironmental Glucose Level.

    Science.gov (United States)

    Felisbino, Marina Barreto; Alves da Costa, Thiago; Gatti, Maria Silvia Viccari; Mello, Maria Luiza Silveira

    2016-10-01

    Diabetes is a complex multifactorial disorder characterized by chronic hyperglycemia due to impaired insulin secretion. Recent observations suggest that the complexity of the disease cannot be entirely accounted for genetic predisposition and a compelling argument for an epigenetic component is rapidly emerging. The use of histone deacetylase inhibitor (HDACi) in clinical setting is an emerging area of investigation. In this study, we have aimed to understand and compare the response of hepatocyte chromatin to valproic acid (VPA) and trichostatin A (TSA) treatments under normoglycemic or hyperglycemic conditions to expand our knowledge about the consequences of HDACi treatment in a diabetes cell model. Under normoglycemic conditions, these treatments promoted chromatin remodeling, as assessed by image analysis and H3K9ac and H3K9me2 abundance. Simultaneously, H3K9ac marks shifted to the nuclear periphery accompanied by HP1 dissociation from the heterochromatin and a G1 cell cycle arrest. More striking changes in the cell cycle progression and mitotic ratios required drastic treatment. Under hyperglycemic conditions, high glucose per se promoted chromatin changes similar to those promoted by VPA and TSA. Nonetheless, these results were not intensified in cells treated with HDACis under hyperglycemic conditions. Despite the absence of morphological changes being promoted, HDACi treatment seems to confer a physiological meaning, ameliorating the cellular hyperglycemic state through reduction of glucose production. These observations allow us to conclude that the glucose level to which the hepatocytes are subjected affects how chromatin responds to HDACi and their action under high-glucose environment might not reflect on chromatin remodeling. J. Cell. Physiol. 231: 2257-2265, 2016. © 2016 Wiley Periodicals, Inc. PMID:26888775

  2. Nuclear envelope proteins and chromatin arrangement: a pathogenic mechanism for laminopathies

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    NM Maraldi

    2009-06-01

    Full Text Available The involvement of the nuclear envelope in the modulation of chromatin organization is strongly suggested by the increasing number of human diseases due to mutations of nuclear envelope proteins. A common feature of these diseases, named laminopathies, is the occurrence of major chromatin defects. Laminopathies share in some instances their clinical features, but each of them is characterized by a phenotype that involves one or multiple tissues.We previously reported that cells from laminopathic patients show an altered nuclear profile, and loss or detachment of heterochromatin from the nuclear envelope. Recent evidence indicates that processing of the lamin A precursor is altered in laminopathies featuring pre-mature aging and/or lipodystrophy phenotype. In these cases, pre-lamin A is accumulated in the nucleus and heterochromatin is severely disorganized. Moreover, altered distribution and solubility properties of heterochromatin-associated proteins such as HP1 are observed. These findings indicate that defects of chromatin remodeling are involved in the cascade of epigenetic events leading to the laminopathic phenotypes. Here we report evidence indicating that pre-lamin A is mis-localized in the nuclei of Emery-Dreifuss muscular dystrophy fibroblasts, either bearing lamin A/C or emerin mutations. Abnornal pre-lamin A-containing structures are formed following treatment with a farnesyl-transferase inhibitor, a drug that causes accumulation of non-farnesylated pre-lamin A. Pre-lamin A-labeled structures co-localize with heterochromatin clumps. These data indicate that in almost all laminopathies the expression of the mutant lamin A precursor disrupts the organization of heterochromatin domains so that affected cells are unable to maintain the silenced chromatin state capable to allow/preserve terminal differentiation. Our results further show that the absence of emerin expression alters the distribution of pre-lamin A and of heterochromatin

  3. Premitotic assembly of human CENPs -T and -W switches centromeric chromatin to a mitotic state.

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    Lisa Prendergast

    2011-06-01

    Full Text Available Centromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They are distinct genetic loci in that their identity in most organisms is determined not by the DNA sequences they are associated with, but through specific chromatin composition and context. The core nucleosomal protein CENP-A/cenH3 plays a primary role in centromere determination in all species and directs assembly of a large complex of associated proteins in vertebrates. While CENP-A itself is stably transmitted from one generation to the next, the nature of the template for centromere replication and its relationship to kinetochore function are as yet poorly understood. Here, we investigate the assembly and inheritance of a histone fold complex of the centromere, the CENP-T/W complex, which is integrated with centromeric chromatin in association with canonical histone H3 nucleosomes. We have investigated the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENPs -T and -W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations, properties reciprocal to those measured for CENP-A. We propose that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. Segregation of the assembly mechanisms for the two allows the cell to switch between chromatin configurations that reciprocally support the replication of the centromere and its conversion to a mitotic state on postreplicative chromatin.

  4. Dynamic chromatin states in human ES cells reveal potential regulatory sequences and genes involved in pluripotency

    Institute of Scientific and Technical Information of China (English)

    R David Hawkins; Zhen Ye; Samantha Kuan; Pengzhi Yu; Hui Liu; Xinmin Zhang; Roland D Green; Victor V Lobanenkov; Ron Stewart; James A Thomson; Bing Ren; Gary C Hon; Chuhu Yang; Jessica E Antosiewicz-Bourget; LeonardKLee; Que-Minh Ngo; Sarit Klugman; Keith A Ching; Lee E Edsall

    2011-01-01

    Pluripotency,the ability of a cell to differentiate and give rise to all embryonic lineages,defines a small number of mammalian cell types such as embryonic stem (ES) cells.While it has been generally held that pluripotency is the product of a transcriptional regulatory network that activates and maintains the expression of key stem cell genes,accumulating evidence is pointing to a critical role for epigenetic processes in establishing and safeguarding the pluripotency of ES cells,as well as maintaining the identity of differentiated cell types.In order to better understand the role of epigenetic mechanisms in pluripotency,we have examined the dynamics of chromatin modifications genomewide in human ES cells (hESCs) undergoing differentiation into a mesendodermal lineage.We found that chromatin modifications at promoters remain largely invariant during differentiation,except at a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression,suggesting a hierarchy in cell fate commitment over most differentially expressed genes.We also mapped over 50 000 potential enhancers,and observed much greater dynamics in chromatin modifications,especially H3K4mel and H3K27ac,which correlate with expression of their potential target genes.Further analysis of these enhancers revealed potentially key transcriptional regulators of pluripotency and a chromatin signature indicative of a poised state that may confer developmental competence in hESCs.Our results provide new evidence supporting the role of chromatin modifications in defining enhancers and pluripotency.

  5. Spatial and temporal plasticity of chromatin during programmed DNA-reorganization in Stylonychia macronuclear development

    Directory of Open Access Journals (Sweden)

    Postberg Jan

    2008-10-01

    Full Text Available Abstract Background: In this study we exploit the unique genome organization of ciliates to characterize the biological function of histone modification patterns and chromatin plasticity for the processing of specific DNA sequences during a nuclear differentiation process. Ciliates are single-cell eukaryotes containing two morphologically and functionally specialized types of nuclei, the somatic macronucleus and the germline micronucleus. In the course of sexual reproduction a new macronucleus develops from a micronuclear derivative. During this process specific DNA sequences are eliminated from the genome, while sequences that will be transcribed in the mature macronucleus are retained. Results: We show by immunofluorescence microscopy, Western analyses and chromatin immunoprecipitation (ChIP experiments that each nuclear type establishes its specific histone modification signature. Our analyses reveal that the early macronuclear anlage adopts a permissive chromatin state immediately after the fusion of two heterochromatic germline micronuclei. As macronuclear development progresses, repressive histone modifications that specify sequences to be eliminated are introduced de novo. ChIP analyses demonstrate that permissive histone modifications are associated with sequences that will be retained in the new macronucleus. Furthermore, our data support the hypothesis that a PIWI-family protein is involved in a transnuclear cross-talk and in the RNAi-dependent control of developmental chromatin reorganization. Conclusion: Based on these data we present a comprehensive analysis of the spatial and temporal pattern of histone modifications during this nuclear differentiation process. Results obtained in this study may also be relevant for our understanding of chromatin plasticity during metazoan embryogenesis.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-08-21

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

  7. CAST-ChIP Maps Cell-Type-Specific Chromatin States in the Drosophila Central Nervous System

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    Tamás Schauer

    2013-10-01

    Full Text Available Chromatin organization and gene activity are responsive to developmental and environmental cues. Although many genes are transcribed throughout development and across cell types, much of gene regulation is highly cell-type specific. To readily track chromatin features at the resolution of cell types within complex tissues, we developed and validated chromatin affinity purification from specific cell types by chromatin immunoprecipitation (CAST-ChIP, a broadly applicable biochemical procedure. RNA polymerase II (Pol II CAST-ChIP identifies ∼1,500 neuronal and glia-specific genes in differentiated cells within the adult Drosophila brain. In contrast, the histone H2A.Z is distributed similarly across cell types and throughout development, marking cell-type-invariant Pol II-bound regions. Our study identifies H2A.Z as an active chromatin signature that is refractory to changes across cell fates. Thus, CAST-ChIP powerfully identifies cell-type-specific as well as cell-type-invariant chromatin states, enabling the systematic dissection of chromatin structure and gene regulation within complex tissues such as the brain.

  8. A Fuzzy-C-Means-Clustering Approach: Quantifying Chromatin Pattern of Non-Neoplastic Cervical Squamous Cells.

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    Jing Rui Tang

    Full Text Available Despite the effectiveness of Pap-smear test in reducing the mortality rate due to cervical cancer, the criteria of the reporting standard of the Pap-smear test are mostly qualitative in nature. This study addresses the issue on how to define the criteria in a more quantitative and definite term. A negative Pap-smear test result, i.e. negative for intraepithelial lesion or malignancy (NILM, is qualitatively defined to have evenly distributed, finely granular chromatin in the nuclei of cervical squamous cells. To quantify this chromatin pattern, this study employed Fuzzy C-Means clustering as the segmentation technique, enabling different degrees of chromatin segmentation to be performed on sample images of non-neoplastic squamous cells. From the simulation results, a model representing the chromatin distribution of non-neoplastic cervical squamous cell is constructed with the following quantitative characteristics: at the best representative sensitivity level 4 based on statistical analysis and human experts' feedbacks, a nucleus of non-neoplastic squamous cell has an average of 67 chromatins with a total area of 10.827 μm2; the average distance between the nearest chromatin pair is 0.508 μm and the average eccentricity of the chromatin is 0.47.

  9. The RSC chromatin remodeling complex has a crucial role in the complete remodeler set for yeast PHO5 promoter opening.

    Science.gov (United States)

    Musladin, Sanja; Krietenstein, Nils; Korber, Philipp; Barbaric, Slobodan

    2014-04-01

    Although yeast PHO5 promoter chromatin opening is a founding model for chromatin remodeling, the complete set of involved remodelers remained unknown for a long time. The SWI/SNF and INO80 remodelers cooperate here, but nonessentially, and none of the many tested single or combined remodeler gene mutations could prevent PHO5 promoter opening. RSC, the most abundant and only remodeler essential for viability, was a controversial candidate for the unrecognized remodeling activity but unassessed in vivo. Now we show that remodels the structure of chromatin (RSC) is crucially involved in PHO5 promoter opening. Further, the isw1 chd1 double deletion also delayed chromatin remodeling. Strikingly, combined absence of RSC and Isw1/Chd1 or Snf2 abolished for the first time promoter opening on otherwise sufficient induction in vivo. Together with previous findings, we recognize now a surprisingly complex network of five remodelers (RSC, SWI/SNF, INO80, Isw1 and Chd1) from four subfamilies (SWI/SNF, INO80, ISWI and CHD) as involved in PHO5 promoter chromatin remodeling. This is likely the first described complete remodeler set for a physiological chromatin transition. RSC was hardly involved at the coregulated PHO8 or PHO84 promoters despite cofactor recruitment by the same transactivator and RSC's presence at all three promoters. Therefore, promoter-specific chromatin rather than transactivators determine remodeler requirements.

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

    Science.gov (United States)

    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.

  11. RBPJ, the major transcriptional effector of Notch signaling, remains associated with chromatin throughout mitosis, suggesting a role in mitotic bookmarking.

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    Robert J Lake

    2014-03-01

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

  12. Modulation of Higher Order Chromatin Conformation in Mammalian Cell Nuclei Can Be Mediated by Polyamines and Divalent Cations.

    Directory of Open Access Journals (Sweden)

    Ashwat Visvanathan

    Full Text Available The organisation of the large volume of mammalian genomic DNA within cell nuclei requires mechanisms to regulate chromatin compaction involving the reversible formation of higher order structures. The compaction state of chromatin varies between interphase and mitosis and is also subject to rapid and reversible change upon ATP depletion/repletion. In this study we have investigated mechanisms that may be involved in promoting the hyper-condensation of chromatin when ATP levels are depleted by treating cells with sodium azide and 2-deoxyglucose. Chromatin conformation was analysed in both live and permeabilised HeLa cells using FLIM-FRET, high resolution fluorescence microscopy and by electron spectroscopic imaging microscopy. We show that chromatin compaction following ATP depletion is not caused by loss of transcription activity and that it can occur at a similar level in both interphase and mitotic cells. Analysis of both live and permeabilised HeLa cells shows that chromatin conformation within nuclei is strongly influenced by the levels of divalent cations, including calcium and magnesium. While ATP depletion results in an increase in the level of unbound calcium, chromatin condensation still occurs even in the presence of a calcium chelator. Chromatin compaction is shown to be strongly affected by small changes in the levels of polyamines, including spermine and spermidine. The data are consistent with a model in which the increased intracellular pool of polyamines and divalent cations, resulting from depletion of ATP, bind to DNA and contribute to the large scale hyper-compaction of chromatin by a charge neutralisation mechanism.

  13. Plasticity of fission yeast CENP-A chromatin driven by relative levels of histone H3 and H4.

    Directory of Open Access Journals (Sweden)

    Araceli G Castillo

    2007-07-01

    Full Text Available The histone H3 variant CENP-A assembles into chromatin exclusively at centromeres. The process of CENP-A chromatin assembly is epigenetically regulated. Fission yeast centromeres are composed of a central kinetochore domain on which CENP-A chromatin is assembled, and this is flanked by heterochromatin. Marker genes are silenced when placed within kinetochore or heterochromatin domains. It is not known if fission yeast CENP-A(Cnp1 chromatin is confined to specific sequences or whether histone H3 is actively excluded. Here, we show that fission yeast CENP-A(Cnp1 can assemble on noncentromeric DNA when it is inserted within the central kinetochore domain, suggesting that in fission yeast CENP-A(Cnp1 chromatin assembly is driven by the context of a sequence rather than the underlying DNA sequence itself. Silencing in the central domain is correlated with the amount of CENP-A(Cnp1 associated with the marker gene and is also affected by the relative level of histone H3. Our analyses indicate that kinetochore integrity is dependent on maintaining the normal ratio of H3 and H4. Excess H3 competes with CENP-A(Cnp1 for assembly into central domain chromatin, resulting in less CENP-A(Cnp1 and other kinetochore proteins at centromeres causing defective kinetochore function, which is manifest as aberrant mitotic chromosome segregation. Alterations in the levels of H3 relative to H4 and CENP-A(Cnp1 influence the extent of DNA at centromeres that is packaged in CENP-A(Cnp1 chromatin and the composition of this chromatin. Thus, CENP-A(Cnp1 chromatin assembly in fission yeast exhibits plasticity with respect to the underlying sequences and is sensitive to the levels of CENP-A(Cnp1 and other core histones.

  14. Plasticity of fission yeast CENP-A chromatin driven by relative levels of histone H3 and H4.

    Science.gov (United States)

    Castillo, Araceli G; Mellone, Barbara G; Partridge, Janet F; Richardson, William; Hamilton, Georgina L; Allshire, Robin C; Pidoux, Alison L

    2007-07-01

    The histone H3 variant CENP-A assembles into chromatin exclusively at centromeres. The process of CENP-A chromatin assembly is epigenetically regulated. Fission yeast centromeres are composed of a central kinetochore domain on which CENP-A chromatin is assembled, and this is flanked by heterochromatin. Marker genes are silenced when placed within kinetochore or heterochromatin domains. It is not known if fission yeast CENP-A(Cnp1) chromatin is confined to specific sequences or whether histone H3 is actively excluded. Here, we show that fission yeast CENP-A(Cnp1) can assemble on noncentromeric DNA when it is inserted within the central kinetochore domain, suggesting that in fission yeast CENP-A(Cnp1) chromatin assembly is driven by the context of a sequence rather than the underlying DNA sequence itself. Silencing in the central domain is correlated with the amount of CENP-A(Cnp1) associated with the marker gene and is also affected by the relative level of histone H3. Our analyses indicate that kinetochore integrity is dependent on maintaining the normal ratio of H3 and H4. Excess H3 competes with CENP-A(Cnp1) for assembly into central domain chromatin, resulting in less CENP-A(Cnp1) and other kinetochore proteins at centromeres causing defective kinetochore function, which is manifest as aberrant mitotic chromosome segregation. Alterations in the levels of H3 relative to H4 and CENP-A(Cnp1) influence the extent of DNA at centromeres that is packaged in CENP-A(Cnp1) chromatin and the composition of this chromatin. Thus, CENP-A(Cnp1) chromatin assembly in fission yeast exhibits plasticity with respect to the underlying sequences and is sensitive to the levels of CENP-A(Cnp1) and other core histones. PMID:17677001

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-07-15

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

  16. Interrogation of allelic chromatin states in human cells by high-density ChIP-genotyping.

    Science.gov (United States)

    Light, Nicholas; Adoue, Véronique; Ge, Bing; Chen, Shu-Huang; Kwan, Tony; Pastinen, Tomi

    2014-09-01

    Allele-specific (AS) assessment of chromatin has the potential to elucidate specific cis-regulatory mechanisms, which are predicted to underlie the majority of the known genetic associations to complex disease. However, development of chromatin landscapes at allelic resolution has been challenging since sites of variable signal strength require substantial read depths not commonly applied in sequencing based approaches. In this study, we addressed this by performing parallel analyses of input DNA and chromatin immunoprecipitates (ChIP) on high-density Illumina genotyping arrays. Allele-specificity for the histone modifications H3K4me1, H3K4me3, H3K27ac, H3K27me3, and H3K36me3 was assessed using ChIP samples generated from 14 lymphoblast and 6 fibroblast cell lines. AS-ChIP SNPs were combined into domains and validated using high-confidence ChIP-seq sites. We observed characteristic patterns of allelic-imbalance for each histone-modification around allele-specifically expressed transcripts. Notably, we found H3K4me1 to be significantly anti-correlated with allelic expression (AE) at transcription start sites, indicating H3K4me1 allelic imbalance as a marker of AE. We also found that allelic chromatin domains exhibit population and cell-type specificity as well as heritability within trios. Finally, we observed that a subset of allelic chromatin domains is regulated by DNase I-sensitive quantitative trait loci and that these domains are significantly enriched for genome-wide association studies hits, with autoimmune disease associated SNPs specifically enriched in lymphoblasts. This study provides the first genome-wide maps of allelic-imbalance for five histone marks. Our results provide new insights into the role of chromatin in cis-regulation and highlight the need for high-depth sequencing in ChIP-seq studies along with the need to improve allele-specificity of ChIP-enrichment.

  17. The binding of [3H]oestradiol-receptor complex to hypothalamic chromatin of male and female mice.

    Science.gov (United States)

    Lopez, A; Burgos, J; Ventanas, J

    1985-01-01

    Histones and masking acidic proteins were removed from hypothalamic chromatin in order to evaluate/measure the number of available acceptor sites for the [3H]oestradiol-receptor complex. This number increases after dehistonizing and unmasking and is lower than published values for comparable preparations. No sex-related difference in [3H]oestradiol-receptor binding to hypothalamic chromatin in vitro was observed. Failure to observe such a difference suggests that sexual differentiation and steroid sensitivity cannot be attributed to marked differences in the degree of chromatin masking.

  18. Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification.

    Science.gov (United States)

    Nguyen, Ngoc-Thuy-Trinh; Saguez, Cyril; Conesa, Christine; Lefebvre, Olivier; Acker, Joël

    2015-02-01

    To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.

  19. MeCP2 Rett mutations affect large scale chromatin organization

    DEFF Research Database (Denmark)

    Gupta, Noopur Agarwal; Becker, Annette; Jost, K Laurence;

    2011-01-01

    Rett syndrome is a neurological, X chromosomal-linked disorder associated with mutations in the MECP2 gene. MeCP2 protein has been proposed to play a role in transcriptional regulation as well as in chromatin architecture. Since MeCP2 mutant cells exhibit surprisingly mild changes in gene...... expression, we have now explored the possibility that Rett mutations may affect the ability of MeCP2 to bind and organize chromatin. We found that all but one of the 21 missense MeCP2 mutants analyzed accumulated at heterochromatin and about half of them were significantly affected. Furthermore, two......-thirds of all mutants showed a significantly decreased ability to cluster heterochromatin. Three mutants containing different proline substitutions (P101H, P101R and P152R) were severely affected only in heterochromatin clustering and located far away from the DNA interface in the MeCP2 methyl-binding domain...

  20. Chromatin organizer SATB1 is an important determinant of T-cell differentiation.

    Science.gov (United States)

    Burute, Mithila; Gottimukkala, Kamal; Galande, Sanjeev

    2012-10-01

    T-cell development and differentiation is coordinated by a multitude of signaling molecules and transcription factors that impart distinct functional properties to progenitors. In this review, we focus on the role of the T lineage-enriched chromatin organizer and regulator SATB1 in T-cell differentiation. SATB1 mediates Wnt signaling by recruiting β-catenin to its genomic targets and coordinates T helper type 2 (T(H)2) differentiation by positively regulating GATA-3. In contrast, maintenance of regulatory T cell (Treg) functions are dependent on inhibition of SATB1-mediated modulation of global chromatin organization. We discuss how regulation of the activity of SATB1 has a critical role in driving these two important differentiation pathways in T cells.

  1. Cytogenetic evidences of genome rearrangement and differential epigenetic chromatin modification in the sea lamprey (Petromyzon marinus).

    Science.gov (United States)

    Covelo-Soto, Lara; Morán, Paloma; Pasantes, Juan J; Pérez-García, Concepción

    2014-12-01

    This work explores both the chromatin loss and the differential genome methylation in the sea lamprey (Petromyzon marinus) from a molecular cytogenetic point of view. Fluorescent in situ hybridization experiments on meiotic bivalents and mitotic chromosomes corroborate the chromatin loss previously observed during the development of the sea lamprey and demonstrate that the elimination affects not only to Germ1 sequences but also to the rpt200 satellite DNA and most part of the major ribosomal DNA present on the germinal line. 5-Methylcytosine immunolocation revealed that the GC-rich heterochromatin is highly methylated in the germ line but significantly less in somatic chromosomes. These findings not only support previous observations about genome rearrangements but also give new information about epigenetic changes in P. marinus. The key position of lampreys in the vertebrate phylogenetic tree makes them an interesting taxon to provide relevant information about genome evolution in vertebrates.

  2. Distinct differences in chromatin structure at subtelomeric X and Y' elements in budding yeast.

    Directory of Open Access Journals (Sweden)

    Xuefeng Zhu

    Full Text Available In Saccharomyces cerevisiae, all ends of telomeric DNA contain telomeric repeats of (TG(1-3, but the number and position of subtelomeric X and Y' repeat elements vary. Using chromatin immunoprecipitation and genome-wide analyses, we here demonstrate that the subtelomeric X and Y' elements have distinct structural and functional properties. Y' elements are transcriptionally active and highly enriched in nucleosomes, whereas X elements are repressed and devoid of nucleosomes. In contrast to X elements, the Y' elements also lack the classical hallmarks of heterochromatin, such as high Sir3 and Rap1 occupancy as well as low levels of histone H4 lysine 16 acetylation. Our analyses suggest that the presence of X and Y' elements govern chromatin structure and transcription activity at individual chromosome ends.

  3. DNA breaks and repair in interstitial telomere sequences: Influence of chromatin structure

    International Nuclear Information System (INIS)

    Interstitial Telomeric Sequences (ITS) are over-involved in spontaneous and radiationinduced chromosome aberrations in chinese hamster cells. We have performed a study to investigate the origin of their instability, spontaneously or after low doses irradiation. Our results demonstrate that ITS have a particular chromatin structure: short nucleotide repeat length, less compaction of the 30 nm chromatin fiber, presence of G-quadruplex structures. These features would modulate breaks production and would favour the recruitment of alternative DNA repair mechanisms, which are prone to produce chromosome aberrations. These pathways could be at the origin of chromosome aberrations in ITS whereas NHEJ and HR Double Strand Break repair pathways are rather required for a correct repair in these regions. (author)

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

    Science.gov (United States)

    Tajbakhsh, Jian

    2011-12-01

    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.

  5. Isolation of chromatin DNA tightly bound to the nuclear envelope of HeLa cells.

    Science.gov (United States)

    Kuvichkin, Vasily Vladimirovich

    2012-11-01

    Recent discovery of the role of nuclear pores in transcription, predicted by our early DNA-membrane complex (DMC) model, makes membrane-bound DNA (MBD) isolation from the cell nucleus and analysis of the MBD actual. The method of MBD isolation proposed by us retains DMC integrity during isolation. We used HeLa cells for DMC extraction. Changing the ionic composition of the isolation medium and replacing DNase I, used commonly for chromatin destruction, with a set of restriction enzymes allowed us to isolate the MBD. Treatment of a nuclear membrane with proteinase K and ultrasound has been used to increase the yield of MBD. Electron microscopic analysis of the purified fraction of isolated DMC supports our previous model of nuclear envelope lipid-chromatin interaction in the nuclear pore assembly.

  6. Evidence for the internucleosomal breakage of chromatin in rat thymocytes irradiated in vitro

    International Nuclear Information System (INIS)

    Low molecular-weight (free) DNA increases in lymphatic cells in association with radiation-induced interphase death. In this report, the size of the free DNA from rat thymocytes irradiated in vitro with 1-kR x rays was analyzed by agarose gel electrophoresis and compared with that of the DNA fragments extracted from micrococcal nuclease-digested thymocyte nuclei and with that of HaeIII-digested phi X-174 RF DNA. Results demonstrated clearly that the free DNA consisted of a series of discretely sized DNA fragments with lengths that were integral multiples of a nucleosomal DNA (180 base pairs), suggesting that internucleosomal chromatin breaks occurred in rat thymocytes after in vitro irradiation. Additional evidence was obtained from an electron microscopic observation on the breakdown of chromatin strands into particle form in the irradiated cells

  7. Genome-wide expression of non-coding RNA and global chromatin modification

    Institute of Scientific and Technical Information of China (English)

    Rukui Zhang; Lan Zhang; Wenqiang Yu

    2012-01-01

    Traditionally,we know that genomic DNA will produce transcripts named messenger RNA and then translate into protein following the instruction of genetic central dogma,and RNA works here as a pass-by messenger.Now increasing evidence shows that RNA is a key regulator as well as a message transmitter.It is discovered by next-generation sequencing techniques that most genomic DNA are generally transcribed to non-coding RNA,highly beyond the percentage of coding mRNA.These non-coding RNAs (ncRNAs),belonging to several groups,have critical roles in many cellular processes,expanding our understanding of the RNA world.We review here the different categories of ncRNA according to genome location and how ncRNAs guide and recruit chromatin modification complex to specific loci of genome to modulate gene expression by affecting chromatin state.

  8. H2 O2-induced higher order chromatin degradation: A novel mechanism of oxidative genotoxicity

    Indian Academy of Sciences (India)

    Gregory W Konat

    2003-02-01

    The genotoxicity of reactive oxygen species (ROS) is well established. The underlying mechanism involves oxidation of DNA by ROS. However, we have recently shown that hydrogen peroxide (H2O2), the major mediator of oxidative stress, can also cause genomic damage indirectly. Thus, H2O2 at pathologically relevant concentrations rapidly induces higher order chromatin degradation (HOCD), i.e. enzymatic excision of chromatin loops and their oligomers at matrix-attachment regions. The activation of endonuclease that catalyzes HOCD is a signalling event triggered specifically by H2O2. The activation is not mediated by an influx of calcium ions, but resting concentrations of intracellular calcium ions are required for the maintenance of the endonuclease in an active form. Although H2O2-induced HOCD can efficiently dismantle the genome leading to cell death, under sublethal oxidative stress conditions H2O2-induced HOCD may be the major source of somatic mutations.

  9. Extensive chromatin remodelling and establishment of transcription factor 'hotspots' during early adipogenesis

    DEFF Research Database (Denmark)

    Siersbæk, Rasmus; Nielsen, Ronni; John, Sam;

    2011-01-01

    Adipogenesis is tightly controlled by a complex network of transcription factors acting at different stages of differentiation. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein (C/EBP) family members are key regulators of this process. We have employed DNase I...... hypersensitive site analysis to investigate the genome-wide changes in chromatin structure that accompany the binding of adipogenic transcription factors. These analyses revealed a dramatic and dynamic modulation of the chromatin landscape during the first hours of adipocyte differentiation that coincides...... with cooperative binding of multiple early transcription factors (including glucocorticoid receptor, retinoid X receptor, Stat5a, C/EBPβ and -δ) to transcription factor 'hotspots'. Our results demonstrate that C/EBPβ marks a large number of these transcription factor 'hotspots' before induction of differentiation...

  10. Replication-coupled chromatin assembly of newly synthesized histones: distinct functions for the histone tail domains.

    Science.gov (United States)

    Ejlassi-Lassallette, Aïda; Thiriet, Christophe

    2012-02-01

    The maintenance of the genome during replication requires the assembly of nucleosomes with newly synthesized histones. Achieving the deposition of newly synthesized histones in chromatin implies their transport from the cytoplasm to the nucleus at the replication sites. Several lines of evidence have revealed critical functions of the histone tail domains in these conserved cellular processes. In this review, we discuss the role of the amino termini of the nucleosome building blocks, H2A/H2B and H3/H4, in different model systems. The experimental data showed that H2A/H2B tails and H3/H4 tails display distinct functions in nuclear import and chromatin assembly. Furthermore, we describe recent studies exploiting the unique properties of the slime mold, Physarum polycephalum , that have advanced understanding of the function of the highly conserved replication-dependent diacetylation of H4.

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

    OpenAIRE

    Van Bortle, Kevin; Corces, Victor G.

    2013-01-01

    Eukaryotic genomes are intricately arranged into highly organized yet dynamic structures that underlie patterns of gene expression and cellular identity. The recent adaptation of novel genomic strategies for assaying nuclear architecture has significantly extended and accelerated our ability to query the nature of genome organization and the players involved. In particular, recent explorations of physical arrangements and chromatin landscapes in higher eukaryotes have demonstrated that chroma...

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

    OpenAIRE

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

    2016-01-01

    Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophi...

  13. Human Lymphoid Translocation Fragile Zones Are Hypomethylated and Have Accessible Chromatin

    OpenAIRE

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

    2015-01-01

    Chromosomal translocations are a hallmark of hematopoietic malignancies. CG motifs within translocation fragile zones (typically 20 to 600 bp in size) are prone to chromosomal translocation in lymphomas. Here we demonstrate that the CG motifs in human translocation fragile zones are hypomethylated relative to the adjacent DNA. Using a methyltransferase footprinting assay on isolated nuclei (in vitro), we find that the chromatin at these fragile zones is accessible. We also examined in vivo ac...

  14. Proteomics to study DNA-bound and chromatin-associated gene regulatory complexes

    Science.gov (United States)

    Wierer, Michael; Mann, Matthias

    2016-01-01

    High-resolution mass spectrometry (MS)-based proteomics is a powerful method for the identification of soluble protein complexes and large-scale affinity purification screens can decode entire protein interaction networks. In contrast, protein complexes residing on chromatin have been much more challenging, because they are difficult to purify and often of very low abundance. However, this is changing due to recent methodological and technological advances in proteomics. Proteins interacting with chromatin marks can directly be identified by pulldowns with synthesized histone tails containing posttranslational modifications (PTMs). Similarly, pulldowns with DNA baits harbouring single nucleotide polymorphisms or DNA modifications reveal the impact of those DNA alterations on the recruitment of transcription factors. Accurate quantitation – either isotope-based or label free – unambiguously pinpoints proteins that are significantly enriched over control pulldowns. In addition, protocols that combine classical chromatin immunoprecipitation (ChIP) methods with mass spectrometry (ChIP-MS) target gene regulatory complexes in their in-vivo context. Similar to classical ChIP, cells are crosslinked with formaldehyde and chromatin sheared by sonication or nuclease digested. ChIP-MS baits can be proteins in tagged or endogenous form, histone PTMs, or lncRNAs. Locus-specific ChIP-MS methods would allow direct purification of a single genomic locus and the proteins associated with it. There, loci can be targeted either by artificial DNA-binding sites and corresponding binding proteins or via proteins with sequence specificity such as TAL or nuclease deficient Cas9 in combination with a specific guide RNA. We predict that advances in MS technology will soon make such approaches generally applicable tools in epigenetics. PMID:27402878

  15. Opposition between PKC Isoforms Regulates Histone Deimination and Neutrophil Extracellular Chromatin Release

    OpenAIRE

    Marko eRadic; Indira eNeeli

    2013-01-01

    In response to inflammation, neutrophils deiminate histones and externalize chromatin. Neutrophil extracellular traps (NETs) are an innate immune defense mechanism, yet NETs also may aggravate chronic inflammatory and autoimmune disorders. Activation of peptidylarginine deiminase IV (PAD4) is associated with NET release (NETosis) but the precise mechanisms of PAD4 regulation are unknown. We observed that, in human neutrophils, calcium ionophore induced histone deimination, whereas phorbol ...

  16. The chromatin remodeler DDM1 promotes hybrid vigor by regulating salicylic acid metabolism

    OpenAIRE

    Zhang, Qingzhu; Li, Yanqiang; Xu, Tao; Srivastava, Ashish Kumar; Dong WANG; Zeng, Liang; Yang, Lan; He, Li; Zhang, Heng; Zheng, Zhimin; Yang, Dong-Lei; Zhao, Cheng; Dong, Juan; Gong, Zhizhong; Liu, Renyi

    2016-01-01

    In plants, hybrid vigor is influenced by genetic and epigenetic mechanisms; however, the molecular pathways are poorly understood. We investigated the potential contributions of epigenetic regulators to heterosis in Arabidposis and found that the chromatin remodeler DECREASED DNA METHYLATION 1 (DDM1) affects early seedling growth heterosis in Col/C24 hybrids. ddm1 mutants showed impaired heterosis and increased expression of non-additively expressed genes related to salicylic acid metabolism....

  17. Topoisomerase II–DNA complexes trapped by ICRF-193 perturb chromatin structure

    OpenAIRE

    Germe, Thomas; Hyrien, Olivier

    2005-01-01

    DNA topoisomerase II (topo II) is involved in unlinking replicating DNA and organizing mitotic chromosomes. Topo II is the target of many antitumour drugs. Topo II inhibition results in extensive catenation of newly replicated DNA and may potentially perturb chromatin assembly. Here, we show that the topo II inhibitor ICRF-193 does not prevent the bulk of nucleosome deposition, but perturbs nucleosome spacing in Xenopus egg extracts. This is due to the trapping of topo II-closed clamps on the...

  18. Influence of chromatin molecular changes on RNA synthesis during embryonic development

    International Nuclear Information System (INIS)

    Two aspects of the chromatin repeat length (rl) are discussed: (i) Why is rl longer for slowly dividing cells than in rapidly dividing cells?, and (ii) Why is the temporal evolution of rl a decreasing function of time (t) in mammalian cortical neurons whereas it is an increasing function of t for granule cells around the time of birth? These questions are discussed in terms of a hypothesis which assumes a correlation between deoxyribonucleic acid (DNA) packaging, transcription, and replication. (author). 27 refs

  19. Brd4 Marks Select Genes on Mitotic Chromatin and Directs Postmitotic Transcription

    OpenAIRE

    Dey, Anup; Nishiyama, Akira; Karpova, Tatiana; McNally, James; Ozato, Keiko

    2009-01-01

    On entry into mitosis, many transcription factors dissociate from chromatin, resulting in global transcriptional shutdown. During mitosis, some genes are marked to ensure the inheritance of their expression in the next generation of cells. The nature of mitotic gene marking, however, has been obscure. Brd4 is a double bromodomain protein that localizes to chromosomes during mitosis and is implicated in holding mitotic memory. In interphase, Brd4 interacts with P-TEFb and functions as a global...

  20. Monoallelic chromatin conformation flanking long-range silenced domains in cancer-derived and normal cells.

    Directory of Open Access Journals (Sweden)

    Domenic Di Paola

    Full Text Available Epigenetic inactivation of chromatin plays an important role in determining cell phenotype in both normal and cancer cells, but our knowledge is still incomplete with respect to any potential monoallelic nature of the phenomenon. We have genotyped DNA isolated from chromatin of two colorectal cancer-derived lines and a culture of normal human intestinal epithelial cells (HIEC, which was immunoprecipitated with antibodies to acetylated vs. methylated histone H3K9, and presented the data as B allele frequency differences over multiple single-nucleotide polymorphism (SNP moving window averages. [B allele is an arbitrary term defined as one of the two alleles at any given SNP, named A and B]. Three different validation tests confirmed that peaks exhibiting differences represented monoallelic domains. These complementary tests confirmed the following: 1 genes in the regions of high B allele frequency difference were expressed monoallelically; 2 in normal cells all five imprinting control regions which carried heterozygous SNPs were characterized by B allele difference peaks; and 3 the haplotypes in the B allele difference peaks were faithfully maintained in the chromatin immunoprecipitated with the respective antibodies. In both samples most of the monoallelic domains were found at the boundaries between regions of open and closed chromatin. With respect to the cancer line, this supports the established concept of conformation spreading, but the results from the normal cells were unexpected. Since these cells were polyclonal, the monoallelic structures were probably not determined by random choice as occurs in X-inactivation, so we propose that epigenetic inactivation in some domains may be heritable and polymorphic in normal human cells.

  1. The autism-associated chromatin modifier CHD8 regulates other autism risk genes during human neurodevelopment

    OpenAIRE

    Cotney, Justin; Muhle, Rebecca A.; Sanders, Stephan J.; Liu, Li; Willsey, A. Jeremy; Niu, Wei; Liu, Wenzhong; Klei, Lambertus; Lei, Jing; Yin, Jun; Reilly, Steven K.; Tebbenkamp, Andrew T.; Bichsel, Candace; Pletikos, Mihovil; Sestan, Nenad

    2015-01-01

    Recent studies implicate chromatin modifiers in autism spectrum disorder (ASD) through the identification of recurrent de novo loss of function mutations in affected individuals. ASD risk genes are co-expressed in human midfetal cortex, suggesting that ASD risk genes converge in specific regulatory networks during neurodevelopment. To elucidate such networks, we identify genes targeted by CHD8, a chromodomain helicase strongly associated with ASD, in human midfetal brain, human neural stem ce...

  2. Heterochromatin and RNAi Are Required to Establish CENP-A Chromatin at Centromeres

    OpenAIRE

    Folco, Hernan Diego; Pidoux, Alison L.; Urano, Takeshi; Allshire, Robin C.

    2008-01-01

    Heterochromatin is defined by distinct posttranslational modifications on histones, such as methylation of histone H3 at lysine 9 (H3K9), which allows heterochromatin protein 1 (HP1)-related chromodomain proteins to bind. Heterochromatin is frequently found near CENP-A chromatin, which is the key determinant of kinetochore assembly. We have discovered that the RNA interference (RNAi)-directed heterochromatin flanking the central kinetochore domain at fission yeast centromeres is required to p...

  3. CENP-C facilitates the recruitment of M18BP1 to centromeric chromatin

    OpenAIRE

    Dambacher, Silvia; Deng, Wen; Hahn, Matthias; Sadic, Dennis; Fröhlich, Jonathan; Nuber, Alexander; Hoischen, Christian; Diekmann, Stephan; Leonhardt, Heinrich; Schotta, Gunnar

    2012-01-01

    Centromeres are important structural constituents of chromosomes that ensure proper chromosome segregation during mitosis by providing defined sites for kinetochore attachment. In higher eukaryotes, centromeres have no specific DNA sequence and thus, they are rather determined through epigenetic mechanisms. A fundamental process in centromere establishment is the incorporation of the histone variant CENP-A into centromeric chromatin, which provides a binding platform for the other centromeric...

  4. Camk2a-Cre-Mediated Conditional Deletion of Chromatin Remodeler Brg1 Causes Perinatal Hydrocephalus

    OpenAIRE

    Cao, Mou; Wu, Jiang I.

    2015-01-01

    Mammalian SWI/SNF-like BAF chromatin remodeling complexes are essential for many aspects of neural development. Mutations in the genes encoding the core subunit Brg1/SmarcA4or other complex components cause neurodevelopmental diseases and are associated with autism. Congenital hydrocephalus is a serious brain disorder often experienced by these patients. We report a role of Brg1 in the pathogenesis of hydrocephalus disorder. We discovered an unexpected early activity of mouse Camk2a-Cre trans...

  5. Effect of combined density gradient centrifugation on X- and Y- sperm separation and chromatin integrity

    OpenAIRE

    Tahereh Esmaeilpour; Leila Elyasi; Soghra Bahmanpour; Alireza Ghannadi; Ahmad Monabbati; Farzaneh Dehghani; Marjaneh Kazerooni

    2012-01-01

    Background: It has been claimed that by using different washing methods, the sperms can be separated according to size, motility, density, chromosomal content and surface markings and charge. These methods also reduce sperm chromatin deficiencies and screen the sperms before applying in assisted reproduction techniques. Objective: This study compared simple density gradient methods and a combined method with albumin density gradient and PureSperm separation (alb/PureSperm) for sex preselectio...

  6. Metabolomics reveals a role for the chromatin-binding protein HMGN5 in glutathione metabolism.

    Directory of Open Access Journals (Sweden)

    Eric D Ciappio

    Full Text Available High mobility group nucleosome-binding protein 5 (HMGN5 is a chromatin architectural protein that binds specifically to nucleosomes and reduces the compaction of the chromatin fiber. The protein is present in most vertebrate tissues however the physiological function of this protein is unknown. To examine the function of HMGN5 in vivo, mice lacking the nucleosome-binding domain of HMGN5 were generated and characterized. Serological analysis revealed that compared to wild-type littermates (Hmgn5(+/Y, mice with a targeted mutation in the HMGN5 gene (Hmgn5(tm1/Y, had elevated serum albumin, non-HDL cholesterol, triglycerides, and alanine transaminase, suggesting mild hepatic abnormalities. Metabolomics analysis of liver extracts and urine revealed clear differences in metabolites between Hmgn5(tm1/Y and their Hmgn5(+/Y littermates. Hmgn5(tm1/Y mice had a significant increase in hepatic glutathione levels and decreased urinary concentrations of betaine, phenylacetylglycine, and creatine, all of which are metabolically related to the glutathione precursor glycine. Microarray and qPCR analysis revealed that expression of two genes affecting glutathione metabolism, glutathione peroxidase 6 (Gpx6 and hexokinase 1 (Hk1, was significantly decreased in Hmgn5(tm1/Y mouse liver tissue. Analysis of chromatin structure by DNase I digestion revealed alterations in the chromatin structure of these genes in the livers of Hmgn5(tm1/Y mice. Thus, functional loss of HMGN5 leads to changes in transcription of Gpx6 and Hk1 that alter glutathione metabolism.

  7. QuIN: A Web Server for Querying and Visualizing Chromatin Interaction Networks.

    Directory of Open Access Journals (Sweden)

    Asa Thibodeau

    2016-06-01

    Full Text Available Recent studies of the human genome have indicated that regulatory elements (e.g. promoters and enhancers at distal genomic locations can interact with each other via chromatin folding and affect gene expression levels. Genomic technologies for mapping interactions between DNA regions, e.g., ChIA-PET and HiC, can generate genome-wide maps of interactions between regulatory elements. These interaction datasets are important resources to infer distal gene targets of non-coding regulatory elements and to facilitate prioritization of critical loci for important cellular functions. With the increasing diversity and complexity of genomic information and public ontologies, making sense of these datasets demands integrative and easy-to-use software tools. Moreover, network representation of chromatin interaction maps enables effective data visualization, integration, and mining. Currently, there is no software that can take full advantage of network theory approaches for the analysis of chromatin interaction datasets. To fill this gap, we developed a web-based application, QuIN, which enables: 1 building and visualizing chromatin interaction networks, 2 annotating networks with user-provided private and publicly available functional genomics and interaction datasets, 3 querying network components based on gene name or chromosome location, and 4 utilizing network based measures to identify and prioritize critical regulatory targets and their direct and indirect interactions.QuIN's web server is available at http://quin.jax.org QuIN is developed in Java and JavaScript, utilizing an Apache Tomcat web server and MySQL database and the source code is available under the GPLV3 license available on GitHub: https://github.com/UcarLab/QuIN/.

  8. Analysis of Chromatin Attachment and Partitioning Functions of Bovine Papillomavirus Type 1 E2 Protein

    OpenAIRE

    Abroi, Aare; Ilves, Ivar; Kivi, Sirje; Ustav, Mart

    2004-01-01

    Recent studies have suggested that the tethering of viral genomes to host cell chromosomes could provide one of the ways to achieve their nuclear retention and partitioning during extrachromosomal maintenance in dividing cells. The data we present here provide firm evidence that the partitioning of the bovine papillomavirus type 1 (BPV1) genome is dependent on the chromatin attachment process mediated by viral E2 protein and its multiple binding sites. On the other hand, the attachment of E2 ...

  9. Analysis of chromatin attachment and partitioning functions of bovine papillomavirus type 1 E2 protein.

    Science.gov (United States)

    Abroi, Aare; Ilves, Ivar; Kivi, Sirje; Ustav, Mart

    2004-02-01

    Recent studies have suggested that the tethering of viral genomes to host cell chromosomes could provide one of the ways to achieve their nuclear retention and partitioning during extrachromosomal maintenance in dividing cells. The data we present here provide firm evidence that the partitioning of the bovine papillomavirus type 1 (BPV1) genome is dependent on the chromatin attachment process mediated by viral E2 protein and its multiple binding sites. On the other hand, the attachment of E2 and the E2-mediated tethering of reporter plasmids to host chromosomes are not necessarily sufficient for efficient partitioning, suggesting that additional E2-dependent activities might be involved in the latter process. The activity of E2 protein in chromatin attachment and partitioning is more sensitive to the point mutations in the N-terminal domain than its transactivation and replication initiation functions. Therefore, at least part of the interactions of the E2 N-terminal domain with its targets during the chromatin attachment and partitioning processes are likely to involve specific receptors not involved in transactivation and replication activities of the protein. The mutational analysis also indicates that the binding of E2 to chromatin is not achieved through interaction of linear N-terminal subsequences of the E2 protein with putative receptors. Instead, the composite surface elements of the N-terminal domain build up the receptor-binding surface of E2. In this regard, the interaction of BPV1 E2 with its chromosomal targets clearly differs from the interactions of LANA1 protein from Kaposi's sarcoma-associated human herpesvirus and EBNA1 from Epstein-Barr virus with their specific receptors. PMID:14747575

  10. Aberrant Neural Stem Cell Proliferation and Increased Adult Neurogenesis in Mice Lacking Chromatin Protein HMGB2

    OpenAIRE

    Abraham, Ariel B; Robert Bronstein; Avanish S Reddy; Mirjana Maletic-Savatic; Adan Aguirre; Tsirka, Stella E.

    2013-01-01

    Neural stem and progenitor cells (NSCs/NPCs) are distinct groups of cells found in the mammalian central nervous system (CNS). Previously we determined that members of the High Mobility Group (HMG) B family of chromatin structural proteins modulate NSC proliferation and self-renewal. Among them HMGB2 was found to be dynamically expressed in proliferating and differentiating NSCs, suggesting that it may regulate NSC maintenance. We report now that Hmgb2(-/-) mice exhibit SVZ hyperproliferation...

  11. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

    Directory of Open Access Journals (Sweden)

    Page Grier P

    2009-04-01

    Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

  12. Comparing genome-wide chromatin profiles using ChIP-chip or ChIP-seq

    OpenAIRE

    Johannes, Frank; Wardenaar, Rene; Colomé Tatché, Maria; Mousson, Florence; de Graaf, Petra; Mokry, Michal; Guryev, Victor; Timmers, H. Th. Marc; Cuppen, Edwin; Ritsert C Jansen; Bateman, Alex

    2010-01-01

    Motivation: ChIP-chip and ChIP-seq technologies provide genomewide measurements of various types of chromatin marks at an unprecedented resolution. With ChIP samples collected from different tissue types and/ or individuals, we can now begin to characterize stochastic or systematic changes in epigenetic patterns during development (intra-individual) or at the population level (inter-individual). This requires statistical methods that permit a simultaneous comparison of multiple ChIP samples o...

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

    OpenAIRE

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

    2012-01-01

    Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein–DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large scale screening purposes. To address these challenges, we report HTChIP,...

  14. Aberrant chromatin at genes encoding stem cell regulators in human mixed-lineage leukemia

    OpenAIRE

    Guenther, Matthew G.; Lawton, Lee N.; Rozovskaia, Tatiana; Frampton, Garrett M.; Levine, Stuart S.; Thomas L Volkert; Croce, Carlo M.; Nakamura, Tatsuya; Canaani, Eli; Young, Richard A.

    2008-01-01

    Mixed-lineage leukemia (MLL) fusion proteins are potent inducers of leukemia, but how these proteins generate aberrant gene expression programs is poorly understood. Here we show that the MLL-AF4 fusion protein occupies developmental regulatory genes important for hematopoietic stem cell identity and self-renewal in human leukemia cells. These MLL-AF4-bound regions have grossly altered chromatin structure, with histone modifications catalyzed by trithorax group proteins and DOT1 extending acr...

  15. Imipramine Treatment and Resiliency Exhibit Similar Chromatin Regulation in the Mouse Nucleus Accumbens in Depression Models

    OpenAIRE

    Wilkinson, Matthew B.; Xiao, Guanghua; Kumar, Arvind; LaPlant, Quincey; Renthal, William; Sikder, Devanjan; Kodadek, Thomas J; Nestler, Eric J.

    2009-01-01

    Though it is a widely studied psychiatric syndrome, major depressive disorder remains a poorly understood illness, especially with regard to the disconnect between treatment initiation and the delayed onset of clinical improvement. We have recently validated chronic social defeat stress in mice as a model in which a depression-like phenotype is reversed by chronic, but not acute, antidepressant administration. Here, we use ChIP-chip assays—chromatin immunoprecipitation (ChIP) followed by geno...

  16. LINE retrotransposon RNA is an essential structural and functional epigenetic component of a core neocentromeric chromatin.

    Directory of Open Access Journals (Sweden)

    Anderly C Chueh

    2009-01-01

    Full Text Available We have previously identified and characterized the phenomenon of ectopic human centromeres, known as neocentromeres. Human neocentromeres form epigenetically at euchromatic chromosomal sites and are structurally and functionally similar to normal human centromeres. Recent studies have indicated that neocentromere formation provides a major mechanism for centromere repositioning, karyotype evolution, and speciation. Using a marker chromosome mardel(10 containing a neocentromere formed at the normal chromosomal 10q25 region, we have previously mapped a 330-kb CENP-A-binding domain and described an increased prevalence of L1 retrotransposons in the underlying DNA sequences of the CENP-A-binding clusters. Here, we investigated the potential role of the L1 retrotransposons in the regulation of neocentromere activity. Determination of the transcriptional activity of a panel of full-length L1s (FL-L1s across a 6-Mb region spanning the 10q25 neocentromere chromatin identified one of the FL-L1 retrotransposons, designated FL-L1b and residing centrally within the CENP-A-binding clusters, to be transcriptionally active. We demonstrated the direct incorporation of the FL-L1b RNA transcripts into the CENP-A-associated chromatin. RNAi-mediated knockdown of the FL-L1b RNA transcripts led to a reduction in CENP-A binding and an impaired mitotic function of the 10q25 neocentromere. These results indicate that LINE retrotransposon RNA is a previously undescribed essential structural and functional component of the neocentromeric chromatin and that retrotransposable elements may serve as a critical epigenetic determinant in the chromatin remodelling events leading to neocentromere formation.

  17. Dysregulation of select ATP-dependent chromatin remodeling factors in high trait anxiety.

    Science.gov (United States)

    Wille, Alexandra; Amort, Thomas; Singewald, Nicolas; Sartori, Simone B; Lusser, Alexandra

    2016-09-15

    Enhanced anxiety is a salient feature of a number of psychiatric disorders including anxiety disorders, trauma-related disorders and depression. Although aberrant expression of various genes has been detected in patients suffering from persistent high anxiety as well as in high anxiety rodent models, the molecular mechanisms responsible for altered transcription regulation have been poorly addressed. Transcription regulation intimately involves the contribution of chromatin modifying processes, such as histone modification and ATP-dependent chromatin remodeling, yet their role in pathological anxiety is not known. Here, we investigated for the first time if altered levels of several ATP-dependent chromatin remodeling factors (ChRFs) and histone deacetylases (HDACs) may be linked to high trait anxiety in mice. While we found protein levels of the ChRFs SNF2H, ATRX, CHD1, CHD3 and CHD5 and of HDACs 1-3 and 6 to be similar in most of the tested brain areas of mice with high (HAB) versus normal (NAB) anxiety-related behavior, we observed distinctly altered regulation of SNF2H in the amygdala, and of CHD3 and CHD5 in the ventral hippocampus. In particular, CHD3 and CHD5 exhibited altered expression of protein but not of mRNA in HAB mice. Since both proteins are components of NuRD-like complexes, these results may indicate an impaired equilibrium between different NuRD-like complexes in the ventral hippocampus. Overall, our data provide novel evidence for localized differences of specific ATP-dependent chromatin remodeling factors in mice with high trait anxiety that may ultimately contribute to altered transcriptional programs resulting in the manifestation of pathological anxiety. PMID:27208790

  18. SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown

    OpenAIRE

    Turgay Y; Champion L; Balazs C; Held M; Toso A; Gerlich DW; Meraldi P; Kutay U.

    2014-01-01

    SUN proteins reside in the inner nuclear membrane and form complexes with KASH proteins of the outer nuclear membrane that connect the nuclear envelope (NE) to the cytoskeleton. These complexes have well-established functions in nuclear anchorage and migration in interphase, but little is known about their involvement in mitotic processes. Our analysis demonstrates that simultaneous depletion of human SUN1 and SUN2 delayed removal of membranes from chromatin during NE breakdown (NEBD) and imp...

  19. Chromatin disruption in the promoter of Bovine Leukemia Virus during transcriptional activation

    OpenAIRE

    Colin, Laurence; Dekoninck, Ann; Reichert, Michal; Calao, Miriam; Merimi, Makram; Van den Broeke, Anne; Vierendeel, Valérie; Cleuter, Yvette; Burny, Arsène; Rohr, Olivier; Van Lint, Carine

    2011-01-01

    Bovine leukemia virus expression relies on its chromatin organization after integration into the host cell genome. Proviral latency, which results from transcriptional repression in vivo, represents a viral strategy to escape the host immune system and likely allows for tumor progression. Here, we discriminated two types of latency: an easily reactivable latent state of the YR2 provirus and a ‘locked’ latent state of the L267 provirus. The defective YR2 provirus was characterized by the prese...

  20. Chromatin modification by PSC occurs at one PSC per nucleosome and does not require the acidic patch of histone H2A.

    Science.gov (United States)

    Lo, Stanley M; McElroy, Kyle A; Francis, Nicole J

    2012-01-01

    Chromatin architecture is regulated through both enzymatic and non-enzymatic activities. For example, the Polycomb Group (PcG) proteins maintain developmental gene silencing using an array of chromatin-based mechanisms. The essential Drosophila PcG protein, Posterior Sex Combs (PSC), compacts chromatin and inhibits chromatin remodeling and transcription through a non-enzymatic mechanism involving nucleosome bridging. Nucleosome bridging is achieved through a combination of nucleosome binding and self-interaction. Precisely how PSC interacts with chromatin to bridge nucleosomes is not known and is the subject of this work. We determine the stoichiometry of PSC-chromatin interactions in compact chromatin (in which nucleosomes are bridged) using Scanning Transmission Electron Microscopy (STEM). We find that full compaction occurs with one PSC per nucleosome. In addition to compacting chromatin, we show that PSC oligomerizes nucleosome arrays. PSC-mediated oligomerization of chromatin occurs at similar stoichiometry as compaction suggesting it may also involve nucleosome bridging. Interactions between the tail of histone H4 and the acidic patch of histone H2A are important for chromatin folding and oligomerization, and several chromatin proteins bind the histone H2A acidic patch. However, mutation of the acidic patch of histone H2A does not affect PSC's ability to inhibit chromatin remodeling or bridge nucleosomes. In fact, PSC does not require nucleosomes for bridging activity but can bridge naked DNA segments. PSC clusters nucleosomes on sparsely assembled templates, suggesting it interacts preferentially with nucleosomes over bare DNA. This may be due to the ability of PSC to bind free histones. Our data are consistent with a model in which each PSC binds a nucleosome and at least one other PSC to directly bridge nucleosomes and compact chromatin, but also suggest that naked DNA can be included in compacted structures. We discuss how our data highlight the diversity

  1. Minor Loops in Major Folds: Enhancer-Promoter Looping, Chromatin Restructuring, and Their Association with Transcriptional Regulation and Disease.

    Science.gov (United States)

    Matharu, Navneet; Ahituv, Nadav

    2015-12-01

    The organization and folding of chromatin within the nucleus can determine the outcome of gene expression. Recent technological advancements have enabled us to study chromatin interactions in a genome-wide manner at high resolution. These studies have increased our understanding of the hierarchy and dynamics of chromatin domains that facilitate cognate enhancer-promoter looping, defining the transcriptional program of different cell types. In this review, we focus on vertebrate chromatin long-range interactions as they relate to transcriptional regulation. In addition, we describe how the alteration of boundaries that mark discrete regions in the genome with high interaction frequencies within them, called topological associated domains (TADs), could lead to various phenotypes, including human diseases, which we term as "TADopathies." PMID:26632825

  2. Minor Loops in Major Folds: Enhancer-Promoter Looping, Chromatin Restructuring, and Their Association with Transcriptional Regulation and Disease.

    Directory of Open Access Journals (Sweden)

    Navneet Matharu

    2015-12-01

    Full Text Available The organization and folding of chromatin within the nucleus can determine the outcome of gene expression. Recent technological advancements have enabled us to study chromatin interactions in a genome-wide manner at high resolution. These studies have increased our understanding of the hierarchy and dynamics of chromatin domains that facilitate cognate enhancer-promoter looping, defining the transcriptional program of different cell types. In this review, we focus on vertebrate chromatin long-range interactions as they relate to transcriptional regulation. In addition, we describe how the alteration of boundaries that mark discrete regions in the genome with high interaction frequencies within them, called topological associated domains (TADs, could lead to various phenotypes, including human diseases, which we term as "TADopathies."

  3. Function of chromatin structure and dynamics in DNA damage, repair and misrepair: γ-rays and protons in action

    International Nuclear Information System (INIS)

    According to their physical characteristics, protons and ion beams promise a revolution in cancer radiotherapy. Curing protocols however reflect rather the empirical knowledge than experimental data on DNA repair. This especially holds for the spatio-temporal organization of repair processes in the context of higher-order chromatin structure—the problematics addressed in this work. The consequences for the mechanism of chromosomal translocations are compared for gamma rays and proton beams. - Highlights: ► The majority of DSBs are repaired individually close to the sites of their origin. ► Decondensation of damaged chromatin domains can potentiate clustering of lesions. ► DSB clustering might increase the risk of chromatin translocation. ► Distances of lesions and higher-order chromatin structure influence DSB clustering. ► The conclusions seem to hold both for DSB damage caused by γ-radiation and protons

  4. Facilitates Chromatin Transcription Complex Is an “Accelerator” of Tumor Transformation and Potential Marker and Target of Aggressive Cancers

    Directory of Open Access Journals (Sweden)

    Henry Garcia

    2013-07-01

    Full Text Available The facilitates chromatin transcription (FACT complex is involved in chromatin remodeling during transcription, replication, and DNA repair. FACT was previously considered to be ubiquitously expressed and not associated with any disease. However, we discovered that FACT is the target of a class of anticancer compounds and is not expressed in normal cells of adult mammalian tissues, except for undifferentiated and stem-like cells. Here, we show that FACT expression is strongly associated with poorly differentiated aggressive cancers with low overall survival. In addition, FACT was found to be upregulated during in vitro transformation and to be necessary, but not sufficient, for driving transformation. FACT also promoted survival and growth of established tumor cells. Genome-wide mapping of chromatin-bound FACT indicated that FACT’s role in cancer most likely involves selective chromatin remodeling of genes that stimulate proliferation, inhibit cell death and differentiation, and regulate cellular stress responses.

  5. DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism.

    Science.gov (United States)

    Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Morano, Annalisa; Zuchegna, Candida; Romano, Antonella; Muller, Mark T; Gottesman, Max E; Porcellini, Antonio; Avvedimento, Enrico V

    2016-01-01

    We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5' and 3' ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells. PMID:27629060

  6. Making Sense of the Tangle: Insights into Chromatin Folding and Gene Regulation.

    Science.gov (United States)

    Chung, Ill-Min; Ketharnathan, Sarada; Kim, Seung-Hyun; Thiruvengadam, Muthu; Rani, Mari Kavitha; Rajakumar, Govindasamy

    2016-01-01

    Proximity ligation assays such as circularized chromosome conformation capture and high-throughput chromosome capture assays have shed light on the structural organization of the interphase genome. Functional topologically associating domains (TADs) that constitute the building blocks of genomic organization are disrupted and reconstructed during the cell cycle. Epigenetic memory, as well as the sequence of chromosomes, regulate TAD reconstitution. Sub-TAD domains that are invariant across cell types have been identified, and contacts between these domains, rather than looping, are speculated to drive chromatin folding. Replication domains are established simultaneously with TADs during the cell cycle and the two correlate well in terms of characteristic features, such as lamin association and histone modifications. CCCTC-binding factor (CTCF) and cohesin cooperate across different cell types to regulate genes and genome organization. CTCF elements that demarcate TAD boundaries are commonly disrupted in cancer and promote oncogene activation. Chromatin looping facilitates interactions between distant promoters and enhancers, and the resulting enhanceosome complex promotes gene expression. Deciphering the chromatin tangle requires comprehensive integrative analyses of DNA- and protein-dependent factors that regulate genomic organization. PMID:27669308

  7. A chromatin link to caste identity in the carpenter ant Camponotus floridanus.

    Science.gov (United States)

    Simola, Daniel F; Ye, Chaoyang; Mutti, Navdeep S; Dolezal, Kelly; Bonasio, Roberto; Liebig, Jürgen; Reinberg, Danny; Berger, Shelley L

    2013-03-01

    In many ant species, sibling larvae follow alternative ontogenetic trajectories that generate striking variation in morphology and behavior among adults. These organism-level outcomes are often determined by environmental rather than genetic factors. Therefore, epigenetic mechanisms may mediate the expression of adult polyphenisms. We produced the first genome-wide maps of chromatin structure in a eusocial insect and found that gene-proximal changes in histone modifications, notably H3K27 acetylation, discriminate two female worker and male castes in Camponotus floridanus ants and partially explain differential gene expression between castes. Genes showing coordinated changes in H3K27ac and RNA implicate muscle development, neuronal regulation, and sensory responses in modulating caste identity. Binding sites of the acetyltransferase CBP harbor the greatest caste variation in H3K27ac, are enriched with motifs for conserved transcription factors, and show evolutionary expansion near developmental and neuronal genes. These results suggest that environmental effects on caste identity may be mediated by differential recruitment of CBP to chromatin. We propose that epigenetic mechanisms that modify chromatin structure may help orchestrate the generation and maintenance of polyphenic caste morphology and social behavior in ants. PMID:23212948

  8. The Costimulatory Receptor OX40 Inhibits Interleukin-17 Expression through Activation of Repressive Chromatin Remodeling Pathways.

    Science.gov (United States)

    Xiao, Xiang; Shi, Xiaomin; Fan, Yihui; Wu, Chenglin; Zhang, Xiaolong; Minze, Laurie; Liu, Wentao; Ghobrial, Rafik M; Lan, Peixiang; Li, Xian Chang

    2016-06-21

    T helper 17 (Th17) cells are prominently featured in multiple autoimmune diseases, but the regulatory mechanisms that control Th17 cell responses are poorly defined. Here we found that stimulation of OX40 triggered a robust chromatin remodeling response and produced a "closed" chromatin structure at interleukin-17 (IL-17) locus to inhibit Th17 cell function. OX40 activated the NF-κB family member RelB, and RelB recruited the histone methyltransferases G9a and SETDB1 to the Il17 locus to deposit "repressive" chromatin marks at H3K9 sites, and consequently repressing IL-17 expression. Unlike its transcriptional activities, RelB acted independently of both p52 and p50 in the suppression of IL-17. In an experimental autoimmune encephalomyelitis (EAE) disease model, we found that OX40 stimulation inhibited IL-17 and reduced EAE. Conversely, RelB-deficient CD4(+) T cells showed enhanced IL-17 induction and exacerbated the disease. Our data uncover a mechanism in the control of Th17 cells that might have important clinic implications. PMID:27317259

  9. Identification of chromatin-associated regulators of MSL complex targeting in Drosophila dosage compensation.

    Directory of Open Access Journals (Sweden)

    Erica Larschan

    Full Text Available Sex chromosome dosage compensation in Drosophila provides a model for understanding how chromatin organization can modulate coordinate gene regulation. Male Drosophila increase the transcript levels of genes on the single male X approximately two-fold to equal the gene expression in females, which have two X-chromosomes. Dosage compensation is mediated by the Male-Specific Lethal (MSL histone acetyltransferase complex. Five core components of the MSL complex were identified by genetic screens for genes that are specifically required for male viability and are dispensable for females. However, because dosage compensation must interface with the general transcriptional machinery, it is likely that identifying additional regulators that are not strictly male-specific will be key to understanding the process at a mechanistic level. Such regulators would not have been recovered from previous male-specific lethal screening strategies. Therefore, we have performed a cell culture-based, genome-wide RNAi screen to search for factors required for MSL targeting or function. Here we focus on the discovery of proteins that function to promote MSL complex recruitment to "chromatin entry sites," which are proposed to be the initial sites of MSL targeting. We find that components of the NSL (Non-specific lethal complex, and a previously unstudied zinc-finger protein, facilitate MSL targeting and display a striking enrichment at MSL entry sites. Identification of these factors provides new insight into how MSL complex establishes the specialized hyperactive chromatin required for dosage compensation in Drosophila.

  10. An Allosteric Interaction Links USP7 to Deubiquitination and Chromatin Targeting of UHRF1

    Directory of Open Access Journals (Sweden)

    Zhi-Min Zhang

    2015-09-01

    Full Text Available The protein stability and chromatin functions of UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1 are regulated in a cell-cycle-dependent manner. We report a structural characterization of the complex between UHRF1 and the deubiquitinase USP7. The first two UBL domains of USP7 bind to the polybasic region (PBR of UHRF1, and this interaction is required for the USP7-mediated deubiquitination of UHRF1. Importantly, we find that the USP7-binding site of the UHRF1 PBR overlaps with the region engaging in an intramolecular interaction with the N-terminal tandem Tudor domain (TTD. We show that the USP7-UHRF1 interaction perturbs the TTD-PBR interaction of UHRF1, thereby shifting the conformation of UHRF1 from a TTD-“occluded” state to a state open for multivalent histone binding. Consistently, introduction of a USP7-interaction-defective mutation to UHRF1 significantly reduces its chromatin association. Together, these results link USP7 interaction to the dynamic deubiquitination and chromatin association of UHRF1.

  11. The Relation Between Promoter Chromatin Status, Xyr1 and Cellulase Ex-pression in Trichoderma reesei.

    Science.gov (United States)

    Mello-de-Sousa, Thiago M; Rassinger, Alice; Derntl, Christian; Poças-Fonseca, Marcio J; Mach, Robert L; Mach-Aigner, Astrid R

    2016-04-01

    The ascomycete Trichoderma reesei is used for the production of plant cell wall-degrading enzymes in industrial scale. The interplay of the transactivator Xyr1 and the repressor Cre1 mainly regulates the expression of these enzymes. During induc-ing conditions, such as in the presence of sophorose, the transcription of the two major cellulase-encoding genes, cbh1 and cbh2, is activated as well as the expression of xyr1. In the presence of D-glucose carbon catabolite repression mediated by Cre1 takes place and the expression of Xyr1 and the plant cell wall-degrading enzymes is down-regulated. In this study we compare the chromatin status of xyr1, cbh1, and cbh2 promoters in the wild-type strain and the Cre1-deficient strain Rut-C30. Chromatin rearrangement occurs in the xyr1 promoter during induction on sophorose. Chromatin opening and protein-DNA interactions in the xyr1 promoter were detected especially in a region located 0.9 kb upstream the translation start co-don, which bears several putative Cre1-binding sites and a CCAAT-box. Moreover, the xyr1 promoter is overall more acces-sible in a cre1-truncated background, no matter which carbon source is present. This makes the xyr1 regulatory sequence a good target for promoter engineering aiming at the enhancement of cellulase production. PMID:27226770

  12. Involvement of chromatin and histone acetylation in theregulation of HIV-LTR by thyroid hormone receptor

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The HIV-1 LTR controls the expression of HIV-1 viral genes and thus is critical for viral propagation and pathology.Numerous host factors have been shown to participate in the regulation of the LTR promoter.Among them is the thyroid hormone (T3) receptor (TR).TR has been shown to bind to the critical region of the promoter that contain the NFκB and Sp1 binding sites.Interestingly,earlier transient transfection studies in tissue culture cells have yielded contradicting conclusions on the role of TR in LTR regulation,likely due to the use of different cell types and/or lack of proper chromatin organization.Here,using the frog oocyte as a model system that allows replication-coupled chromatin assembly,mimicking that in somatic cells,we demonstrate that unliganded heterodimers of TR and RXR (9-cis retinoic acid receptor) repress LTR while the addition of T3 relieves the repression and further activates the promoter.More importantly,we show that chromatin and unliganded TR/RXR synergize to repress the promoter in a histone deacetylase-dependent manner.

  13. Fission yeast Scm3: A CENP-A receptor required for integrity of subkinetochore chromatin.

    Science.gov (United States)

    Pidoux, Alison L; Choi, Eun Shik; Abbott, Johanna K R; Liu, Xingkun; Kagansky, Alexander; Castillo, Araceli G; Hamilton, Georgina L; Richardson, William; Rappsilber, Juri; He, Xiangwei; Allshire, Robin C

    2009-02-13

    The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin. PMID:19217404

  14. Heterochromatin and RNAi are required to establish CENP-A chromatin at centromeres.

    Science.gov (United States)

    Folco, Hernan Diego; Pidoux, Alison L; Urano, Takeshi; Allshire, Robin C

    2008-01-01

    Heterochromatin is defined by distinct posttranslational modifications on histones, such as methylation of histone H3 at lysine 9 (H3K9), which allows heterochromatin protein 1 (HP1)-related chromodomain proteins to bind. Heterochromatin is frequently found near CENP-A chromatin, which is the key determinant of kinetochore assembly. We have discovered that the RNA interference (RNAi)-directed heterochromatin flanking the central kinetochore domain at fission yeast centromeres is required to promote CENP-A(Cnp1) and kinetochore assembly over the central domain. The H3K9 methyltransferase Clr4 (Suv39); the ribonuclease Dicer, which cleaves heterochromatic double-stranded RNA to small interfering RNA (siRNA); Chp1, a component of the RNAi effector complex (RNA-induced initiation of transcriptional gene silencing; RITS); and Swi6 (HP1) are required to establish CENP-A(Cnp1) chromatin on naïve templates. Once assembled, CENP-A(Cnp1) chromatin is propagated by epigenetic means in the absence of heterochromatin. Thus, another, potentially conserved, role for centromeric RNAi-directed heterochromatin has been identified. PMID:18174443

  15. Impaired methylation modifications of FZD3 alter chromatin accessibility and are involved in congenital hydrocephalus pathogenesis.

    Science.gov (United States)

    Wang, Li; Shangguan, Shaofang; Chang, Shaoyan; Wang, Zhen; Lu, Xiaolin; Wu, Lihua; Li, Rui; Bao, Yihua; Qiu, Zhiyong; Niu, Bo; Zhang, Ting

    2014-06-20

    Congenital hydrocephalus is heterogeneous in its etiology, and in addition to a genetic component, has been shown to be caused by environmental factors. Until now, however, no methylation alterations of target genes have been connected with congenital hydrocephalus in humans. Frizzled 3(FZD3) is a planar cell polarity (PCP) gene required for PCP signaling. Partial restoration of frizzled 3 activities in FZD3 mutant mice results in hydrocephalus. To analyze the possible roles of epigenetic modifications of the FZD3 gene in congenital hydrocephalus pathogenesis, DNA methylation in the promoter region of FZD3 was assayed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Gene expression and chromatin accessibility were also determined to assess the role of methylation alterations. Our study found methylation levels of the FZD3 gene were increased in congenital hydrocephalus, especially in males (10.57 ± 3.90 vs. 7.08 ± 0.94, p=0.001). Hypermethylation of FZD3 increased congenital hydrocephalus risk, with an odds ratio of 10.125 (p=0.003). Aberrant methylation modification of FZD3 altered both chromatin structure in this region and FZD3 expression levels. Totally, aberrant methylation modification of the FZD3 gene increases the risk of congenital hydrocephalus by altering chromatin structure and disturbing gene expression.

  16. Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Johanna Sebald

    Full Text Available The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster.

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

    International Nuclear Information System (INIS)

    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

  18. Making Sense of the Tangle: Insights into Chromatin Folding and Gene Regulation

    Directory of Open Access Journals (Sweden)

    Ill-Min Chung

    2016-09-01

    Full Text Available Proximity ligation assays such as circularized chromosome conformation capture and high-throughput chromosome capture assays have shed light on the structural organization of the interphase genome. Functional topologically associating domains (TADs that constitute the building blocks of genomic organization are disrupted and reconstructed during the cell cycle. Epigenetic memory, as well as the sequence of chromosomes, regulate TAD reconstitution. Sub-TAD domains that are invariant across cell types have been identified, and contacts between these domains, rather than looping, are speculated to drive chromatin folding. Replication domains are established simultaneously with TADs during the cell cycle and the two correlate well in terms of characteristic features, such as lamin association and histone modifications. CCCTC-binding factor (CTCF and cohesin cooperate across different cell types to regulate genes and genome organization. CTCF elements that demarcate TAD boundaries are commonly disrupted in cancer and promote oncogene activation. Chromatin looping facilitates interactions between distant promoters and enhancers, and the resulting enhanceosome complex promotes gene expression. Deciphering the chromatin tangle requires comprehensive integrative analyses of DNA- and protein-dependent factors that regulate genomic organization.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  1. Chromatin remodeling during the in vivo glial differentiation in early Drosophila embryos

    Science.gov (United States)

    Ye, Youqiong; Gu, Liang; Chen, Xiaolong; Shi, Jiejun; Zhang, Xiaobai; Jiang, Cizhong

    2016-01-01

    Chromatin remodeling plays a critical role in gene regulation and impacts many biological processes. However, little is known about the relationship between chromatin remodeling dynamics and in vivo cell lineage commitment. Here, we reveal the patterns of histone modification change and nucleosome positioning dynamics and their epigenetic regulatory roles during the in vivo glial differentiation in early Drosophila embryos. The genome-wide average H3K9ac signals in promoter regions are decreased in the glial cells compared to the neural progenitor cells. However, H3K9ac signals are increased in a group of genes that are up-regulated in glial cells and involved in gliogenesis. There occurs extensive nucleosome remodeling including shift, loss, and gain. Nucleosome depletion regions (NDRs) form in both promoters and enhancers. As a result, the associated genes are up-regulated. Intriguingly, NDRs form in two fashions: nucleosome shift and eviction. Moreover, the mode of NDR formation is independent of the original chromatin state of enhancers in the neural progenitor cells. PMID:27634414

  2. Impaired methylation modifications of FZD3 alter chromatin accessibility and are involved in congenital hydrocephalus pathogenesis.

    Science.gov (United States)

    Wang, Li; Shangguan, Shaofang; Chang, Shaoyan; Wang, Zhen; Lu, Xiaolin; Wu, Lihua; Li, Rui; Bao, Yihua; Qiu, Zhiyong; Niu, Bo; Zhang, Ting

    2014-06-20

    Congenital hydrocephalus is heterogeneous in its etiology, and in addition to a genetic component, has been shown to be caused by environmental factors. Until now, however, no methylation alterations of target genes have been connected with congenital hydrocephalus in humans. Frizzled 3(FZD3) is a planar cell polarity (PCP) gene required for PCP signaling. Partial restoration of frizzled 3 activities in FZD3 mutant mice results in hydrocephalus. To analyze the possible roles of epigenetic modifications of the FZD3 gene in congenital hydrocephalus pathogenesis, DNA methylation in the promoter region of FZD3 was assayed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Gene expression and chromatin accessibility were also determined to assess the role of methylation alterations. Our study found methylation levels of the FZD3 gene were increased in congenital hydrocephalus, especially in males (10.57 ± 3.90 vs. 7.08 ± 0.94, p=0.001). Hypermethylation of FZD3 increased congenital hydrocephalus risk, with an odds ratio of 10.125 (p=0.003). Aberrant methylation modification of FZD3 altered both chromatin structure in this region and FZD3 expression levels. Totally, aberrant methylation modification of the FZD3 gene increases the risk of congenital hydrocephalus by altering chromatin structure and disturbing gene expression. PMID:24796881

  3. Human-Chromatin-Related Protein Interactions Identify a Demethylase Complex Required for Chromosome Segregation

    Directory of Open Access Journals (Sweden)

    Edyta Marcon

    2014-07-01

    Full Text Available Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.

  4. A chromatin link to caste identity in the carpenter ant Camponotus floridanus.

    Science.gov (United States)

    Simola, Daniel F; Ye, Chaoyang; Mutti, Navdeep S; Dolezal, Kelly; Bonasio, Roberto; Liebig, Jürgen; Reinberg, Danny; Berger, Shelley L

    2013-03-01

    In many ant species, sibling larvae follow alternative ontogenetic trajectories that generate striking variation in morphology and behavior among adults. These organism-level outcomes are often determined by environmental rather than genetic factors. Therefore, epigenetic mechanisms may mediate the expression of adult polyphenisms. We produced the first genome-wide maps of chromatin structure in a eusocial insect and found that gene-proximal changes in histone modifications, notably H3K27 acetylation, discriminate two female worker and male castes in Camponotus floridanus ants and partially explain differential gene expression between castes. Genes showing coordinated changes in H3K27ac and RNA implicate muscle development, neuronal regulation, and sensory responses in modulating caste identity. Binding sites of the acetyltransferase CBP harbor the greatest caste variation in H3K27ac, are enriched with motifs for conserved transcription factors, and show evolutionary expansion near developmental and neuronal genes. These results suggest that environmental effects on caste identity may be mediated by differential recruitment of CBP to chromatin. We propose that epigenetic mechanisms that modify chromatin structure may help orchestrate the generation and maintenance of polyphenic caste morphology and social behavior in ants.

  5. Radioiodination of chicken erythrocyte histones H4 and H5 in chromatin.

    Science.gov (United States)

    Griffiths, G R; Huang, P C

    1979-08-25

    The conformational state of histones in isolated chicken erythrocyte chromatin was studied using procedures developed for probing surface proteins on membranes. Under controlled conditions, only exposed tyrosyl residues react with iodide radicals, generated either by the oxidant, chloramine-T (paratoluenesulfonyl chloramide), or the enzyme lactoperoxidase, giving monoidotyrosine. Using 125-iodine, this study compared the reactive tyrosines in free and bound histones H4, and H5. The relative extent of iodination of these histones within (H4) and outside (H5) of the nucleosomes was measured after extraction and gel electrophoresis. Each of the histones was further analyzed for the extent of specific tyrosine iodination by separating the tryptic peptides by high voltage electrophoresis. The identity of the labeled peptide was determined by dansylation of the amino acids present in each hydrolyzed peptide. The results show that there is a difference in the conformational arrangement of these histones on chromatin and in the free forms, since in chromatin not all tyrosine residues are as accessible for iodination as in the denatured state. Residue 53 of histone H5 for instance is more reactive than residues 28 and 58, indicating that the segments containing the latter residues are involved in either protein-DNA or protein-protein interactions. In histone H4, preferential labeling of 2 of the 4 tyrosines present was also observed.

  6. Chromatin Landscape of the IRF Genes and Role of the Epigenetic Reader BRD4.

    Science.gov (United States)

    Bachu, Mahesh; Dey, Anup; Ozato, Keiko

    2016-07-01

    Histone post-translational modification patterns represent epigenetic states of genomic genes and denote the state of their transcription, past history, and future potential in gene expression. Genome-wide chromatin modification patterns reported from various laboratories are assembled in the ENCODE database, providing a fertile ground for understanding epigenetic regulation of any genes of interest across many cell types. The IRF family genes critically control innate immunity as they direct expression and activities of interferons. While these genes have similar structural and functional traits, their chromatin landscapes and epigenetic features have not been systematically evaluated. Here, by mining ENCODE database using an imputational approach, we summarize chromatin modification patterns for 6 of 9 IRF genes and show characteristic features that connote their epigenetic states. BRD4 is a BET bromodomain protein that "reads and translates" epigenetic marks into transcription. We review recent findings that BRD4 controls constitutive and signal-dependent transcription of many genes, including IRF genes. BRD4 dynamically binds to various genomic genes with a spatial and temporal specificity. Of particular importance, BRD4 is shown to critically regulate IRF-dependent anti-pathogen protection, inflammatory responses triggered by NF-κB, and the growth and spread of many cancers. The advent of small molecule inhibitors that disrupt binding of BET bromdomain to acetylated histone marks has opened new therapeutic possibilities for cancer and inflammatory diseases.

  7. Increased exchange rate of histone H1 on chromatin by exogenous myogenin expression

    Institute of Scientific and Technical Information of China (English)

    MING; GONG; JU; HUA; NI; HONG; TI; JIA

    2002-01-01

    To explore the molecular mechanism of chromatin remodeling involved in the regulation of transcriptionalactivation of specific genes by a myogenic regulatory factor Myogenin, we used NIH3T3 fibroblasts with astably integrated H1.1-GFP fusion protein to monitor histone H1 movement directly by fluorescence recov-ery after photobleaching (FRAP) in living cells. The observation from FRAP experiments with myogenintransfected fibroblasts showed that the exchange rate of histone H1 in chromatin was obviously increased,indicating that forced expression of exogenous Myogenin can induce chromatin remodeling. The hyper-acetylation of histones H3 and H4 from myogenin transfected fibroblasts was detected by triton-acid-urea(TAU)/SDS (2-D) electrophoresis and Western blot with specific antibodies against acetylated N-termini ofhistones H3 and H4. RT-PCR analysis indicated that the nAChR α-subunit gene was expressed in the trans-fected fibroblasts. These results suggest that the expression of exogenous Myogenin can induce chromatinremodeling and activate the transcription of Myogenin-targeted gene in non-muscle cells.

  8. Induction of systemic lupus erythematosus syndrome in BALB/c mice by immunization with active chromatin

    Institute of Scientific and Technical Information of China (English)

    Hong LI; Yun-yi ZHANG; Ya-nan SUN; Xi-yi HUANG; Yong-feng JIA; Duan LI

    2004-01-01

    AIM: To establish an animal model for systemic lupus erythematosus (SLE)-like syndrome in mice. METHODS:BALB/c mice were immunized with active chromatin isolated from ConA-actived syngeneic spleno-lymphocytes.Plasma samples of mice were tested by enzyme-linked immunosorbent assays (ELISA) for the presence of IgG anti-dsDNA, -ssDNA, and anti-histone antibodies. Tumor necrosis factor-α (TNF-α) in serum was measured by ELISA. Spleno-lymphocyte proliferation assays and the levels of interferon-γ (IFN-γ) in supernatants were tested respectively. Proteinuria was measured. Kidneys were examined by direct immunohistochemical method and light microscopy. RESULTS: Anti-ds DNA, ssDNA, and histone antibodies were induced in active chromatin-immunized mice, the proliferation response of splenocytes to ConA and LPS were reduced, levels of interferon-γ in supernatants and TNF-α in serum were lowered. Lupus nephritis was assessed by the presence of Ig deposits,glomerular pathology and proteinuria. CONCLUSION: The active chromatin-induced SLE-like mouse model was similar to idiopathic SLE in human.

  9. RNAi-mediated gene silencing reveals involvement of Arabidopsis chromatin-related genes in Agrobacterium-mediated root transformation

    OpenAIRE

    Crane, Yan Ma; Gelvin, Stanton B

    2007-01-01

    We investigated the effect of RNAi-mediated gene silencing of 109 Arabidopsis thaliana chromatin-related genes (termed “chromatin genes” hereafter) on Agrobacterium-mediated root transformation. Each of the RNAi lines contains a single- or low-copy-number insertion of a hairpin construction that silences the endogenous copy of the target gene. We used three standard transient and stable transformation assays to screen 340 independent RNAi lines, representing 109 target genes, for the rat (res...

  10. The Chromatin-Modifying Enzyme Ezh2 Is Critical for the Maintenance of Regulatory T Cell Identity after Activation

    OpenAIRE

    DuPage, Michel; Chopra, Gaurav; Quiros, Jason; Rosenthal, Wendy L.; Morar, Malika M.; Holohan, Dan; Zhang, Ruan; Turka, Laurence; Marson, Alexander; Bluestone, Jeffrey A.

    2015-01-01

    Regulatory T cells (Treg cells) are required for immune homeostasis. Chromatin remodeling is essential for establishing diverse cellular identities, but how the epigenetic program in Treg cells is maintained throughout the dynamic activation process remains unclear. Here we have shown that CD28 co-stimulation, an extracellular cue intrinsically required for Treg cell maintenance, induced the chromatin-modifying enzyme, Ezh2. Treg-specific ablation of Ezh2 resulted in spontaneous autoimmunity ...

  11. A Map of General and Specialized Chromatin Readers in Mouse Tissues Generated by Label-free Interaction Proteomics

    DEFF Research Database (Denmark)

    Eberl, H.C.; Mann, M.; Spruijt, C.G.;

    2013-01-01

    Posttranslational modifications on core histones can serve as binding scaffolds for chromatin-associated proteins. Proteins that specifically bind to or "read" these modifications were previously identified in mass spectrometry-based proteomics screens based on stable isotope-labeling in cell lines...... the chromatin interaction landscape in mouse tissues, our workflow can be used for peptides with different modifications and cell types of any organism. © 2013 Elsevier Inc....

  12. Chromatin-bound NLS proteins recruit membrane vesicles and nucleoporins for nuclear envelope assembly via importin-α/β

    Institute of Scientific and Technical Information of China (English)

    Quanlong Lu; Zhigang Lu; Qinying Liu; Li Guo; He Ren; Jingyan Fu; Qing Jiang; Paul R Clarke; Chuanmao Zhang

    2012-01-01

    The mechanism for nuclear envelope (NE) assembly is not fully understood.Importin-β and the small GTPase Ran have been implicated in the spatial regulation of NE assembly process.Here we report that chromatin-bound NLS (nuclear localization sequence) proteins provide docking sites for the NE precursor membrane vesicles and nucleoporins via importin-α and -β during NE assembly in Xenopus egg extracts.We show that along with the fast recruitment of the abundant NLS proteins such as nucleoplasmin and histones to the demembranated sperm chromatin in the extracts,importin-α binds the chromatin NLS proteins rapidly.Meanwhile,importin-β binds cytoplasmic NE precursor membrane vesicles and nucleoporins.Through interacting with importin-α on the chromatin NLS proteins,importin-β targets the membrane vesicles and nucleoporins to the chromatin surface.Once encountering RanGTP on the chromatin generated by RCC1,importin-β preferentially binds Ran-GTP and releases the membrane vesicles and nucleoporins for NE assembly.NE assembly is disrupted by blocking the interaction between importin-α and NLS proteins with excess soluble NLS proteins or by depletion of importin-β from the extract.Our findings reveal a novel molecular mechanism for NE assembly in Xenopus egg extracts.

  13. From the chromatin interaction network to the organization of the human genome into replication N/U-domains

    International Nuclear Information System (INIS)

    The three-dimensional (3D) architecture of the mammalian nucleus is now being unraveled thanks to the recent development of chromatin conformation capture (3C) technologies. Here we report the results of a combined multiscale analysis of genome-wide mean replication timing and chromatin conformation data that reveal some intimate relationships between chromatin folding and human DNA replication. We previously described megabase replication N/U-domains as mammalian multiorigin replication units, and showed that their borders are ‘master’ replication initiation zones that likely initiate cascades of origin firing responsible for the stereotypic replication of these domains. Here, we demonstrate that replication N/U-domains correspond to the structural domains of self-interacting chromatin, and that their borders act as insulating regions both in high-throughput 3C (Hi-C) data and high-resolution 3C (4C) experiments. Further analyses of Hi-C data using a graph-theoretical approach reveal that N/U-domain borders are long-distance, interconnected hubs of the chromatin interaction network. Overall, these results and the observation that a well-defined ordering of chromatin states exists from N/U-domain borders to centers suggest that ‘master’ replication initiation zones are at the heart of a high-order, epigenetically controlled 3D organization of the human genome. (paper)

  14. Dose-related Increased Binding of Nickel to Chromatin Proteins; and Changes to DNA Concentration in the Liver of Guinea Pigs Treated with Nigerian Light Crude Oil

    Directory of Open Access Journals (Sweden)

    Lauretta Idabor

    2007-09-01

    Full Text Available The alteration in nuclear DNA concentration and the concomitant binding of xenobiotics (alkylating agents, heavy metals, etc. to chromatin constituents may adversely affect gene structure and/or function, and thus initiate carcinogenesis. Binding of nickel to chromatin DNA has been reported to cause DNA damage (cross-links, single-strand breaks, and although many soluble nickel compounds and complexes have been shown to bind to chromatin, porphyrin-complexed nickel (PCN in crude oils has not been studied. We have determined the doserelated increases in total and chromatin DNA concentrations, and the differential distribution (binding of PCN (crude oil nickel-CON to chromatin constituents in livers of adult male guinea pigs treated with 1.25, 2.50 and 5.0 ml/kg bw Nigerian Bonny light crude oil (BLCO by intraperitoneal injection. The results showed large BLCO-induced increases in total DNA concentrations of 424%, 632% and 436% at 1.25, 2.50 and 5.0 ml/kg bw BLCO respectively over the untreated controls; while it induced equally large increases in chromatin DNA concentrations of 585% and 200% at 2.50 and 5.0 ml/kg bw respectively. In both cases, maximum increases occurred at 2.50 ml/kg bw BLCO. The distribution of PCN in BLCO between chromatin DNA and chromatin proteins (histones and non-histones showed that at 2.50 and 5.0 ml/kg bw BLCO, nickel content in chromatin DNA reduced by 25% and 12.5% respectively over the controls; while its content in chromatin proteins also reduced by 26%; but increased by 166% at 2.50 and 5.0 ml/kg bw BLCO, respectively over the untreated controls. However, in intra-chromatin comparison, 38.8% more PCN bound to chromatin DNA than to chromatin proteins at 2.50 ml/kg bw; but at 5.0 ml/kg bw BLCO, 90.4% more PCN bound to chromatin proteins than to chromatin DNA. These results show a greater affinity of PCN in BLCO for chromatin proteins over chromatin DNA which may have played a role in the increased DNA concentrations

  15. Flightless I (Drosophila) homolog facilitates chromatin accessibility of the estrogen receptor α target genes in MCF-7 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Kwang Won, E-mail: kwjeong@gachon.ac.kr

    2014-04-04

    Highlights: • H3K4me3 and Pol II binding at TFF1 promoter were reduced in FLII-depleted MCF-7 cells. • FLII is required for chromatin accessibility of the enhancer of ERalpha target genes. • Depletion of FLII causes inhibition of proliferation of MCF-7 cells. - Abstract: The coordinated activities of multiple protein complexes are essential to the remodeling of chromatin structure and for the recruitment of RNA polymerase II (Pol II) to the promoter in order to facilitate the initiation of transcription in nuclear receptor-mediated gene expression. Flightless I (Drosophila) homolog (FLII), a nuclear receptor coactivator, is associated with the SWI/SNF-chromatin remodeling complex during estrogen receptor (ER)α-mediated transcription. However, the function of FLII in estrogen-induced chromatin opening has not been fully explored. Here, we show that FLII plays a critical role in establishing active histone modification marks and generating the open chromatin structure of ERα target genes. We observed that the enhancer regions of ERα target genes are heavily occupied by FLII, and histone H3K4me3 and Pol II binding induced by estrogen are decreased in FLII-depleted MCF-7 cells. Furthermore, formaldehyde-assisted isolation of regulatory elements (FAIRE)-quantitative polymerase chain reaction (qPCR) experiments showed that depletion of FLII resulted in reduced chromatin accessibility of multiple ERα target genes. These data suggest FLII as a key regulator of ERα-mediated transcription through its role in regulating chromatin accessibility for the binding of RNA Polymerase II and possibly other transcriptional coactivators.

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

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    Yolanda Stypula-Cyrus

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  18. Silencing of renal DNaseI in murine lupus nephritis imposes exposure of large chromatin fragments and activation of Toll like receptors and the Clec4e

    DEFF Research Database (Denmark)

    Thiyagarajan, Dhivya; Fismen, Silje; Seredkina, Natalya;

    2012-01-01

    Recent studies demonstrate that transformation of mild lupus nephritis into end-stage disease is imposed by silencing of renal DNaseI gene expression in (NZBxNZW)F1 mice. Down-regulation of DNaseI results in reduced chromatin fragmentation, and in deposition of extracellular chromatin-IgG complexes...... in glomerular basement membranes in individuals that produce IgG anti-chromatin antibodies. The main focus of the present study is to describe the biological consequences of renal DNaseI shut-down and reduced chromatin fragmentation with a particular focus on whether exposed large chromatin fragments activate...... Toll like receptors and the necrosis-related Clec4e receptor in murine and human lupus nephritis. Furthermore, analyses where performed to determine if matrix metalloproteases are up-regulated as a consequence of chromatin-mediated Toll like receptors/Clec4e stimulation. Mouse and human mRNA expression...

  19. Chromatin Dynamics and the Development of the TCRα and TCRδ Repertoires.

    Science.gov (United States)

    Carico, Zachary; Krangel, Michael S

    2015-01-01

    The adaptive immune system allows vertebrates to orchestrate highly specific responses to a virtually unlimited milieu of antigens. Effective adaptive immune responses depend on the capacity of T and B lymphocytes to generate diverse repertoires of antigen receptors through the recombination of variable (V), diversity (D), and joining (J) gene segments at antigen receptor loci. V(D)J recombination must be carefully regulated during the early stages of T and B lymphocyte development to ensure the proper development of lymphocyte subsets and to maximize antigen receptor combinatorial diversity. Among all T cell receptor (TCR) and immunoglobulin loci, the TCRα/δ (Tcra/Tcrd) locus is unique in its complexity since it undergoes recombination at two distinct stages of T cell development to create distinct TCR proteins that are used by different lineages of T cells. Here, we review the mechanisms that regulate V(D)J recombination at the Tcra/Tcrd locus, with a focus on the dynamic chromatin environment and how it instructs the assembly of the Tcra and Tcrd repertoires. We discuss the dynamics of Tcra and Tcrd repertoire formation in the context of T cell development, and we consider how the recombination program is directed by localized changes in chromatin structure that regulate the accessibility of Tcra and Tcrd gene segments to the V(D)J recombinase. We then move beyond local to address spatial relationships in the nucleus, emphasizing the three-dimensional organization of the Tcra/Tcrd locus as a critical player in understanding long-distance interactions between chromatin regulatory elements as well as long-distance interactions between recombination substrates. PMID:26477370

  20. Replication domains are self-interacting structural chromatin units of human chromosomes

    Science.gov (United States)

    Arneodo, Alain

    2011-03-01

    In higher eukaryotes, the absence of specific sequence motifs marking the origins of replication has been a serious hindrance to the understanding of the mechanisms that regulate the initiation and the maintenance of the replication program in different cell types. In silico analysis of nucleotide compositional skew has predicted the existence, in the germline, of replication N-domains bordered by putative replication origins and where the skew decreases rather linearly as the signature of a progressive inversion of the average fork polarity. Here, from the demonstration that the average fork polarity can be directly extracted from the derivative of replication timing profiles, we develop a wavelet-based pattern recognition methodology to delineate replication U-domains where the replication timing profile is shaped as a U and its derivative as a N. Replication U-domains are robustly found in seven cell lines as covering a significant portion (40-50%) of the human genome where the replication timing data actually displays some plasticity between cell lines. The early replication initiation zones at U-domains borders are found to be hypersensitive to DNase I cleavage, to be associated with transcriptional activity and to present a significant enrichment in insular-binding proteins CTCF, the hallmark of an open chromatin structure. A comparative analysis of genome-wide chromatin interaction (HiC) data shows that replication-U domains correspond to self-interacting structural high order chromatin units of megabase characteristic size. Taken together, these findings provide evidence that the epigenetic compartmentalization of the human genome into autonomous replication U-domains comes along with an extensive remodelling of the threedimensional chromosome architecture during development or in specific diseases. The observed cell specific conservation of the replication timing between the human and mouse genomes strongly suggests that this chromosome organization into