Sample records for affecting chromatin structure

  1. Nucleosome-free DNA regions differentially affect distant communication in chromatin

    PubMed Central

    Nizovtseva, Ekaterina V.; Clauvelin, Nicolas; Todolli, Stefjord; Kulaeva, Olga I.; Wengrzynek, Scott

    2017-01-01

    Abstract Communication between distantly spaced genomic regions is one of the key features of gene regulation in eukaryotes. Chromatin per se can stimulate efficient enhancer-promoter communication (EPC); however, the role of chromatin structure and dynamics in this process remains poorly understood. Here we show that nucleosome spacing and the presence of nucleosome-free DNA regions can modulate chromatin structure/dynamics and, in turn, affect the rate of EPC in vitro and in silico. Increasing the length of internucleosomal linker DNA from 25 to 60 bp results in more efficient EPC. The presence of longer nucleosome-free DNA regions can positively or negatively affect the rate of EPC, depending upon the length and location of the DNA region within the chromatin fiber. Thus the presence of histone-free DNA regions can differentially affect the efficiency of EPC, suggesting that gene regulation over a distance could be modulated by changes in the length of internucleosomal DNA spacers. PMID:27940560

  2. Perturbation of Chromatin Structure Globally Affects Localization and Recruitment of Splicing Factors

    PubMed Central

    Risso, Guillermo J.; Pawellek, Andrea; Ule, Jernej; Lamond, Angus I.; Kornblihtt, Alberto R.

    2012-01-01

    Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3′ splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ∼20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin

  3. Comparative study of cyanotoxins affecting cytoskeletal and chromatin structures in CHO-K1 cells.

    PubMed

    Gácsi, Mariann; Antal, Otilia; Vasas, Gábor; Máthé, Csaba; Borbély, György; Saker, Martin L; Gyori, János; Farkas, Anna; Vehovszky, Agnes; Bánfalvi, Gáspár

    2009-06-01

    In this study we compared the effects of the two frequently occuring and most dangerous cyanobacterial toxins on the cellular organization of microfilaments, microtubules and on the chromatin structure in Chinese hamster ovary (CHO-K1) cells. These compounds are the widely known microcystin-LR (MC-LR) and cylindrospermopsin (CYN) classified as the highest-priority cyanotoxin. Toxic effects were tested in a concentration and time dependent manner. The hepatotoxic MC-LR did not cause significant cytotoxicity on CHO-K1 cells under 20 microM, but caused apoptotic changes at higher concentrations. Apoptotic shrinkage was associated with the shortening and loss of actin filaments and with a concentration dependent depolymerization of microtubules. No necrosis was observed over the concentration range (1-50 microM MC-LR) tested. Cylindrospermopsin did cause apoptosis at low concentrations (1-2 microM) and over short exposure periods (12h). Necrosis was observed at higher concentrations (5-10 microM) and following longer exposure periods (24 or 48h). Cyanotoxins also affected the chromatin structure. The condensation process was inhibited by MC-LR at a later stage and manifested as broken elongated prechromosomes. CYN inhibited chromatin condensation at the early fibrillary stage leading to blurred fluorescent images of apoptotic bodies and preventing the formation of metaphase chromosomes. Cylindrospermopsin exhibited a more pronounced toxic effect causing cytoskeletal and nuclear changes as well as apoptotic and necrotic alterations.

  4. Kinases and chromatin structure

    PubMed Central

    Miotto, Benoit

    2013-01-01

    Chromatin structure is regulated by families of proteins that are able to covalently modify the histones and the DNA, as well as to regulate the spacing of nucleosomes along the DNA. Over the years, these chromatin remodeling factors have been proven to be essential to a variety of processes, including gene expression, DNA replication, and chromosome cohesion. The function of these remodeling factors is regulated by a number of chemical and developmental signals and, in turn, changes in the chromatin structure eventually contribute to the response to changes in the cellular environment. Exciting new research findings by the laboratories of Sharon Dent and Steve Jackson indicate, in two different contexts, that changes in the chromatin structure may, in reverse, signal to intracellular signaling pathways to regulate cell fate. The discoveries clearly challenge our traditional view of ‘epigenetics’, and may have important implications in human health. PMID:23917692

  5. Statistical physics of nucleosome positioning and chromatin structure

    NASA Astrophysics Data System (ADS)

    Morozov, Alexandre

    2012-02-01

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

  6. Computational strategies to address chromatin structure problems

    NASA Astrophysics Data System (ADS)

    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.

  7. Regulating the chromatin landscape: structural and mechanistic perspectives.

    PubMed

    Bartholomew, Blaine

    2014-01-01

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

  8. Capturing Structural Heterogeneity in Chromatin Fibers.

    PubMed

    Ekundayo, Babatunde; Richmond, Timothy J; Schalch, Thomas

    2017-10-13

    Chromatin fiber organization is implicated in processes such as transcription, DNA repair and chromosome segregation, but how nucleosomes interact to form higher-order structure remains poorly understood. We solved two crystal structures of tetranucleosomes with approximately 11-bp DNA linker length at 5.8 and 6.7 Å resolution. Minimal intramolecular nucleosome-nucleosome interactions result in a fiber model resembling a flat ribbon that is compatible with a two-start helical architecture, and that exposes histone and DNA surfaces to the environment. The differences in the two structures combined with electron microscopy reveal heterogeneous structural states, and we used site-specific chemical crosslinking to assess the diversity of nucleosome-nucleosome interactions through identification of structure-sensitive crosslink sites that provide a means to characterize fibers in solution. The chromatin fiber architectures observed here provide a basis for understanding heterogeneous chromatin higher-order structures as they occur in a genomic context. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    PubMed

    Eirín-López, José M

    2013-01-01

    The study of chromatin constitutes one of the most active research fields in life sciences, being subject to constant revisions that continuously redefine the state of the art in its knowledge. As every other rapidly changing field, chromatin biology requires clear and straightforward educational strategies able to efficiently translate such a vast body of knowledge to the classroom. With this aim, the present work describes a multidisciplinary computer lab designed to introduce undergraduate students to the dynamic nature of chromatin, within the context of the one semester course "Chromatin: Structure, Function and Evolution." This exercise is organized in three parts including (a) molecular evolutionary biology of histone families (using the H1 family as example), (b) histone structure and variation across different animal groups, and (c) effect of histone diversity on nucleosome structure and chromatin dynamics. By using freely available bioinformatic tools that can be run on common computers, the concept of chromatin dynamics is interactively illustrated from a comparative/evolutionary perspective. At the end of this computer lab, students are able to translate the bioinformatic information into a biochemical context in which the relevance of histone primary structure on chromatin dynamics is exposed. During the last 8 years this exercise has proven to be a powerful approach for teaching chromatin structure and dynamics, allowing students a higher degree of independence during the processes of learning and self-assessment. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  10. A DEK Domain-Containing Protein Modulates Chromatin Structure and Function in Arabidopsis[W][OPEN

    PubMed Central

    Waidmann, Sascha; Kusenda, Branislav; Mayerhofer, Juliane; Mechtler, Karl; Jonak, Claudia

    2014-01-01

    Chromatin is a major determinant in the regulation of virtually all DNA-dependent processes. Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. The evolutionarily conserved DEK domain-containing protein is implicated in important chromatin-related processes in animals, but little is known about its DNA targets and protein interaction partners. In plants, the role of DEK has remained elusive. In this work, we identified DEK3 as a chromatin-associated protein in Arabidopsis thaliana. DEK3 specifically binds histones H3 and H4. Purification of other proteins associated with nuclear DEK3 also established DNA topoisomerase 1α and proteins of the cohesion complex as in vivo interaction partners. Genome-wide mapping of DEK3 binding sites by chromatin immunoprecipitation followed by deep sequencing revealed enrichment of DEK3 at protein-coding genes throughout the genome. Using DEK3 knockout and overexpressor lines, we show that DEK3 affects nucleosome occupancy and chromatin accessibility and modulates the expression of DEK3 target genes. Furthermore, functional levels of DEK3 are crucial for stress tolerance. Overall, data indicate that DEK3 contributes to modulation of Arabidopsis chromatin structure and function. PMID:25387881

  11. On the chromatin structure of eukaryotic telomeres

    PubMed Central

    Vaquero-Sedas, María I

    2011-01-01

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

  12. Molecular structures guide the engineering of chromatin

    PubMed Central

    Tekel, Stefan J.

    2017-01-01

    Abstract Chromatin is a system of proteins, RNA, and DNA that interact with each other to organize and regulate genetic information within eukaryotic nuclei. Chromatin proteins carry out essential functions: packing DNA during cell division, partitioning DNA into sub-regions within the nucleus, and controlling levels of gene expression. There is a growing interest in manipulating chromatin dynamics for applications in medicine and agriculture. Progress in this area requires the identification of design rules for the chromatin system. Here, we focus on the relationship between the physical structure and function of chromatin proteins. We discuss key research that has elucidated the intrinsic properties of chromatin proteins and how this information informs design rules for synthetic systems. Recent work demonstrates that chromatin-derived peptide motifs are portable and in some cases can be customized to alter their function. Finally, we present a workflow for fusion protein design and discuss best practices for engineering chromatin to assist scientists in advancing the field of synthetic epigenetics. PMID:28609787

  13. Regulation of chromatin structure in the cardiovascular system.

    PubMed

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

    2013-01-01

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

  14. Molecular structures guide the engineering of chromatin.

    PubMed

    Tekel, Stefan J; Haynes, Karmella A

    2017-07-27

    Chromatin is a system of proteins, RNA, and DNA that interact with each other to organize and regulate genetic information within eukaryotic nuclei. Chromatin proteins carry out essential functions: packing DNA during cell division, partitioning DNA into sub-regions within the nucleus, and controlling levels of gene expression. There is a growing interest in manipulating chromatin dynamics for applications in medicine and agriculture. Progress in this area requires the identification of design rules for the chromatin system. Here, we focus on the relationship between the physical structure and function of chromatin proteins. We discuss key research that has elucidated the intrinsic properties of chromatin proteins and how this information informs design rules for synthetic systems. Recent work demonstrates that chromatin-derived peptide motifs are portable and in some cases can be customized to alter their function. Finally, we present a workflow for fusion protein design and discuss best practices for engineering chromatin to assist scientists in advancing the field of synthetic epigenetics. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Chromatin Structure and the Cell Cycle

    PubMed Central

    Pederson, Thoru

    1972-01-01

    Pancreatic DNase I is used to probe the structure of chromatin isolated from synchronized HeLa cells. The degree to which DNA in chromatin is protected from DNase attack varies during the G1, S, and G2 phases of the cell cycle. In addition, the DNase sensitivity of chromatin from contact-inhibited African green monkey kidney cells differs from that of actively dividing, subconfluent cultures. These cell cycle-dependent chromatin changes were observed consistently at all enzyme concentrations (5000-fold range) and incubation times (15 min-2 hr) tested. The results indicate that the degree of complexing between DNA and chromosomal proteins changes during interphase, and they suggest that the chromosome coiling cycle of visible mitosis may extend in more subtle form over the entire cell cycle. PMID:4626402

  16. Sanguinarine interacts with chromatin, modulates epigenetic modifications, and transcription in the context of chromatin.

    PubMed

    Selvi B, Ruthrotha; Pradhan, Suman Kalyan; Shandilya, Jayasha; Das, Chandrima; Sailaja, Badi Sri; Shankar G, Naga; Gadad, Shrikanth S; Reddy, Ashok; Dasgupta, Dipak; Kundu, Tapas K

    2009-02-27

    DNA-binding anticancer agents cause alteration in chromatin structure and dynamics. We report the dynamic interaction of the DNA intercalator and potential anticancer plant alkaloid, sanguinarine (SGR), with chromatin. Association of SGR with different levels of chromatin structure was enthalpy driven with micromolar dissociation constant. Apart from DNA, it binds with comparable affinity with core histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation-dependent chromatin transcription. SGR-mediated repression of epigenetic marks and the alteration of chromatin geography (nucleography) also result in the modulation of global gene expression. These data, conclusively, show an anticancer DNA binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context.

  17. Chromatin Structure and Replication Origins: Determinants Of Chromosome Replication And Nuclear Organization

    PubMed Central

    Smith, Owen K.; Aladjem, Mirit I.

    2014-01-01

    The DNA replication program is, in part, determined by the epigenetic landscape that governs local chromosome architecture and directs chromosome duplication. Replication must coordinate with other biochemical processes occurring concomitantly on chromatin, such as transcription and remodeling, to insure accurate duplication of both genetic and epigenetic features and to preserve genomic stability. The importance of genome architecture and chromatin looping in coordinating cellular processes on chromatin is illustrated by two recent sets of discoveries. First, chromatin-associated proteins that are not part of the core replication machinery were shown to affect the timing of DNA replication. These chromatin-associated proteins could be working in concert, or perhaps in competition, with the transcriptional machinery and with chromatin modifiers to determine the spatial and temporal organization of replication initiation events. Second, epigenetic interactions are mediated by DNA sequences that determine chromosomal replication. In this review we summarize recent findings and current models linking spatial and temporal regulation of the replication program with epigenetic signaling. We discuss these issues in the context of the genome’s three-dimensional structure with an emphasis on events occurring during the initiation of DNA replication. PMID:24905010

  18. A new fractionation assay, based on the size of formaldehyde-crosslinked, mildly sheared chromatin, delineates the chromatin structure at promoter regions

    PubMed Central

    Ishihara, Satoru; Varma, Rajat; Schwartz, Ronald H.

    2010-01-01

    To explore the higher order structure of transcribable chromatin in vivo, its local configuration was assessed through the accessibility of the chromatin to crosslinking with formaldehyde. The application of crosslinked and mildly sheared chromatin to sedimentation velocity centrifugation followed by size-fractionation of the DNA enabled us to biochemically distinguish between chromatin with heavily versus sparsely crosslinkable structures. The separated fractions showed a good correlation with gene expression profiles. Genes with poor crosslinking around the promoter region were actively transcribed, while transcripts were hardly detected from genes with extensive crosslinking in their promoter regions. For the inducible gene, Il2, the distribution of the promoter shifted in the gradient following T-cell receptor stimulation, consistent with a change in structure at this locus during activation. The kinetics of this switch preceded the chromatin change observed in a DNase I accessibility assay. Thus, this new chromatin fractionation technique has revealed a change in chromatin structure that has not been previously characterized. PMID:20371521

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Rodríguez-Campos, Antonio; Azorín, Fernando

    2007-11-14

    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.

  1. RNA Is an Integral Component of Chromatin that Contributes to Its Structural Organization

    PubMed Central

    Rodríguez-Campos, Antonio; Azorín, Fernando

    2007-01-01

    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 α-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. PMID:18000552

  2. Structured illumination to spatially map chromatin motions.

    PubMed

    Bonin, Keith; Smelser, Amanda; Moreno, Naike Salvador; Holzwarth, George; Wang, Kevin; Levy, Preston; Vidi, Pierre-Alexandre

    2018-05-01

    We describe a simple optical method that creates structured illumination of a photoactivatable probe and apply this method to characterize chromatin motions in nuclei of live cells. A laser beam coupled to a diffractive optical element at the back focal plane of an excitation objective generates an array of near diffraction-limited beamlets with FWHM of 340  ±  30  nm, which simultaneously photoactivate a 7  ×  7 matrix pattern of GFP-labeled histones, with spots 1.70  μm apart. From the movements of the photoactivated spots, we map chromatin diffusion coefficients at multiple microdomains of the cell nucleus. The results show correlated motions of nearest chromatin microdomain neighbors, whereas chromatin movements are uncorrelated at the global scale of the nucleus. The method also reveals a DNA damage-dependent decrease in chromatin diffusion. The diffractive optical element instrumentation can be easily and cheaply implemented on commercial inverted fluorescence microscopes to analyze adherent cell culture models. A protocol to measure chromatin motions in nonadherent human hematopoietic stem and progenitor cells is also described. We anticipate that the method will contribute to the identification of the mechanisms regulating chromatin mobility, which influences most genomic processes and may underlie the biogenesis of genomic translocations associated with hematologic malignancies. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

  3. Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration.

    PubMed

    Matysiak, Julien; Lesbats, Paul; Mauro, Eric; Lapaillerie, Delphine; Dupuy, Jean-William; Lopez, Angelica P; Benleulmi, Mohamed Salah; Calmels, Christina; Andreola, Marie-Line; Ruff, Marc; Llano, Manuel; Delelis, Olivier; Lavigne, Marc; Parissi, Vincent

    2017-07-28

    Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.

  4. p53 targets chromatin structure alteration to repress alpha-fetoprotein gene expression.

    PubMed

    Ogden, S K; Lee, K C; Wernke-Dollries, K; Stratton, S A; Aronow, B; Barton, M C

    2001-11-09

    Many of the functions ascribed to p53 tumor suppressor protein are mediated through transcription regulation. We have shown that p53 represses hepatic-specific alpha-fetoprotein (AFP) gene expression by direct interaction with a composite HNF-3/p53 DNA binding element. Using solid-phase, chromatin-assembled AFP DNA templates and analysis of chromatin structure and transcription in vitro, we find that p53 binds DNA and alters chromatin structure at the AFP core promoter to regulate transcription. Chromatin assembled in the presence of hepatoma extracts is activated for AFP transcription with an open, accessible core promoter structure. Distal (-850) binding of p53 during chromatin assembly, but not post-assembly, reverses transcription activation concomitant with promoter inaccessibility to restriction enzyme digestion. Inhibition of histone deacetylase activity by trichostatin-A (TSA) addition, prior to and during chromatin assembly, activated chromatin transcription in parallel with increased core promoter accessibility. Chromatin immunoprecipitation analyses showed increased H3 and H4 acetylated histones at the core promoter in the presence of TSA, while histone acetylation remained unchanged at the site of distal p53 binding. Our data reveal that p53 targets chromatin structure alteration at the core promoter, independently of effects on histone acetylation, to establish repressed AFP gene expression.

  5. Concerted Flexibility of Chromatin Structure, Methylome, and Histone Modifications along with Plant Stress Responses

    PubMed Central

    Santos, Ana Paula; Ferreira, Liliana J.; Oliveira, M. Margarida

    2017-01-01

    The spatial organization of chromosome structure within the interphase nucleus, as well as the patterns of methylome and histone modifications, represent intersecting layers that influence genome accessibility and function. This review is focused on the plastic nature of chromatin structure and epigenetic marks in association to stress situations. The use of chemical compounds (epigenetic drugs) or T-DNA-mediated mutagenesis affecting epigenetic regulators (epi-mutants) are discussed as being important tools for studying the impact of deregulated epigenetic backgrounds on gene function and phenotype. The inheritability of epigenetic marks and chromatin configurations along successive generations are interpreted as a way for plants to “communicate” past experiences of stress sensing. A mechanistic understanding of chromatin and epigenetics plasticity in plant response to stress, including tissue- and genotype-specific epigenetic patterns, may help to reveal the epigenetics contributions for genome and phenotype regulation. PMID:28275209

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

    PubMed Central

    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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lebedev, D. V., E-mail: isaev@omrb.pnpi.spb.ru; Filatov, M. V.; Kuklin, A. I.

    The chromatin organization in chicken erythrocyte nuclei was studied by small-angle neutron scattering in the scattering-vector range from 1.5 x 10{sup -1} to 10{sup -4} A{sup -1} with the use of the contrast-variation technique. This scattering-vector range corresponds to linear dimensions from 4 nm to 6 {mu}m and covers the whole hierarchy of chromatin structures, from the nucleosomal structure to the entire nucleus. The results of the present study allowed the following conclusions to be drawn: (1) both the chromatin-protein structure and the structure of the nucleic acid component in chicken erythrocyte nuclei have mass-fractal properties, (2) the structure ofmore » 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.« less

  8. Differential Chromatin Structure Encompassing Replication Origins in Transformed and Normal Cells

    PubMed Central

    Di Paola, Domenic; Rampakakis, Emmanouil; Chan, Man Kid

    2012-01-01

    This study examines the chromatin structure encompassing replication origins in transformed and normal cells. Analysis of the global levels of histone H3 acetylated at K9&14 (open chromatin) and histone H3 trimethylated at K9 (closed chromatin) revealed a higher ratio of open to closed chromatin in the transformed cells. Also, the trithorax and polycomb group proteins, Brg-1 and Bmi-1, respectively, were overexpressed and more abundantly bound to chromatin in the transformed cells. Quantitative comparative analyses of episomal and in situ chromosomal replication origin activity as well as chromatin immunoprecipitation (ChIP) assays, using specific antibodies targeting members of the pre-replication complex (pre-RC) as well as open/closed chromatin markers encompassing both episomal and chromosomal origins, revealed that episomal origins had similar levels of in vivo activity, nascent DNA abundance, pre-RC protein association, and elevated open chromatin structure at the origin in both cell types. In contrast, the chromosomal origins corresponding to 20mer1, 20mer2, and c-myc displayed a 2- to 3-fold higher activity and pre-RC protein abundance as well as higher ratios of open to closed chromatin and of Brg-1 to Bmi-1 in the transformed cells, whereas the origin associated with the housekeeping lamin B2 gene exhibited similar levels of activity, pre-RC protein abundance, and higher ratios of open to closed chromatin and of Brg-1 to Bmi-1 in both cell types. Nucleosomal positioning analysis, using an MNase-Southern blot assay, showed that all the origin regions examined were situated within regions of inconsistently positioned nucleosomes, with the nucleosomes being spaced farther apart from each other prior to the onset of S phase in both cell types. Overall, the results indicate that cellular transformation is associated with differential epigenetic regulation, whereby chromatin structure is more open, rendering replication origins more accessible to initiator

  9. Macronuclear chromatin structure dynamics in Colpoda inflata (Protista, Ciliophora) resting encystment.

    PubMed

    Tiano, L; Chessa, M G; Carrara, S; Tagliafierro, G; Delmonte Corrado, M U

    1999-01-01

    The chromatin structure dynamics of the Colpoda inflata macronucleus have been investigated in relation to its functional condition, concerning chromatin body extrusion regulating activity. Samples of 2- and 25-day-old resting cysts derived from a standard culture, and of 1-year-old resting cysts derived from a senescent culture, were examined by means of histogram analysis performed on acquired optical microscopy images. Three groups of histograms were detected in each sample. Histogram classification, clustering and matching were assessed in order to obtain the mean histogram of each group. Comparative analysis of the mean histogram showed a similarity in the grey level range of 25-day- and 1-year-old cysts, unlike the wider grey level range found in 2-day-old cysts. Moreover, the respective mean histograms of the three cyst samples appeared rather similar in shape. All this implies that macronuclear chromatin structural features of 1-year-old cysts are common to both cyst standard cultures. The evaluation of the acquired images and their respective histograms evidenced a dynamic state of the macronuclear chromatin, appearing differently condensed in relation to the chromatin body extrusion regulating activity of the macronucleus. The coexistence of a chromatin-decondensed macronucleus with a pycnotic extrusion body suggests that chromatin unable to decondense, thus inactive, is extruded. This finding, along with the presence of chromatin structural features common to standard and senescent cyst populations, supports the occurrence of 'rejuvenated' cell lines from 1-year-old encysted senescent cells, a phenomenon which could be a result of accomplished macronuclear renewal.

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

    PubMed Central

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

    2014-01-01

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

  11. Role of Histone Acetylation in the Assembly and Modulation of Chromatin Structures

    PubMed Central

    Annunziato, Anthony T.; Hansen, Jeffrey C.

    2000-01-01

    The acetylation of the core histone N-terminal “tail” domains is now recognized as a highly conserved mechanism for regulating chromatin functional states. The following article examines possible roles of acetylation in two critically important cellular processes: replication-coupled nucleosome assembly, and reversible transitions in chromatin higher order structure. After a description of the acetylation of newly synthesized histones, and of the likely acetyltransferases involved, an overview of histone octamer assembly is presented. Our current understanding of the factors thought to assemble chromatin in vivo is then described. Genetic and biochemical investigations of the function the histone tails, and their acetylation, in nucleosome assembly are detailed, followed by an analysis of the importance of histone deacetylation in the maturation of newly replicated chromatin. In the final section the involvement of the histone tail domains in chromatin higher order structures is addressed, along with the role of histone acetylation in chromatin folding. Suggestions for future research are offered in the concluding remarks. PMID:11097424

  12. Generic Features of Tertiary Chromatin Structure as Detected in Natural Chromosomes

    PubMed Central

    Müller, Waltraud G.; Rieder, Dietmar; Kreth, Gregor; Cremer, Christoph; Trajanoski, Zlatko; McNally, James G.

    2004-01-01

    Knowledge of tertiary chromatin structure in mammalian interphase chromosomes is largely derived from artificial tandem arrays. In these model systems, light microscope images reveal fibers or beaded fibers after high-density targeting of transactivators to insertional domains spanning several megabases. These images of fibers have lent support to chromonema fiber models of tertiary structure. To assess the relevance of these studies to natural mammalian chromatin, we identified two different ∼400-kb regions on human chromosomes 6 and 22 and then examined light microscope images of interphase tertiary chromatin structure when the regions were transcriptionally active and inactive. When transcriptionally active, these natural chromosomal regions elongated, yielding images characterized by a series of adjacent puncta or “beads”, referred to hereafter as beaded images. These elongated structures required transcription for their maintenance. Thus, despite marked differences in the density and the mode of transactivation, the natural and artificial systems showed similarities, suggesting that beaded images are generic features of transcriptionally active tertiary chromatin. We show here, however, that these images do not necessarily favor chromonema fiber models but can also be explained by a radial-loop model or even a simple nucleosome affinity, random-chain model. Thus, light microscope images of tertiary structure cannot distinguish among competing models, although they do impose key constraints: chromatin must be clustered to yield beaded images and then packaged within each cluster to enable decondensation into adjacent clusters. PMID:15485905

  13. Nucleosomal Barrier to Transcription: Structural Determinants and Changes in Chromatin Structure

    PubMed Central

    Studitsky, Vasily M.; Nizovtseva, Ekaterina V.; Shaytan, Alexey K.; Luse, Donal S.

    2016-01-01

    Packaging of DNA into chromatin affects all processes on DNA. Nucleosomes present a strong barrier to transcription, raising important questions about the nature and the mechanisms of overcoming the barrier. Recently it was shown that DNA sequence, DNA–histone interactions and backtracking by RNA polymerase II (Pol II) all contribute to formation of the barrier. After partial uncoiling of nucleosomal DNA from histone octamer by Pol II and backtracking of the enzyme, nucleosomal DNA recoils on the octamer, locking Pol II in the arrested state. Histone chaperones and transcription factors TFIIS, TFIIF and FACT facilitate transcription through chromatin using different molecular mechanisms. PMID:27754494

  14. NOTCH-mediated non-cell autonomous regulation of chromatin structure during senescence.

    PubMed

    Parry, Aled J; Hoare, Matthew; Bihary, Dóra; Hänsel-Hertsch, Robert; Smith, Stephen; Tomimatsu, Kosuke; Mannion, Elizabeth; Smith, Amy; D'Santos, Paula; Russell, I Alasdair; Balasubramanian, Shankar; Kimura, Hiroshi; Samarajiwa, Shamith A; Narita, Masashi

    2018-05-09

    Senescent cells interact with the surrounding microenvironment achieving diverse functional outcomes. We have recently identified that NOTCH1 can drive 'lateral induction' of a unique senescence phenotype in adjacent cells by specifically upregulating the NOTCH ligand JAG1. Here we show that NOTCH signalling can modulate chromatin structure autonomously and non-autonomously. In addition to senescence-associated heterochromatic foci (SAHF), oncogenic RAS-induced senescent (RIS) cells exhibit a massive increase in chromatin accessibility. NOTCH signalling suppresses SAHF and increased chromatin accessibility in this context. Strikingly, NOTCH-induced senescent cells, or cancer cells with high JAG1 expression, drive similar chromatin architectural changes in adjacent cells through cell-cell contact. Mechanistically, we show that NOTCH signalling represses the chromatin architectural protein HMGA1, an association found in multiple human cancers. Thus, HMGA1 is involved not only in SAHFs but also in RIS-driven chromatin accessibility. In conclusion, this study identifies that the JAG1-NOTCH-HMGA1 axis mediates the juxtacrine regulation of chromatin architecture.

  15. Altered sperm chromatin structure in mice exposed to sodium fluoride through drinking water.

    PubMed

    Sun, Zilong; Niu, Ruiyan; Wang, Bin; Wang, Jundong

    2014-06-01

    This study investigated the effects of sodium fluoride (NaF) on sperm abnormality, sperm chromatin structure, protamine 1 and protamine 2 (P1 and P2) mRNA expression, and histones expression in sperm in male mice. NaF was orally administrated to male mice at 30, 70, and 150 mg/l for 49 days (more than one spermatogenic cycle). Sperm head and tail abnormalities were significantly enhanced at middle and high doses. Similarly, sperm chromatin structure was also adversely affected by NaF exposure, indicating DNA integrity damage. Furthermore, middle and high NaF significantly reduced the mRNA expressions of P1 and P2, and P1/P2 ratio, whereas the sperm histones level was increased, suggesting the abnormal histone-protamine replacement. Therefore, we concluded that the mechanism by which F induced mice sperm abnormality and DNA integrity damage may involved in the alterations in P1, P2, and histones expression in sperm of mice. Copyright © 2012 Wiley Periodicals, Inc.

  16. [Automated morphometric evaluation of the chromatin structure of liver cell nuclei after vagotomy].

    PubMed

    Butusova, N N; Zhukotskiĭ, A V; Sherbo, I V; Gribkov, E N; Dubovaia, T K

    1989-05-01

    The morphometric analysis of the interphase chromatine structure of the hepatic cells nuclei was carried out on the automated TV installation for the quantitative analysis of images "IBAS-2" (by the OPTON firm, the FRG) according to 50 optical and geometric parameters during various periods (1.2 and 4 weeks) after the vagotomy operation. It is determined that upper-molecular organisation of chromatine undergoes the biggest changes one week after operation, and changes of granular component are more informative than changes of the nongranular component (with the difference 15-20%). It was also revealed that chromatine components differ in tinctorial properties, which are evidently dependent on physicochemical characteristics of the chromatine under various functional conditions of the cell. As a result of the correlation analysis the group of morphometric indices of chromatine structure was revealed, which are highly correlated with level of transcription activity of chromatine during various terms after denervation. The correlation quotient of these parameters is 0.85-0.97. The summing up: vagus denervation of the liver causes changes in the morphofunctional organisation of the chromatine.

  17. Ubiquitin Utilizes an Acidic Surface Patch to Alter Chromatin Structure

    PubMed Central

    Debelouchina, Galia T.; Gerecht, Karola; Muir, Tom W.

    2016-01-01

    Ubiquitylation of histone H2B, associated with gene activation, leads to chromatin decompaction through an unknown mechanism. We used a hydrogen-deuterium exchange strategy coupled with nuclear magnetic resonance spectroscopy to map the ubiquitin surface responsible for its structural effects on chromatin. Our studies revealed that a previously uncharacterized acidic patch on ubiquitin comprising residues Glu16 and Glu18 is essential for decompaction. These residues mediate promiscuous electrostatic interactions with the basic histone proteins, potentially positioning the ubiquitin moiety as a dynamic “wedge” that prevents the intimate association of neighboring nucleosomes. Using two independent cross-linking strategies and an oligomerization assay, we also showed that ubiquitin-ubiquitin contacts occur in the chromatin environment and are important for the solubilization of the chromatin polymers. Our work highlights a novel, chromatin-related aspect of the “ubiquitin code”, and sheds light on how the information rich ubiquitin modification can orchestrate different biochemical outcomes using different surface features. PMID:27870837

  18. Disconnect between alcohol-induced alterations in chromatin structure and gene transcription in a mouse embryonic stem cell model of exposure.

    PubMed

    Veazey, Kylee J; Wang, Haiqing; Bedi, Yudhishtar S; Skiles, William M; Chang, Richard Cheng-An; Golding, Michael C

    2017-05-01

    Alterations to chromatin structure induced by environmental insults have become an attractive explanation for the persistence of exposure effects into subsequent life stages. However, a growing body of work examining the epigenetic impact that alcohol and other drugs of abuse exert consistently notes a disconnection between induced changes in chromatin structure and patterns of gene transcription. Thus, an important question is whether perturbations in the 'histone code' induced by prenatal exposures to alcohol implicitly subvert gene expression, or whether the hierarchy of cellular signaling networks driving development is such that they retain control over the transcriptional program. To address this question, we examined the impact of ethanol exposure in mouse embryonic stem cells cultured under 2i conditions, where the transcriptional program is rigidly enforced through the use of small molecule inhibitors. We find that ethanol-induced changes in post-translational histone modifications are dose-dependent, unique to the chromatin modification under investigation, and that the extent and direction of the change differ between the period of exposure and the recovery phase. Similar to in vivo models, we find post-translational modifications affecting histone 3 lysine 9 are the most profoundly impacted, with the signature of exposure persisting long after alcohol has been removed. These changes in chromatin structure associate with dose-dependent alterations in the levels of transcripts encoding Dnmt1, Uhrf1, Tet1, Tet2, Tet3, and Polycomb complex members Eed and Ezh2. However, in this model, ethanol-induced changes to the chromatin template do not consistently associate with changes in gene transcription, impede the process of differentiation, or affect the acquisition of monoallelic patterns of expression for the imprinted gene Igf2R. These findings question the inferred universal relevance of epigenetic changes induced by drugs of abuse and suggest that changes

  19. RNA polymerase III transcription - regulated by chromatin structure and regulator of nuclear chromatin organization.

    PubMed

    Pascali, Chiara; Teichmann, Martin

    2013-01-01

    RNA polymerase III (Pol III) transcription is regulated by modifications of the chromatin. DNA methylation and post-translational modifications of histones, such as acetylation, phosphorylation and methylation have been linked to Pol III transcriptional activity. In addition to being regulated by modifications of DNA and histones, Pol III genes and its transcription factors have been implicated in the organization of nuclear chromatin in several organisms. In yeast, the ability of the Pol III transcription system to contribute to nuclear organization seems to be dependent on direct interactions of Pol III genes and/or its transcription factors TFIIIC and TFIIIB with the structural maintenance of chromatin (SMC) protein-containing complexes cohesin and condensin. In human cells, Pol III genes and transcription factors have also been shown to colocalize with cohesin and the transcription regulator and genome organizer CCCTC-binding factor (CTCF). Furthermore, chromosomal sites have been identified in yeast and humans that are bound by partial Pol III machineries (extra TFIIIC sites - ETC; chromosome organizing clamps - COC). These ETCs/COC as well as Pol III genes possess the ability to act as boundary elements that restrict spreading of heterochromatin.

  20. Using local chromatin structure to improve CRISPR/Cas9 efficiency in zebrafish.

    PubMed

    Chen, Yunru; Zeng, Shiyang; Hu, Ruikun; Wang, Xiangxiu; Huang, Weilai; Liu, Jiangfang; Wang, Luying; Liu, Guifen; Cao, Ying; Zhang, Yong

    2017-01-01

    Although the CRISPR/Cas9 has been successfully applied in zebrafish, considerable variations in efficiency have been observed for different gRNAs. The workload and cost of zebrafish mutant screening is largely dependent on the mutation rate of injected embryos; therefore, selecting more effective gRNAs is especially important for zebrafish mutant construction. Besides the sequence features, local chromatin structures may have effects on CRISPR/Cas9 efficiency, which remain largely unexplored. In the only related study in zebrafish, nucleosome organization was not found to have an effect on CRISPR/Cas9 efficiency, which is inconsistent with recent studies in vitro and in mammalian cell lines. To understand the effects of local chromatin structure on CRISPR/Cas9 efficiency in zebrafish, we first determined that CRISPR/Cas9 introduced genome editing mainly before the dome stage. Based on this observation, we reanalyzed our published nucleosome organization profiles and generated chromatin accessibility profiles in the 256-cell and dome stages using ATAC-seq technology. Our study demonstrated that chromatin accessibility showed positive correlation with CRISPR/Cas9 efficiency, but we did not observe a clear correlation between nucleosome organization and CRISPR/Cas9 efficiency. We constructed an online database for zebrafish gRNA selection based on local chromatin structure features that could prove beneficial to zebrafish homozygous mutant construction via CRISPR/Cas9.

  1. Structure of chromatin and the linking number of DNA.

    PubMed Central

    Worcel, A; Strogatz, S; Riley, D

    1981-01-01

    Recent observations suggest that the basic supranucleosomal structure of chromatin is a zigzag helical ribbon with a repeat unit made of two nucleosomes connected by a relaxed spacer DNA. A remarkable feature of one particular ribbon is that it solves the apparent paradox between the number of DNA turns per nucleosome and the total linking number of a nucleosome-containing closed circular DNA molecule. We show here that the repeat unit of the proposed structure, which contains two nucleosomes with -1 3/4 DNA turns per nucleosome and one spacer crossover per repeat, contributes -2 to the linking number of closed circular DNA. Space-filling models show that the cylindrical 250-A chromatin fiber can be generated by twisting the ribbon. Images PMID:6940168

  2. Preservation of large-scale chromatin structure in FISH experiments

    PubMed Central

    Hepperger, Claudia; Otten, Simone; von Hase, Johann

    2006-01-01

    The nuclear organization of specific endogenous chromatin regions can be investigated only by fluorescence in situ hybridization (FISH). One of the two fixation procedures is typically applied: (1) buffered formaldehyde or (2) hypotonic shock with methanol acetic acid fixation followed by dropping of nuclei on glass slides and air drying. In this study, we compared the effects of these two procedures and some variations on nuclear morphology and on FISH signals. We analyzed mouse erythroleukemia and mouse embryonic stem cells because their clusters of subcentromeric heterochromatin provide an easy means to assess preservation of chromatin. Qualitative and quantitative analyses revealed that formaldehyde fixation provided good preservation of large-scale chromatin structures, while classical methanol acetic acid fixation after hypotonic treatment severely impaired nuclear shape and led to disruption of chromosome territories, heterochromatin structures, and large transgene arrays. Our data show that such preparations do not faithfully reflect in vivo nuclear architecture. Electronic supplementary material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00412-006-0084-2 and is accessible for authorized users. PMID:17119992

  3. Structure of transcribed chromatin is a sensor of DNA damage

    PubMed Central

    Pestov, Nikolay A.; Gerasimova, Nadezhda S.; Kulaeva, Olga I.; Studitsky, Vasily M.

    2015-01-01

    Early detection and repair of damaged DNA is essential for cell functioning and survival. Although multiple cellular systems are involved in the repair of single-strand DNA breaks (SSBs), it remains unknown how SSBs present in the nontemplate strand (NT-SSBs) of DNA organized in chromatin are detected. The effect of NT-SSBs on transcription through chromatin by RNA polymerase II was studied. NT-SSBs localized in the promoter-proximal region of nucleosomal DNA and hidden in the nucleosome structure can induce a nearly quantitative arrest of RNA polymerase downstream of the break, whereas more promoter-distal SSBs moderately facilitate transcription. The location of the arrest sites on nucleosomal DNA suggests that formation of small intranucleosomal DNA loops causes the arrest. This mechanism likely involves relief of unconstrained DNA supercoiling accumulated during transcription through chromatin by NT-SSBs. These data suggest the existence of a novel chromatin-specific mechanism that allows the detection of NT-SSBs by the transcribing enzyme. PMID:26601207

  4. Binding of DNA-bending non-histone proteins destabilizes regular 30-nm chromatin structure

    PubMed Central

    Bajpai, Gaurav; Jain, Ishutesh; Inamdar, Mandar M.; Das, Dibyendu; Padinhateeri, Ranjith

    2017-01-01

    Why most of the in vivo experiments do not find the 30-nm chromatin fiber, well studied in vitro, is a puzzle. Two basic physical inputs that are crucial for understanding the structure of the 30-nm fiber are the stiffness of the linker DNA and the relative orientations of the DNA entering/exiting nucleosomes. Based on these inputs we simulate chromatin structure and show that the presence of non-histone proteins, which bind and locally bend linker DNA, destroys any regular higher order structures (e.g., zig-zag). Accounting for the bending geometry of proteins like nhp6 and HMG-B, our theory predicts phase-diagram for the chromatin structure as a function of DNA-bending non-histone protein density and mean linker DNA length. For a wide range of linker lengths, we show that as we vary one parameter, that is, the fraction of bent linker region due to non-histone proteins, the steady-state structure will show a transition from zig-zag to an irregular structure—a structure that is reminiscent of what is observed in experiments recently. Our theory can explain the recent in vivo observation of irregular chromatin having co-existence of finite fraction of the next-neighbor (i + 2) and neighbor (i + 1) nucleosome interactions. PMID:28135276

  5. High-Resolution Mapping of Chromatin Conformation in Cardiac Myocytes Reveals Structural Remodeling of the Epigenome in Heart Failure

    PubMed Central

    Rosa-Garrido, Manuel; Chapski, Douglas J.; Schmitt, Anthony D.; Kimball, Todd H.; Karbassi, Elaheh; Monte, Emma; Balderas, Enrique; Pellegrini, Matteo; Shih, Tsai-Ting; Soehalim, Elizabeth; Liem, David; Ping, Peipei; Galjart, Niels J.; Ren, Shuxun; Wang, Yibin; Ren, Bing

    2017-01-01

    Background: Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined. Methods: To investigate the mechanisms of epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload–induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes. Results: Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements. Conclusions: These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy. PMID

  6. High-Resolution Mapping of Chromatin Conformation in Cardiac Myocytes Reveals Structural Remodeling of the Epigenome in Heart Failure.

    PubMed

    Rosa-Garrido, Manuel; Chapski, Douglas J; Schmitt, Anthony D; Kimball, Todd H; Karbassi, Elaheh; Monte, Emma; Balderas, Enrique; Pellegrini, Matteo; Shih, Tsai-Ting; Soehalim, Elizabeth; Liem, David; Ping, Peipei; Galjart, Niels J; Ren, Shuxun; Wang, Yibin; Ren, Bing; Vondriska, Thomas M

    2017-10-24

    Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined. To investigate the mechanisms of epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload-induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes. Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements. These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy. © 2017 The Authors.

  7. The higher structure of chromatin in the LCR of the beta-globin locus changes during development.

    PubMed

    Fang, Xiangdong; Yin, Wenxuan; Xiang, Ping; Han, Hemei; Stamatoyannopoulos, George; Li, Qiliang

    2009-11-27

    The beta-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant beta-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.

  8. Disconnect between alcohol-induced alterations in chromatin structure and gene transcription in a mouse embryonic stem cell model of exposure

    PubMed Central

    Veazey, Kylee J.; Wang, Haiqing; Behdi, Yudhishtar S.; Skiles, William M.; Chang, Richard Cheng-An; Golding, Michael C.

    2017-01-01

    Alterations to chromatin structure induced by environmental insults have become an attractive explanation for the persistence of exposure effects into subsequent life stages. However, a growing body of work examining the epigenetic impact alcohol and other drugs of abuse exert consistently note a disconnect between induced changes in chromatin structure and patterns of gene transcription. Thus, an important question is whether perturbations in the ‘histone code’ induced by prenatal exposures to alcohol implicitly subvert gene expression, or if the hierarchy of cellular signaling networks driving development is such that they retain control over the transcriptional program. To address this question, we examined the impact of ethanol exposure in mouse embryonic stem cells cultured under 2i conditions, where the transcriptional program is rigidly enforced through the use of small molecule inhibitors. We find that ethanol-induced changes in post-translational histone modifications are dose-dependent, unique to the chromatin modification under investigation, and that the extent and direction of the change differ between the period of exposure and the recovery phase. Similar to in vivo models, we find post-translational modifications affecting histone 3 lysine 9 are the most profoundly impacted, with the signature of exposure persisting long after alcohol has been removed. These changes in chromatin structure associate with dose-dependent alterations in the levels of transcripts encoding Dnmt1, Uhrf1, Tet1, Tet2, Tet3, and Polycomb complex members Eed and Ezh2. However, in this model, ethanol-induced changes to the chromatin template do not consistently associate with changes in gene transcription, impede the process of differentiation or impact the acquisition of monoallelic patterns of expression for the imprinted gene Igf2R. These findings question the inferred universal relevance of epigenetic changes induced by drugs of abuse and suggest changes in chromatin

  9. Polymer physics predicts the effects of structural variants on chromatin architecture.

    PubMed

    Bianco, Simona; Lupiáñez, Darío G; Chiariello, Andrea M; Annunziatella, Carlo; Kraft, Katerina; Schöpflin, Robert; Wittler, Lars; Andrey, Guillaume; Vingron, Martin; Pombo, Ana; Mundlos, Stefan; Nicodemi, Mario

    2018-05-01

    Structural variants (SVs) can result in changes in gene expression due to abnormal chromatin folding and cause disease. However, the prediction of such effects remains a challenge. Here we present a polymer-physics-based approach (PRISMR) to model 3D chromatin folding and to predict enhancer-promoter contacts. PRISMR predicts higher-order chromatin structure from genome-wide chromosome conformation capture (Hi-C) data. Using the EPHA4 locus as a model, the effects of pathogenic SVs are predicted in silico and compared to Hi-C data generated from mouse limb buds and patient-derived fibroblasts. PRISMR deconvolves the folding complexity of the EPHA4 locus and identifies SV-induced ectopic contacts and alterations of 3D genome organization in homozygous or heterozygous states. We show that SVs can reconfigure topologically associating domains, thereby producing extensive rewiring of regulatory interactions and causing disease by gene misexpression. PRISMR can be used to predict interactions in silico, thereby providing a tool for analyzing the disease-causing potential of SVs.

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

    PubMed Central

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

    2015-01-01

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

  11. Aldosterone alters the chromatin structure of the murine endothelin-1 gene.

    PubMed

    Welch, Amanda K; Jeanette Lynch, I; Gumz, Michelle L; Cain, Brian D; Wingo, Charles S

    2016-08-15

    Aldosterone increases sodium reabsorption in the renal collecting duct and systemic blood pressure. Paradoxically, aldosterone also induces transcription of the endothelin-1 (Edn1) gene to increase protein (ET-1) levels, which inhibits sodium reabsorption. Here we investigated changes in the chromatin structure of the Edn1 gene of collecting duct cell lines in response to aldosterone treatment. The Edn1 gene has a CpG island that encompasses the transcription start site and four sites in the 5' regulatory region previously linked to transcriptional regulation. The chromatin structure of the Edn1 gene was investigated using a quantitative PCR-based DNaseI hypersensitivity assay in murine hepatocyte (AML12), renal cortical collecting duct (mpkCCDC14), outer medullary collecting duct1 (OMCD1), and inner medullary collecting duct-3 (IMCD-3) cell lines. The CpG island was uniformly accessible. One calcium-responsive NFAT element remained at low chromatin accessibility in all cell lines under all conditions tested. However, the second calcium responsive NFAT element located at -1563bp upstream became markedly more accessible in IMCD-3 cells exposed to aldosterone. Importantly, one established aldosterone hormone response element HRE at -671bp relative to the transcription start site was highly accessible, and another HRE (-551bp) became more accessible in aldosterone-treated IMCD-3 and OMCD1 cells. The evidence supports a model in which aldosterone activation of the mineralocorticoid receptor (MR) results in the MR-hormone complex binding at HRE at -671bp to open chromatin structure around other regulatory elements in the Edn1 gene. Published by Elsevier Inc.

  12. Chromatin accessibility and guide sequence secondary structure affect CRISPR-Cas9 gene editing efficiency.

    PubMed

    Jensen, Kristopher Torp; Fløe, Lasse; Petersen, Trine Skov; Huang, Jinrong; Xu, Fengping; Bolund, Lars; Luo, Yonglun; Lin, Lin

    2017-07-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) systems have emerged as the method of choice for genome editing, but large variations in on-target efficiencies continue to limit their applicability. Here, we investigate the effect of chromatin accessibility on Cas9-mediated gene editing efficiency for 20 gRNAs targeting 10 genomic loci in HEK293T cells using both SpCas9 and the eSpCas9(1.1) variant. Our study indicates that gene editing is more efficient in euchromatin than in heterochromatin, and we validate this finding in HeLa cells and in human fibroblasts. Furthermore, we investigate the gRNA sequence determinants of CRISPR-Cas9 activity using a surrogate reporter system and find that the efficiency of Cas9-mediated gene editing is dependent on guide sequence secondary structure formation. This knowledge can aid in the further improvement of tools for gRNA design. © 2017 Federation of European Biochemical Societies.

  13. Segregation distortion and genome-wide digenic interactions affect transmission of introgressed chromatin from wild cotton species.

    PubMed

    Chandnani, Rahul; Wang, Baohua; Draye, Xavier; Rainville, Lisa K; Auckland, Susan; Zhuang, Zhimin; Lubbers, Edward L; May, O Lloyd; Chee, Peng W; Paterson, Andrew H

    2017-10-01

    This study reports transmission genetics of chromosomal segments into Gossypium hirsutum from its most distant euploid relative, Gossypium mustelinum . Mutilocus interactions and structural rearrangements affect introgression and segregation of donor chromatin. Wild allotetraploid relatives of cotton are a rich source of genetic diversity that can be used in genetic improvement, but linkage drag and non-Mendelian transmission genetics are prevalent in interspecific crosses. These problems necessitate knowledge of transmission patterns of chromatin from wild donor species in cultivated recipient species. From an interspecific cross, Gossypium hirsutum × Gossypium mustelinum, we studied G. mustelinum (the most distant tetraploid relative of Upland cotton) allele retention in 35 BC 3 F 1 plants and segregation patterns in BC 3 F 2 populations totaling 3202 individuals, using 216 DNA marker loci. The average retention of donor alleles across BC 3 F 1 plants was higher than expected and the average frequency of G. mustelinum alleles in BC 3 F 2 segregating families was less than expected. Despite surprisingly high retention of G. mustelinum alleles in BC 3 F 1 , 46 genomic regions showed no introgression. Regions on chromosomes 3 and 15 lacking introgression were closely associated with possible small inversions previously reported. Nonlinear two-locus interactions are abundant among loci with single-locus segregation distortion, and among loci originating from one of the two subgenomes. Comparison of the present results with those of prior studies indicates different permeability of Upland cotton for donor chromatin from different allotetraploid relatives. Different contributions of subgenomes to two-locus interactions suggest different fates of subgenomes in the evolution of allotetraploid cottons. Transmission genetics of G. hirsutum × G. mustelinum crosses reveals allelic interactions, constraints on fixation and selection of donor alleles, and challenges with

  14. SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells

    PubMed Central

    Barutcu, A. Rasim; Lajoie, Bryan R.; Fritz, Andrew J.; McCord, Rachel P.; Nickerson, Jeffrey A.; van Wijnen, Andre J.; Lian, Jane B.; Stein, Janet L.; Dekker, Job; Stein, Gary S.; Imbalzano, Anthony N.

    2016-01-01

    The packaging of DNA into chromatin plays an important role in transcriptional regulation and nuclear processes. Brahma-related gene-1 SMARCA4 (also known as BRG1), the essential ATPase subunit of the mammalian SWI/SNF chromatin remodeling complex, uses the energy from ATP hydrolysis to disrupt nucleosomes at target regions. Although the transcriptional role of SMARCA4 at gene promoters is well-studied, less is known about its role in higher-order genome organization. SMARCA4 knockdown in human mammary epithelial MCF-10A cells resulted in 176 up-regulated genes, including many related to lipid and calcium metabolism, and 1292 down-regulated genes, some of which encode extracellular matrix (ECM) components that can exert mechanical forces and affect nuclear structure. ChIP-seq analysis of SMARCA4 localization and SMARCA4-bound super-enhancers demonstrated extensive binding at intergenic regions. Furthermore, Hi-C analysis showed extensive SMARCA4-mediated alterations in higher-order genome organization at multiple resolutions. First, SMARCA4 knockdown resulted in clustering of intra- and inter-subtelomeric regions, demonstrating a novel role for SMARCA4 in telomere organization. SMARCA4 binding was enriched at topologically associating domain (TAD) boundaries, and SMARCA4 knockdown resulted in weakening of TAD boundary strength. Taken together, these findings provide a dynamic view of SMARCA4-dependent changes in higher-order chromatin organization and gene expression, identifying SMARCA4 as a novel component of chromatin organization. PMID:27435934

  15. CHD chromatin remodelers and the transcription cycle

    PubMed Central

    Murawska, Magdalena

    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 also 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. PMID:22223048

  16. Do chromatin changes around a nascent double strand DNA break spread spherically into linearly non-adjacent chromatin?

    PubMed

    Savic, Velibor

    2013-01-01

    In the last decade, a lot has been done in elucidating the sequence of events that occur at the nascent double strand DNA break. Nevertheless, the overall structure formed by the DNA damage response (DDR) factors around the break site, the repair focus, remains poorly understood. Although most of the data presented so far only address events that occur in chromatin in cis around the break, there are strong indications that in mammalian systems it may also occur in trans, analogous to the recent findings showing this if budding yeast. There have been attempts to address the issue but the final proof is still missing due to lack of a proper experimental system. If found to be true, the spatial distribution of DDR factors would have a major impact on the neighboring chromatin both in cis and in trans, significantly affecting local chromatin function; gene transcription and potentially other functions.

  17. Telomere Chromatin Condensation Assay (TCCA): a novel approach to study structural telomere integrity.

    PubMed

    Gonzalez-Vasconcellos, Iria; Alonso-Rodríguez, Silvia; López-Baltar, Isidoro; Fernández, José Luis

    2015-01-01

    Telomeres, the DNA-protein complexes located at the end of linear eukaryotic chromosomes are essential for genome stability. Improper higher-order chromatin organization at the chromosome ends can give rise to telomeric recombination and genomic instability. We report the development of an assay to quantify differences in the condensation of telomeric chromatin, thereby offering new opportunities to study telomere biology and stability. We have combined a DNA nuclease digestion with a quantitative PCR (qPCR) assay of telomeric DNA, which we term the Telomere Chromatin Condensation Assay (TCCA). By quantifying the relative quantities of telomeric DNA that are progressively digested with the exonuclease Bal 31 the method can discriminate between different levels of telomeric chromatin condensation. The structural chromatin packaging at telomeres shielded against exonuclease digestion delivered an estimate, which we term Chromatin Protection Factor (CPF) that ranged from 1.7 to 2.3 fold greater than that present in unpacked DNA. The CPF was significantly decreased when cell cultures were incubated with the DNA hypomethylating agent 5-azacytidine, demonstrating the ability of the TCCA assay to discriminate between packaging levels of telomeric DNA. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Higher order chromatin structure: bridging physics and biology

    PubMed Central

    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 interphase chromatin organization. By taking into account topological constraints acting on the chromatin fiber, recently-developed polymer models of interphase chromatin can reproduce the observed scaling of distances between genomic loci, chromosomal territories, and probabilities of contacts between loci measured by chromosome conformation capture methods. Polymer models provide a framework for the interpretation of experimental data as ensembles of conformations rather than collections of loops, and will be crucial for untangling functional implications of chromosomal organization. PMID:22360992

  19. Integrating epigenomic data and 3D genomic structure with a new measure of chromatin assortativity.

    PubMed

    Pancaldi, Vera; Carrillo-de-Santa-Pau, Enrique; Javierre, Biola Maria; Juan, David; Fraser, Peter; Spivakov, Mikhail; Valencia, Alfonso; Rico, Daniel

    2016-07-08

    Network analysis is a powerful way of modeling chromatin interactions. Assortativity is a network property used in social sciences to identify factors affecting how people establish social ties. We propose a new approach, using chromatin assortativity, to integrate the epigenomic landscape of a specific cell type with its chromatin interaction network and thus investigate which proteins or chromatin marks mediate genomic contacts. We use high-resolution promoter capture Hi-C and Hi-Cap data as well as ChIA-PET data from mouse embryonic stem cells to investigate promoter-centered chromatin interaction networks and calculate the presence of specific epigenomic features in the chromatin fragments constituting the nodes of the network. We estimate the association of these features with the topology of four chromatin interaction networks and identify features localized in connected areas of the network. Polycomb group proteins and associated histone marks are the features with the highest chromatin assortativity in promoter-centered networks. We then ask which features distinguish contacts amongst promoters from contacts between promoters and other genomic elements. We observe higher chromatin assortativity of the actively elongating form of RNA polymerase 2 (RNAPII) compared with inactive forms only in interactions between promoters and other elements. Contacts among promoters and between promoters and other elements have different characteristic epigenomic features. We identify a possible role for the elongating form of RNAPII in mediating interactions among promoters, enhancers, and transcribed gene bodies. Our approach facilitates the study of multiple genome-wide epigenomic profiles, considering network topology and allowing the comparison of chromatin interaction networks.

  20. Roles of chromatin insulator proteins in higher-order chromatin organization and transcription regulation

    PubMed Central

    Vogelmann, Jutta; Valeri, Alessandro; Guillou, Emmanuelle; Cuvier, Olivier; Nollmann, Marcelo

    2013-01-01

    Eukaryotic chromosomes are condensed into several hierarchical levels of complexity: DNA is wrapped around core histones to form nucleosomes, nucleosomes form a higher-order structure called chromatin, and chromatin is subsequently compartmentalized in part by the combination of multiple specific or unspecific long-range contacts. The conformation of chromatin at these three levels greatly influences DNA metabolism and transcription. One class of chromatin regulatory proteins called insulator factors may organize chromatin both locally, by setting up barriers between heterochromatin and euchromatin, and globally by establishing platforms for long-range interactions. Here, we review recent data revealing a global role of insulator proteins in the regulation of transcription through the formation of clusters of long-range interactions that impact different levels of chromatin organization. PMID:21983085

  1. Higher-order chromatin structure: bridging physics and biology.

    PubMed

    Fudenberg, Geoffrey; Mirny, Leonid A

    2012-04-01

    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 higher-order interphase chromatin organization. By taking into account topological constraints acting on the chromatin fiber, recently developed polymer models of interphase chromatin can reproduce the observed scaling of distances between genomic loci, chromosomal territories, and probabilities of contacts between loci measured by chromosome conformation capture methods. Polymer models provide a framework for the interpretation of experimental data as ensembles of conformations rather than collections of loops, and will be crucial for untangling functional implications of chromosomal organization. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Do chromatin changes around a nascent double strand DNA break spread spherically into linearly non-adjacent chromatin?

    PubMed Central

    Savic, Velibor

    2013-01-01

    In the last decade, a lot has been done in elucidating the sequence of events that occur at the nascent double strand DNA break. Nevertheless, the overall structure formed by the DNA damage response (DDR) factors around the break site, the repair focus, remains poorly understood. Although most of the data presented so far only address events that occur in chromatin in cis around the break, there are strong indications that in mammalian systems it may also occur in trans, analogous to the recent findings showing this if budding yeast. There have been attempts to address the issue but the final proof is still missing due to lack of a proper experimental system. If found to be true, the spatial distribution of DDR factors would have a major impact on the neighboring chromatin both in cis and in trans, significantly affecting local chromatin function; gene transcription and potentially other functions. PMID:23882282

  3. Steroid hormone receptor status defines the MMTV promoter chromatin structure in vivo.

    PubMed

    Archer, T K; Fryer, C J; Lee, H L; Zaniewski, E; Liang, T; Mymryk, J S

    1995-06-01

    The ability to respond to small signalling molecules such as steroid hormones is important for many physiological processes. Steroid hormones act through a group of high affinity receptors that regulate transcription by binding to hormone response elements (HREs) located within the promoters of target genes, which themselves are organized with nuclear proteins to form chromatin. To dissect the mechanisms(s) of steroid hormone action we have used the steroid inducible mouse mammary tumor virus (MMTV) promoter as a model system. The MMTV promoter is assembled into a phased array of nucleosomes that are specifically positioned in rodent cells. Induction of transcription by glucocorticoids is accompanied by the appearance of a hypersensitive region in the proximal promoter which allows the hormone dependent assembly of a preinitiation complex including transcription factors such as nuclear factor 1 (NF1) and the octamer transcription factor (OTF). Surprisingly, when introduced by transient transfection, the progesterone receptor (PR) is unable to activate this promoter in vivo, a finding that may result from its inability to alter MMTV promoter chromatin. In an attempt to investigate the failure of the PR to activate the promoter, we have stably introduced the MMTV promoter into human T47D breast cancer cells that express high levels of the PR. In contrast to what has been observed previously in rodent cells, the MMTV templates resident in human breast cancer cells adopt a novel and constitutively open chromatin structure. The constitutively open chromatin structure is accompanied by the hormone independent loading of transcription factors including the PR and NF1. In T47D cells that stably express the glucocorticoid receptor, the MMTV promoter responds to glucocorticoids, but not progestins, and displays glucocorticoid induced restriction enzyme hypersensitivity and transcription factor loading. These findings suggest that the organization of the MMTV chromatin

  4. Chromatin Structure and Radiation-Induced Intrachromosome Exchange

    NASA Technical Reports Server (NTRS)

    Mangala; Zhang, Ye; Hada, Megumi; Cucinotta, Francis A.; Wu, Honglu

    2011-01-01

    We have recently investigated the location of breaks involved in intrachromosomal type exchange events, using the multicolor banding in situ hybridization (mBAND) technique for human chromosome 3. In human epithelial cells exposed to both low- and high-LET radiations in vitro, intrachromosome exchanges were found to occur preferentially between a break in the 3p21 and one in the 3q11. Exchanges were also observed between a break in 3p21 and one in 3q26, but few exchanges were observed between breaks in 3q11 and 3q26, even though the two regions were on the same arm of the chromosome. To explore the relationships between intrachromosome exchanges and chromatin structure, we used probes that hybridize the three regions of 3p21, 3q11 and 3q26, and measured the distance between two of the three regions in interphase cells. We further analyzed fragile sites on the chromosome that have been identified in various types of cancers. Our results demonstrated that the distribution of breaks involved in radiation-induced intrachromosome aberrations depends upon both the location of fragile sites and the folding of chromatins

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

    PubMed Central

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

    2014-01-01

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

  6. Chromatin insulator bodies are nuclear structures that form in response to osmotic stress and cell death

    PubMed Central

    Schoborg, Todd; Rickels, Ryan; Barrios, Josh

    2013-01-01

    Chromatin insulators assist in the formation of higher-order chromatin structures by mediating long-range contacts between distant genomic sites. It has been suggested that insulators accomplish this task by forming dense nuclear foci termed insulator bodies that result from the coalescence of multiple protein-bound insulators. However, these structures remain poorly understood, particularly the mechanisms triggering body formation and their role in nuclear function. In this paper, we show that insulator proteins undergo a dramatic and dynamic spatial reorganization into insulator bodies during osmostress and cell death in a high osmolarity glycerol–p38 mitogen-activated protein kinase–independent manner, leading to a large reduction in DNA-bound insulator proteins that rapidly repopulate chromatin as the bodies disassemble upon return to isotonicity. These bodies occupy distinct nuclear territories and contain a defined structural arrangement of insulator proteins. Our findings suggest insulator bodies are novel nuclear stress foci that can be used as a proxy to monitor the chromatin-bound state of insulator proteins and provide new insights into the effects of osmostress on nuclear and genome organization. PMID:23878275

  7. Structure and Dynamic Properties of a Glucocorticoid Receptor-Induced Chromatin Transition

    PubMed Central

    Fletcher, Terace M.; Ryu, Byung-Woo; Baumann, Christopher T.; Warren, Barbour S.; Fragoso, Gilberto; John, Sam; Hager, Gordon L.

    2000-01-01

    Activation of the mouse mammary tumor virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). Recent evidence indicates that this transition extends upstream of the B nucleosome, encompassing a region larger than a single nucleosome (G. Fragoso, W. D. Pennie, S. John, and G. L. Hager, Mol. Cell. Biol. 18:3633–3644). We have reconstituted MMTV LTR DNA into a polynucleosome array using Drosophila embryo extracts. We show binding of purified GR to specific GR elements within a large, multinucleosome array and describe a GR-induced nucleoprotein transition that is dependent on ATP and a HeLa nuclear extract. Previously uncharacterized GR binding sites in the upstream C nucleosome region are involved in the extended region of chromatin remodeling. We also show that GR-dependent chromatin remodeling is a multistep process; in the absence of ATP, GR binds to multiple sites on the chromatin array and prevents restriction enzyme access to recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzymes sites within the transition region. These findings suggest a dynamic model in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template. This model is consistent with the recent description of a “hit-and-run” mechanism for GR action in living cells (J. G. McNally, W. G. Müller, D. Walker, and G. L. Hager, Science 287:1262–1264, 2000). PMID:10938123

  8. Chromatin histone modifications and rigidity affect nuclear morphology independent of lamins

    PubMed Central

    Stephens, Andrew D.; Liu, Patrick Z.; Banigan, Edward J.; Almassalha, Luay M.; Backman, Vadim; Adam, Stephen A.; Goldman, Robert D.; Marko, John F.

    2018-01-01

    Nuclear shape and architecture influence gene localization, mechanotransduction, transcription, and cell function. Abnormal nuclear morphology and protrusions termed “blebs” are diagnostic markers for many human afflictions including heart disease, aging, progeria, and cancer. Nuclear blebs are associated with both lamin and chromatin alterations. A number of prior studies suggest that lamins dictate nuclear morphology, but the contributions of altered chromatin compaction remain unclear. We show that chromatin histone modification state dictates nuclear rigidity, and modulating it is sufficient to both induce and suppress nuclear blebs. Treatment of mammalian cells with histone deacetylase inhibitors to increase euchromatin or histone methyltransferase inhibitors to decrease heterochromatin results in a softer nucleus and nuclear blebbing, without perturbing lamins. Conversely, treatment with histone demethylase inhibitors increases heterochromatin and chromatin nuclear rigidity, which results in reduced nuclear blebbing in lamin B1 null nuclei. Notably, increased heterochromatin also rescues nuclear morphology in a model cell line for the accelerated aging disease Hutchinson–Gilford progeria syndrome caused by mutant lamin A, as well as cells from patients with the disease. Thus, chromatin histone modification state is a major determinant of nuclear blebbing and morphology via its contribution to nuclear rigidity. PMID:29142071

  9. DNA breaks and chromatin structural changes enhance the transcription of autoimmune regulator target genes.

    PubMed

    Guha, Mithu; Saare, Mario; Maslovskaja, Julia; Kisand, Kai; Liiv, Ingrid; Haljasorg, Uku; Tasa, Tõnis; Metspalu, Andres; Milani, Lili; Peterson, Pärt

    2017-04-21

    The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. DNA breaks and chromatin structural changes enhance the transcription of autoimmune regulator target genes

    PubMed Central

    Guha, Mithu; Saare, Mario; Maslovskaja, Julia; Kisand, Kai; Liiv, Ingrid; Haljasorg, Uku; Tasa, Tõnis; Metspalu, Andres; Milani, Lili; Peterson, Pärt

    2017-01-01

    The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes. PMID:28242760

  11. Divergence of Mammalian Higher Order Chromatin Structure Is Associated with Developmental Loci

    PubMed Central

    Chambers, Emily V.; Bickmore, Wendy A.; Semple, Colin A.

    2013-01-01

    Several recent studies have examined different aspects of mammalian higher order chromatin structure – replication timing, lamina association and Hi-C inter-locus interactions — and have suggested that most of these features of genome organisation are conserved over evolution. However, the extent of evolutionary divergence in higher order structure has not been rigorously measured across the mammalian genome, and until now little has been known about the characteristics of any divergent loci present. Here, we generate a dataset combining multiple measurements of chromatin structure and organisation over many embryonic cell types for both human and mouse that, for the first time, allows a comprehensive assessment of the extent of structural divergence between mammalian genomes. Comparison of orthologous regions confirms that all measurable facets of higher order structure are conserved between human and mouse, across the vast majority of the detectably orthologous genome. This broad similarity is observed in spite of many loci possessing cell type specific structures. However, we also identify hundreds of regions (from 100 Kb to 2.7 Mb in size) showing consistent evidence of divergence between these species, constituting at least 10% of the orthologous mammalian genome and encompassing many hundreds of human and mouse genes. These regions show unusual shifts in human GC content, are unevenly distributed across both genomes, and are enriched in human subtelomeric regions. Divergent regions are also relatively enriched for genes showing divergent expression patterns between human and mouse ES cells, implying these regions cause divergent regulation. Particular divergent loci are strikingly enriched in genes implicated in vertebrate development, suggesting important roles for structural divergence in the evolution of mammalian developmental programmes. These data suggest that, though relatively rare in the mammalian genome, divergence in higher order chromatin

  12. Chromatin immunoprecipitation of mouse embryos.

    PubMed

    Voss, Anne K; Dixon, Mathew P; McLennan, Tamara; Kueh, Andrew J; Thomas, Tim

    2012-01-01

    During prenatal development, a large number of different cell types are formed, the vast majority of which contain identical genetic material. The basis of the great variety in cell phenotype and function is the differential expression of the approximately 25,000 genes in the mammalian genome. Transcriptional activity is regulated at many levels by proteins, including members of the basal transcriptional apparatus, DNA-binding transcription factors, and chromatin-binding proteins. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency, with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method to assess if chromatin modifications or proteins are present at a specific locus. ChIP involves the cross linking of DNA and associated proteins and immunoprecipitation using specific antibodies to DNA-associated proteins followed by examination of the co-precipitated DNA sequences or proteins. In the last few years, ChIP has become an essential technique for scientists studying transcriptional regulation and chromatin structure. Using ChIP on mouse embryos, we can document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development. Here, we describe a ChIP technique adapted for mouse embryos.

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

    ERIC Educational Resources Information Center

    Eirin-Lopez, Jose M.

    2013-01-01

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

  14. Chromatin Ring Formation at Plant Centromeres.

    PubMed

    Schubert, Veit; Ruban, Alevtina; Houben, Andreas

    2016-01-01

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

  15. Chromatin Ring Formation at Plant Centromeres

    PubMed Central

    Schubert, Veit; Ruban, Alevtina; Houben, Andreas

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    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.

  17. Structural studies of chromatin and chromosomes. Progress report, March 15--September 15, 1997

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bradbury, E.M.

    This study focused on the following: (1) the structure of chromatin and chromosomes by neutron and x-ray scatter and atomic force microscope; (2) the architecture of human sperm and the structure of sperm by atomic force microscopy (AFM); (3) genome-architecture and higher-order structures in human sperm nuclei; and (4) the effects of histone modifications on the structure of nucleosomes by protein DNA crosslinking method.

  18. Replication domains are self-interacting structural chromatin units of human chromosomes

    NASA Astrophysics Data System (ADS)

    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

  19. Global Quantitative Modeling of Chromatin Factor Interactions

    PubMed Central

    Zhou, Jian; Troyanskaya, Olga G.

    2014-01-01

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

  20. Probing chromatin structure with nuclease sensitivity assays.

    PubMed

    Gregory, R I; Khosla, S; Feil, R

    2001-01-01

    To further our understanding of genomic imprinting it will be essential to identify key control elements, and to investigate their regulation by both epigenetic modifications (such as DNA methylation) and trans-acting factors. So far, sequence elements that regulate parental allele-specific gene expression have been identified in a number of imprinted loci, either because of their differential DNA methylation or through functional studies in transgenic mice (1,2). A systematic search for allele-specific chromatin features constitutes an alternative strategy to identify elements that regulate imprinting. The validity of such an in vivo chromatin approach derives from the fact that in several known imprinting control-elements, a specialized organization of chromatin characterized by nuclease hypersensitivity is present on only one of the two parental chromosome (3). For example, the differentially methylated 5 -portion of the human SNRPN gene-a sequence element that controls imprinting in the Prader-Willi and Angelman syndromes' domain on chromosome 15q11- q13-has strong DNase-I hypersensitive sites on the unmethylated paternal chromosome (4). A differentially methylated region that regulates the imprinting of H19 and that of the neighboring insulin-like growth factor-2 gene on mouse chromosome 7 was also found to have parental chromosome-specific hypersensitive sites (5,6). The precise nature of the allelic nuclease hypersensitivity in these and other imprinted loci remains to be determined in more detail, for example, by applying complementary chromatin methodologies (7,8). However, it is commonly observed that a nuclease hypersensitive site corresponds to a small region where nucleosomes are absent or partially disrupted.

  1. Facilitated diffusion in chromatin lattices: mechanistic diversity and regulatory potential.

    PubMed

    Kampmann, Martin

    2005-08-01

    The interaction between a protein and a specific DNA site is the molecular basis for vital processes in all organisms. Location of the DNA target site by the protein commonly involves facilitated diffusion. Mechanisms of facilitated diffusion vary among proteins; they include one- and two-dimensional sliding along DNA, direct transfer between uncorrelated sites, as well as combinations of these mechanisms. Facilitated diffusion has almost exclusively been studied in vitro. This review discusses facilitated diffusion in the context of the living cell and proposes a theoretical model for facilitated diffusion in chromatin lattices. Chromatin structure differentially affects proteins in different modes of diffusion. The interplay of facilitated diffusion and chromatin structure can determine the rate of protein association with the target site, the frequency of association-dissociation events at the target site, and, under particular conditions, the occupancy of the target site. Facilitated diffusion is required in vivo for efficient DNA repair and bacteriophage restriction and has potential roles in fine-tuning gene regulatory networks and kinetically compartmentalizing the eukaryotic nucleus.

  2. Aging by epigenetics-A consequence of chromatin damage?

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sedivy, John M.; Banumathy, Gowrishankar; Adams, Peter D.

    Chromatin structure is not fixed. Instead, chromatin is dynamic and is subject to extensive developmental and age-associated remodeling. In some cases, this remodeling appears to counter the aging and age-associated diseases, such as cancer, and extend organismal lifespan. However, stochastic non-deterministic changes in chromatin structure might, over time, also contribute to the break down of nuclear, cell and tissue function, and consequently aging and age-associated diseases.

  3. Chromatin in embryonic stem cell neuronal differentiation.

    PubMed

    Meshorer, E

    2007-03-01

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

  4. Chromatin and Transcription in Yeast

    PubMed Central

    Rando, Oliver J.; Winston, Fred

    2012-01-01

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

  5. Expression and chromatin structures of cellulolytic enzyme gene regulated by heterochromatin protein 1.

    PubMed

    Zhang, Xiujun; Qu, Yinbo; Qin, Yuqi

    2016-01-01

    Heterochromatin protein 1 (HP1, homologue HepA in Penicillium oxalicum ) binding is associated with a highly compact chromatin state accompanied by gene silencing or repression. HP1 loss leads to the derepression of gene expression. We investigated HepA roles in regulating cellulolytic enzyme gene expression, as an increasingly number of studies have suggested that cellulolytic enzyme gene expression is not only regulated by transcription factors, but is also affected by the chromatin status. Among the genes that exhibited significant differences between the hepA deletion strain (Δ hepA ) and the wild type (WT), most (95.0 %) were upregulated in Δ hepA compared with WT. The expression of the key transcription factor for cellulolytic enzyme gene (e.g., repressor CreA and activator ClrB) increased significantly. However, the deletion of hepA led to downregulation of prominent extracellular cellulolytic enzyme genes. Among the top 10 extracellular glycoside hydrolases (Amy15A, Amy13A, Cel7A/CBHI, Cel61A, Chi18A, Cel3A/BGLI, Xyn10A, Cel7B/EGI, Cel5B/EGII, and Cel6A/CBHII), in which secretion amount is from the highest to the tenth in P . oxalicum secretome, eight genes, including two amylase genes ( amy15A and amy13A ), all five cellulase genes ( cel7A / cbh1 , cel6A / cbh2 , cel7B / eg1 , cel5B / eg2 , and cel3A / bgl1 ), and the cellulose-active LPMO gene ( cel61A ) expression were downregulated. Results of chromatin accessibility real-time PCR (CHART-PCR) showed that the chromatin of all three tested upstream regions opened specifically because of the deletion of hepA in the case of two prominent cellulase genes cel7A/cbh1 and cel7B/eg1 . However, the open chromatin status did not occur along with the activation of cellulolytic enzyme gene expression. The overexpression of hepA upregulated the cellulolytic enzyme gene expression without chromatin modification. The overexpression of hepA remarkably activated the cellulolytic enzyme synthesis, not only in WT (~150

  6. ATP-dependent chromatin assembly is functionally distinct from chromatin remodeling

    PubMed Central

    Torigoe, Sharon E; Patel, Ashok; Khuong, Mai T; Bowman, Gregory D; Kadonaga, James T

    2013-01-01

    Chromatin assembly involves the combined action of ATP-dependent motor proteins and histone chaperones. Because motor proteins in chromatin assembly also function as chromatin remodeling factors, we investigated the relationship between ATP-driven chromatin assembly and chromatin remodeling in the generation of periodic nucleosome arrays. We found that chromatin remodeling-defective Chd1 motor proteins are able to catalyze ATP-dependent chromatin assembly. The resulting nucleosomes are not, however, spaced in periodic arrays. Wild-type Chd1, but not chromatin remodeling-defective Chd1, can catalyze the conversion of randomly-distributed nucleosomes into periodic arrays. These results reveal a functional distinction between ATP-dependent nucleosome assembly and chromatin remodeling, and suggest a model for chromatin assembly in which randomly-distributed nucleosomes are formed by the nucleosome assembly function of Chd1, and then regularly-spaced nucleosome arrays are generated by the chromatin remodeling activity of Chd1. These findings uncover an unforeseen level of specificity in the role of motor proteins in chromatin assembly. DOI: http://dx.doi.org/10.7554/eLife.00863.001 PMID:23986862

  7. Interaction of chromatin with NaCl and MgCl2. Solubility and binding studies, transition to and characterization of the higher-order structure.

    PubMed

    Ausio, J; Borochov, N; Seger, D; Eisenberg, H

    1984-08-15

    Chicken erythrocyte chromatin containing histones H1 and H5 was carefully separated into a number of well-characterized fractions. A distinction could be made between chromatin insoluble in NaCl above about 80 mM, and chromatin soluble at all NaCl concentrations. Both chromatin forms were indistinguishable electrophoretically and both underwent the transition from the low salt "10 nm" coil to the "30 nm" higher-order structure solenoid by either raising the MgCl2 concentration to about 0.3 mM or the NaCl concentration to about 75 mM. The transitions were examined in detail by elastic light-scattering procedures. It could be shown that the 10 nm form is a flexible coil. For the 30 nm solenoid, the assumption of a rigid cylindrical structure was in good agreement with 5.7 nucleosomes per helical turn. However, disagreement of calculated frictional parameters with values derived from quasielastic light-scattering and sedimentation introduced the possibility that the higher-order structure, under these conditions, is more extended, flexible, or perhaps a mixture of structures. Values for density and refractive index increments of chromatin are also given. To understand the interaction of chromatin with NaCl and with MgCl2, a number of experiments were undertaken to study solubility, precipitation, conformational transitions and binding of ions over a wide range of experimental conditions, including chromatin concentration.

  8. HiTAD: detecting the structural and functional hierarchies of topologically associating domains from chromatin interactions

    PubMed Central

    Wang, Xiao-Tao; Cui, Wang

    2017-01-01

    Abstract A current question in the high-order organization of chromatin is whether topologically associating domains (TADs) are distinct from other hierarchical chromatin domains. However, due to the unclear TAD definition in tradition, the structural and functional uniqueness of TAD is not well studied. In this work, we refined TAD definition by further constraining TADs to the optimal separation on global intra-chromosomal interactions. Inspired by this constraint, we developed a novel method, called HiTAD, to detect hierarchical TADs from Hi-C chromatin interactions. HiTAD performs well in domain sensitivity, replicate reproducibility and inter cell-type conservation. With a novel domain-based alignment proposed by us, we defined several types of hierarchical TAD changes which were not systematically studied previously, and subsequently used them to reveal that TADs and sub-TADs differed statistically in correlating chromosomal compartment, replication timing and gene transcription. Finally, our work also has the implication that the refinement of TAD definition could be achieved by only utilizing chromatin interactions, at least in part. HiTAD is freely available online. PMID:28977529

  9. Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures

    PubMed Central

    Naughton, Catherine; Avlonitis, Nicolaos; Corless, Samuel; Prendergast, James G.; Mati, Ioulia K.; Eijk, Paul P.; Cockroft, Scott L.; Bradley, Mark; Ylstra, Bauke; Gilbert, Nick

    2013-01-01

    DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling we used biotinylated-trimethylpsoralen as a DNA structure probe to show the genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF binding sites. Under-wound domains are transcriptionally active, enriched in topoisomerase I, “open” chromatin fibers and DNaseI sites, but are depleted of topoisomerase II. Furthermore DNA supercoiling impacts on additional levels of chromatin compaction as under-wound domains are cytologically decondensed, topologically constrained, and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation providing an evolutionary purpose for clustering genes along chromosomes. PMID:23416946

  10. Characterizing the molecular architectures of chromatin-modifying complexes.

    PubMed

    Setiaputra, Dheva T; Yip, Calvin K

    2017-11-01

    Eukaryotic cells package their genome in the form of a DNA-protein complex known as chromatin. This organization not only condenses the genome to fit within the confines of the nucleus, but also provides a platform for a cell to regulate accessibility to different gene sequences. The basic packaging element of chromatin is the nucleosome, which consists of 146 base pairs of DNA wrapped around histone proteins. One major means that a cell regulates chromatin structure is by depositing post-translational modifications on nucleosomal histone proteins, and thereby altering internucleosomal interactions and/or binding to different chromatin associated factors. These chromatin modifications are often catalyzed by multi-subunit enzyme complexes, whose large size, sophisticated composition, and inherent conformational flexibility pose significant technical challenges to their biochemical and structural characterization. Multiple structural approaches including nuclear magnetic resonance spectroscopy, X-ray crystallography, single-particle electron microscopy, and crosslinking coupled to mass spectrometry are often used synergistically to probe the overall architecture, subunit organization, and catalytic mechanisms of these macromolecular assemblies. In this review, we highlight several recent chromatin-modifying complexes studies that embodies this multipronged structural approach, and explore common themes amongst them. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed

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

    1978-05-01

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

  12. HMGN proteins modulate chromatin regulatory sites and gene expression during activation of naïve B cells

    PubMed Central

    Zhang, Shaofei; Zhu, Iris; Deng, Tao; Furusawa, Takashi; Rochman, Mark; Vacchio, Melanie S.; Bosselut, Remy; Yamane, Arito; Casellas, Rafael; Landsman, David; Bustin, Michael

    2016-01-01

    The activation of naïve B lymphocyte involves rapid and major changes in chromatin organization and gene expression; however, the complete repertoire of nuclear factors affecting these genomic changes is not known. We report that HMGN proteins, which bind to nucleosomes and affect chromatin structure and function, co-localize with, and maintain the intensity of DNase I hypersensitive sites genome wide, in resting but not in activated B cells. Transcription analyses of resting and activated B cells from wild-type and Hmgn−/− mice, show that loss of HMGNs dampens the magnitude of the transcriptional response and alters the pattern of gene expression during the course of B-cell activation; defense response genes are most affected at the onset of activation. Our study provides insights into the biological function of the ubiquitous HMGN chromatin binding proteins and into epigenetic processes that affect the fidelity of the transcriptional response during the activation of B cell lymphocytes. PMID:27112571

  13. [Changes in the chromatin structure of hepatocyte nuclei of rats trained to hypoxia].

    PubMed

    Domkina, L K; Bresler, V M; Simanovskiĭ, L N

    1976-03-01

    Structure of chromatin in the nuclei of the isolated surviving hepatocytes and in the isolated nuclei of hepatocytes were studied by fluorochroming with acridine orange and by microfluorimetry of fluorescenc connected with the stain chromatin at 530 and 590 nm in intact rats and in the animals trained to hypoxia in a pressure chamber for 60 days. The nuclei of hepatocytes of intact rats were distributed by fluorescence at 530 nm into three classes with the intensity ratio of 1:2:4; as to the nuclei of hepatocytes of the rats trained to hypoxia - they formed a single class corresponding to the second class of control. In intact rats the ratio of the fluorescence intensity at 590 nm to such at 530 nm (alpha coefficient) formed normal distribution; in trained rats - a bimodal distribution with a shift of the maximum in the direction of reduction and increase of alpha in comparison with control. It is supposed that in hypoxia there is a repression of one and depression of other genes in the chromatine of the nuclei of the liver.

  14. The structure of the core NuRD repression complex provides insights into its interaction with chromatin

    PubMed Central

    Millard, Christopher J; Varma, Niranjan; Saleh, Almutasem; Morris, Kyle; Watson, Peter J; Bottrill, Andrew R; Fairall, Louise; Smith, Corinne J; Schwabe, John WR

    2016-01-01

    The NuRD complex is a multi-protein transcriptional corepressor that couples histone deacetylase and ATP-dependent chromatin remodelling activities. The complex regulates the higher-order structure of chromatin, and has important roles in the regulation of gene expression, DNA damage repair and cell differentiation. HDACs 1 and 2 are recruited by the MTA1 corepressor to form the catalytic core of the complex. The histone chaperone protein RBBP4, has previously been shown to bind to the carboxy-terminal tail of MTA1. We show that MTA1 recruits a second copy of RBBP4. The crystal structure reveals an extensive interface between MTA1 and RBBP4. An EM structure, supported by SAXS and crosslinking, reveals the architecture of the dimeric HDAC1:MTA1:RBBP4 assembly which forms the core of the NuRD complex. We find evidence that in this complex RBBP4 mediates interaction with histone H3 tails, but not histone H4, suggesting a mechanism for recruitment of the NuRD complex to chromatin. DOI: http://dx.doi.org/10.7554/eLife.13941.001 PMID:27098840

  15. Nucleoporins and chromatin metabolism.

    PubMed

    Ptak, Christopher; Wozniak, Richard W

    2016-06-01

    Mounting evidence has implicated a group of proteins termed nucleoporins, or Nups, in various processes that regulate chromatin structure and function. Nups were first recognized as building blocks for nuclear pore complexes, but several members of this group of proteins also reside in the cytoplasm and within the nucleus. Moreover, many are dynamic and move between these various locations. Both at the nuclear envelope, as part of nuclear pore complexes, and within the nucleoplasm, Nups interact with protein complexes that function in gene transcription, chromatin remodeling, DNA repair, and DNA replication. Here, we review recent studies that provide further insight into the molecular details of these interactions and their role in regulating the activity of chromatin modifying factors. Copyright © 2016. Published by Elsevier Ltd.

  16. Genome-wide Hi-C analysis reveals extensive hierarchical chromatin interactions in rice.

    PubMed

    Dong, Qianli; Li, Ning; Li, Xiaochong; Yuan, Zan; Xie, Dejian; Wang, Xiaofei; Li, Jianing; Yu, Yanan; Wang, Jinbin; Ding, Baoxu; Zhang, Zhibin; Li, Changping; Bian, Yao; Zhang, Ai; Wu, Ying; Liu, Bao; Gong, Lei

    2018-06-01

    The non-random spatial packing of chromosomes in the nucleus plays a critical role in orchestrating gene expression and genome function. Here, we present a Hi-C analysis of the chromatin interaction patterns in rice (Oryza sativa L.) at hierarchical architectural levels. We confirm that rice chromosomes occupy their own territories with certain preferential inter-chromosomal associations. Moderate compartment delimitation and extensive TADs (Topologically Associated Domains) were determined to be associated with heterogeneous genomic compositions and epigenetic marks in the rice genome. We found subtle features including chromatin loops, gene loops, and off-/near-diagonal intensive interaction regions. Gene chromatin loops associated with H3K27me3 could be positively involved in gene expression. In addition to insulated enhancing effects for neighbor gene expression, the identified rice gene loops could bi-directionally (+/-) affect the expression of looped genes themselves. Finally, web-interleaved off-diagonal IHIs/KEEs (Interactive Heterochromatic Islands or KNOT ENGAGED ELEMENTs) could trap transposable elements (TEs) via the enrichment of silencing epigenetic marks. In parallel, the near-diagonal FIREs (Frequently Interacting Regions) could positively affect the expression of involved genes. Our results suggest that the chromatin packing pattern in rice is generally similar to that in Arabidopsis thaliana but with clear differences at specific structural levels. We conclude that genomic composition, epigenetic modification, and transcriptional activity could act in combination to shape global and local chromatin packing in rice. Our results confirm recent observations in rice and A. thaliana but also provide additional insights into the patterns and features of chromatin organization in higher plants. © 2018 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

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

    PubMed

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

    2008-12-01

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

  18. Assembly of transcriptionally inactive chromatin in vitro.

    PubMed

    Shanahan, M M; Kmiec, E B

    1989-07-01

    We have successfully uncoupled the previously interlocked activities of chromatin assembly and in vitro transcription promoted by the Xenopus oocyte S-150 cell-free extract. Our isolated fraction catalyzes extensive chromatin assembly measured both by changes in DNA topology and Micrococcal nuclease digestions. The assembly of chromatin is slowed by the exogenous addition of ATP. In the absence of exogenously added ATP, the fraction forms a chromatin template that is transcriptionally inert. Addition of small amounts of the HeLa cell extract (S-100) converts these templates into transcriptionally active ones without disrupting the chromatin structure. Our protocol defines a method for the isolation of a fraction from the Xenopus cell free extract that catalyzes the assembly of transcriptionally inactive chromatin. We characterize this reaction and establish conditions for the transcriptional activation of these inactive minichromosomes.

  19. Histone H4 acetylation required for chromatin decompaction during DNA replication.

    PubMed

    Ruan, Kun; Yamamoto, Takaharu G; Asakawa, Haruhiko; Chikashige, Yuji; Kimura, Hiroshi; Masukata, Hisao; Haraguchi, Tokuko; Hiraoka, Yasushi

    2015-07-30

    Faithful DNA replication is a prerequisite for cell proliferation. Several cytological studies have shown that chromosome structures alter in the S-phase of the cell cycle. However, the molecular mechanisms behind the alteration of chromosome structures associated with DNA replication have not been elucidated. Here, we investigated chromatin structures and acetylation of specific histone residues during DNA replication using the meiotic nucleus of the fission yeast Schizosaccharomyces pombe. The S. pombe meiotic nucleus provides a unique opportunity for measuring the levels of compaction of chromatin along the chromosome in a defined orientation. By direct measurement of chromatin compaction in living cells, we demonstrated that decompaction of chromatin occurs during meiotic DNA replication. This chromatin decompaction was suppressed by depletion of histone acetyltransferase Mst1 or by arginine substitution of specific lysine residues (K8 and K12) of histone H4. These results suggest that acetylation of histone H4 residues K8 and K12 plays a critical role in loosening chromatin structures during DNA replication.

  20. Mesoscale Modeling of Chromatin Folding

    NASA Astrophysics Data System (ADS)

    Schlick, Tamar

    2009-03-01

    Eukaryotic chromatin is the fundamental protein/nucleic acid unit that stores the genetic material. Understanding how chromatin fibers fold and unfold in physiological conditions is important for interpreting fundamental biological processes like DNA replication and transcription regulation. Using a mesoscopic model of oligonucleosome chains and tailored sampling protocols, we elucidate the energetics of oligonucleosome folding/unfolding and the role of each histone tail, linker histones, and divalent ions in regulating chromatin structure. The resulting compact topologies reconcile features of the zigzag model with straight linker DNAs with the solenoid model with bent linker DNAs for optimal fiber organization and reveal dynamic and energetic aspects involved.

  1. Effect of Chromatin Structure on the Extent and Distribution of DNA Double Strand Breaks Produced by Ionizing Radiation; Comparative Study of hESC and Differentiated Cells Lines.

    PubMed

    Venkatesh, Priyanka; Panyutin, Irina V; Remeeva, Evgenia; Neumann, Ronald D; Panyutin, Igor G

    2016-01-02

    Chromatin structure affects the extent of DNA damage and repair. Thus, it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here, we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC, heterochromatin is confined to distinct regions, while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives, normal human fibroblasts; and one cancer cell line. At the same time, the number of DNA repair foci were not statistically different among these cells. We showed that in hESC, DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells, and to a lesser extent in IMR90 normal fibroblasts, but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC.

  2. Epigenetics: Beyond Chromatin Modifications and Complex Genetic Regulation1

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Tiana, Guido; Amitai, Assaf; Pollex, Tim; Piolot, Tristan; Holcman, David; Heard, Edith; Giorgetti, Luca

    2016-03-29

    Experiments based on chromosome conformation capture have shown that mammalian genomes are partitioned into topologically associating domains (TADs), within which the chromatin fiber preferentially interacts. TADs may provide three-dimensional scaffolds allowing genes to contact their appropriate distal regulatory DNA sequences (e.g., enhancers) and thus to be properly regulated. Understanding the cell-to-cell and temporal variability of the chromatin fiber within TADs, and what determines them, is thus of great importance to better understand transcriptional regulation. We recently described an equilibrium polymer model that can accurately predict cell-to-cell variation of chromosome conformation within single TADs, from chromosome conformation capture-based data. Here we further analyze the conformational and energetic properties of our model. We show that the chromatin fiber within TADs can easily fluctuate between several conformational states, which are hierarchically organized and are not separated by important free energy barriers, and that this is facilitated by the fact that the chromatin fiber within TADs is close to the onset of the coil-globule transition. We further show that in this dynamic state the properties of the chromatin fiber, and its contact probabilities in particular, are determined in a nontrivial manner not only by site-specific interactions between strongly interacting loci along the fiber, but also by nonlocal correlations between pairs of contacts. Finally, we use live-cell experiments to measure the dynamics of the chromatin fiber in mouse embryonic stem cells, in combination with dynamical simulations, and predict that conformational changes within one TAD are likely to occur on timescales that are much shorter than the duration of one cell cycle. This suggests that genes and their regulatory elements may come together and disassociate several times during a cell cycle. These results have important implications for transcriptional

  4. TOPICAL REVIEW: The physics of chromatin

    NASA Astrophysics Data System (ADS)

    Schiessel, Helmut

    2003-05-01

    Recent progress has been made in the understanding of the physical properties of chromatin - the dense complex of DNA and histone proteins that occupies the nuclei of plant and animal cells. Here I will focus on the two lowest levels of the hierarchy of DNA folding into the chromatin complex. (i) The nucleosome, the chromatin repeating unit consisting of a globular aggregate of eight histone proteins with the DNA wrapped around it: its overcharging, the DNA unwrapping transition, the 'sliding' of the octamer along the DNA. (ii) The 30 nm chromatin fibre, the necklace-like structure of nucleosomes connected via linker DNA: its geometry, its mechanical properties under stretching and its response to changing ionic conditions. I will stress that chromatin combines two seemingly contradictory features: (1) high compaction of DNA within the nuclear envelope and, at the same time, (2) accessibility to genes, promoter regions and gene regulatory sequences.

  5. Chromatin organization at the nuclear periphery as revealed by image analysis of structured illumination microscopy data.

    PubMed

    Fišerová, Jindřiška; Efenberková, Michaela; Sieger, Tomáš; Maninová, Miloslava; Uhlířová, Jana; Hozák, Pavel

    2017-06-15

    The nuclear periphery (NP) plays a substantial role in chromatin organization. Heterochromatin at the NP is interspersed with active chromatin surrounding nuclear pore complexes (NPCs); however, details of the peripheral chromatin organization are missing. To discern the distribution of epigenetic marks at the NP of HeLa nuclei, we used structured illumination microscopy combined with a new MATLAB software tool for automatic NP and NPC detection, measurements of fluorescent intensity and statistical analysis of measured data. Our results show that marks for both active and non-active chromatin associate differentially with NPCs. The incidence of heterochromatin marks, such as H3K27me2 and H3K9me2, was significantly lower around NPCs. In contrast, the presence of marks of active chromatin such as H3K4me2 was only decreased very slightly around the NPCs or not at all (H3K9Ac). Interestingly, the histone demethylases LSD1 (also known as KDM1A) and KDM2A were enriched within the NPCs, suggesting that there was a chromatin-modifying mechanism at the NPCs. Inhibition of transcription resulted in a larger drop in the distribution of H1, H3K9me2 and H3K23me2, which implies that transcription has a role in the organization of heterochromatin at the NP. © 2017. Published by The Company of Biologists Ltd.

  6. Chromatin Computation

    PubMed Central

    Bryant, Barbara

    2012-01-01

    In living cells, DNA is packaged along with protein and RNA into chromatin. Chemical modifications to nucleotides and histone proteins are added, removed and recognized by multi-functional molecular complexes. Here I define a new computational model, in which chromatin modifications are information units that can be written onto a one-dimensional string of nucleosomes, analogous to the symbols written onto cells of a Turing machine tape, and chromatin-modifying complexes are modeled as read-write rules that operate on a finite set of adjacent nucleosomes. I illustrate the use of this “chromatin computer” to solve an instance of the Hamiltonian path problem. I prove that chromatin computers are computationally universal – and therefore more powerful than the logic circuits often used to model transcription factor control of gene expression. Features of biological chromatin provide a rich instruction set for efficient computation of nontrivial algorithms in biological time scales. Modeling chromatin as a computer shifts how we think about chromatin function, suggests new approaches to medical intervention, and lays the groundwork for the engineering of a new class of biological computing machines. PMID:22567109

  7. The Chromatin Remodeler SPLAYED Regulates Specific Stress Signaling Pathways

    PubMed Central

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

    2008-01-01

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

  8. Histone modifications influence mediator interactions with chromatin

    PubMed Central

    Zhu, Xuefeng; Zhang, Yongqiang; Bjornsdottir, Gudrun; Liu, Zhongle; Quan, Amy; Costanzo, Michael; Dávila López, Marcela; Westholm, Jakub Orzechowski; Ronne, Hans; Boone, Charles; Gustafsson, Claes M.; Myers, Lawrence C.

    2011-01-01

    The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. Genome wide localization studies have demonstrated that Mediator occupancy not only correlates with high levels of transcription, but that the complex also is present at transcriptionally silenced locations. We provide evidence that Mediator localization is guided by an interaction with histone tails, and that this interaction is regulated by their post-translational modifications. A quantitative, high-density genetic interaction map revealed links between Mediator components and factors affecting chromatin structure, especially histone deacetylases. Peptide binding assays demonstrated that pure wild-type Mediator forms stable complexes with the tails of Histone H3 and H4. These binding assays also showed Mediator—histone H4 peptide interactions are specifically inhibited by acetylation of the histone H4 lysine 16, a residue critical in transcriptional silencing. Finally, these findings were validated by tiling array analysis that revealed a broad correlation between Mediator and nucleosome occupancy in vivo, but a negative correlation between Mediator and nucleosomes acetylated at histone H4 lysine 16. Our studies show that chromatin structure and the acetylation state of histones are intimately connected to Mediator localization. PMID:21742760

  9. Megabase replication domains along the human genome: relation to chromatin structure and genome organisation.

    PubMed

    Audit, Benjamin; Zaghloul, Lamia; Baker, Antoine; Arneodo, Alain; Chen, Chun-Long; d'Aubenton-Carafa, Yves; Thermes, Claude

    2013-01-01

    In higher eukaryotes, the absence of specific sequence motifs, marking the origins of replication has been a serious hindrance to the understanding of (i) the mechanisms that regulate the spatio-temporal replication program, and (ii) the links between origins activation, chromatin structure and transcription. In this chapter, we review the partitioning of the human genome into megabased-size replication domains delineated as N-shaped motifs in the strand compositional asymmetry profiles. They collectively span 28.3% of the genome and are bordered by more than 1,000 putative replication origins. We recapitulate the comparison of this partition of the human genome with high-resolution experimental data that confirms that replication domain borders are likely to be preferential replication initiation zones in the germline. In addition, we highlight the specific distribution of experimental and numerical chromatin marks along replication domains. Domain borders correspond to particular open chromatin regions, possibly encoded in the DNA sequence, and around which replication and transcription are highly coordinated. These regions also present a high evolutionary breakpoint density, suggesting that susceptibility to breakage might be linked to local open chromatin fiber state. Altogether, this chapter presents a compartmentalization of the human genome into replication domains that are landmarks of the human genome organization and are likely to play a key role in genome dynamics during evolution and in pathological situations.

  10. Loss of neuronal 3D chromatin organization causes transcriptional and behavioural deficits related to serotonergic dysfunction.

    PubMed

    Ito, Satomi; Magalska, Adriana; Alcaraz-Iborra, Manuel; Lopez-Atalaya, Jose P; Rovira, Victor; Contreras-Moreira, Bruno; Lipinski, Michal; Olivares, Roman; Martinez-Hernandez, Jose; Ruszczycki, Blazej; Lujan, Rafael; Geijo-Barrientos, Emilio; Wilczynski, Grzegorz M; Barco, Angel

    2014-07-18

    The interior of the neuronal cell nucleus is a highly organized three-dimensional (3D) structure where regions of the genome that are linearly millions of bases apart establish sub-structures with specialized functions. To investigate neuronal chromatin organization and dynamics in vivo, we generated bitransgenic mice expressing GFP-tagged histone H2B in principal neurons of the forebrain. Surprisingly, the expression of this chimeric histone in mature neurons caused chromocenter declustering and disrupted the association of heterochromatin with the nuclear lamina. The loss of these structures did not affect neuronal viability but was associated with specific transcriptional and behavioural deficits related to serotonergic dysfunction. Overall, our results demonstrate that the 3D organization of chromatin within neuronal cells provides an additional level of epigenetic regulation of gene expression that critically impacts neuronal function. This in turn suggests that some loci associated with neuropsychiatric disorders may be particularly sensitive to changes in chromatin architecture.

  11. Immune subversion by chromatin manipulation: a 'new face' of host-bacterial pathogen interaction.

    PubMed

    Arbibe, Laurence

    2008-08-01

    Bacterial pathogens have evolved various strategies to avoid immune surveillance, depending of their in vivo'lifestyle'. The identification of few bacterial effectors capable to enter the nucleus and modifying chromatin structure in host raises the fascinating questions of how pathogens modulate chromatin structure and why. Chromatin is a dynamic structure that maintains the stability and accessibility of the host DNA genome to the transcription machinery. This review describes the various strategies used by pathogens to interface with host chromatin. In some cases, chromatin injury can be a strategy to take control of major cellular functions, such as the cell cycle. In other cases, manipulation of chromatin structure at specific genomic locations by modulating epigenetic information provides a way for the pathogen to impose its own transcriptional signature onto host cells. This emerging field should strongly influence our understanding of chromatin regulation at interphase nucleus and may provide invaluable openings to the control of immune gene expression in inflammatory and infectious diseases.

  12. Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Peng, Jamy C.

    Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) thatmore » binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those

  13. Chromatin Condensing Functions of the Linker Histone C-terminal Domain are mediated by Specific Amino Acid Composition and Intrinsic Protein Disorder†

    PubMed Central

    Lu, Xu; Hamkalo, Barbara; Parseghian, Missag H.; Hansen, Jeffrey C.

    2009-01-01

    Linker histones bind to the nucleosomes and linker DNA of chromatin fibers, causing changes in linker DNA structure and stabilization of higher order folded and oligomeric chromatin structures. Linker histones affect chromatin structure acting primarily through their ~100 residue C-terminal domain (CTD). We have previously shown that the ability of the linker histone H1° to alter chromatin structure was localized to two discontinuous 24-/25-residue CTD regions (Lu, X., and Hansen, J. C. (2004) J Biol Chem 279, 8701–8707). To determine the biochemical basis for these results, we have characterized chromatin model systems assembled with endogenous mouse somatic H1 isoforms, or recombinant H1° CTD mutants in which the primary sequence has been scrambled, the amino acid composition mutated, or the location of various CTD regions swapped. Our results indicate that specific amino acid composition plays a fundamental role in molecular recognition and function by the H1 CTD. Additionally, these experiments support a new molecular model for CTD function, and provide a biochemical basis for the redundancy observed in H1 isoform knockout experiments in vivo. PMID:19072710

  14. Superresolution microscopy reveals structural mechanisms driving the nanoarchitecture of a viral chromatin tether.

    PubMed

    Grant, Margaret J; Loftus, Matthew S; Stoja, Aiola P; Kedes, Dean H; Smith, M Mitchell

    2018-05-08

    By tethering their circular genomes (episomes) to host chromatin, DNA tumor viruses ensure retention and segregation of their genetic material during cell divisions. Despite functional genetic and crystallographic studies, there is little information addressing the 3D structure of these tethers in cells, issues critical for understanding persistent infection by these viruses. Here, we have applied direct stochastic optical reconstruction microscopy (dSTORM) to establish the nanoarchitecture of tethers within cells latently infected with the oncogenic human pathogen, Kaposi's sarcoma-associated herpesvirus (KSHV). Each KSHV tether comprises a series of homodimers of the latency-associated nuclear antigen (LANA) that bind with their C termini to the tandem array of episomal terminal repeats (TRs) and with their N termini to host chromatin. Superresolution imaging revealed that individual KSHV tethers possess similar overall dimensions and, in aggregate, fold to occupy the volume of a prolate ellipsoid. Using plasmids with increasing numbers of TRs, we found that tethers display polymer power law scaling behavior with a scaling exponent characteristic of active chromatin. For plasmids containing a two-TR tether, we determined the size, separation, and relative orientation of two distinct clusters of bound LANA, each corresponding to a single TR. From these data, we have generated a 3D model of the episomal half of the tether that integrates and extends previously established findings from epifluorescent, crystallographic, and epigenetic approaches. Our findings also validate the use of dSTORM in establishing novel structural insights into the physical basis of molecular connections linking host and pathogen genomes.

  15. Cohesin organizes chromatin loops at DNA replication factories

    PubMed Central

    Guillou, Emmanuelle; Ibarra, Arkaitz; Coulon, Vincent; Casado-Vela, Juan; Rico, Daniel; Casal, Ignacio; Schwob, Etienne; Losada, Ana; Méndez, Juan

    2010-01-01

    Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication. PMID:21159821

  16. The elusive structural role of ubiquitinated histones.

    PubMed

    Moore, Susan C; Jason, Laure; Ausió, Juan

    2002-01-01

    It is increasingly apparent that histone posttranslational modifications are important in chromatin structure and dynamics. However, histone ubiquitination has received little attention. Histones H1, H3, H2A, and H2B can be ubiquitinated in vivo, but the most prevalent are uH2A and uH2B. The size of this modification suggests some sort of structural impact. Physiological observations suggest that ubiquitinated histones may have multiple functions and structural effects. Ubiquitinated histones have been correlated with transcriptionally active DNA, implying that it may prevent chromatin folding or help maintain an open conformation. Also, in some organisms during spermiogenesis, a process involving extensive chromatin remodeling, uH2A levels increase just prior to histone replacement by protamines. Determination of chromatin's structural changes resulting from histone ubiquitination is therefore important. Recent work using reconstituted nucleosomes and chromatin fibers containing uH2A indicate that in the absence of linker histones, ubiquitination has little structural impact. DNase I digests and analytical ultracentrifugation of reconstituted ubiquitinated nucleosomes show no structural differences. Solubility assays using reconstituted chromatin fibers in the presence of divalent ions demonstrate that uH2A fibers are slightly more prone to aggregation than controls, and analytical ultracentrifugation results with different MgCl2 and NaCl concentrations determined that chromatin folding is not affected by this modification. Additional work to assess possible synergistic affects with histone acetylation also precludes any structural implications. Protamine displacement experiments concluded that the presence of uH2A does not significantly affect the ability of the protamines to displace histones. In addition, uH2A does not interfere with histone H1 binding to the nucleosome. While work with uH2B remains insufficient to come to any definitive conclusions about its

  17. Monte Carlo simulation of ionizing radiation induced DNA strand breaks utilizing coarse grained high-order chromatin structures.

    PubMed

    Liang, Ying; Yang, Gen; Liu, Feng; Wang, Yugang

    2016-01-07

    Ionizing radiation threatens genome integrity by causing DNA damage. Monte Carlo simulation of the interaction of a radiation track structure with DNA provides a powerful tool for investigating the mechanisms of the biological effects. However, the more or less oversimplification of the indirect effect and the inadequate consideration of high-order chromatin structures in current models usually results in discrepancies between simulations and experiments, which undermine the predictive role of the models. Here we present a biophysical model taking into consideration factors that influence indirect effect to simulate radiation-induced DNA strand breaks in eukaryotic cells with high-order chromatin structures. The calculated yields of single-strand breaks and double-strand breaks (DSBs) for photons are in good agreement with the experimental measurements. The calculated yields of DSB for protons and α particles are consistent with simulations by the PARTRAC code, whereas an overestimation is seen compared with the experimental results. The simulated fragment size distributions for (60)Co γ irradiation and α particle irradiation are compared with the measurements accordingly. The excellent agreement with (60)Co irradiation validates our model in simulating photon irradiation. The general agreement found in α particle irradiation encourages model applicability in the high linear energy transfer range. Moreover, we demonstrate the importance of chromatin high-order structures in shaping the spectrum of initial damage.

  18. Predictive Computational Modeling of Chromatin Folding

    NASA Astrophysics Data System (ADS)

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

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

  19. Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations

    PubMed Central

    Szczurek, Aleksander; Klewes, Ludger; Xing, Jun; Gourram, Amine; Birk, Udo; Knecht, Hans; Dobrucki, Jurek W.; Mai, Sabine

    2017-01-01

    Abstract Advanced light microscopy is an important tool for nanostructure analysis of chromatin. In this report we present a general concept for Single Molecule localization Microscopy (SMLM) super-resolved imaging of DNA-binding dyes based on modifying the properties of DNA and the dye. By careful adjustment of the chemical environment leading to local, reversible DNA melting and hybridization control over the fluorescence signal of the DNA-binding dye molecules can be introduced. We postulate a transient binding as the basis for our variation of binding-activated localization microscopy (BALM). We demonstrate that several intercalating and minor-groove binding DNA dyes can be used to register (optically isolate) only a few DNA-binding dye signals at a time. To highlight this DNA structure fluctuation-assisted BALM (fBALM), we applied it to measure, for the first time, nanoscale differences in nuclear architecture in model ischemia with an anticipated structural resolution of approximately 50 nm. Our data suggest that this approach may open an avenue for the enhanced microscopic analysis of chromatin nano-architecture and hence the microscopic analysis of nuclear structure aberrations occurring in various pathological conditions. It may also become possible to analyse nuclear nanostructure differences in different cell types, stages of development or environmental stress conditions. PMID:28082388

  20. Chromatin ionic atmosphere analyzed by a mesoscale electrostatic approach.

    PubMed

    Gan, Hin Hark; Schlick, Tamar

    2010-10-20

    Characterizing the ionic distribution around chromatin is important for understanding the electrostatic forces governing chromatin structure and function. Here we develop an electrostatic model to handle multivalent ions and compute the ionic distribution around a mesoscale chromatin model as a function of conformation, number of nucleosome cores, and ionic strength and species using Poisson-Boltzmann theory. This approach enables us to visualize and measure the complex patterns of counterion condensation around chromatin by examining ionic densities, free energies, shielding charges, and correlations of shielding charges around the nucleosome core and various oligonucleosome conformations. We show that: counterions, especially divalent cations, predominantly condense around the nucleosomal and linker DNA, unburied regions of histone tails, and exposed chromatin surfaces; ionic screening is sensitively influenced by local and global conformations, with a wide ranging net nucleosome core screening charge (56-100e); and screening charge correlations reveal conformational flexibility and interactions among chromatin subunits, especially between the histone tails and parental nucleosome cores. These results provide complementary and detailed views of ionic effects on chromatin structure for modest computational resources. The electrostatic model developed here is applicable to other coarse-grained macromolecular complexes. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  1. Distinct structural transitions of chromatin topological domains correlate with coordinated hormone-induced gene regulation

    PubMed Central

    Le Dily, François; Baù, Davide; Pohl, Andy; Vicent, Guillermo P.; Serra, François; Soronellas, Daniel; Castellano, Giancarlo; Wright, Roni H.G.; Ballare, Cecilia; Filion, Guillaume; Marti-Renom, Marc A.

    2014-01-01

    The human genome is segmented into topologically associating domains (TADs), but the role of this conserved organization during transient changes in gene expression is not known. Here we describe the distribution of progestin-induced chromatin modifications and changes in transcriptional activity over TADs in T47D breast cancer cells. Using ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing), Hi-C (chromosome capture followed by high-throughput sequencing), and three-dimensional (3D) modeling techniques, we found that the borders of the ∼2000 TADs in these cells are largely maintained after hormone treatment and that up to 20% of the TADs could be considered as discrete regulatory units where the majority of the genes are either transcriptionally activated or repressed in a coordinated fashion. The epigenetic signatures of the TADs are homogeneously modified by hormones in correlation with the transcriptional changes. Hormone-induced changes in gene activity and chromatin remodeling are accompanied by differential structural changes for activated and repressed TADs, as reflected by specific and opposite changes in the strength of intra-TAD interactions within responsive TADs. Indeed, 3D modeling of the Hi-C data suggested that the structure of TADs was modified upon treatment. The differential responses of TADs to progestins and estrogens suggest that TADs could function as “regulons” to enable spatially proximal genes to be coordinately transcribed in response to hormones. PMID:25274727

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

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Wong, Michael; Hada, Megumi; Wu, Honglu

    2015-01-01

    Microgravity has been shown to alter global gene expression patterns and protein levels both in cultured cells and animal models. It has been suggested that the packaging of chromatin fibers in the interphase nucleus is closely related to genome function, and the changes in transcriptional activity are tightly correlated with changes in chromatin folding. This study explores the changes of chromatin conformation and chromatin-chromatin interactions in the simulated microgravity environment, and investigates their correlation to the expression of genes located at different regions of the chromosome. To investigate the folding of chromatin in interphase under various culture conditions, human epithelial cells, fibroblasts, and lymphocytes were fixed in the G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome as separate colors. After images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multi-mega base pair scale. In order to determine the effects of microgravity on chromosome conformation and orientation, measures such as distance between homologous pairs, relative orientation of chromosome arms about a shared midpoint, and orientation of arms within individual chromosomes were all considered as potentially impacted by simulated microgravity conditions. The studies revealed non-random folding of chromatin in interphase, and suggested an association of interphase chromatin folding with radiation-induced chromosome aberration hotspots. Interestingly, the distributions of genes with expression changes over chromosome 3 in cells cultured under microgravity environment are apparently clustered on specific loci and chromosomes. This data provides important insights into how mammalian cells respond to microgravity at molecular level.

  3. Chromatin hydrodynamics.

    PubMed

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

    2014-05-06

    Following recent observations of large scale correlated motion of chromatin inside the nuclei of live differentiated cells, we present a hydrodynamic theory-the two-fluid model-in which the content of a nucleus is described as a chromatin solution with the nucleoplasm playing the role of the solvent and the chromatin fiber that of a solute. This system is subject to both passive thermal fluctuations and active scalar and vector events that are associated with free energy consumption, such as ATP hydrolysis. Scalar events drive the longitudinal viscoelastic modes (where the chromatin fiber moves relative to the solvent) while vector events generate the transverse modes (where the chromatin fiber moves together with the solvent). Using linear response methods, we derive explicit expressions for the response functions that connect the chromatin density and velocity correlation functions to the corresponding correlation functions of the active sources and the complex viscoelastic moduli of the chromatin solution. We then derive general expressions for the flow spectral density of the chromatin velocity field. We use the theory to analyze experimental results recently obtained by one of the present authors and her co-workers. We find that the time dependence of the experimental data for both native and ATP-depleted chromatin can be well-fitted using a simple model-the Maxwell fluid-for the complex modulus, although there is some discrepancy in terms of the wavevector dependence. Thermal fluctuations of ATP-depleted cells are predominantly longitudinal. ATP-active cells exhibit intense transverse long wavelength velocity fluctuations driven by force dipoles. Fluctuations with wavenumbers larger than a few inverse microns are dominated by concentration fluctuations with the same spectrum as thermal fluctuations but with increased intensity. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  4. Chromatin Hydrodynamics

    PubMed Central

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

    2014-01-01

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

  5. Chromatin isolation by RNA purification (ChIRP).

    PubMed

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

    2012-03-25

    Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression (1,2,3,4,5,6,7). The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion (8,9). While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation (10,11). However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide( 3,12,13), but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex (14); HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex (13). Prior studies mapping RNA occupancy at chromatin have revealed substantial insights (15,16), but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution (17). This method, Chromatin Isolation by

  6. Chromatin Immunoprecipitation in Early Mouse Embryos.

    PubMed

    García-González, Estela G; Roque-Ramirez, Bladimir; Palma-Flores, Carlos; Hernández-Hernández, J Manuel

    2018-01-01

    Epigenetic regulation is achieved at many levels by different factors such as tissue-specific transcription factors, members of the basal transcriptional apparatus, chromatin-binding proteins, and noncoding RNAs. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method that allows elucidating gene regulation at the molecular level by assessing if chromatin modifications or proteins are present at a specific locus. Initially, the majority of ChIP experiments were performed on cultured cell lines and more recently this technique has been adapted to a variety of tissues in different model organisms. Using ChIP on mouse embryos, it is possible to document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development and to get biological meaning from observations made on tissue culture analyses. We describe here a ChIP protocol on freshly isolated mouse embryonic somites for in vivo analysis of muscle specific transcription factor binding on chromatin. This protocol has been easily adapted to other mouse embryonic tissues and has also been successfully scaled up to perform ChIP-Seq.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Banerjee, Amrita; Sanyal, Sulagna; Majumder, Parijat

    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) andmore » 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.« less

  8. Sedimentation Velocity Analysis of Large Oligomeric Chromatin Complexes Using Interference Detection.

    PubMed

    Rogge, Ryan A; Hansen, Jeffrey C

    2015-01-01

    Sedimentation velocity experiments measure the transport of molecules in solution under centrifugal force. Here, we describe a method for monitoring the sedimentation of very large biological molecular assemblies using the interference optical systems of the analytical ultracentrifuge. The mass, partial-specific volume, and shape of macromolecules in solution affect their sedimentation rates as reflected in the sedimentation coefficient. The sedimentation coefficient is obtained by measuring the solute concentration as a function of radial distance during centrifugation. Monitoring the concentration can be accomplished using interference optics, absorbance optics, or the fluorescence detection system, each with inherent advantages. The interference optical system captures data much faster than these other optical systems, allowing for sedimentation velocity analysis of extremely large macromolecular complexes that sediment rapidly at very low rotor speeds. Supramolecular oligomeric complexes produced by self-association of 12-mer chromatin fibers are used to illustrate the advantages of the interference optics. Using interference optics, we show that chromatin fibers self-associate at physiological divalent salt concentrations to form structures that sediment between 10,000 and 350,000S. The method for characterizing chromatin oligomers described in this chapter will be generally useful for characterization of any biological structures that are too large to be studied by the absorbance optical system. © 2015 Elsevier Inc. All rights reserved.

  9. Structural domains and conformational changes in nuclear chromatin: a quantitative thermodynamic approach by differential scanning calorimetry.

    PubMed

    Balbi, C; Abelmoschi, M L; Gogioso, L; Parodi, S; Barboro, P; Cavazza, B; Patrone, E

    1989-04-18

    A good deal of information on the thermodynamic properties of chromatin was derived in the last few years from optical melting experiments. The structural domains of the polynucleosomal chain, the linker, and the core particle denature as independent units. The differential scanning calorimetry profile of isolated chromatin is made up of three endotherms, at approximately 74, 90, and 107 degrees C, having an almost Gaussian shape. Previous work on this matter, however, was mainly concerned with the dependence of the transition enthalpy on external parameters, such as the ionic strength, or with the melting of nuclei from different sources. In this paper we report the structural assignment of the transitions of rat liver nuclei, observed at 58, 66, 75, 92, and 107 degrees C. They are representative of the quiescent state of the cell. The strategy adopted in this work builds on the method developed for the investigation of complex biological macromolecules. The heat absorption profile of the nucleus was related to the denaturation of isolated nuclear components; electron microscopy and electrophoretic techniques were used for their morphological and molecular characterization. The digestion of chromatin by endogenous nuclease mimics perfectly the decondensation of the higher order structure and represented the source of several misinterpretations. This point was carefully examined in order to define unambiguously the thermal profile of native nuclei. The low-temperature transitions, centered around 58 and 66 degrees C, arise from the melting of scaffolding structures and of the proteins associated with heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. The Centromere: Chromatin Foundation for the Kinetochore Machinery

    PubMed Central

    Fukagawa, Tatsuo; Earnshaw, William C.

    2014-01-01

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

  11. Dietary polyphenols and chromatin remodeling.

    PubMed

    Russo, Gian Luigi; Vastolo, Viviana; Ciccarelli, Marco; Albano, Luigi; Macchia, Paolo Emidio; Ungaro, Paola

    2017-08-13

    Polyphenols are the most abundant phytochemicals in fruits, vegetables, and plant-derived beverages. Recent findings suggest that polyphenols display the ability to reverse adverse epigenetic regulation involved in pathological conditions, such as obesity, metabolic disorder, cardiovascular and neurodegenerative diseases, and various forms of cancer. Epigenetics, defined as heritable changes to the transcriptome, independent from those occurring in the genome, includes DNA methylation, histone modifications, and posttranscriptional gene regulation by noncoding RNAs. Sinergistically and cooperatively, these processes regulate gene expression by changing chromatin organization and DNA accessibility. Such induced epigenetic changes can be inherited during cell division, resulting in permanent maintenance of the acquired phenotype, but they may also occur throughout an individual life-course and may ultimately influence phenotypic outcomes (health and disease risk). In the last decade, a number of studies have shown that nutrients can affect metabolic traits by altering the structure of chromatin and directly regulate both transcription and translational processes. In this context, dietary polyphenol-targeted epigenetics becomes an attractive approach for disease prevention and intervention. Here, we will review how polyphenols, including flavonoids, curcuminoids, and stilbenes, modulate the establishment and maintenance of key epigenetic marks, thereby influencing gene expression and, hence, disease risk and health.

  12. Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin

    PubMed Central

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

    2016-01-01

    SUMMARY 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, and 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 become more accessible to telomere-associated proteins and accumulate DDR signals. 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

  13. Processing of DNA double strand breaks by alternative non-homologous end-joining in hyperacetylated chromatin.

    PubMed

    Manova, Vasilissa; Singh, Satyendra K; Iliakis, George

    2012-08-22

    Mammalian cells employ at least two subpathways of non-homologous end-joining for the repair of ionizing radiation induced DNA double strand breaks: The canonical DNA-PK-dependent form of non-homologous end-joining (D-NHEJ) and an alternative, slowly operating, error-prone backup pathway (B-NHEJ). In contrast to D-NHEJ, which operates with similar efficiency throughout the cell cycle, B-NHEJ operates more efficiently in G2-phase. Notably, B-NHEJ also shows strong and as of yet unexplained dependency on growth activity and is markedly compromised in serum-deprived cells, or in cells that enter the plateau-phase of growth. The molecular mechanisms underpinning this response remain unknown. Since chromatin structure or changes in chromatin structure are prime candidate-B-NHEJ-modulators, we study here the role of chromatin hyperacetylation, either by HDAC2 knockdown or treatment with the HDAC inhibitor TSA, on the repair by B-NHEJ of IR-induced DSBs. siRNA-mediated knockdown of HDAC2 fails to provoke histone hyperacetylation in Lig4-/- MEFs and has no detectable effect on B-NHEJ function. Treatment with TSA that inhibits multiple HDACs causes efficient, reversible chromatin hyperacetylation in Lig4-/- MEFs, as well as in human HCT116 Lig4-/- cells and the human glioma cell line M059K. The IR yield of DSBs in TSA-treated cells remains similar to that of untreated cells despite the expected chromatin relaxation. In addition, chromatin hyperacetylation leaves unchanged repair of DSBs by B-NHEJ in irradiated exponentially growing, or plateau-phase cells. Notably, under the experimental conditions employed here, chromatin hyperacetylation fails to detectably modulate B-NHEJ in M059K cells as well. In summary, the results show that chromatin acetylation or deacetylation does not affect the kinetics of alternative NHEJ in all types of cells examined both in exponentially growing and serum deprived cultures. We conclude that parameters beyond chromatin acetylation determine B

  14. Processing of DNA double strand breaks by alternative non-homologous end-joining in hyperacetylated chromatin

    PubMed Central

    2012-01-01

    Background Mammalian cells employ at least two subpathways of non-homologous end-joining for the repair of ionizing radiation induced DNA double strand breaks: The canonical DNA-PK-dependent form of non-homologous end-joining (D-NHEJ) and an alternative, slowly operating, error-prone backup pathway (B-NHEJ). In contrast to D-NHEJ, which operates with similar efficiency throughout the cell cycle, B-NHEJ operates more efficiently in G2-phase. Notably, B-NHEJ also shows strong and as of yet unexplained dependency on growth activity and is markedly compromised in serum-deprived cells, or in cells that enter the plateau-phase of growth. The molecular mechanisms underpinning this response remain unknown. Since chromatin structure or changes in chromatin structure are prime candidate-B-NHEJ-modulators, we study here the role of chromatin hyperacetylation, either by HDAC2 knockdown or treatment with the HDAC inhibitor TSA, on the repair by B-NHEJ of IR-induced DSBs. Results siRNA-mediated knockdown of HDAC2 fails to provoke histone hyperacetylation in Lig4-/- MEFs and has no detectable effect on B-NHEJ function. Treatment with TSA that inhibits multiple HDACs causes efficient, reversible chromatin hyperacetylation in Lig4-/- MEFs, as well as in human HCT116 Lig4-/- cells and the human glioma cell line M059K. The IR yield of DSBs in TSA-treated cells remains similar to that of untreated cells despite the expected chromatin relaxation. In addition, chromatin hyperacetylation leaves unchanged repair of DSBs by B-NHEJ in irradiated exponentially growing, or plateau-phase cells. Notably, under the experimental conditions employed here, chromatin hyperacetylation fails to detectably modulate B-NHEJ in M059K cells as well. Conclusions In summary, the results show that chromatin acetylation or deacetylation does not affect the kinetics of alternative NHEJ in all types of cells examined both in exponentially growing and serum deprived cultures. We conclude that parameters beyond

  15. Sperm Chromatin Immaturity Observed in Short Abstinence Ejaculates Affects DNA Integrity and Longevity In Vitro

    PubMed Central

    Salian, Sujith Raj; Kumar, Dayanidhi; Singh, Vikram Jeet; D’Souza, Fiona; Kalthur, Guruprasad; Kamath, Asha; Adiga, Satish Kumar

    2016-01-01

    Background The influence of ejaculatory abstinence (EA) on semen parameters and subsequent reproductive outcome is still debatable; hence understanding the impact of EA on sperm structural and functional integrity may provide a valuable information on predicting successful clinical outcome. Objective To understand the influence of EA on sperm chromatin maturity, integrity, longevity and global methylation status. Methods This experimental prospective study included 76 ejaculates from 19 healthy volunteers who provided ejaculates after observing 1, 3, 5 and 7 days of abstinence. Sperm chromatin maturity, DNA integrity and global methylation status were assessed in the neat ejaculate. Sperm motility, DNA integrity and longevity were assessed in the processed fraction of the fresh and frozen-thawed ejaculates to determine their association with the length of EA. Results Spermatozoa from 1 day ejaculatory abstinence (EA-1) displayed significantly higher level of sperm chromatin immaturity in comparison to EA-3 (P < 0.05) and EA-5 (P < 0.01) whereas; the number of 5-methyl cytosine immunostained spermatozoa did not vary significantly across groups. On the other hand, in vitro incubation of processed ejaculate from EA-1 resulted in approximately 20 and 40 fold increase in the DNA fragmented spermatozoa at the end of 6 and 24h respectively (P < 0.01–0.001). Conclusion Use of short-term EA for therapeutic fertilization would be a clinically valuable strategy to improve the DNA quality. However, use of such spermatozoa after prolonged incubation in vitro should be avoided as it can carry a substantial risk of transmitting DNA fragmentation to the oocytes. PMID:27043437

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  17. Chromatin Configuration Determines Cell Responses to Hormone Stimuli | Center for Cancer Research

    Cancer.gov

    Ever since selective gene expression was established as the central driver of cell behavior, researchers have been working to understand the forces that control gene transcription. Aberrant gene expression can cause or promote many diseases, including cancer, and alterations in gene expression are the goal of many therapeutic agents. Recent work has focused on the potential role of chromatin structure as a contributor to gene regulation. Chromatin can exist in a tightly packed/inaccessible or loose/accessible configuration depending on the interactions between DNA and its associated proteins. Patterns of chromatin structure can differ between cell types and can also change within cells in response to certain signals. Cancer researchers are particularly interested in the role of chromatin in gene regulation because many of the genomic regions found to be associated with cancer risk are in open chromatin structures.

  18. The polymorphisms of the chromatin fiber

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  19. Chromatin immunoprecipitation with fixed animal tissues and preparation for high-throughput sequencing.

    PubMed

    Cotney, Justin L; Noonan, James P

    2015-02-02

    Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) is a powerful method used to identify genome-wide binding patterns of transcription factors and distribution of various histone modifications associated with different chromatin states. In most published studies, ChIP-Seq has been performed on cultured cells grown under controlled conditions, allowing generation of large amounts of material in a homogeneous biological state. Although such studies have provided great insight into the dynamic landscapes of animal genomes, they do not allow the examination of transcription factor binding and chromatin states in adult tissues, developing embryonic structures, or tumors. Such knowledge is critical to understanding the information required to create and maintain a complex biological tissue and to identify noncoding regions of the genome directly involved in tissues affected by complex diseases such as autism. Studying these tissue types with ChIP-Seq can be challenging due to the limited availability of tissues and the lack of complex biological states able to be achieved in culture. These inherent differences require alterations of standard cross-linking and chromatin extraction typically used in cell culture. Here we describe a general approach for using small amounts of animal tissue to perform ChIP-Seq directed at histone modifications and transcription factors. Tissue is homogenized before treatment with formaldehyde to ensure proper cross-linking, and a two-step nuclear isolation is performed to increase extraction of soluble chromatin. Small amounts of soluble chromatin are then used for immunoprecipitation (IP) and prepared for multiplexed high-throughput sequencing. © 2015 Cold Spring Harbor Laboratory Press.

  20. Structural organization of chromatin during the cell cycle of Entamoeba histolytica trophozoites.

    PubMed

    Argüello, C; Valenzuela, B; Rangel, E

    1992-01-01

    The nuclear division of E. histolytica trophozoites was analyzed by using specific stains for DNA, with the aim to define the sequential changes of chromatin during its life cycle. Furthermore, we characterized the internal structural arrangements of microtubules in the microtubular organizing center (MTOC) and determined the number of chromosomes and its association with the spindle. The MTOC is formed by multiple microtubule-nucleating centers, that are involved in the displacement of DNA during nuclear division. We found the existence of a single MTOC in one pole of the nucleus at early anaphase. Our results lead us to propose a new hypothesis in which it is suggested that metaphase corresponds to the arrangement of condensed DNA bodies, or "chromosomes" around the MTOC and, through the assembly of microtubules, one set of uncondensed chromatin is displaced to the opposite pole of the nucleus, while the other remains condensed and associated to the original MTOC. We observed six chromosomes in our preparations, corroborating previous observations (2,3). Whether or not a new MTOC is formed during nuclear division remains to be clarified.

  1. TDP-43 Promotes Neurodegeneration by Impairing Chromatin Remodeling.

    PubMed

    Berson, Amit; Sartoris, Ashley; Nativio, Raffaella; Van Deerlin, Vivianna; Toledo, Jon B; Porta, Sílvia; Liu, Shichong; Chung, Chia-Yu; Garcia, Benjamin A; Lee, Virginia M-Y; Trojanowski, John Q; Johnson, F Brad; Berger, Shelley L; Bonini, Nancy M

    2017-12-04

    Regulation of chromatin structure is critical for brain development and function. However, the involvement of chromatin dynamics in neurodegeneration is less well understood. Here we find, launching from Drosophila models of amyotrophic lateral sclerosis and frontotemporal dementia, that TDP-43 impairs the induction of multiple key stress genes required to protect from disease by reducing the recruitment of the chromatin remodeler Chd1 to chromatin. Chd1 depletion robustly enhances TDP-43-mediated neurodegeneration and promotes the formation of stress granules. Conversely, upregulation of Chd1 restores nucleosomal dynamics, promotes normal induction of protective stress genes, and rescues stress sensitivity of TDP-43-expressing animals. TDP-43-mediated impairments are conserved in mammalian cells, and, importantly, the human ortholog CHD2 physically interacts with TDP-43 and is strikingly reduced in level in temporal cortex of human patient tissue. These findings indicate that TDP-43-mediated neurodegeneration causes impaired chromatin dynamics that prevents appropriate expression of protective genes through compromised function of the chromatin remodeler Chd1/CHD2. Enhancing chromatin dynamics may be a treatment approach to amyotrophic lateral scleorosis (ALS)/frontotemporal dementia (FTD). Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Cooperativeness of the higher chromatin structure of the beta-globin locus revealed by the deletion mutations of DNase I hypersensitive site 3 of the LCR.

    PubMed

    Fang, Xiangdong; Xiang, Ping; Yin, Wenxuan; Stamatoyannopoulos, George; Li, Qiliang

    2007-01-05

    High-level transcription of the globin genes requires the enhancement by a distant element, the locus control region (LCR). Such long-range regulation in vivo involves spatial interaction between transcriptional elements, with intervening chromatin looping out. It has been proposed that the clustering of the HS sites of the LCR, the active globin genes, as well as the remote 5' hypersensitive sites (HSs) (HS-60/-62 in mouse, HS-110 in human) and 3'HS1 forms a specific spatial chromatin structure, termed active chromatin hub (ACH). Here we report the effects of the HS3 deletions of the LCR on the spatial chromatin structure of the beta-globin locus as revealed by the chromatin conformation capture (3C) technology. The small HS3 core deletion (0.23 kb), but not the large HS3 deletion (2.3 kb), disrupted the spatial interactions among all the HS sites of the LCR, the beta-globin gene and 3'HS1. We have previously demonstrated that the large HS3 deletion barely impairs the structure of the LCR holocomplex, while the structure is significantly disrupted by the HS3 core deletion. Taken together, these results suggest that the formation of the ACH is dependent on a largely intact LCR structure. We propose that the ACH indeed is an extension of the LCR holocomplex.

  3. Chromatin-unstable boar spermatozoa have little chance of reaching oocytes in vivo.

    PubMed

    Ardón, Florencia; Helms, Dietmar; Sahin, Evrim; Bollwein, Heinrich; Töpfer-Petersen, Edda; Waberski, Dagmar

    2008-04-01

    In the present study, the prevalence of chromatin instability in the fertilizing-competent sperm population in the porcine oviduct in vivo was examined through qualitative analysis of the chromatin structure status of accessory boar sperm found in in vivo-derived embryos. The binding of chromatin-unstable sperm to oviductal epithelium in vitro was also studied. To examine the sperm chromatin state, a modified fluorescence microscopic sperm chromatin structure assay was used. Among a population of 173 fertile boars, individuals were selected for according to their chromatin status: 25 animals showed more than 5% of chromatin-unstable sperm in their ejaculates, and 7 showed consistently elevated percentages of chromatin-unstable sperm in three successively collected semen samples. A positive correlation was found between incidence of chromatin instability and attached cytoplasmic droplets (r=0.44, P<0.01). Analyses of accessory spermatozoa from in vivo-derived embryos demonstrated that the proportion of chromatin-unstable sperm was significantly (P<0.05) reduced in the population of fertilizing-competent sperm in the oviduct compared with the inseminated sperm. Populations of sperm bound to the oviduct in vitro had significantly (P<0.05) lower percentages of chromatin instability than in the original diluted semen sample. In conclusion, numbers of sperm with unstable chromatin are reduced in the oviductal sperm reservoir, possibly because of associated changes in the plasma membrane that prevent sperm from binding to the oviductal epithelium. We conclude that in vivo the likelihood that sperm with unstable chromatin will reach the egg and fertilize it is low.

  4. Chromatin Switches during Neural Cell Differentiation and Their Dysregulation by Prenatal Alcohol Exposure.

    PubMed

    Gavin, David P; Grayson, Dennis R; Varghese, Sajoy P; Guizzetti, Marina

    2017-05-11

    Prenatal alcohol exposure causes persistent neuropsychiatric deficits included under the term fetal alcohol spectrum disorders (FASD). Cellular identity emerges from a cascade of intrinsic and extrinsic (involving cell-cell interactions and signaling) processes that are partially initiated and maintained through changes in chromatin structure. Prenatal alcohol exposure influences neuronal and astrocyte development, permanently altering brain connectivity. Prenatal alcohol exposure also alters chromatin structure through histone and DNA modifications. However, the data linking alcohol-induced differentiation changes with developmental alterations in chromatin structure remain to be elucidated. In the first part of this review, we discuss the sequence of chromatin structural changes involved in neural cell differentiation during normal development. We then discuss the effects of prenatal alcohol on developmental histone modifications and DNA methylation in the context of neurogenesis and astrogliogenesis. We attempt to synthesize the developmental literature with the FASD literature, proposing that alcohol-induced changes to chromatin structure account for altered neurogenesis and astrogliogenesis as well as altered neuron and astrocyte differentiation. Together these changes may contribute to the cognitive and behavioral abnormalities in FASD. Future studies using standardized alcohol exposure paradigms at specific developmental stages will advance the understanding of how chromatin structural changes impact neural cell fate and maturation in FASD.

  5. Chromatin Switches during Neural Cell Differentiation and Their Dysregulation by Prenatal Alcohol Exposure

    PubMed Central

    Gavin, David P.; Grayson, Dennis R.; Varghese, Sajoy P.; Guizzetti, Marina

    2017-01-01

    Prenatal alcohol exposure causes persistent neuropsychiatric deficits included under the term fetal alcohol spectrum disorders (FASD). Cellular identity emerges from a cascade of intrinsic and extrinsic (involving cell-cell interactions and signaling) processes that are partially initiated and maintained through changes in chromatin structure. Prenatal alcohol exposure influences neuronal and astrocyte development, permanently altering brain connectivity. Prenatal alcohol exposure also alters chromatin structure through histone and DNA modifications. However, the data linking alcohol-induced differentiation changes with developmental alterations in chromatin structure remain to be elucidated. In the first part of this review, we discuss the sequence of chromatin structural changes involved in neural cell differentiation during normal development. We then discuss the effects of prenatal alcohol on developmental histone modifications and DNA methylation in the context of neurogenesis and astrogliogenesis. We attempt to synthesize the developmental literature with the FASD literature, proposing that alcohol-induced changes to chromatin structure account for altered neurogenesis and astrogliogenesis as well as altered neuron and astrocyte differentiation. Together these changes may contribute to the cognitive and behavioral abnormalities in FASD. Future studies using standardized alcohol exposure paradigms at specific developmental stages will advance the understanding of how chromatin structural changes impact neural cell fate and maturation in FASD. PMID:28492482

  6. Dose-dependent alcohol-induced alterations in chromatin structure persist beyond the window of exposure and correlate with fetal alcohol syndrome birth defects.

    PubMed

    Veazey, Kylee J; Parnell, Scott E; Miranda, Rajesh C; Golding, Michael C

    2015-01-01

    In recent years, we have come to recognize that a multitude of in utero exposures have the capacity to induce the development of congenital and metabolic defects. As most of these encounters manifest their effects beyond the window of exposure, deciphering the mechanisms of teratogenesis is incredibly difficult. For many agents, altered epigenetic programming has become suspect in transmitting the lasting signature of exposure leading to dysgenesis. However, while several chemicals can perturb chromatin structure acutely, for many agents (particularly alcohol) it remains unclear if these modifications represent transient responses to exposure or heritable lesions leading to pathology. Here, we report that mice encountering an acute exposure to alcohol on gestational Day-7 exhibit significant alterations in chromatin structure (histone 3 lysine 9 dimethylation, lysine 9 acetylation, and lysine 27 trimethylation) at Day-17, and that these changes strongly correlate with the development of craniofacial and central nervous system defects. Using a neural cortical stem cell model, we find that the epigenetic changes arising as a consequence of alcohol exposure are heavily dependent on the gene under investigation, the dose of alcohol encountered, and that the signatures arising acutely differ significantly from those observed after a 4-day recovery period. Importantly, the changes observed post-recovery are consistent with those modeled in vivo, and associate with alterations in transcripts encoding multiple homeobox genes directing neurogenesis. Unexpectedly, we do not observe a correlation between alcohol-induced changes in chromatin structure and alterations in transcription. Interestingly, the majority of epigenetic changes observed occur in marks associated with repressive chromatin structure, and we identify correlative disruptions in transcripts encoding Dnmt1, Eed, Ehmt2 (G9a), EzH2, Kdm1a, Kdm4c, Setdb1, Sod3, Tet1 and Uhrf1. These observations suggest that the

  7. Osmotic modulation of chromatin impacts on efficiency and kinetics of cell fate modulation.

    PubMed

    Lima, A F; May, G; Colunga, J; Pedreiro, S; Paiva, A; Ferreira, L; Enver, T; Iborra, F J; Pires das Neves, R

    2018-05-08

    Chromatin structure is a major regulator of transcription and gene expression. Herein we explore the use of osmotic modulation to modify the chromatin structure and reprogram gene expression. In this study we use the extracellular osmotic pressure as a chromatin structure and transcriptional modulator. Hyposmotic modulation promotes chromatin loosening and induces changes in RNA polymerase II (Pol II) activity. The chromatin decondensation opens space for higher amounts of DNA engaged RNA Pol II. Hyposmotic modulation constitutes an alternative route to manipulate cell fate decisions. This technology was tested in model protocols of induced pluripotency and transdifferentiation in cells growing in suspension and adherent to substrates, CD34 + umbilical-cord-blood (UCB), fibroblasts and B-cells. The efficiency and kinetics of these cell fate modulation processes were improved by transient hyposmotic modulation of the cell environment.

  8. Dependence of the Linker Histone and Chromatin Condensation on the Nucleosome Environment.

    PubMed

    Perišić, Ognjen; Schlick, Tamar

    2017-08-24

    The linker histone (LH), an auxiliary protein that can bind to chromatin and interact with the linker DNA to form stem motifs, is a key element of chromatin compaction. By affecting the chromatin condensation level, it also plays an active role in gene expression. However, the presence and variable concentration of LH in chromatin fibers with different DNA linker lengths indicate that its folding and condensation are highly adaptable and dependent on the immediate nucleosome environment. Recent experimental studies revealed that the behavior of LH in mononucleosomes markedly differs from that in small nucleosome arrays, but the associated mechanism is unknown. Here we report a structural analysis of the behavior of LH in mononucleosomes and oligonucleosomes (2-6 nucleosomes) using mesoscale chromatin simulations. We show that the adapted stem configuration heavily depends on the strength of electrostatic interactions between LH and its parental DNA linkers, and that those interactions tend to be asymmetric in small oligonucleosome systems. Namely, LH in oligonucleosomes dominantly interacts with one DNA linker only, as opposed to mononucleosomes where LH has similar interactions with both linkers and forms a highly stable nucleosome stem. Although we show that the LH condensation depends sensitively on the electrostatic interactions with entering and exiting DNA linkers, other interactions, especially by nonparental cores and nonparental linkers, modulate the structural condensation by softening LH and thus making oligonucleosomes more flexible, in comparison to to mono- and dinucleosomes. We also find that the overall LH/chromatin interactions sensitively depend on the linker length because the linker length determines the maximal nucleosome stem length. For mononucleosomes with DNA linkers shorter than LH, LH condenses fully, while for DNA linkers comparable or longer than LH, the LH extension in mononucleosomes strongly follows the length of DNA linkers

  9. Heritable Individual-Specific and Allele-Specific Chromatin Signatures in Humans

    PubMed Central

    McDaniell, Ryan; Lee, Bum-Kyu; Song, Lingyun; Liu, Zheng; Boyle, Alan P.; Erdos, Michael R.; Scott, Laura J.; Morken, Mario A.; Kucera, Katerina S.; Battenhouse, Anna; Keefe, Damian; Collins, Francis S.; Willard, Huntington F.; Lieb, Jason D.; Furey, Terrence S.; Crawford, Gregory E.; Iyer, Vishwanath R.; Birney, Ewan

    2010-01-01

    The extent to which variation in chromatin structure and transcription factor binding may influence gene expression, and thus underlie or contribute to variation in phenotype, is unknown. To address this question, we cataloged both individual-to-individual variation and differences between homologous chromosomes within the same individual (allele-specific variation) in chromatin structure and transcription factor binding in lymphoblastoid cells derived from individuals of geographically diverse ancestry. Ten percent of active chromatin sites were individual-specific; a similar proportion were allele-specific. Both individual-specific and allele-specific sites were commonly transmitted from parent to child, which suggests that they are heritable features of the human genome. Our study shows that heritable chromatin status and transcription factor binding differ as a result of genetic variation and may underlie phenotypic variation in humans. PMID:20299549

  10. General method for rapid purification of native chromatin fragments.

    PubMed

    Kuznetsov, Vyacheslav I; Haws, Spencer A; Fox, Catherine A; Denu, John M

    2018-05-24

    Biochemical, proteomic and epigenetic studies of chromatin rely on the efficient ability to isolate native nucleosomes in high yield and purity. However, isolation of native chromatin suitable for many downstream experiments remains a challenging task. This is especially true for the budding yeast Saccharomyces cerevisiae, which continues to serve as an important model organism for the study of chromatin structure and function. Here, we developed a time- and cost-efficient universal protocol for isolation of native chromatin fragments from yeast, insect, and mammalian cells. The resulting protocol preserves histone posttranslational modification in the native chromatin state, and is applicable for both parallel multi-sample spin-column purification and large scale isolation. This protocol is based on the efficient and stable purification of polynucleosomes, features a combination of optimized cell lysis and purification conditions, three options for chromatin fragmentation, and a novel ion-exchange chromatographic purification strategy.  The procedure will aid chromatin researchers interested in isolating native chromatin material for biochemical studies, and as a mild, acid- and detergent-free sample preparation method for mass-spectrometry analysis. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

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

    PubMed

    Linnemann, Amelia K; Krawetz, Stephen A

    2009-01-01

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

  12. Activation of the alpha-globin gene expression correlates with dramatic upregulation of nearby non-globin genes and changes in local and large-scale chromatin spatial structure.

    PubMed

    Ulianov, Sergey V; Galitsyna, Aleksandra A; Flyamer, Ilya M; Golov, Arkadiy K; Khrameeva, Ekaterina E; Imakaev, Maxim V; Abdennur, Nezar A; Gelfand, Mikhail S; Gavrilov, Alexey A; Razin, Sergey V

    2017-07-11

    In homeotherms, the alpha-globin gene clusters are located within permanently open genome regions enriched in housekeeping genes. Terminal erythroid differentiation results in dramatic upregulation of alpha-globin genes making their expression comparable to the rRNA transcriptional output. Little is known about the influence of the erythroid-specific alpha-globin gene transcription outburst on adjacent, widely expressed genes and large-scale chromatin organization. Here, we have analyzed the total transcription output, the overall chromatin contact profile, and CTCF binding within the 2.7 Mb segment of chicken chromosome 14 harboring the alpha-globin gene cluster in cultured lymphoid cells and cultured erythroid cells before and after induction of terminal erythroid differentiation. We found that, similarly to mammalian genome, the chicken genomes is organized in TADs and compartments. Full activation of the alpha-globin gene transcription in differentiated erythroid cells is correlated with upregulation of several adjacent housekeeping genes and the emergence of abundant intergenic transcription. An extended chromosome region encompassing the alpha-globin cluster becomes significantly decompacted in differentiated erythroid cells, and depleted in CTCF binding and CTCF-anchored chromatin loops, while the sub-TAD harboring alpha-globin gene cluster and the upstream major regulatory element (MRE) becomes highly enriched with chromatin interactions as compared to lymphoid and proliferating erythroid cells. The alpha-globin gene domain and the neighboring loci reside within the A-like chromatin compartment in both lymphoid and erythroid cells and become further segregated from the upstream gene desert upon terminal erythroid differentiation. Our findings demonstrate that the effects of tissue-specific transcription activation are not restricted to the host genomic locus but affect the overall chromatin structure and transcriptional output of the encompassing

  13. The effect of cigarette smoking on human seminal parameters, sperm chromatin structure and condensation.

    PubMed

    Mostafa, R M; Nasrallah, Y S; Hassan, M M; Farrag, A F; Majzoub, A; Agarwal, A

    2018-04-01

    Considerable debate still exists regarding the effects of cigarette smoking on male fertility. This work aimed to explore effects of cigarette smoking on semen parameters and DNA fragmentation on 95 infertile patients who were divided into infertile male nonsmokers (45) and infertile male smokers (50). Smokers were subdivided according to a number of cigarettes smoked per day into mild (≤10), moderate (11-20) and heavy smokers (≥21). Semen analysis, sperm chromatin condensation integrity with aniline blue staining and sperm viability were compared between the study groups. A significant decrease has been shown in sperm count (p = .006), progressive motility (p = <.001), percentage of normal forms (p = <.001) and viability (p = .002) between infertile nonsmoker and infertile smokers. The percentage of abnormal sperm chromatin condensation was significantly higher in smokers compared to nonsmokers (p = <.001). A linear correlation was detected between the extent of cigarette smoking and the degree of worsening in progressive motility (p = .001), total motility (p < .001), viability (p < .001) and normal morphology (p < .001). These results indicate that cigarette smoking has detrimental effects on semen parameters. It negatively affected all conventional semen parameters in addition to sperm chromatin condensation and sperm viability. These abnormalities were also proportional to the number of cigarettes smoked per day and to the duration of smoking. © 2017 Blackwell Verlag GmbH.

  14. In silico approaches reveal the potential for DNA sequence-dependent histone octamer affinity to influence chromatin structure in vivo.

    PubMed

    Fraser, Ross M; Allan, James; Simmen, Martin W

    2006-12-08

    Nucleosome positioning signals embedded within the DNA sequence have the potential to influence the detailed structure of the higher-order chromatin fibre. In two previous studies of long stretches of DNA, encompassing the chicken beta-globin and ovine beta-lactoglobulin genes, respectively, we mapped the relative affinity of every site for the core histone octamer. In both cases a periodic arrangement of the in vitro positioning sites suggests that they might influence the folding of a nucleosome chain into higher-order structure; this hypothesis was borne out in the case of the beta-lactoglobulin gene, where the distribution of the in vitro positioning sites is related to the positions nucleosomes actually occupy in sheep liver cells. Here, we have exploited the in vitro nucleosome positioning datasets to simulate nucleosomal organisation using in silico approaches. We use the high-resolution, quantitative positioning maps to define a one-dimensional positioning energy lattice, which can be populated with a defined number of nucleosomes. Monte Carlo techniques are employed to simulate the behaviour of the model at equilibrium to produce a set of configurations, which provide a probability-based occupancy map. Employing a variety of techniques we show that the occupancy maps are a sensitive function of the histone octamer density (nucleosome repeat length) and find that a minimal change in this property can produce dramatic localised changes in structure. Although simulations generally give rise to regular periodic nucleosomal arrangements, they often show octamer density-dependent discontinuities, which tend to co-localise with sequences that adopt distinctive chromatin structure in vivo. Furthermore, the overall organisation of simulated chromatin structures are more closely related to the situation in vivo than is the original in vitro positioning data, particularly at a nucleosome density corresponding to the in vivo state. Although our model is simplified, we

  15. Ectopically tethered CP190 induces large-scale chromatin decondensation

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

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

    PubMed

    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.

  17. DNA repair goes hip-hop: SMARCA and CHD chromatin remodellers join the break dance.

    PubMed

    Rother, Magdalena B; van Attikum, Haico

    2017-10-05

    Proper signalling and repair of DNA double-strand breaks (DSB) is critical to prevent genome instability and diseases such as cancer. The packaging of DNA into chromatin, however, has evolved as a mere obstacle to these DSB responses. Posttranslational modifications and ATP-dependent chromatin remodelling help to overcome this barrier by modulating nucleosome structures and allow signalling and repair machineries access to DSBs in chromatin. Here we recap our current knowledge on how ATP-dependent SMARCA- and CHD-type chromatin remodellers alter chromatin structure during the signalling and repair of DSBs and discuss how their dysfunction impacts genome stability and human disease.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Authors.

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

    PubMed Central

    Linnemann, Amelia K.; Krawetz, Stephen A.

    2010-01-01

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

  19. Chromatin-Specific Regulation of Mammalian rDNA Transcription by Clustered TTF-I Binding Sites

    PubMed Central

    Diermeier, Sarah D.; Németh, Attila; Rehli, Michael; Grummt, Ingrid; Längst, Gernot

    2013-01-01

    Enhancers and promoters often contain multiple binding sites for the same transcription factor, suggesting that homotypic clustering of binding sites may serve a role in transcription regulation. Here we show that clustering of binding sites for the transcription termination factor TTF-I downstream of the pre-rRNA coding region specifies transcription termination, increases the efficiency of transcription initiation and affects the three-dimensional structure of rRNA genes. On chromatin templates, but not on free rDNA, clustered binding sites promote cooperative binding of TTF-I, loading TTF-I to the downstream terminators before it binds to the rDNA promoter. Interaction of TTF-I with target sites upstream and downstream of the rDNA transcription unit connects these distal DNA elements by forming a chromatin loop between the rDNA promoter and the terminators. The results imply that clustered binding sites increase the binding affinity of transcription factors in chromatin, thus influencing the timing and strength of DNA-dependent processes. PMID:24068958

  20. Chromatin regulators as a guide for cancer treatment choice

    PubMed Central

    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

    2016-01-01

    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 impact resistance mechanisms. Here, we explore how the mis-expression of chromatin regulators—factors involved in the establishment and maintenance of functional chromatin domains—can inform about the extent of docetaxel response. We exploit gene Affymetrix and NanoString gene expression data for a set of chromatin regulators generated from breast cancer patient-derived xenograft (PDX) 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 towards chromatin regulators as biomarkers for drug response as well as therapeutic targets to sensitize patients towards docetaxel and combat drug resistance. PMID:27196757

  1. [Correcting influence of vitamin E short chain derivatives on lipid peroxidation, liver cell membrane, and chromatin structure when rats are exposed to embichin].

    PubMed

    Kovalenko, V M; Byshovets', T F; Hubs'kyĭ, Iu I; Levyts'kyĭ, Ie L; Shaiakhmetova, H M; Marchenko, O M; Voloshyna, O S; Saĭfetdinova, H A; Okhrimenko, V O; Donchenko, H V

    2000-01-01

    Embikhin causes activation of LPO processes in endoplasmic reticulum and in nuclear chromatine fractions of rat liver cells. The latter is accompanied by the impairment of repressive and active nuclear chromatine fractions structure. Derivate of vitamin E in these conditions renders correcting action on parameters of lipid peroxidation in the investigated subcellular structures, testifying its positive influence on the cell heredity apparatus state. The normalizing action of tocopherol derivative on cytochromes P450 and b5 levels is shown.

  2. Micron-scale coherence in interphase chromatin dynamics

    PubMed Central

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

    2013-01-01

    Chromatin structure and dynamics control all aspects of DNA biology yet are poorly understood, especially at large length scales. We developed an approach, displacement correlation spectroscopy based on time-resolved image correlation analysis, to map chromatin dynamics simultaneously across the whole nucleus in cultured human cells. This method revealed that chromatin movement was coherent across large regions (4–5 µm) for several seconds. Regions of coherent motion extended beyond the boundaries of single-chromosome territories, suggesting elastic coupling of motion over length scales much larger than those of genes. These large-scale, coupled motions were ATP dependent and unidirectional for several seconds, perhaps accounting for ATP-dependent directed movement of single genes. Perturbation of major nuclear ATPases such as DNA polymerase, RNA polymerase II, and topoisomerase II eliminated micron-scale coherence, while causing rapid, local movement to increase; i.e., local motions accelerated but became uncoupled from their neighbors. We observe similar trends in chromatin dynamics upon inducing a direct DNA damage; thus we hypothesize that this may be due to DNA damage responses that physically relax chromatin and block long-distance communication of forces. PMID:24019504

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Persson, Jenna; Ekwall, Karl, E-mail: karl.ekwall@ki.se; School of Life Sciences, University College Sodertorn, NOVUM, Huddinge

    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 canmore » 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.« less

  4. DNA Looping Facilitates Targeting of a Chromatin Remodeling Enzyme

    PubMed Central

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

    2013-01-01

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

  5. Genetic Rearrangements Can Modify Chromatin Features at Epialleles

    PubMed Central

    Foerster, Andrea M.; Dinh, Huy Q.; Sedman, Laura; Wohlrab, Bonnie; Mittelsten Scheid, Ortrun

    2011-01-01

    Analogous to genetically distinct alleles, epialleles represent heritable states of different gene expression from sequence-identical genes. Alleles and epialleles both contribute to phenotypic heterogeneity. While alleles originate from mutation and recombination, the source of epialleles is less well understood. We analyze active and inactive epialleles that were found at a transgenic insert with a selectable marker gene in Arabidopsis. Both converse expression states are stably transmitted to progeny. The silent epiallele was previously shown to change its state upon loss-of-function of trans-acting regulators and drug treatments. We analyzed the composition of the epialleles, their chromatin features, their nuclear localization, transcripts, and homologous small RNA. After mutagenesis by T-DNA transformation of plants carrying the silent epiallele, we found new active alleles. These switches were associated with different, larger or smaller, and non-overlapping deletions or rearrangements in the 3′ regions of the epiallele. These cis-mutations caused different degrees of gene expression stability depending on the nature of the sequence alteration, the consequences for transcription and transcripts, and the resulting chromatin organization upstream. This illustrates a tight dependence of epigenetic regulation on local structures and indicates that sequence alterations can cause epigenetic changes at some distance in regions not directly affected by the mutation. Similar effects may also be involved in gene expression and chromatin changes in the vicinity of transposon insertions or excisions, recombination events, or DNA repair processes and could contribute to the origin of new epialleles. PMID:22028669

  6. Minor Groove Binder Distamycin Remodels Chromatin but Inhibits Transcription

    PubMed Central

    Majumder, Parijat; Banerjee, Amrita; Shandilya, Jayasha; Senapati, Parijat; Chatterjee, Snehajyoti; Kundu, Tapas K.; Dasgupta, Dipak

    2013-01-01

    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. PMID:23460895

  7. The nucleosome: orchestrating DNA damage signaling and repair within chromatin.

    PubMed

    Agarwal, Poonam; Miller, Kyle M

    2016-10-01

    DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling events that extensively modulates chromatin structure and organization to coordinate DDR factor recruitment to the break and repair, whilst also promoting the maintenance of normal chromatin functions within the damaged region. For example, DDR pathways must avoid conflicts between other DNA-based processes that function within the context of chromatin, including transcription and replication. The molecular mechanisms governing the recognition, target specificity, and recruitment of DDR factors and enzymes to the fundamental repeating unit of chromatin, i.e., the nucleosome, are poorly understood. Here we present our current view of how chromatin recognition by DDR factors is achieved at the level of the nucleosome. Emerging evidence suggests that the nucleosome surface, including the nucleosome acidic patch, promotes the binding and activity of several DNA damage factors on chromatin. Thus, in addition to interactions with damaged DNA and histone modifications, nucleosome recognition by DDR factors plays a key role in orchestrating the requisite chromatin response to maintain both genome and epigenome integrity.

  8. Physical constraints in the condensation of eukaryotic chromosomes. Local concentration of DNA versus linear packing ratio in higher order chromatin structures.

    PubMed

    Daban, J R

    2000-04-11

    The local concentration of DNA in metaphase chromosomes of different organisms has been determined in several laboratories. The average of these measurements is 0.17 g/mL. In the first level of chromosome condensation, DNA is wrapped around histones forming nucleosomes. This organization limits the DNA concentration in nucleosomes to 0. 3-0.4 g/mL. Furthermore, in the structural models suggested in different laboratories for the 30-40 nm chromatin fiber, the estimated DNA concentration is significantly reduced; it ranges from 0.04 to 0.27 g/mL. The DNA concentration is further reduced when the fiber is folded into the successive higher order structures suggested in different models for metaphase chromosomes; the estimated minimum decrease of DNA concentration represents an additional 40%. These observations suggest that most of the models proposed for the 30-40 nm chromatin fiber are not dense enough for the construction of metaphase chromosomes. In contrast, it is well-known that the linear packing ratio increases dramatically in each level of DNA folding in chromosomes. Thus, the consideration of the linear packing ratio is not enough for the study of chromatin condensation; the constraint resulting from the actual DNA concentration in metaphase chromosomes must be considered for the construction of models for condensed chromatin.

  9. Chromatin organization and radio resistance in the bacterium Gemmata obscuriglobus.

    PubMed

    Lieber, Arnon; Leis, Andrew; Kushmaro, Ariel; Minsky, Abraham; Medalia, Ohad

    2009-03-01

    The organization of chromatin has a major impact on cellular activities, such as gene expression. For bacteria, it was suggested that the spatial organization of the genetic material correlates with transcriptional levels, implying a specific architecture of the chromosome within the cytoplasm. Accordingly, recent technological advances have emphasized the organization of the genetic material within nucleoid structures. Gemmata obscuriglobus, a member of the phylum Planctomycetes, exhibits a distinctive nucleoid structure in which chromatin is encapsulated within a discrete membrane-bound compartment. Here, we show that this soil and freshwater bacterium tolerates high doses of UV and ionizing radiation. Cryoelectron tomography of frozen hydrated sections and electron microscopy of freeze-substituted cells have indicated a more highly ordered condensed-chromatin organization in actively dividing and stationary-phase G. obscuriglobus cells. These three-dimensional analyses revealed a complex network of double membranes that engulf the condensed DNA. Bioinformatics analysis has revealed the existence of a putative component involved in nonhomologous DNA end joining that presumably plays a role in maintaining chromatin integrity within the bacterium. Thus, our observations further support the notion that packed chromatin organization enhances radiation tolerance.

  10. CHD3 and CHD4 recruitment and chromatin remodeling activity at DNA breaks is promoted by early poly(ADP-ribose)-dependent chromatin relaxation.

    PubMed

    Smith, Rebecca; Sellou, Hafida; Chapuis, Catherine; Huet, Sébastien; Timinszky, Gyula

    2018-05-04

    One of the first events to occur upon DNA damage is the local opening of the compact chromatin architecture, facilitating access of repair proteins to DNA lesions. This early relaxation is triggered by poly(ADP-ribosyl)ation by PARP1 in addition to ATP-dependent chromatin remodeling. CHD4 recruits to DNA breaks in a PAR-dependent manner, although it lacks any recognizable PAR-binding domain, and has the ability to relax chromatin structure. However, its role in chromatin relaxation at the site of DNA damage has not been explored. Using a live cell fluorescence three-hybrid assay, we demonstrate that the recruitment of CHD4 to DNA damage, while being poly(ADP-ribosyl)ation-dependent, is not through binding poly(ADP-ribose). Additionally, we show that CHD3 is recruited to DNA breaks in the same manner as CHD4 and that both CHD3 and CHD4 play active roles in chromatin remodeling at DNA breaks. Together, our findings reveal a two-step mechanism for DNA damage induced chromatin relaxation in which PARP1 and the PAR-binding remodeler activities of Alc1/CHD1L induce an initial chromatin relaxation phase that promotes the subsequent recruitment of CHD3 and CHD4 via binding to DNA for further chromatin remodeling at DNA breaks.

  11. Novel chromatin texture features for the classification of pap smears

    NASA Astrophysics Data System (ADS)

    Bejnordi, Babak E.; Moshavegh, Ramin; Sujathan, K.; Malm, Patrik; Bengtsson, Ewert; Mehnert, Andrew

    2013-03-01

    This paper presents a set of novel structural texture features for quantifying nuclear chromatin patterns in cells on a conventional Pap smear. The features are derived from an initial segmentation of the chromatin into bloblike texture primitives. The results of a comprehensive feature selection experiment, including the set of proposed structural texture features and a range of different cytology features drawn from the literature, show that two of the four top ranking features are structural texture features. They also show that a combination of structural and conventional features yields a classification performance of 0.954±0.019 (AUC±SE) for the discrimination of normal (NILM) and abnormal (LSIL and HSIL) slides. The results of a second classification experiment, using only normal-appearing cells from both normal and abnormal slides, demonstrates that a single structural texture feature measuring chromatin margination yields a classification performance of 0.815±0.019. Overall the results demonstrate the efficacy of the proposed structural approach and that it is possible to detect malignancy associated changes (MACs) in Papanicoloau stain.

  12. Links between genome replication and chromatin landscapes.

    PubMed

    Sequeira-Mendes, Joana; Gutierrez, Crisanto

    2015-07-01

    Post-embryonic organogenesis in plants requires the continuous production of cells in the organ primordia, their expansion and a coordinated exit to differentiation. Genome replication is one of the most important processes that occur during the cell cycle, as the maintenance of genomic integrity is of primary relevance for development. As it is chromatin that must be duplicated, a strict coordination occurs between DNA replication, the deposition of new histones, and the introduction of histone modifications and variants. In turn, the chromatin landscape affects several stages during genome replication. Thus, chromatin accessibility is crucial for the initial stages and to specify the location of DNA replication origins with different chromatin signatures. The chromatin landscape also determines the timing of activation during the S phase. Genome replication must occur fully, but only once during each cell cycle. The re-replication avoidance mechanisms rely primarily on restricting the availability of certain replication factors; however, the presence of specific histone modifications are also revealed as contributing to the mechanisms that avoid re-replication, in particular for heterochromatin replication. We provide here an update of genome replication mostly focused on data from Arabidopsis, and the advances that genomic approaches are likely to provide in the coming years. The data available, both in plants and animals, point to the relevance of the chromatin landscape in genome replication, and require a critical evaluation of the existing views about the nature of replication origins, the mechanisms of origin specification and the relevance of epigenetic modifications for genome replication. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  13. Effects of short peptides on lymphocyte chromatin in senile subjects.

    PubMed

    Khavinson, V Kh; Lezhava, T A; Malinin, V V

    2004-01-01

    Effects of synthetic short peptides (Vilon, Epithalon, Livagen, Prostamax, and Cortagen) on activity of ribosome genes, parameters of common heterochromatin melting, polymorphism of structural heterochromatin (C segments) of chromosomes 1, 9, and 16, and variability of facultative heterochromatin were studied in leukocytes of subjects aged 75-88 years. All the studied peptides induced activation of ribosome genes, decondensation of densely packed chromatin fibrils, and release of genes repressed as a result of age-specific condensation of the cellular euchromatin regions (deheterochromatinization of facultative chromatin). Treatment with Epithalon, Livagen, and Prostamax led to decondensation of chromosome 1 pericentromeric structural chromatin, while Epithalon and Livagen treatment led to changes in chromosome 9 as well. Hence, short peptides activate heterochromatin and heterochromatinized regions of cell chromosomes in senile subjects.

  14. The Arabidopsis BRAHMA Chromatin-Remodeling ATPase Is Involved in Repression of Seed Maturation Genes in Leaves1[W][OA

    PubMed Central

    Tang, Xurong; Hou, Anfu; Babu, Mohan; Nguyen, Vi; Hurtado, Lidia; Lu, Qing; Reyes, Jose C.; Wang, Aiming; Keller, Wilfred A.; Harada, John J.; Tsang, Edward W.T.; Cui, Yuhai

    2008-01-01

    Synthesis and accumulation of seed storage proteins (SSPs) is an important aspect of the seed maturation program. Genes encoding SSPs are specifically and highly expressed in the seed during maturation. However, the mechanisms that repress the expression of these genes in leaf tissue are not well understood. To gain insight into the repression mechanisms, we performed a genetic screen for mutants that express SSPs in leaves. Here, we show that mutations affecting BRAHMA (BRM), a SNF2 chromatin-remodeling ATPase, cause ectopic expression of a subset of SSPs and other embryogenesis-related genes in leaf tissue. Consistent with the notion that such SNF2-like ATPases form protein complexes in vivo, we observed similar phenotypes for mutations of AtSWI3C, a BRM-interacting partner, and BSH, a SNF5 homolog and essential SWI/SNF subunit. Chromatin immunoprecipitation experiments show that BRM is recruited to the promoters of a number of embryogenesis genes in wild-type leaves, including the 2S genes, expressed in brm leaves. Consistent with its role in nucleosome remodeling, BRM appears to affect the chromatin structure of the At2S2 promoter. Thus, the BRM-containing chromatin-remodeling ATPase complex involved in many aspects of plant development mediates the repression of SSPs in leaf tissue. PMID:18508955

  15. Budding yeast chromatin is dispersed in a crowded nucleoplasm in vivo

    PubMed Central

    Chen, Chen; Lim, Hong Hwa; Shi, Jian; Tamura, Sachiko; Maeshima, Kazuhiro; Surana, Uttam; Gan, Lu

    2016-01-01

    Chromatin organization has an important role in the regulation of eukaryotic systems. Although recent studies have refined the three-dimensional models of chromatin organization with high resolution at the genome sequence level, little is known about how the most fundamental units of chromatin—nucleosomes—are positioned in three dimensions in vivo. Here we use electron cryotomography to study chromatin organization in the budding yeast Saccharomyces cerevisiae. Direct visualization of yeast nuclear densities shows no evidence of 30-nm fibers. Aside from preribosomes and spindle microtubules, few nuclear structures are larger than a tetranucleosome. Yeast chromatin does not form compact structures in interphase or mitosis and is consistent with being in an “open” configuration that is conducive to high levels of transcription. From our study and those of others, we propose that yeast can regulate its transcription using local nucleosome–nucleosome associations. PMID:27605704

  16. Chromatin dynamics during interphase explored by single-particle tracking.

    PubMed

    Levi, Valeria; Gratton, Enrico

    2008-01-01

    Our view of the structure and function of the interphase nucleus has changed drastically in recent years. It is now widely accepted that the nucleus is a well organized and highly compartmentalized organelle and that this organization is intimately related to nuclear function. In this context, chromatin-initially considered a randomly entangled polymer-has also been shown to be structurally organized in interphase and its organization was found to be very important to gene regulation. Relevant and not completely answered questions are how chromatin organization is achieved and what mechanisms are responsible for changes in the positions of chromatin loci in the nucleus. A significant advance in the field resulted from tagging chromosome sites with bacterial operator sequences, and visualizing these tags using green fluorescent protein fused with the appropriate repressor protein. Simultaneously, fluorescence imaging techniques evolved significantly during recent years, allowing observation of the time evolution of processes in living specimens. In this context, the motion of the tagged locus was observed and analyzed to extract quantitative information regarding its dynamics. This review focuses on recent advances in our understanding of chromatin dynamics in interphase with the emphasis placed on the information obtained from single-particle tracking (SPT) experiments. We introduce the basis of SPT methods and trajectory analysis, and summarize what has been learnt by using this new technology in the context of chromatin dynamics. Finally, we briefly describe a method of SPT in a two-photon excitation microscope that has several advantages over methods based on conventional microscopy and review the information obtained using this novel approach to study chromatin dynamics.

  17. Chromatin dynamics during interphase explored by single particle tracking

    PubMed Central

    Levi, Valeria; Gratton, Enrico

    2009-01-01

    Our view of the structure and function of the interphase nucleus has drastically changed in the last years. It is now widely accepted that the nucleus is a well organized and highly compartmentalized organelle and that this organization is intimately related to nuclear function. In this context, chromatin -initially considered a randomly entangled polymer- has also been shown to be structurally organized in interphase and its organization was found to be very important to gene regulation. Relevant and not completely answered questions are how chromatin organization is achieved and what mechanisms are responsible for changes in the positions of chromatin loci in the nucleus. A significant advance in the field resulted from tagging chromosome sites with bacterial operator sequences, and visualizing these tags using green fluorescent protein fused with the appropriate repressor protein. Simultaneously, fluorescence imaging techniques significantly evolved during the last years allowing the observation of the time evolution of processes in living specimens. In this context, the motion of the tagged locus was observed and analyzed to extract quantitative information regarding its dynamics. This review focuses on recent advances in our understanding of chromatin dynamics in interphase with the emphasis placed on the information obtained from single particle tracking (SPT) experiments. We introduce the basis of SPT methods and trajectories analysis, and summarize what has been learnt by using this new technology in the context of chromatin dynamics. Finally, we briefly describe a method of SPT in a two-photon excitation microscope that has several advantages over methods based on conventional microscopy and review the information obtained by using this novel approach to study chromatin dynamics. PMID:18461483

  18. DDB2 promotes chromatin decondensation at UV-induced DNA damage

    PubMed Central

    Lindh, Michael; Acs, Klara; Vrouwe, Mischa G.; Pines, Alex; van Attikum, Haico; Mullenders, Leon H.

    2012-01-01

    Nucleotide excision repair (NER) is the principal pathway that removes helix-distorting deoxyribonucleic acid (DNA) damage from the mammalian genome. Recognition of DNA lesions by xeroderma pigmentosum group C (XPC) protein in chromatin is stimulated by the damaged DNA-binding protein 2 (DDB2), which is part of a CUL4A–RING ubiquitin ligase (CRL4) complex. In this paper, we report a new function of DDB2 in modulating chromatin structure at DNA lesions. We show that DDB2 elicits unfolding of large-scale chromatin structure independently of the CRL4 ubiquitin ligase complex. Our data reveal a marked adenosine triphosphate (ATP)–dependent reduction in the density of core histones in chromatin containing UV-induced DNA lesions, which strictly required functional DDB2 and involved the activity of poly(adenosine diphosphate [ADP]–ribose) polymerase 1. Finally, we show that lesion recognition by XPC, but not DDB2, was strongly reduced in ATP-depleted cells and was regulated by the steady-state levels of poly(ADP-ribose) chains. PMID:22492724

  19. Chromatin Insulators and Topological Domains: Adding New Dimensions to 3D Genome Architecture

    PubMed Central

    Matharu, Navneet K.; Ahanger, Sajad H.

    2015-01-01

    The spatial organization of metazoan genomes has a direct influence on fundamental nuclear processes that include transcription, replication, and DNA repair. It is imperative to understand the mechanisms that shape the 3D organization of the eukaryotic genomes. Chromatin insulators have emerged as one of the central components of the genome organization tool-kit across species. Recent advancements in chromatin conformation capture technologies have provided important insights into the architectural role of insulators in genomic structuring. Insulators are involved in 3D genome organization at multiple spatial scales and are important for dynamic reorganization of chromatin structure during reprogramming and differentiation. In this review, we will discuss the classical view and our renewed understanding of insulators as global genome organizers. We will also discuss the plasticity of chromatin structure and its re-organization during pluripotency and differentiation and in situations of cellular stress. PMID:26340639

  20. Condensins exert force on chromatin-nuclear envelope tethers to mediate nucleoplasmic reticulum formation in Drosophila melanogaster.

    PubMed

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

    2014-12-30

    Although the nuclear envelope is known primarily for its role as a boundary between the nucleus and cytoplasm in eukaryotes, it plays a vital and dynamic role in many cellular processes. Studies of nuclear structure have revealed tissue-specific changes in nuclear envelope architecture, suggesting that its three-dimensional structure contributes to its functionality. Despite the importance of the nuclear envelope, the factors that regulate and maintain nuclear envelope shape remain largely unexplored. The nuclear envelope makes extensive and dynamic interactions with the underlying chromatin. Given this inexorable link between chromatin and the nuclear envelope, it is possible that local and global chromatin organization reciprocally impact nuclear envelope form and function. In this study, we use Drosophila salivary glands to show that the three-dimensional structure of the nuclear envelope can be altered with condensin II-mediated chromatin condensation. Both naturally occurring and engineered chromatin-envelope interactions are sufficient to allow chromatin compaction forces to drive distortions of the nuclear envelope. Weakening of the nuclear lamina further enhanced envelope remodeling, suggesting that envelope structure is capable of counterbalancing chromatin compaction forces. Our experiments reveal that the nucleoplasmic reticulum is born of the nuclear envelope and remains dynamic in that they can be reabsorbed into the nuclear envelope. We propose a model where inner nuclear envelope-chromatin tethers allow interphase chromosome movements to change nuclear envelope morphology. Therefore, interphase chromatin compaction may be a normal mechanism that reorganizes nuclear architecture, while under pathological conditions, such as laminopathies, compaction forces may contribute to defects in nuclear morphology. Copyright © 2015 Bozler et al.

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

    PubMed Central

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

    2014-01-01

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

  2. Nanoscale chromatin structure characterization for optical applications: a transmission electron microscopy study (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Li, Yue; Cherkezyan, Lusik; Zhang, Di; Almassalha, Luay; Roth, Eric; Chandler, John; Bleher, Reiner; Subramanian, Hariharan; Dravid, Vinayak P.; Backman, Vadim

    2017-02-01

    Structural and biological origins of light scattering in cells and tissue are still poorly understood. We demonstrate how this problem might be addressed through the use of transmission electron microscopy (TEM). For biological samples, TEM image intensity is proportional to mass-density, and thus proportional to refractive index (RI). By calculating the autocorrelation function (ACF) of TEM image intensity of a thin-section of cells, we essentially maintain the nanoscale ACF of the 3D cellular RI distribution, given that the RI distribution is statistically isotropic. Using this nanoscale 3D RI ACF, we can simulate light scattering through biological samples, and thus guiding many optical techniques to quantify specific structures. In this work, we chose to use Partial Wave Spectroscopy (PWS) microscopy as a one of the nanoscale-sensitive optical techniques. Hela cells were prepared using standard protocol to preserve nanoscale ultrastructure, and a 50-nm slice was sectioned for TEM imaging at 6 nm resolution. The ACF was calculated for chromatin, and the PWS mean sigma was calculated by summing over the power spectral density in the visible light frequency of a random medium generated to match the ACF. A 1-µm slice adjacent to the 50-nm slice was sectioned for PWS measurement to guarantee identical chromatin structure. For 33 cells, we compared the calculated PWS mean sigma from TEM and the value measured directly, and obtained a strong correlation of 0.69. This example indicates the great potential of using TEM measured RI distribution to better understand the quantification of cellular nanostructure by optical methods.

  3. [Biochemical characterization of fractionated rat liver chromatin in experimental D-hypovitaminosis and after administration of steroidal drugs].

    PubMed

    Levitskiĭ, E L; Kholodova, Iu D; Gubskiĭ, Iu I; Primak, R G; Chabannyĭ, V N; Kindruk, N L; Mozzhukhina, T G; Lenchevskaia, L K; Mironova, V N; Saad, L M

    1993-01-01

    Marked changes in the structural and functional characteristics of liver nuclear chromatin fractions are observed under experimental D-hypovitaminosis, which differ in the degree of transcriptional activity. DNA-polymerase activity and activity of the fraction, enriched with RNA-polymerase I, increases in the active fraction. Free radical LPO reactions are modified in the chromatin fraction with low activity and to the less degree in the active one. Disturbances of chromatine structural properties are caused with the change in the protein and lipid components of chromatin. Administration of ecdysterone preparations (separately and together with vitamin D3) has a partial corrective effect on structural and functional organization of nuclear chromatine. At the action of ecdysterone normalization of LPO reactions modified by pathological changes is observed in the chromatin fraction with low activity and to the less degree in the active one. This kind of influence corrects to the less degree chromatin functional activity and quantitative and qualitative modifications of its protein component. Simultaneous influence of ecdysterone and vitamin D3 leads to the partial normalization of the biochemical indices studied (except for those which characterize LPO reactions) mainly in the active chromatin fraction.

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

    PubMed

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

    2003-10-24

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

  5. Radiation breakage of DNA: a model based on random-walk chromatin structure

    NASA Technical Reports Server (NTRS)

    Ponomarev, A. L.; Sachs, R. K.

    2001-01-01

    Monte Carlo computer software, called DNAbreak, has recently been developed to analyze observed non-random clustering of DNA double strand breaks in chromatin after exposure to densely ionizing radiation. The software models coarse-grained configurations of chromatin and radiation tracks, small-scale details being suppressed in order to obtain statistical results for larger scales, up to the size of a whole chromosome. We here give an analytic counterpart of the numerical model, useful for benchmarks, for elucidating the numerical results, for analyzing the assumptions of a more general but less mechanistic "randomly-located-clusters" formalism, and, potentially, for speeding up the calculations. The equations characterize multi-track DNA fragment-size distributions in terms of one-track action; an important step in extrapolating high-dose laboratory results to the much lower doses of main interest in environmental or occupational risk estimation. The approach can utilize the experimental information on DNA fragment-size distributions to draw inferences about large-scale chromatin geometry during cell-cycle interphase.

  6. Spectroscopic detection of etoposide binding to chromatin components: The role of histone proteins

    NASA Astrophysics Data System (ADS)

    Chamani, Elham; Rabbani-Chadegani, Azra; Zahraei, Zohreh

    2014-12-01

    Chromatin has been introduced as a main target for most anticancer drugs. Etoposide is known as a topoisomerase II inhibitor, but its effect on chromatin components is unknown. This report, for the first time, describes the effect of etoposide on DNA, histones and DNA-histones complex in the structure of nucleosomes employing thermal denaturation, fluorescence, UV absorbance and circular dichroism spectroscopy techniques. The results showed that the binding of etoposide decreased UV absorbance and fluorescence emission intensity, altered secondary structure of chromatin and hypochromicity was occurred in thermal denaturation profiles. The drug exhibited higher affinity to chromatin compared to DNA. Quenching of drug chromophores with tyrosine residues of histones indicated that globular domain of histones is the site of etoposide binding. Moreover, the binding of etoposide to histones altered their secondary structure accompanied with hypochromicity revealing compaction of histones in the presence of the drug. From the results it is concludes that apart from topoisomerase II, chromatin components especially its protein moiety can be introduced as a new site of etoposide binding and histone proteins especially H1 play a fundamental role in this process and anticancer activity of etoposide.

  7. Architectural roles of multiple chromatin insulators at the human apolipoprotein gene cluster

    PubMed Central

    Mishiro, Tsuyoshi; Ishihara, Ko; Hino, Shinjiro; Tsutsumi, Shuichi; Aburatani, Hiroyuki; Shirahige, Katsuhiko; Kinoshita, Yoshikazu; Nakao, Mitsuyoshi

    2009-01-01

    Long-range regulatory elements and higher-order chromatin structure coordinate the expression of multiple genes in cluster, and CTCF/cohesin-mediated chromatin insulator may be a key in this regulation. The human apolipoprotein (APO) A1/C3/A4/A5 gene region, whose alterations increase the risk of dyslipidemia and atherosclerosis, is partitioned at least by three CTCF-enriched sites and three cohesin protein RAD21-enriched sites (two overlap with the CTCF sites), resulting in the formation of two transcribed chromatin loops by interactions between insulators. The C3 enhancer and APOC3/A4/A5 promoters reside in the same loop, where the APOC3/A4 promoters are pointed towards the C3 enhancer, whereas the APOA1 promoter is present in the different loop. The depletion of either CTCF or RAD21 disrupts the chromatin loop structure, together with significant changes in the APO expression and the localization of transcription factor hepatocyte nuclear factor (HNF)-4α and transcriptionally active form of RNA polymerase II at the APO promoters. Thus, CTCF/cohesin-mediated insulators maintain the chromatin loop formation and the localization of transcriptional apparatus at the promoters, suggesting an essential role of chromatin insulation in controlling the expression of clustered genes. PMID:19322193

  8. Chromatin potentiates transcription

    PubMed Central

    Nagai, Shigeki; Davis, Ralph E.; Mattei, Pierre Jean; Eagen, Kyle Patrick; Kornberg, Roger D.

    2017-01-01

    Chromatin isolated from the chromosomal locus of the PHO5 gene of yeast in a transcriptionally repressed state was transcribed with 12 pure proteins (80 polypeptides): RNA polymerase II, six general transcription factors, TFIIS, the Pho4 gene activator protein, and the SAGA, SWI/SNF, and Mediator complexes. Contrary to expectation, a nucleosome occluding the TATA box and transcription start sites did not impede transcription but rather, enhanced it: the level of chromatin transcription was at least sevenfold greater than that of naked DNA, and chromatin gave patterns of transcription start sites closely similar to those occurring in vivo, whereas naked DNA gave many aberrant transcripts. Both histone acetylation and trimethylation of H3K4 (H3K4me3) were important for chromatin transcription. The nucleosome, long known to serve as a general gene repressor, thus also performs an important positive role in transcription. PMID:28137832

  9. Epigenetic switch from repressive to permissive chromatin in response to cold stress

    PubMed Central

    Park, Junghoon; Lim, Chae Jin; Shen, Mingzhe; Park, Hee Jin; Cha, Joon-Yung; Iniesto, Elisa; Rubio, Vicente; Mengiste, Tesfaye; Bressan, Ray A.; Lee, Sang Yeol; Lee, Byeong-ha; Kim, Woe-Yeon; Yun, Dae-Jin

    2018-01-01

    Switching from repressed to active status in chromatin regulation is part of the critical responses that plants deploy to survive in an ever-changing environment. We previously reported that HOS15, a WD40-repeat protein, is involved in histone deacetylation and cold tolerance in Arabidopsis. However, it remained unknown how HOS15 regulates cold responsive genes to affect cold tolerance. Here, we show that HOS15 interacts with histone deacetylase 2C (HD2C) and both proteins together associate with the promoters of cold-responsive COR genes, COR15A and COR47. Cold induced HD2C degradation is mediated by the CULLIN4 (CUL4)-based E3 ubiquitin ligase complex in which HOS15 acts as a substrate receptor. Interference with the association of HD2C and the COR gene promoters by HOS15 correlates with increased acetylation levels of histone H3. HOS15 also interacts with CBF transcription factors to modulate cold-induced binding to the COR gene promoters. Our results here demonstrate that cold induces HOS15-mediated chromatin modifications by degrading HD2C. This switches the chromatin structure status and facilitates recruitment of CBFs to the COR gene promoters. This is an apparent requirement to acquire cold tolerance. PMID:29784800

  10. Chromatin organization regulated by EZH2-mediated H3K27me3 is required for OPN-induced migration of bone marrow-derived mesenchymal stem cells.

    PubMed

    Liu, Lingling; Luo, Qing; Sun, Jinghui; Ju, Yang; Morita, Yasuyuki; Song, Guanbin

    2018-03-01

    Osteopontin (OPN) is a chemokine-like extracellular matrix-associated protein involved in the migration of bone marrow-derived mesenchymal stem cells (BMSCs). An increasing number of studies have found that chromatin organization may affect cellular migration. However, whether OPN regulates chromatin organization is not understood, nor are the underlying molecular mechanisms. In this study, we investigated the link between chromatin organization and BMSC migration and demonstrated that OPN-mediated BMSC migration leads to elevated levels of heterochromatin marker histone H3 lysine 27 trimethylation (H3K27me3) through the methyltransferase EZH2. The expression of EZH2 reorganizes the chromatin structure of BMSCs. Pharmacological inhibition or depletion of EZH2 blocks BMSC migration. Moreover, using an atomic force microscope (AFM), we found that chromatin decondensation alters the mechanical properties of the nucleus. In addition, inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signals represses OPN-promoted chromatin condensation and cell migration. Thus, our results identify a mechanism by which ERK1/2 signalling drives specific chromatin modifications in BMSCs, which alters chromatin organization and thereby enables OPN-mediated BMSC migration. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Epigenetic regulation and chromatin remodeling in learning and memory.

    PubMed

    Kim, Somi; Kaang, Bong-Kiun

    2017-01-13

    Understanding the underlying mechanisms of memory formation and maintenance has been a major goal in the field of neuroscience. Memory formation and maintenance are tightly controlled complex processes. Among the various processes occurring at different levels, gene expression regulation is especially crucial for proper memory processing, as some genes need to be activated while some genes must be suppressed. Epigenetic regulation of the genome involves processes such as DNA methylation and histone post-translational modifications. These processes edit genomic properties or the interactions between the genome and histone cores. They then induce structural changes in the chromatin and lead to transcriptional changes of different genes. Recent studies have focused on the concept of chromatin remodeling, which consists of 3D structural changes in chromatin in relation to gene regulation, and is an important process in learning and memory. In this review, we will introduce three major epigenetic processes involved in memory regulation: DNA methylation, histone methylation and histone acetylation. We will also discuss general mechanisms of long-term memory storage and relate the epigenetic control of learning and memory to chromatin remodeling. Finally, we will discuss how epigenetic mechanisms can contribute to the pathologies of neurological disorders and cause memory-related symptoms.

  12. Linker DNA accessibility in chromatin fibers of different conformations: a reevaluation.

    PubMed Central

    Zlatanova, J; Leuba, S H; Yang, G; Bustamante, C; van Holde, K

    1994-01-01

    New studies on chromatin fiber morphology, using the technique of scanning force microscopy (SFM), have caused us to reexamine recent analysis of nuclease digestion of chromatin. Chicken erythrocyte chromatin fibers, glutaraldehyde-fixed at 0, 10, and 80 mM NaCl, were imaged with the help of SFM. The chromatin fibers possessed a loose three-dimensional 30-nm structure even in the absence of added salt. This structure slightly condensed upon addition of 10 mM NaCl, and highly compacted, irregularly segmented fibers were observed at 80 mM NaCl. This sheds new light upon our previously reported analysis of the kinetics of digestion by soluble and membrane-immobilized micrococcal nuclease [Leuba, S. H., Zlatanova, J. & van Holde, K. (1994) J. Mol. Biol. 235, 871-880]. While the low-ionic-strength fibers were readily digested, the highly compacted structure formed at 80 mM NaCl was refractory to nuclease attack, implying that the linkers were fully accessible in the low-ionic-strength conformation but not in the condensed fibers. We now find that cleavage of the linker DNA by a small molecule, methidiumpropyl-EDTA-Fe(II), proceeds for all types of conformations at similar rates. Thus, steric hindrance is responsible for the lack of accessibility to micrococcal nuclease in the condensed fiber. Taken in total the data suggest that reexamination of existing models of chromatin conformation is warranted. Images PMID:8202481

  13. Correlation Between Interphase Chromatin Structure and - and High-Let Radiation-Induced - and Intra-Chromosome Exchange Hotspots

    NASA Astrophysics Data System (ADS)

    Zhang, Ye; Wu, Honglu; Mangala, Lingegowda; Asaithamby, Aroumougame; Chen, David

    2012-07-01

    CORRELATION BETWEEN INTERPHASE CHROMATIN STRUCTURE AND LOW- AND HIGH-LET RADIATION-INDUCED INTER- AND INTRA-CHROMOSOME EXCHANGE HOTSPOTS Ye Zhang1,2, Lingegowda S. Mangala1,3, Aroumougame Asaithamby4, David J. Chen4, and Honglu Wu1 1 NASA Johnson Space Center, Houston, Texas, USA 2 Wyle Integrated Science and Engineering Group, Houston, Texas, USA 3 University of Houston Clear Lake, Houston, Texas, USA 4 University of Texas, Southwestern Medical Center, Dallas, Texas, USA To investigate the relationship between chromosome aberrations induced by low- and high-LET radiation and chromatin folding, we reconstructed the three dimensional structure of chromosome 3 and measured the physical distances between different regions of this chromosome. Previously, we investigated the location of breaks involved in inter- and intrachromosomal type exchange events in chromosome 3 of human epithelial cells, using the multicolor banding in situ hybridization (mBAND) technique. After exposure to both low- and high-LET radiations in vitro, intra-chromosome exchanges occurred preferentially between a break in the 3p21 and one in the 3q11 regions, and the breaks involved in inter-chromosome exchanges occurred in two regions near the telomeres of the chromosome. In this study, human epithelial cells were fixed in G1 phase and interphase chromosomes hybridized with an mBAND probe for chromosome 3 were captured with a laser scanning confocal microscope. The 3-dimensional structure of interphase chromosome 3 with different colored regions was reconstructed, and the distance between different regions was measured. We show that, in most of the G1 cells, the regions containing 3p21 and 3q11 are colocalized in the center of the chromosome domain, whereas, the regions towards the telomeres of the chromosome are located in the peripherals of the chromosome domain. Our results demonstrate that the distribution of breaks involved in radiation-induced inter and intra-chromosome aberrations depends

  14. Oxidative stress signaling to chromatin in health and disease

    PubMed Central

    Kreuz, Sarah; Fischle, Wolfgang

    2016-01-01

    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. PMID:27319358

  15. Common ground: small RNA programming and chromatin modifications.

    PubMed

    Lejeune, Erwan; Allshire, Robin C

    2011-06-01

    Epigenetic mechanisms regulate genome structure and expression profiles in eukaryotes. RNA interference (RNAi) and other small RNA-based chromatin-modifying activities can act to reset the epigenetic landscape at defined chromatin domains. Centromeric heterochromatin assembly is a RNAi-dependent process in the fission yeast Schizosaccharomyces pombe, and provides a paradigm for detailed examination of such epigenetic processes. Here we review recent progress in understanding the mechanisms that underpin RNAi-mediated heterochromatin formation in S. pombe. We discuss recent analyses of the events that trigger RNAi and manipulations which uncouple RNAi and chromatin modification. Finally we provide an overview of similar molecular machineries across species where related small RNA pathways appear to drive the epigenetic reprogramming in germ cells and/or during early development in metazoans. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Plant chromatin warms up in Madrid

    PubMed Central

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

    2014-01-01

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

  17. Chromatin: Its history, current research, and the seminal researchers and their philosophy.

    PubMed

    Deichmann, Ute

    2015-01-01

    The concept of chromatin as a complex of nucleic acid and proteins in the cell nucleus was developed by cytologists and biochemists in the late 19th century. It was the starting point for biochemical research on DNA and nuclear proteins. Although interest in chromatin declined rapidly at the beginning of the 20th century, a few decades later a new focus on chromatin emerged, which was not only related to its structure, but also to its function in gene regulatory processes in the development of higher organisms. Since the late 20th century, research on chromatin modifications has also been conducted under the label of epigenetics. This article highlights the major phases of chromatin research until the present time and introduces major investigators and their scientific and philosophical outlooks.

  18. Ftx is a non-coding RNA which affects Xist expression and chromatin structure within the X-inactivation center region.

    PubMed

    Chureau, Corinne; Chantalat, Sophie; Romito, Antonio; Galvani, Angélique; Duret, Laurent; Avner, Philip; Rougeulle, Claire

    2011-02-15

    X chromosome inactivation (XCI) is an essential epigenetic process which involves several non-coding RNAs (ncRNAs), including Xist, the master regulator of X-inactivation initiation. Xist is flanked in its 5' region by a large heterochromatic hotspot, which contains several transcription units including a gene of unknown function, Ftx (five prime to Xist). In this article, we describe the characterization and functional analysis of murine Ftx. We present evidence that Ftx produces a conserved functional long ncRNA, and additionally hosts microRNAs (miR) in its introns. Strikingly, Ftx partially escapes X-inactivation and is upregulated specifically in female ES cells at the onset of X-inactivation, an expression profile which closely follows that of Xist. We generated Ftx null ES cells to address the function of this gene. In these cells, only local changes in chromatin marks are detected within the hotspot, indicating that Ftx is not involved in the global maintenance of the heterochromatic structure of this region. The Ftx mutation, however, results in widespread alteration of transcript levels within the X-inactivation center (Xic) and particularly important decreases in Xist RNA levels, which were correlated with increased DNA methylation at the Xist CpG island. Altogether our results indicate that Ftx is a positive regulator of Xist and lead us to propose that Ftx is a novel ncRNA involved in XCI.

  19. Sperm chromatin maturity and integrity correlated to zygote development in ICSI program.

    PubMed

    Asmarinah; Syauqy, Ahmad; Umar, Liya Agustin; Lestari, Silvia Werdhy; Mansyur, Eliza; Hestiantoro, Andon; Paradowszka-Dogan, Agnieszka

    2016-10-01

    This study aimed to evaluate sperm chromatin maturity and integrity of that injected into good-quality oocytes in an in vitro fertilization-intra cytoplasmic sperm injection (IVF-ICSI) program. A cut-off value of sperm chromatin maturity and integrity was developed as a function of their correlation to the zygote development, i.e., embryo formation and cleavage rate. The study assessed sperm chromatin maturity using aniline blue (AB) staining, whereas toluidine blue (TB) staining was used to assess sperm chromatin integrity. Ejaculates from 59 patients undergoing ICSI and 46 fertile normozoospermic donors for determination of normal values of sperm chromatin status were used in this study. Embryo formation and cleavage rates were observed for the period of 3 days after ICSI. There was a significant difference in the percentage of sperm with mature chromatin between ejaculate from ICSI patients and fertile donor (p=0.020); while there was no significant difference in sperm chromatin integrity of both samples (p=0.120). There was no significant correlation between sperm chromatin maturity and either embryo formation or cleavage rate; as well as sperm chromatin integrity to both parameters of zygote development (p>0.05). Furthermore, we found that the cut-off value of sperm chromatin maturity and integrity of the fertile normozoospermic ejaculates were 87.2% and 80.2%, respectively. Using the cut-offs, we found that low sperm chromatin maturity at the level of <87% correlated significantly with the cleavage rate of the zygote (p=0.022; r=0.371); whereas poor sperm chromatin integrity at the level of <80% correlated with embryo formation (p=0.048; r=0,485). In conclusion, this study showed that poor maturity and integrity of sperm chromatin (AB<87% and TB<80%, respectively), could affect zygote development following ICSI. AB: aniline blue; CMA3: chromomycin A3; ICSI: intra cytoplasmic sperm injection; IVF: in vitro fertilization; PBS: phosphate buffer saline; SPSS

  20. Structural consequences of disease-causing mutations in the ATRX-DNMT3-DNMT3L (ADD) domain of the chromatin-associated protein ATRX.

    PubMed

    Argentaro, Anthony; Yang, Ji-Chun; Chapman, Lynda; Kowalczyk, Monika S; Gibbons, Richard J; Higgs, Douglas R; Neuhaus, David; Rhodes, Daniela

    2007-07-17

    The chromatin-associated protein ATRX was originally identified because mutations in the ATRX gene cause a severe form of syndromal X-linked mental retardation associated with alpha-thalassemia. Half of all of the disease-associated missense mutations cluster in a cysteine-rich region in the N terminus of ATRX. This region was named the ATRX-DNMT3-DNMT3L (ADD) domain, based on sequence homology with a family of DNA methyltransferases. Here, we report the solution structure of the ADD domain of ATRX, which consists of an N-terminal GATA-like zinc finger, a plant homeodomain finger, and a long C-terminal alpha-helix that pack together to form a single globular domain. Interestingly, the alpha-helix of the GATA-like finger is exposed and highly basic, suggesting a DNA-binding function for ATRX. The disease-causing mutations fall into two groups: the majority affect buried residues and hence affect the structural integrity of the ADD domain; another group affects a cluster of surface residues, and these are likely to perturb a potential protein interaction site. The effects of individual point mutations on the folding state and stability of the ADD domain correlate well with the levels of mutant ATRX protein in patients, providing insights into the molecular pathophysiology of ATR-X syndrome.

  1. On the physical and chemical dynamics of chromatin

    NASA Astrophysics Data System (ADS)

    Apratim, Manjul

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

  2. Effect of capsid confinement on the chromatin organization of the SV40 minichromosome

    PubMed Central

    Saper, Gadiel; Kler, Stanislav; Asor, Roi; Oppenheim, Ariella; Raviv, Uri; Harries, Daniel

    2013-01-01

    Using small-angle X-ray scattering, we determined the three-dimensional packing architecture of the minichromosome confined within the SV40 virus. In solution, the minichromosome, composed of closed circular dsDNA complexed in nucleosomes, was shown to be structurally similar to cellular chromatin. In contrast, we find a unique organization of the nanometrically encapsidated chromatin, whereby minichromosomal density is somewhat higher at the center of the capsid and decreases towards the walls. This organization is in excellent agreement with a coarse-grained computer model, accounting for tethered nucleosomal interactions under viral capsid confinement. With analogy to confined liquid crystals, but contrary to the solenoid structure of cellular chromatin, our simulations indicate that the nucleosomes within the capsid lack orientational order. Nucleosomes in the layer adjacent to the capsid wall, however, align with the boundary, thereby inducing a ‘molten droplet’ state of the chromatin. These findings indicate that nucleosomal interactions suffice to predict the genome organization in polyomavirus capsids and underscore the adaptable nature of the eukaryotic chromatin architecture to nanoscale confinement. PMID:23258701

  3. Effect of capsid confinement on the chromatin organization of the SV40 minichromosome.

    PubMed

    Saper, Gadiel; Kler, Stanislav; Asor, Roi; Oppenheim, Ariella; Raviv, Uri; Harries, Daniel

    2013-02-01

    Using small-angle X-ray scattering, we determined the three-dimensional packing architecture of the minichromosome confined within the SV40 virus. In solution, the minichromosome, composed of closed circular dsDNA complexed in nucleosomes, was shown to be structurally similar to cellular chromatin. In contrast, we find a unique organization of the nanometrically encapsidated chromatin, whereby minichromosomal density is somewhat higher at the center of the capsid and decreases towards the walls. This organization is in excellent agreement with a coarse-grained computer model, accounting for tethered nucleosomal interactions under viral capsid confinement. With analogy to confined liquid crystals, but contrary to the solenoid structure of cellular chromatin, our simulations indicate that the nucleosomes within the capsid lack orientational order. Nucleosomes in the layer adjacent to the capsid wall, however, align with the boundary, thereby inducing a 'molten droplet' state of the chromatin. These findings indicate that nucleosomal interactions suffice to predict the genome organization in polyomavirus capsids and underscore the adaptable nature of the eukaryotic chromatin architecture to nanoscale confinement.

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

    NASA Astrophysics Data System (ADS)

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

    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.

  5. Dissecting the chromatin interactome of microRNA genes.

    PubMed

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

    2014-03-01

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

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

    PubMed Central

    Brosch, Gerald; Loidl, Peter; Graessle, Stefan

    2008-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2002-08-01

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

  8. Topology of zigzag chromatin.

    PubMed

    Strogatz, S

    1983-08-21

    An enormous length of DNA is packaged in the nuclei of eukaryotic cells. This is achieved through several intermediate levels of compaction, ranging from the double helix to the chromosome. The nucleosome is now firmly established as the first level of chromatin structure. Next it appears that the nucleosomes are themselves stacked in a two-track array, with a dinucleosome repeat. Several winding patterns of DNA are compatible with such a structure. It is shown here that, compared to other feasible DNA paths, the observed winding pattern has remarkable topological properties. The possible biological significance of this peculiarity is discussed.

  9. Gal4-VP16 directs ATP-independent chromatin reorganization in a yeast chromatin assembly system.

    PubMed

    Robinson, Karen M; Schultz, Michael C

    2005-03-22

    Major insights into the regulation of chromatin organization have stemmed from biochemical studies using Gal4-VP16, a chimeric transcriptional activator in which the DNA binding domain of Gal4p is fused to the activation domain of viral protein VP16. Unexpectedly, given previous intensive efforts to understand how Gal4-VP16 functions in the context of chromatin, we have uncovered a new mode of chromatin reorganization that is dependent on Gal4-VP16. This reorganization is performed by an activity in a crude DEAE (CD) fraction from budding yeast which also supports ATP-dependent assembly of physiologically spaced nucleosome arrays. Biochemical analysis reveals that the activity tightly associates with chromatin and reorganizes nucleosome arrays by a mechanism which is insensitive to ATP depletion after nucleosome assembly. It generates a chromatin organization in which a nucleosome is stably positioned immediately adjacent to Gal4p binding sites in the template DNA. Individual deletion of genes previously implicated in chromatin assembly and remodeling, namely, the histone chaperones NAP1, ASF1, and CAC1 and the SNF2-like DEAD/H ATPases SNF2, ISW1, ISW2, CHD1, SWR1, YFR038w, and SPT20, does not significantly perturb reorganization. Therefore, Gal4-VP16-directed chromatin reorganization in yeast can occur by an ATP-independent mechanism that does not require SAGA, SWI/SNF, Isw1, or Isw2 chromatin remodeling complexes.

  10. The chromatin basis of neurodevelopmental disorders: Rethinking dysfunction along the molecular and temporal axes.

    PubMed

    Gabriele, Michele; Lopez Tobon, Alejandro; D'Agostino, Giuseppe; Testa, Giuseppe

    2018-06-08

    The complexity of the human brain emerges from a long and finely tuned developmental process orchestrated by the crosstalk between genome and environment. Vis à vis other species, the human brain displays unique functional and morphological features that result from this extensive developmental process that is, unsurprisingly, highly vulnerable to both genetically and environmentally induced alterations. One of the most striking outcomes of the recent surge of sequencing-based studies on neurodevelopmental disorders (NDDs) is the emergence of chromatin regulation as one of the two domains most affected by causative mutations or Copy Number Variations besides synaptic function, whose involvement had been largely predicted for obvious reasons. These observations place chromatin dysfunction at the top of the molecular pathways hierarchy that ushers in a sizeable proportion of NDDs and that manifest themselves through synaptic dysfunction and recurrent systemic clinical manifestation. Here we undertake a conceptual investigation of chromatin dysfunction in NDDs with the aim of systematizing the available evidence in a new framework: first, we tease out the developmental vulnerabilities in human corticogenesis as a structuring entry point into the causation of NDDs; second, we provide a much needed clarification of the multiple meanings and explanatory frameworks revolving around "epigenetics", highlighting those that are most relevant for the analysis of these disorders; finally we go in-depth into paradigmatic examples of NDD-causing chromatin dysregulation, with a special focus on human experimental models and datasets. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Epigenetic switch from repressive to permissive chromatin in response to cold stress.

    PubMed

    Park, Junghoon; Lim, Chae Jin; Shen, Mingzhe; Park, Hee Jin; Cha, Joon-Yung; Iniesto, Elisa; Rubio, Vicente; Mengiste, Tesfaye; Zhu, Jian-Kang; Bressan, Ray A; Lee, Sang Yeol; Lee, Byeong-Ha; Jin, Jing Bo; Pardo, Jose M; Kim, Woe-Yeon; Yun, Dae-Jin

    2018-06-05

    Switching from repressed to active status in chromatin regulation is part of the critical responses that plants deploy to survive in an ever-changing environment. We previously reported that HOS15, a WD40-repeat protein, is involved in histone deacetylation and cold tolerance in Arabidopsis However, it remained unknown how HOS15 regulates cold responsive genes to affect cold tolerance. Here, we show that HOS15 interacts with histone deacetylase 2C (HD2C) and both proteins together associate with the promoters of cold-responsive COR genes, COR15A and COR47 Cold induced HD2C degradation is mediated by the CULLIN4 (CUL4)-based E3 ubiquitin ligase complex in which HOS15 acts as a substrate receptor. Interference with the association of HD2C and the COR gene promoters by HOS15 correlates with increased acetylation levels of histone H3. HOS15 also interacts with CBF transcription factors to modulate cold-induced binding to the COR gene promoters. Our results here demonstrate that cold induces HOS15-mediated chromatin modifications by degrading HD2C. This switches the chromatin structure status and facilitates recruitment of CBFs to the COR gene promoters. This is an apparent requirement to acquire cold tolerance. Copyright © 2018 the Author(s). Published by PNAS.

  12. The ''self-stirred'' genome: Bulk and surface dynamics of the chromatin globule

    NASA Astrophysics Data System (ADS)

    Zidovska, Alexandra

    Chromatin structure and dynamics control all aspects of DNA biology yet are poorly understood. In interphase, time between two cell divisions, chromatin fills the cell nucleus in its minimally condensed polymeric state. Chromatin serves as substrate to a number of biological processes, e.g. gene expression and DNA replication, which require it to become locally restructured. These are energy-consuming processes giving rise to non-equilibrium dynamics. Chromatin dynamics has been traditionally studied by imaging of fluorescently labeled nuclear proteins and single DNA-sites, thus focusing only on a small number of tracer particles. Recently, we developed an approach, displacement correlation spectroscopy (DCS) based on time-resolved image correlation analysis, to map chromatin dynamics simultaneously across the whole nucleus in cultured human cells. DCS revealed that chromatin movement was coherent across large regions (4-5 μm) for several seconds. Regions of coherent motion extended beyond the boundaries of single-chromosome territories, suggesting elastic coupling of motion over length scales much larger than those of genes. These large-scale, coupled motions were ATP-dependent and unidirectional for several seconds. Following these observations, we developed a hydrodynamic theory of active chromatin dynamics, using the two-fluid model and describing the content of cell nucleus as a chromatin solution, which is subject to both passive thermal fluctuations and active (ATP-consuming) scalar and vector events. In this work we continue in our efforts to elucidate the mechanism and function of the chromatin dynamics in interphase. We investigate the chromatin interactions with the nuclear envelope and compare the surface dynamics of the chromatin globule with its bulk dynamics.

  13. Chromatin condensation during terminal erythropoiesis.

    PubMed

    Zhao, Baobing; Yang, Jing; Ji, Peng

    2016-09-02

    Mammalian terminal erythropoiesis involves gradual but dramatic chromatin condensation steps that are essential for cell differentiation. Chromatin and nuclear condensation is followed by a unique enucleation process, which is believed to liberate more spaces for hemoglobin enrichment and enable the generation of a physically flexible mature red blood cell. Although these processes have been known for decades, the mechanisms are still unclear. Our recent study reveals an unexpected nuclear opening formation during mouse terminal erythropoiesis that requires caspase-3 activity. Major histones, except H2AZ, are partially released from the opening, which is important for chromatin condensation. Block of the nuclear opening through caspase inhibitor or knockdown of caspase-3 inhibits chromatin condensation and enucleation. We also demonstrate that nuclear opening and histone release are cell cycle regulated. These studies reveal a novel mechanism for chromatin condensation in mammalia terminal erythropoiesis.

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

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Hada, Megumi; Wu, Honglu

    2014-01-01

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

  15. Intestinal Master Transcription Factor CDX2 Controls Chromatin Access for Partner Transcription Factor Binding

    PubMed Central

    Verzi, Michael P.; Shin, Hyunjin; San Roman, Adrianna K.

    2013-01-01

    Tissue-specific gene expression requires modulation of nucleosomes, allowing transcription factors to occupy cis elements that are accessible only in selected tissues. Master transcription factors control cell-specific genes and define cellular identities, but it is unclear if they possess special abilities to regulate cell-specific chromatin and if such abilities might underlie lineage determination and maintenance. One prevailing view is that several transcription factors enable chromatin access in combination. The homeodomain protein CDX2 specifies the embryonic intestinal epithelium, through unknown mechanisms, and partners with transcription factors such as HNF4A in the adult intestine. We examined enhancer chromatin and gene expression following Cdx2 or Hnf4a excision in mouse intestines. HNF4A loss did not affect CDX2 binding or chromatin, whereas CDX2 depletion modified chromatin significantly at CDX2-bound enhancers, disrupted HNF4A occupancy, and abrogated expression of neighboring genes. Thus, CDX2 maintains transcription-permissive chromatin, illustrating a powerful and dominant effect on enhancer configuration in an adult tissue. Similar, hierarchical control of cell-specific chromatin states is probably a general property of master transcription factors. PMID:23129810

  16. The Core Subunit of A Chromatin-Remodeling Complex, ZmCHB101, Plays Essential Roles in Maize Growth and Development.

    PubMed

    Yu, Xiaoming; Jiang, Lili; Wu, Rui; Meng, Xinchao; Zhang, Ai; Li, Ning; Xia, Qiong; Qi, Xin; Pang, Jinsong; Xu, Zheng-Yi; Liu, Bao

    2016-12-05

    ATP-dependent chromatin remodeling complexes play essential roles in the regulation of diverse biological processes by formulating a DNA template that is accessible to the general transcription apparatus. Although the function of chromatin remodelers in plant development has been studied in A. thaliana, how it affects growth and development of major crops (e.g., maize) remains uninvestigated. Combining genetic, genomic and bioinformatic analyses, we show here that the maize core subunit of chromatin remodeling complex, ZmCHB101, plays essential roles in growth and development of maize at both vegetative and reproductive stages. Independent ZmCHB101 RNA interference plant lines displayed abaxially curling leaf phenotype due to increase of bulliform cell numbers, and showed impaired development of tassel and cob. RNA-seq-based transcriptome profiling revealed that ZmCHB101 dictated transcriptional reprogramming of a significant set of genes involved in plant development, photosynthesis, metabolic regulation, stress response and gene expressional regulation. Intriguingly, we found that ZmCHB101 was required for maintaining normal nucleosome density and 45 S rDNA compaction. Our findings suggest that the SWI3 protein, ZmCHB101, plays pivotal roles in maize normal growth and development via regulation of chromatin structure.

  17. Histone H3 Lysine 14 (H3K14) Acetylation Facilitates DNA Repair in a Positioned Nucleosome by Stabilizing the Binding of the Chromatin Remodeler RSC (Remodels Structure of Chromatin)*

    PubMed Central

    Duan, Ming-Rui; Smerdon, Michael J.

    2014-01-01

    Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage. PMID:24515106

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

    PubMed Central

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

    2013-01-01

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

  19. Investigation of the mechanism of meiotic DNA cleavage by VMA1-derived endonuclease uncovers a meiotic alteration in chromatin structure around the target site.

    PubMed

    Fukuda, Tomoyuki; Ohta, Kunihiro; Ohya, Yoshikazu

    2006-06-01

    VMA1-derived endonuclease (VDE), a homing endonuclease in Saccharomyces cerevisiae, is encoded by the mobile intein-coding sequence within the nuclear VMA1 gene. VDE recognizes and cleaves DNA at the 31-bp VDE recognition sequence (VRS) in the VMA1 gene lacking the intein-coding sequence during meiosis to insert a copy of the intein-coding sequence at the cleaved site. The mechanism underlying the meiosis specificity of VMA1 intein-coding sequence homing remains unclear. We studied various factors that might influence the cleavage activity in vivo and found that VDE binding to the VRS can be detected only when DNA cleavage by VDE takes place, implying that meiosis-specific DNA cleavage is regulated by the accessibility of VDE to its target site. As a possible candidate for the determinant of this accessibility, we analyzed chromatin structure around the VRS and revealed that local chromatin structure near the VRS is altered during meiosis. Although the meiotic chromatin alteration exhibits correlations with DNA binding and cleavage by VDE at the VMA1 locus, such a chromatin alteration is not necessarily observed when the VRS is embedded in ectopic gene loci. This suggests that nucleosome positioning or occupancy around the VRS by itself is not the sole mechanism for the regulation of meiosis-specific DNA cleavage by VDE and that other mechanisms are involved in the regulation.

  20. Investigation of the Mechanism of Meiotic DNA Cleavage by VMA1-Derived Endonuclease Uncovers a Meiotic Alteration in Chromatin Structure around the Target Site

    PubMed Central

    Fukuda, Tomoyuki; Ohta, Kunihiro; Ohya, Yoshikazu

    2006-01-01

    VMA1-derived endonuclease (VDE), a homing endonuclease in Saccharomyces cerevisiae, is encoded by the mobile intein-coding sequence within the nuclear VMA1 gene. VDE recognizes and cleaves DNA at the 31-bp VDE recognition sequence (VRS) in the VMA1 gene lacking the intein-coding sequence during meiosis to insert a copy of the intein-coding sequence at the cleaved site. The mechanism underlying the meiosis specificity of VMA1 intein-coding sequence homing remains unclear. We studied various factors that might influence the cleavage activity in vivo and found that VDE binding to the VRS can be detected only when DNA cleavage by VDE takes place, implying that meiosis-specific DNA cleavage is regulated by the accessibility of VDE to its target site. As a possible candidate for the determinant of this accessibility, we analyzed chromatin structure around the VRS and revealed that local chromatin structure near the VRS is altered during meiosis. Although the meiotic chromatin alteration exhibits correlations with DNA binding and cleavage by VDE at the VMA1 locus, such a chromatin alteration is not necessarily observed when the VRS is embedded in ectopic gene loci. This suggests that nucleosome positioning or occupancy around the VRS by itself is not the sole mechanism for the regulation of meiosis-specific DNA cleavage by VDE and that other mechanisms are involved in the regulation. PMID:16757746

  1. In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins.

    PubMed

    del Valle, Luis J

    2005-06-01

    Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg-jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø(o) (sperm-specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca(2+), pH, and this activity could be a serine-proteinase. Thermal denaturalization of the oocyte extracts (80 degrees C, 10-15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø(o)) will be a first condition in the process of sperm chromatin remodeling.

  2. DNA replication through a chromatin environment.

    PubMed

    Bellush, James M; Whitehouse, Iestyn

    2017-10-05

    Compaction of the genome into the nuclear space is achieved by wrapping DNA around octameric assemblies of histone proteins to form nucleosomes, the fundamental repeating unit of chromatin. Aside from providing a means by which to fit larger genomes into the cell, chromatinization of DNA is a crucial means by which the cell regulates access to the genome. While the complex role that chromatin plays in gene transcription has been appreciated for a long time, it is now also apparent that crucial aspects of DNA replication are linked to the biology of chromatin. This review will focus on recent advances in our understanding of how the chromatin environment influences key aspects of DNA replication.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Author(s).

  3. On the topology of chromatin fibres

    PubMed Central

    Barbi, Maria; Mozziconacci, Julien; Victor, Jean-Marc; Wong, Hua; Lavelle, Christophe

    2012-01-01

    The ability of cells to pack, use and duplicate DNA remains one of the most fascinating questions in biology. To understand DNA organization and dynamics, it is important to consider the physical and topological constraints acting on it. In the eukaryotic cell nucleus, DNA is organized by proteins acting as spools on which DNA can be wrapped. These proteins can subsequently interact and form a structure called the chromatin fibre. Using a simple geometric model, we propose a general method for computing topological properties (twist, writhe and linking number) of the DNA embedded in those fibres. The relevance of the method is reviewed through the analysis of magnetic tweezers single molecule experiments that revealed unexpected properties of the chromatin fibre. Possible biological implications of these results are discussed. PMID:24098838

  4. Human Genome Replication Proceeds through Four Chromatin States

    PubMed Central

    Julienne, Hanna; Zoufir, Azedine; Audit, Benjamin; Arneodo, Alain

    2013-01-01

    Advances in genomic studies have led to significant progress in understanding the epigenetically controlled interplay between chromatin structure and nuclear functions. Epigenetic modifications were shown to play a key role in transcription regulation and genome activity during development and differentiation or in response to the environment. Paradoxically, the molecular mechanisms that regulate the initiation and the maintenance of the spatio-temporal replication program in higher eukaryotes, and in particular their links to epigenetic modifications, still remain elusive. By integrative analysis of the genome-wide distributions of thirteen epigenetic marks in the human cell line K562, at the 100 kb resolution of corresponding mean replication timing (MRT) data, we identify four major groups of chromatin marks with shared features. These states have different MRT, namely from early to late replicating, replication proceeds though a transcriptionally active euchromatin state (C1), a repressive type of chromatin (C2) associated with polycomb complexes, a silent state (C3) not enriched in any available marks, and a gene poor HP1-associated heterochromatin state (C4). When mapping these chromatin states inside the megabase-sized U-domains (U-shaped MRT profile) covering about 50% of the human genome, we reveal that the associated replication fork polarity gradient corresponds to a directional path across the four chromatin states, from C1 at U-domains borders followed by C2, C3 and C4 at centers. Analysis of the other genome half is consistent with early and late replication loci occurring in separate compartments, the former correspond to gene-rich, high-GC domains of intermingled chromatin states C1 and C2, whereas the latter correspond to gene-poor, low-GC domains of alternating chromatin states C3 and C4 or long C4 domains. This new segmentation sheds a new light on the epigenetic regulation of the spatio-temporal replication program in human and provides a

  5. Quantitative analysis of single-molecule force spectroscopy on folded chromatin fibers

    PubMed Central

    Meng, He; Andresen, Kurt; van Noort, John

    2015-01-01

    Single-molecule techniques allow for picoNewton manipulation and nanometer accuracy measurements of single chromatin fibers. However, the complexity of the data, the heterogeneity of the composition of individual fibers and the relatively large fluctuations in extension of the fibers complicate a structural interpretation of such force-extension curves. Here we introduce a statistical mechanics model that quantitatively describes the extension of individual fibers in response to force on a per nucleosome basis. Four nucleosome conformations can be distinguished when pulling a chromatin fiber apart. A novel, transient conformation is introduced that coexists with single wrapped nucleosomes between 3 and 7 pN. Comparison of force-extension curves between single nucleosomes and chromatin fibers shows that embedding nucleosomes in a fiber stabilizes the nucleosome by 10 kBT. Chromatin fibers with 20- and 50-bp linker DNA follow a different unfolding pathway. These results have implications for accessibility of DNA in fully folded and partially unwrapped chromatin fibers and are vital for understanding force unfolding experiments on nucleosome arrays. PMID:25779043

  6. Analysis of DNA replication associated chromatin decondensation: in vivo assay for understanding chromatin remodeling mechanisms of selected proteins.

    PubMed

    Borysov, Sergiy; Bryant, Victoria L; Alexandrow, Mark G

    2015-01-01

    Of critical importance to many of the events underlying transcriptional control of gene expression are modifications to core and linker histones that regulate the accessibility of trans-acting factors to the DNA substrate within the context of chromatin. Likewise, control over the initiation of DNA replication, as well as the ability of the replication machinery to proceed during elongation through the multiple levels of chromatin condensation that are likely to be encountered, is known to involve the creation of chromatin accessibility. In the latter case, chromatin access will likely need to be a transient event so as to prevent total genomic unraveling of the chromatin that would be deleterious to cells. While there are many molecular and biochemical approaches in use to study histone changes and their relationship to transcription and chromatin accessibility, few techniques exist that allow a molecular dissection of the events underlying DNA replication control as it pertains to chromatin changes and accessibility. Here, we outline a novel experimental strategy for addressing the ability of specific proteins to induce large-scale chromatin unfolding (decondensation) in vivo upon site-specific targeting to an engineered locus. Our laboratory has used this powerful system in novel ways to directly address the ability of DNA replication proteins to create chromatin accessibility, and have incorporated modifications to the basic approach that allow for a molecular genetic analysis of the mechanisms and associated factors involved in causing chromatin decondensation by a protein of interest. Alternative approaches involving co-expression of other proteins (competitors or stimulators), concurrent drug treatments, and analysis of co-localizing histone modifications are also addressed, all of which are illustrative of the utility of this experimental system for extending basic findings to physiologically relevant mechanisms. Although used by our group to analyze

  7. Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in Its Dissociation from Chromatin and the Nuclear Matrix.

    PubMed

    Kubota, Sho; Morii, Mariko; Yuki, Ryuzaburo; Yamaguchi, Noritaka; Yamaguchi, Hiromi; Aoyama, Kazumasa; Kuga, Takahisa; Tomonaga, Takeshi; Yamaguchi, Naoto

    2015-04-24

    Protein-tyrosine phosphorylation regulates a wide variety of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine kinases induce global nuclear structure changes, which we called chromatin structural changes. However, the mechanisms are not fully understood. In this study we identify protein kinase A anchoring protein 8 (AKAP8/AKAP95), which associates with chromatin and the nuclear matrix, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of AKAP8 is induced by several tyrosine kinases, such as Src, Fyn, and c-Abl but not Syk. Nucleus-targeted Lyn and c-Src strongly dissociate AKAP8 from chromatin and the nuclear matrix in a kinase activity-dependent manner. The levels of tyrosine phosphorylation of AKAP8 are decreased by substitution of multiple tyrosine residues on AKAP8 into phenylalanine. Importantly, the phenylalanine mutations of AKAP8 inhibit its dissociation from nuclear structures, suggesting that the association/dissociation of AKAP8 with/from nuclear structures is regulated by its tyrosine phosphorylation. Furthermore, the phenylalanine mutations of AKAP8 suppress the levels of nuclear tyrosine kinase-induced chromatin structural changes. In contrast, AKAP8 knockdown increases the levels of chromatin structural changes. Intriguingly, stimulation with hydrogen peroxide induces chromatin structural changes accompanied by the dissociation of AKAP8 from nuclear structures. These results suggest that AKAP8 is involved in the regulation of chromatin structural changes through nuclear tyrosine phosphorylation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    PubMed

    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.

  9. Comparison of the effect of UV laser radiation and of a radiomimetic substance on chromatin

    NASA Astrophysics Data System (ADS)

    Radulescu, Irina; Radu, Liliana; Serbanescu, Ruxandra; Nelea, V. D.; Martin, C.; Mihailescu, Ion N.

    1998-07-01

    The damages of the complex of deoxyribonucleic acid (DNA) and proteins from chromatin, produced by the UV laser radiation and/or by treatment with a radiomimetic substance, bleomycin, were compared. The laser radiation and bleomycin effects on chromatin structure were determined by the static and dynamic fluorimetry of chromatin complexes with the DNA specific ligand-- proflavine and by the analysis of tryptophan chromatin intrinsic fluorescence. Time resolved spectroscopy is a sensitive technique which allows to determine the excited state lifetimes of chromatin--proflavine complexes. Also, the percentage contributions to the fluorescence of proflavine, bound and unbound to chromatin DNA, were evaluated. The damages produced by the UV laser radiation on chromatin are similar with those of radiomimetic substance action and consists in DNA and proteins destruction. The DNA damage degree has been determined. The obtained results may constitute some indications in the laser utilization in radiochimiotherapy.

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

    PubMed

    Saha, Anjanabha; Wittmeyer, Jacqueline; Cairns, Bradley R

    2006-06-01

    Chromatin remodellers are specialized multi-protein machines that enable access to nucleosomal DNA by altering the structure, composition and positioning of nucleosomes. All remodellers have a catalytic ATPase subunit that is similar to known DNA-translocating motor proteins, suggesting DNA translocation as a unifying aspect of their mechanism. Here, we explore the diversity and specialization of chromatin remodellers, discuss how nucleosome modifications regulate remodeller activity and consider a model for the exposure of nucleosomal DNA that involves the use of directional DNA translocation to pump 'DNA waves' around the nucleosome.

  11. Histone Variant Regulates DNA Repair via Chromatin Condensation | Center for Cancer Research

    Cancer.gov

    Activating the appropriate DNA repair pathway is essential for maintaining the stability of the genome after a break in both strands of DNA. How a pathway is selected, however, is not well understood. Since these double strand breaks (DSBs) occur while DNA is packaged as chromatin, changes in its organization are necessary for repair to take place. Numerous alterations have been associated with DSBs, including modifications of histone tails and exchange of histone variants, some increasing chromatin accessibility, others reducing it. In fact, distinct domains flanking a single DSB have been observed that are bound by opposing repair pathway proteins 53BP1and BRCA1, which promote non-homologous end joining (NHEJ) and homologous recombination (HR), respectively. To investigate whether DSB-proximal chromatin reorganization affects repair pathway selection, Philipp Oberdoerffer, Ph.D., of CCR’s Laboratory of Receptor Biology and Gene Expression, and his colleagues performed a high-throughput RNA interference (RNAi) screen for chromatin-related genes that modulate HR.

  12. Comparative analysis of metazoan chromatin organization.

    PubMed

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

    2014-08-28

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

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

    PubMed Central

    2011-01-01

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

  14. The globular domain of histone H5 is internally located in the 30 nm chromatin fiber: an immunochemical study.

    PubMed Central

    Dimitrov, S I; Russanova, V R; Pashev, I G

    1987-01-01

    The location of the globular domain of histone H5 relative to the axis of the 30 nm chromatin fiber was investigated by following the accessibility of this region of the molecule in chicken erythrocyte chromatin to specific antibodies as a function of chromatin structure. Antibodies to the globular domain of H5 as well as their Fab fragments were found to react with chromatin at ionic strengths ranging from 1-80 mM NaCl, the reaction gradually decreasing upon increase of salt concentration. If, however, Fab fragments were conjugated to ferritin, no reaction of the complex with chromatin was observed at salt concentrations higher than 20 mM. The accessibility of the globular part of H5 in unfolded chromatin to the Fab-ferritin complex was also demonstrated with trypsin-digested chromatin. The experiments were carried out by both solid-phase immunoassay and inhibition experiments. The data obtained are consistent with a structure in which the globular domain of H5 is internally located in the 30 nm chromatin fiber. Images Fig. 1. Fig. 2. PMID:2444434

  15. Titration and hysteresis in epigenetic chromatin silencing

    NASA Astrophysics Data System (ADS)

    Dayarian, Adel; Sengupta, Anirvan M.

    2013-06-01

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

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

    PubMed Central

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

    2015-01-01

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

  17. New insights into chromatin folding and dynamics from multi-scale modeling

    NASA Astrophysics Data System (ADS)

    Olson, Wilma

    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 roughly 150 DNA base pairs and eight histone proteins-found on chromatin fibers. We have developed 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 with 3-25 evenly spaced nucleosomes. The correspondence between the predicted and observed effects of nucleosome composition, spacing, and numbers on long-range communication between regulatory proteins bound to the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We have extracted effective nucleosome-nucleosome potentials from the mesoscale simulations and introduced the potentials in a larger scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable influence of nucleosome spacing on chromatin flexibility. Small changes in the length of the DNA fragments linking successive nucleosomes introduce marked changes in the local interactions of the nucleosomes and in the spatial configurations of the fiber as a whole. The changes in nucleosome positioning influence the statistical properties of longer chromatin constructs with 100-10,000 nucleosomes. We are investigating the extent to which the `local' interactions of regularly spaced nucleosomes contribute to the corresponding interactions in chains with mixed spacings as a step toward the treatment of fibers with nucleosomes positioned at the sites mapped at base-pair resolution on genomic sequences. Support of the work by USPHS R01 GM 34809 is gratefully acknowledged.

  18. [The biological aspects of chromatin diminution].

    PubMed

    Akif'ev, A P; Grishanin, A K

    1993-01-01

    The chromatine diminution (CD), first discovered by Boveri (1887) in ascarids, represents programmed elimination of a part of genetic material in the nuclei of the somatic cells in cyclops and ascarids, and in the protist macronuclei. The CD can be considered as a macromutation sharply changing chromosomal structure, though minimally effecting the phenotype. The analysis of CD is of significance for discussing mechanisms of origin of chromosomal organization, transformation of genome molecular structure in eucaryote evolution, role of the extra DNA.

  19. Chromatin- and temperature-dependent modulation of radiation-induced double-strand breaks.

    PubMed

    Elmroth, K; Nygren, J; Stenerlöw, B; Hultborn, R

    2003-10-01

    To investigate the influence of chromatin organization and scavenging capacity in relation to irradiation temperature on the induction of double-strand breaks (DSB) in structures derived from human diploid fibroblasts. Agarose plugs with different chromatin structures (intact cells+/-wortmannin, permeabilized cells with condensed chromatin, nucleoids and DNA) were prepared and irradiated with X-rays at 2 or 37 degrees C and lysed using two different lysis protocols (new ice-cold lysis or standard lysis at 37 degrees C). Induction of DSB was determined by constant-field gel electrophoresis. The dose-modifying factor (DMF(temp)) for irradiation at 37 compared with 2 degrees C was 0.92 in intact cells (i.e. more DSB induced at 2 degrees C), but gradually increased to 1.5 in permeabilized cells, 2.2 in nucleoids and 2.6 in naked DNA, suggesting a role of chromatin organization for temperature modulation of DNA damage. In addition, DMF(temp) was influenced by the presence of 0.1 M DMSO or 30 mM glutathione, but not by post-irradiation temperature. The protective effect of low temperature was correlated to the indirect effects of ionizing radiation and was not dependent on post-irradiation temperature. Reasons for a dose modifying factor <1 in intact cells are discussed.

  20. Chromatin-bound RNA and the neurobiology of psychiatric disease.

    PubMed

    Tushir, J S; Akbarian, S

    2014-04-04

    A large, and still rapidly expanding literature on epigenetic regulation in the nervous system has provided fundamental insights into the dynamic regulation of DNA methylation and post-translational histone modifications in the context of neuronal plasticity in health and disease. Remarkably, however, very little is known about the potential role of chromatin-bound RNAs, including many long non-coding transcripts and various types of small RNAs. Here, we provide an overview on RNA-mediated regulation of chromatin structure and function, with focus on histone lysine methylation and psychiatric disease. Examples of recently discovered chromatin-bound long non-coding RNAs important for neuronal health and function include the brain-derived neurotrophic factor antisense transcript (Bdnf-AS) which regulates expression of the corresponding sense transcript, and LOC389023 which is associated with human-specific histone methylation signatures at the chromosome 2q14.1 neurodevelopmental risk locus by regulating expression of DPP10, an auxillary subunit for voltage-gated K(+) channels. We predict that the exploration of chromatin-bound RNA will significantly advance our current knowledge base in neuroepigenetics and biological psychiatry. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  1. Effects of chromatin decondensation on alternative NHEJ.

    PubMed

    Moscariello, Mario; Iliakis, George

    2013-11-01

    In cells of higher eukaryotes, repair of DNA double strand breaks (DSBs) utilizes different forms of potentially error-prone non-homologous end joining (NHEJ): canonical DNA-PK-dependent (C-NHEJ) and alternative backup pathways (A-NHEJ). In contrast to C-NHEJ, A-NHEJ shows pronounced efficiency fluctuations throughout the cell cycle and is severely compromised as cells cease proliferating and enter the plateau phase (Windhofer et al., 2007 [23]). The molecular mechanisms underpinning this response remain unknown but changes in chromatin structure are prime candidate-A-NHEJ-modulators. Since parameters beyond chromatin acetylation appear to determine A-NHEJ efficiency (Manova et al., 2012 [42,76]), we study here the role of chromatin decondensation mediated either by treatment with 5'-aza-2'-deoxycytidine (AzadC) or growth in hypotonic conditions, on A-NHEJ. We report that both treatments have no detectable effect on C-NHEJ but provoke, specifically for A-NHEJ, cell-growth-dependent effects. These results uncover for the first time a link between A-NHEJ and chromatin organization and provide means for understanding the regulatory mechanisms underpinning the growth-state dependency of A-NHEJ. A-NHEJ is implicated in the formation of chromosomal translocations and in chromosome fusions that underlie genomic instability and carcinogenesis. The observations reported here may therefore contribute to the development of drug-based A-NHEJ suppression-strategies aiming at optimizing cancer treatment outcomes and possibly also at suppressing carcinogenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    PubMed Central

    2014-01-01

    DNA is the blueprint upon which life is based and transmitted, yet the manner in which chromatin, the dynamic complex of nucleic acids and proteins, is packaged and behaves within the cellular nucleus has only begun to be investigated. 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 and chromatin modifications and higher order chromatin structure. PMID:24091454

  3. Ribonucleic Acid Synthesis by Cucumber Chromatin

    PubMed Central

    Johnson, Kenneth D.; Purves, William K.

    1970-01-01

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

  4. Chromatin Remodeling Factors Isw2 and Ino80 Regulate Checkpoint Activity and Chromatin Structure in S Phase

    PubMed Central

    Lee, Laura; Rodriguez, Jairo; Tsukiyama, Toshio

    2015-01-01

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

  5. Understanding RNA-Chromatin Interactions Using Chromatin Isolation by RNA Purification (ChIRP).

    PubMed

    Chu, Ci; Chang, Howard Y

    2016-01-01

    ChIRP is a novel and easy-to-use technique for studying long noncoding RNA (lncRNA)-chromatin interactions. RNA and chromatin are cross-linked in vivo using formaldehyde or glutaraldehyde, and purified using biotinylated antisense oligonucleotides that hybridize to the target RNA. Co-precipitated DNA is then purified and analyzed by quantitative PCR (qPCR) or high-throughput sequencing.

  6. A role for the nucleoporin Nup170p in chromatin structure and gene silencing

    PubMed Central

    Van de Vosse, David W.; Wan, Yakun; Lapetina, Diego L.; Chen, Wei-Ming; Chiang, Jung-Hsien; Aitchison, John D.; Wozniak, Richard W.

    2013-01-01

    Embedded in the nuclear envelope, nuclear pore complexes (NPCs) not only regulate nuclear transport, but also interface with transcriptionally active euchromatin, largely silenced heterochromatin, as well as the boundaries between these regions. It is unclear what functional role NPCs play in establishing or maintaining these distinct chromatin domains. We report that the yeast NPC protein Nup170p interacts with regions of the genome containing ribosomal protein and subtelomeric genes. Here, it functions in nucleosome positioning and as a repressor of transcription. We show that the role of Nup170p in subtelomeric gene silencing is linked to its association with the RSC chromatin-remodeling complex and the silencing factor Sir4p, and that the binding of Nup170p and Sir4p to subtelomeric chromatin is cooperative and necessary for the association of telomeres with the nuclear envelope. Our results establish the NPC as an active participant in silencing and the formation of peripheral heterochromatin. PMID:23452847

  7. Telomere fusion in Drosophila: The role of subtelomeric chromatin

    PubMed Central

    Marzullo, Marta; Gatti, Maurizio

    2015-01-01

    Drosophila telomeres are maintained by transposition to chromosome ends of the HeT-A, TART and TAHRE retrotransposons, collectively designated as HTT. Although all Drosophila telomeres terminate with HTT arrays and are capped by the terminin complex, they differ in the type of subtelomeric chromatin. The HTT sequences of YS, YL, XR, and 4L are juxtaposed to constitutive heterochromatin, while the HTTs of the other telomeres are linked to either the TAS repeat-associated chromatin (XL, 2L, 2R, 3L, 3R) or to the specialized 4R chromatin. We found that mutations in pendolino (peo) cause (telomeric fusions) that preferentially involve the heterochromatin-associated telomeres (Ha-telomeres), a telomeric fusion pattern never observed in the other 10 telomere-capping mutants characterized so far. Peo, is homologous to the E2 variant ubiquitin-conjugating enzymes and is required for DNA replication. Our analyses lead us to hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in Ha-telomeres. These data provide the first demonstration that subtelomeres can affect telomere fusion. PMID:26786804

  8. Chromatin-associated regulation of sorbitol synthesis in flower buds of peach.

    PubMed

    Lloret, Alba; Martínez-Fuentes, Amparo; Agustí, Manuel; Badenes, María Luisa; Ríos, Gabino

    2017-11-01

    PpeS6PDH gene is postulated to mediate sorbitol synthesis in flower buds of peach concomitantly with specific chromatin modifications. Perennial plants have evolved an adaptive mechanism involving protection of meristems within specialized structures named buds in order to survive low temperatures and water deprivation during winter. A seasonal period of dormancy further improves tolerance of buds to environmental stresses through specific mechanisms poorly known at the molecular level. We have shown that peach PpeS6PDH gene is down-regulated in flower buds after dormancy release, concomitantly with changes in the methylation level at specific lysine residues of histone H3 (H3K27 and H3K4) in the chromatin around the translation start site of the gene. PpeS6PDH encodes a NADPH-dependent sorbitol-6-phosphate dehydrogenase, the key enzyme for biosynthesis of sorbitol. Consistently, sorbitol accumulates in dormant buds showing higher PpeS6PDH expression. Moreover, PpeS6PDH gene expression is affected by cold and water deficit stress. Particularly, its expression is up-regulated by low temperature in buds and leaves, whereas desiccation treatment induces PpeS6PDH in buds and represses the gene in leaves. These data reveal the concurrent participation of chromatin modification mechanisms, transcriptional regulation of PpeS6PDH and sorbitol accumulation in flower buds of peach. In addition to its role as a major translocatable photosynthate in Rosaceae species, sorbitol is a widespread compatible solute and cryoprotectant, which suggests its participation in tolerance to environmental stresses in flower buds of peach.

  9. Insulation of the Chicken β-Globin Chromosomal Domain from a Chromatin-Condensing Protein, MENT

    PubMed Central

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

    2003-01-01

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

  10. Multi-scale chromatin state annotation using a hierarchical hidden Markov model

    NASA Astrophysics Data System (ADS)

    Marco, Eugenio; Meuleman, Wouter; Huang, Jialiang; Glass, Kimberly; Pinello, Luca; Wang, Jianrong; Kellis, Manolis; Yuan, Guo-Cheng

    2017-04-01

    Chromatin-state analysis is widely applied in the studies of development and diseases. However, existing methods operate at a single length scale, and therefore cannot distinguish large domains from isolated elements of the same type. To overcome this limitation, we present a hierarchical hidden Markov model, diHMM, to systematically annotate chromatin states at multiple length scales. We apply diHMM to analyse a public ChIP-seq data set. diHMM not only accurately captures nucleosome-level information, but identifies domain-level states that vary in nucleosome-level state composition, spatial distribution and functionality. The domain-level states recapitulate known patterns such as super-enhancers, bivalent promoters and Polycomb repressed regions, and identify additional patterns whose biological functions are not yet characterized. By integrating chromatin-state information with gene expression and Hi-C data, we identify context-dependent functions of nucleosome-level states. Thus, diHMM provides a powerful tool for investigating the role of higher-order chromatin structure in gene regulation.

  11. Macrogenomic engineering via modulation of the scaling of chromatin packing density.

    PubMed

    Almassalha, Luay M; Bauer, Greta M; Wu, Wenli; Cherkezyan, Lusik; Zhang, Di; Kendra, Alexis; Gladstein, Scott; Chandler, John E; VanDerway, David; Seagle, Brandon-Luke L; Ugolkov, Andrey; Billadeau, Daniel D; O'Halloran, Thomas V; Mazar, Andrew P; Roy, Hemant K; Szleifer, Igal; Shahabi, Shohreh; Backman, Vadim

    2017-11-01

    Many human diseases result from the dysregulation of the complex interactions between tens to thousands of genes. However, approaches for the transcriptional modulation of many genes simultaneously in a predictive manner are lacking. Here, through the combination of simulations, systems modelling and in vitro experiments, we provide a physical regulatory framework based on chromatin packing-density heterogeneity for modulating the genomic information space. Because transcriptional interactions are essentially chemical reactions, they depend largely on the local physical nanoenvironment. We show that the regulation of the chromatin nanoenvironment allows for the predictable modulation of global patterns in gene expression. In particular, we show that the rational modulation of chromatin density fluctuations can lead to a decrease in global transcriptional activity and intercellular transcriptional heterogeneity in cancer cells during chemotherapeutic responses to achieve near-complete cancer cell killing in vitro. Our findings represent a 'macrogenomic engineering' approach to modulating the physical structure of chromatin for whole-scale transcriptional modulation.

  12. Predicting chromatin architecture from models of polymer physics.

    PubMed

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

    2017-03-01

    We review the picture of chromatin large-scale 3D organization emerging from the analysis of Hi-C data and polymer modeling. In higher mammals, Hi-C contact maps reveal a complex higher-order organization, extending from the sub-Mb to chromosomal scales, hierarchically folded in a structure of domains-within-domains (metaTADs). The domain folding hierarchy is partially conserved throughout differentiation, and deeply correlated to epigenomic features. Rearrangements in the metaTAD topology relate to gene expression modifications: in particular, in neuronal differentiation models, topologically associated domains (TADs) tend to have coherent expression changes within architecturally conserved metaTAD niches. To identify the nature of architectural domains and their molecular determinants within a principled approach, we discuss models based on polymer physics. We show that basic concepts of interacting polymer physics explain chromatin spatial organization across chromosomal scales and cell types. The 3D structure of genomic loci can be derived with high accuracy and its molecular determinants identified by crossing information with epigenomic databases. In particular, we illustrate the case of the Sox9 locus, linked to human congenital disorders. The model in-silico predictions on the effects of genomic rearrangements are confirmed by available 5C data. That can help establishing new diagnostic tools for diseases linked to chromatin mis-folding, such as congenital disorders and cancer.

  13. Quantitative analysis of the chromatin of lymphocytes: an assay on comparative structuralism.

    PubMed

    Meyer, F

    1980-01-01

    With 26 letters we can form all the words we use, and with a few words it is possible to form an infinite number of different meaningful sentences. In our case, the letters will be a few simple neighborhood image transformations and area measurements. The paper shows how, by iterating these transformations, it is possible to obtain a good quantitative description of the nuclear structure of Feulgen-stained lymphocytes (CLL and normal). The fact that we restricted ourselves to a small number of image transformations made it possible to construct an image analysis system (TAS) able to do these transformations very quickly. We will see, successively, how to segment the nucleus itself, the chromatin, and the interchromatinic channels, how openings and closings lead to size and spatial distribution curves, and how skeletons may be used for measuring the lengths of interchromatinic channels.

  14. BRDT is an essential epigenetic regulator for proper chromatin organization, silencing of sex chromosomes and crossover formation in male meiosis

    PubMed Central

    Oh, Min Young; Garyn, Corey

    2018-01-01

    The double bromodomain and extra-terminal domain (BET) proteins are critical epigenetic readers that bind to acetylated histones in chromatin and regulate transcriptional activity and modulate changes in chromatin structure and organization. The testis-specific BET member, BRDT, is essential for the normal progression of spermatogenesis as mutations in the Brdt gene result in complete male sterility. Although BRDT is expressed in both spermatocytes and spermatids, loss of the first bromodomain of BRDT leads to severe defects in spermiogenesis without overtly compromising meiosis. In contrast, complete loss of BRDT blocks the progression of spermatocytes into the first meiotic division, resulting in a complete absence of post-meiotic cells. Although BRDT has been implicated in chromatin remodeling and mRNA processing during spermiogenesis, little is known about its role in meiotic processes. Here we report that BRDT is an essential regulator of chromatin organization and reprograming during prophase I of meiosis. Loss of BRDT function disrupts the epigenetic state of the meiotic sex chromosome inactivation in spermatocytes, affecting the synapsis and silencing of the X and Y chromosomes. We also found that BRDT controls the global chromatin organization and histone modifications of the chromatin attached to the synaptonemal complex. Furthermore, the homeostasis of crossover formation and localization during pachynema was altered, underlining a possible epigenetic mechanism by which crossovers are regulated and differentially established in mammalian male genomes. Our observations reveal novel findings about the function of BRDT in meiosis and provide insight into how epigenetic regulators modulate the progression of male mammalian meiosis and the formation of haploid gametes. PMID:29513658

  15. Interaction of a common painkiller piroxicam and copper-piroxicam with chromatin causes structural alterations accompanied by modulation at the epigenomic/genomic level.

    PubMed

    Goswami, Sathi; Sanyal, Sulagna; Chakraborty, Payal; Das, Chandrima; Sarkar, Munna

    2017-08-01

    NSAIDs are the most common class of painkillers and anti-inflammatory agents. They also show other functions like chemoprevention and chemosuppression for which they act at the protein but not at the genome level since they are mostly anions at physiological pH, which prohibit their approach to the poly-anionic DNA. Complexing the drugs with bioactive metal obliterate their negative charge and allow them to bind to the DNA, thereby, opening the possibility of genome level interaction. To test this hypothesis, we present the interaction of a traditional NSAID, Piroxicam and its copper complex with core histone and chromatin. Spectroscopy, DLS, and SEM studies were applied to see the effect of the interaction on the structure of histone/chromatin. This was coupled with MTT assay, immunoblot analysis, confocal microscopy, micro array analysis and qRT-PCR. The interaction of Piroxicam and its copper complex with histone/chromatin results in structural alterations. Such structural alterations can have different biological manifestations, but to test our hypothesis, we have focused only on the accompanied modulations at the epigenomic/genomic level. The complex, showed alteration of key epigenetic signatures implicated in transcription in the global context, although Piroxicam caused no significant changes. We have correlated such alterations caused by the complex with the changes in global gene expression and validated the candidate gene expression alterations. Our results provide the proof of concept that DNA binding ability of the copper complexes of a traditional NSAID, opens up the possibility of modulations at the epigenomic/genomic level. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. In vivo dynamical behavior of yeast chromatin modeled as an entangled polymer network with constraint release

    NASA Astrophysics Data System (ADS)

    Wang, Chenxi; Kilfoil, Maria L.

    2013-03-01

    The high fidelity segregation of chromatin is the central problem in cell mitosis. The role of mechanics underlying this, however, is undetermined. Work in this area has largely focused on cytoskeletal elements of the process. Preliminary work in our lab suggests the mechanical properties of chromatin are fundamental in this process. Nevertheless, the mechanical properties of chromatin in the cellular context are not well-characterized. For better understanding of the role of mechanics in this cellular process, and of the chromatin mechanics in vivo generally, a systematic dynamical description of chromatin in vivo is required. Accordingly, we label specific sites on chromatin with fluorescent proteins of different wave lengths, enabling us to detect multiple spots separately in 3D and track their displacements in time inside living yeast cells. We analyze the pairwise cross-correlated motion between spots as a function of relative distance along the DNA contour. Comparison between the reptation model and our data serves to test our conjecture that chromatin in the cell is basically an entangled polymer network under constraints to thermal motion, and removal of constraints by non-thermal cellular processes is expected to affect its dynamic behavior.

  17. Chromatin reprogramming in breast cancer.

    PubMed

    Swinstead, Erin E; Paakinaho, Ville; Hager, Gordon

    2018-04-24

    Reprogramming of the chromatin landscape is a critical component to the transcriptional response in breast cancer. Effects of sex hormones such as estrogens and progesterone have been well described to have a critical impact on breast cancer proliferation. However, the complex network of the chromatin landscape, enhancer regions, and mode of function of steroid receptors (SRs) and other transcription factors (TFs), is an intricate web of signaling and functional processes that is still largely misunderstood at the mechanistic level. In this review, we describe what is currently known about the dynamic interplay between TFs with chromatin and the reprogramming of enhancer elements. Emphasis has been placed on characterizing the different modes of action of TFs in regulating enhancer activity, specifically, how different SRs target enhancer regions and reprogram chromatin in breast cancer cells. In addition, we discuss current techniques employed to study enhancer function at a genome-wide level. Further, we have noted recent advances in live cell imaging technology. These single cell approaches enable the coupling of population based assays with real-time studies to address many unsolved questions about SRs and chromatin dynamics in breast cancer.

  18. [A densitometric analysis of the chromatin structural characteristics of the nuclei in the peripheral blood lymphocytes of breast cancer patients].

    PubMed

    Nalieskina, L A

    1995-01-01

    Alterations in optical structural characteristics of nuclear chromatin, in comparison with healthy individuals (10) and patients with fibroadenoma (29), were detected in 57 patients with a breast cancer by densitometric investigation of peripheral blood lymphocytes. The degree of these alterations are closely associated with the level of malignancy in the initial neoplasia and the aggregation of oncopathology in pedigrees.

  19. Chromatin Dynamics in Genome Stability: Roles in Suppressing Endogenous DNA Damage and Facilitating DNA Repair

    PubMed Central

    Nair, Nidhi; Shoaib, Muhammad

    2017-01-01

    Genomic DNA is compacted into chromatin through packaging with histone and non-histone proteins. Importantly, DNA accessibility is dynamically regulated to ensure genome stability. This is exemplified in the response to DNA damage where chromatin relaxation near genomic lesions serves to promote access of relevant enzymes to specific DNA regions for signaling and repair. Furthermore, recent data highlight genome maintenance roles of chromatin through the regulation of endogenous DNA-templated processes including transcription and replication. Here, we review research that shows the importance of chromatin structure regulation in maintaining genome integrity by multiple mechanisms including facilitating DNA repair and directly suppressing endogenous DNA damage. PMID:28698521

  20. A Method for Visualization of Incoming Adenovirus Chromatin Complexes in Fixed and Living Cells

    PubMed Central

    Komatsu, Tetsuro; Dacheux, Denis; Kreppel, Florian; Nagata, Kyosuke; Wodrich, Harald

    2015-01-01

    Inside the adenovirus virion, the genome forms a chromatin-like structure with viral basic core proteins. Core protein VII is the major DNA binding protein and was shown to remain associated with viral genomes upon virus entry even after nuclear delivery. It has been suggested that protein VII plays a regulatory role in viral gene expression and is a functional component of viral chromatin complexes in host cells. As such, protein VII could be used as a maker to track adenoviral chromatin complexes in vivo. In this study, we characterize a new monoclonal antibody against protein VII that stains incoming viral chromatin complexes following nuclear import. Furthermore, we describe the development of a novel imaging system that uses Template Activating Factor-I (TAF-I/SET), a cellular chromatin protein tightly bound to protein VII upon infection. This setup allows us not only to rapidly visualize protein VII foci in fixed cells but also to monitor their movement in living cells. These powerful tools can provide novel insights into the spatio-temporal regulation of incoming adenoviral chromatin complexes. PMID:26332038

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

    PubMed

    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 chromatin integrity, these are not associated with different in vitro fertility of the bulls involved.

  2. ChIP-less analysis of chromatin states.

    PubMed

    Su, Zhangli; Boersma, Melissa D; Lee, Jin-Hee; Oliver, Samuel S; Liu, Shichong; Garcia, Benjamin A; Denu, John M

    2014-01-01

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

  3. [Changes in the chromatin structure of lymphoid cells under the influence of low-intensity extremely high-frequency electromagnetic radiation against the background of inflammatory process].

    PubMed

    Gapeev, A B; Romanova, N A; Chemeris, N K

    2011-01-01

    Using the alkaline single cell gel electrophoresis technique (comet assay), changes in chromatin structure of peripheral blood leukocytes and peritoneal neutrophils have been studied in mice exposed to low-intensity extremely high-frequency electromagnetic radiation (42.2 GHz, 0.1 mW/cm2, 20 min at 1 h after induction of inflammation) against the background of the systemic inflammatory process. It was revealed that the exposure of mice with the developing inflammation leads to a pronounced decrease in the level of DNA damage to peripheral blood leukocytes and peritoneal neutrophils. It is supposed that the changes in the chromatin structure of lymphoid cells have a genoprotective character in the inflammatory process and can underlie the mechanisms of realization of antiinflammatory effects of the electromagnetic radiation.

  4. Identification of hierarchical chromatin domains

    PubMed Central

    Weinreb, Caleb; Raphael, Benjamin J.

    2016-01-01

    Motivation: The three-dimensional structure of the genome is an important regulator of many cellular processes including differentiation and gene regulation. Recently, technologies such as Hi-C that combine proximity ligation with high-throughput sequencing have revealed domains of self-interacting chromatin, called topologically associating domains (TADs), in many organisms. Current methods for identifying TADs using Hi-C data assume that TADs are non-overlapping, despite evidence for a nested structure in which TADs and sub-TADs form a complex hierarchy. Results: We introduce a model for decomposition of contact frequencies into a hierarchy of nested TADs. This model is based on empirical distributions of contact frequencies within TADs, where positions that are far apart have a greater enrichment of contacts than positions that are close together. We find that the increase in contact enrichment with distance is stronger for the inner TAD than for the outer TAD in a TAD/sub-TAD pair. Using this model, we develop the TADtree algorithm for detecting hierarchies of nested TADs. TADtree compares favorably with previous methods, finding TADs with a greater enrichment of chromatin marks such as CTCF at their boundaries. Availability and implementation: A python implementation of TADtree is available at http://compbio.cs.brown.edu/software/ Contact: braphael@cs.brown.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26315910

  5. Identification of a crenarchaeal orthologue of Elf1: implications for chromatin and transcription in Archaea.

    PubMed

    Daniels, Jan-Peter; Kelly, Steven; Wickstead, Bill; Gull, Keith

    2009-07-29

    The transcription machineries of Archaea and eukaryotes are similar in many aspects, but little is understood about archaeal chromatin and its role in transcription. Here, we describe the identification in hyperthermophilic Crenarchaeota and a Korarchaeon of an orthologue of the eukaryotic transcription elongation factor Elf1, which has been shown to function in chromatin structure maintenance of actively transcribed templates. Our discovery has implications for the relationship of chromatin and transcription in Archaea and the evolution of these processes in eukaryotes.

  6. Chromatin structure of the LCR in the human β-globin locus transcribing the adult δ- and β-globin genes.

    PubMed

    Kim, Seoyeon; Kim, Yea Woon; Shim, Sung Han; Kim, Chul Geun; Kim, Aeri

    2012-03-01

    The β-like globin genes are transcribed in a developmental stage specific fashion in erythroid cells. The specific transcription of globin genes is conferred by the locus control region (LCR), but the chromatin structure of the LCR in the human adult β-globin locus transcribing the δ- and β-globin genes is not clear. Here, we employed hybrid MEL cells that contain a human chromosome 11. The δ- and β-globin genes were highly transcribed in hybrid MEL/ch11 cells after transcriptional induction. LCR HS3 and HS2 were strongly occupied by erythroid specific transcriptional activators and co-factors in the induced locus. These HSs, but not HS4 and HS1, were in close proximity with the active globin genes as revealed by high resolution 3C experiments. The active features at HS3 were markedly established after transcriptional induction, while HS2 was in a relatively active conformation before the induction. Unexpectedly, HS1 did not show notable active features except histone hyperacetylation. Taken together, the LCR of the human β-globin locus transcribing the adult δ- and β-globin genes has HS specific chromatin structure. The structure at each HS, which is different from the locus transcribing the fetal globin genes, might relate to its role in transcribing the adult genes. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    PubMed Central

    Spagnol, Stephen T.; Dahl, Kris Noel

    2016-01-01

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

  8. Retention of the Native Epigenome in Purified Mammalian Chromatin

    PubMed Central

    Ehrensberger, Andreas H.; Franchini, Don-Marc; East, Philip; George, Roger; Matthews, Nik; Maslen, Sarah L.; Svejstrup, Jesper Q.

    2015-01-01

    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. PMID:26248330

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

    PubMed

    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.

  10. Chromatin insulation by a transcriptional activator

    PubMed Central

    Sutter, Nathan B.; Scalzo, David; Fiering, Steven; Groudine, Mark; Martin, David I. K.

    2003-01-01

    In eukaryotic genomes, transcriptionally active regions are interspersed with silent chromatin that may repress genes in its vicinity. Chromatin insulators are elements that can shield a locus from repressive effects of flanking chromatin. Few such elements have been characterized in higher eukaryotes, but transcriptional activating elements are an invariant feature of active loci and have been shown to suppress transgene silencing. Hence, we have assessed the ability of a transcriptional activator to cause chromatin insulation, i.e., to relieve position effects at transgene integration sites in cultured cells. The transgene contained a series of binding sites for the metal-inducible transcriptional activator MTF, linked to a GFP reporter. Clones carrying single integrated transgenes were derived without selection for expression, and in most clones the transgene was silent. Induction of MTF resulted in transition of the transgene from the silent to the active state, prolongation of the active state, and a marked narrowing of the range of expression levels at different genomic sites. At one genomic site, prolonged induction of MTF resulted in suppression of transgene silencing that persisted after withdrawal of the induction stimulus. These results are consistent with MTF acting as a chromatin insulator and imply that transcriptional activating elements can insulate active loci against chromatin repression. PMID:12547916

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

    PubMed

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

    2012-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  13. Quantification of chromatin condensation level by image processing.

    PubMed

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

    2014-03-01

    The level of chromatin condensation is related to the silencing/activation of chromosomal territories and therefore impacts on gene expression. Chromatin condensation changes during cell cycle, progression and differentiation, and is influenced by various physicochemical and epigenetic factors. This study describes a validated experimental technique to quantify chromatin condensation. A novel image processing procedure is developed using Sobel edge detection to quantify the level of chromatin condensation from nuclei images taken by confocal microscopy. The algorithm was developed in MATLAB and used to quantify different levels of chromatin condensation in chondrocyte nuclei achieved through alteration in osmotic pressure. The resulting chromatin condensation parameter (CCP) is in good agreement with independent multi-observer qualitative visual assessment. This image processing technique thereby provides a validated unbiased parameter for rapid and highly reproducible quantification of the level of chromatin condensation. Copyright © 2013 IPEM. Published by Elsevier Ltd. All rights reserved.

  14. A Temporal Chromatin Signature in Human Embryonic Stem Cells Identifies Regulators of Cardiac Development

    PubMed Central

    Paige, Sharon L.; Thomas, Sean; Stoick-Cooper, Cristi L.; Wang, Hao; Maves, Lisa; Sandstrom, Richard; Pabon, Lil; Reinecke, Hans; Pratt, Gabriel; Keller, Gordon; Moon, Randall T.; Stamatoyannopoulos, John; Murry, Charles E.

    2012-01-01

    Summary Directed differentiation of human embryonic stem cells (ESCs) into cardiovascular cells provides a model for studying molecular mechanisms of human cardiovascular development. Though it is known that chromatin modification patterns in ESCs differ markedly from those in lineage-committed progenitors and differentiated cells, the temporal dynamics of chromatin alterations during differentiation along a defined lineage have not been studied. We show that differentiation of human ESCs into cardiovascular cells is accompanied by programmed temporal alterations in chromatin structure that distinguish key regulators of cardiovascular development from other genes. We used this temporal chromatin signature to identify regulators of cardiac development, including the homeobox gene MEIS2. We demonstrate using the zebrafish model that MEIS2 is critical for proper heart tube formation and subsequent cardiac looping. Temporal chromatin signatures should be broadly applicable to other models of stem cell differentiation to identify regulators and provide key insights into major developmental decisions. PMID:22981225

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

    PubMed

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

    2015-12-17

    Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Systematic Determination of Replication Activity Type Highlights Interconnections between Replication, Chromatin Structure and Nuclear Localization

    PubMed Central

    Polten, Andreas; Hezroni, Hadas; Eldar, Yonina C.; Meshorer, Eran; Yakhini, Zohar; Simon, Itamar

    2012-01-01

    DNA replication is a highly regulated process, with each genomic locus replicating at a distinct time of replication (ToR). Advances in ToR measurement technology enabled several genome-wide profiling studies that revealed tight associations between ToR and general genomic features and a remarkable ToR conservation in mammals. Genome wide studies further showed that at the hundreds kb-to-megabase scale the genome can be divided into constant ToR regions (CTRs) in which the replication process propagates at a faster pace due to the activation of multiple origins and temporal transition regions (TTRs) in which the replication process propagates at a slower pace. We developed a computational tool that assigns a ToR to every measured locus and determines its replication activity type (CTR versus TTR). Our algorithm, ARTO (Analysis of Replication Timing and Organization), uses signal processing methods to fit a constant piece-wise linear curve to the measured raw data. We tested our algorithm and provide performance and usability results. A Matlab implementation of ARTO is available at http://bioinfo.cs.technion.ac.il/people/zohar/ARTO/. Applying our algorithm to ToR data measured in multiple mouse and human samples allowed precise genome-wide ToR determination and replication activity type characterization. Analysis of the results highlighted the plasticity of the replication program. For example, we observed significant ToR differences in 10–25% of the genome when comparing different tissue types. Our analyses also provide evidence for activity type differences in up to 30% of the probes. Integration of the ToR data with multiple aspects of chromosome organization characteristics suggests that ToR plays a role in shaping the regional chromatin structure. Namely, repressive chromatin marks, are associated with late ToR both in TTRs and CTRs. Finally, characterization of the differences between TTRs and CTRs, with matching ToR, revealed that TTRs are associated with

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

    EPA Science Inventory

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

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

    PubMed Central

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

    2015-01-01

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

  19. The energy components of stacked chromatin layers explain the morphology, dimensions and mechanical properties of metaphase chromosomes

    PubMed Central

    Daban, Joan-Ramon

    2014-01-01

    The measurement of the dimensions of metaphase chromosomes in different animal and plant karyotypes prepared in different laboratories indicates that chromatids have a great variety of sizes which are dependent on the amount of DNA that they contain. However, all chromatids are elongated cylinders that have relatively similar shape proportions (length to diameter ratio approx. 13). To explain this geometry, it is considered that chromosomes are self-organizing structures formed by stacked layers of planar chromatin and that the energy of nucleosome–nucleosome interactions between chromatin layers inside the chromatid is approximately 3.6 × 10−20 J per nucleosome, which is the value reported by other authors for internucleosome interactions in chromatin fibres. Nucleosomes in the periphery of the chromatid are in contact with the medium; they cannot fully interact with bulk chromatin within layers and this generates a surface potential that destabilizes the structure. Chromatids are smooth cylinders because this morphology has a lower surface energy than structures having irregular surfaces. The elongated shape of chromatids can be explained if the destabilizing surface potential is higher in the telomeres (approx. 0.16 mJ m−2) than in the lateral surface (approx. 0.012 mJ m−2). The results obtained by other authors in experimental studies of chromosome mechanics have been used to test the proposed supramolecular structure. It is demonstrated quantitatively that internucleosome interactions between chromatin layers can justify the work required for elastic chromosome stretching (approx. 0.1 pJ for large chromosomes). The high amount of work (up to approx. 10 pJ) required for large chromosome extensions is probably absorbed by chromatin layers through a mechanism involving nucleosome unwrapping. PMID:24402918

  20. Restraint of angiogenesis by zinc finger transcription factor CTCF-dependent chromatin insulation

    PubMed Central

    Tang, Ming; Chen, Bo; Pardo, Carolina; Pampo, Christine; Chen, Jing; Lien, Ching-Ling; Wu, Lizi; Wang, Heiman; Yao, Kai; Oh, S. Paul; Seto, Edward; Smith, Lois E. H.; Siemann, Dietmar W.; Kladde, Michael P.; Cepko, Constance L.; Lu, Jianrong

    2011-01-01

    Angiogenesis is meticulously controlled by a fine balance between positive and negative regulatory activities. Vascular endothelial growth factor (VEGF) is a predominant angiogenic factor and its dosage is precisely regulated during normal vascular formation. In cancer, VEGF is commonly overproduced, resulting in abnormal neovascularization. VEGF is induced in response to various stimuli including hypoxia; however, very little is known about the mechanisms that confine its induction to ensure proper angiogenesis. Chromatin insulation is a key transcription mechanism that prevents promiscuous gene activation by interfering with the action of enhancers. Here we show that the chromatin insulator-binding factor CTCF binds to the proximal promoter of VEGF. Consistent with the enhancer-blocking mode of chromatin insulators, CTCF has little effect on basal expression of VEGF but specifically affects its activation by enhancers. CTCF knockdown cells are sensitized for induction of VEGF and exhibit elevated proangiogenic potential. Cancer-derived CTCF missense mutants are mostly defective in blocking enhancers at the VEGF locus. Moreover, during mouse retinal development, depletion of CTCF causes excess angiogenesis. Therefore, CTCF-mediated chromatin insulation acts as a crucial safeguard against hyperactivation of angiogenesis. PMID:21896759

  1. Quantitative analysis of nucleolar chromatin distribution in the complex convoluted nucleoli of Didinium nasutum (Ciliophora).

    PubMed

    Leonova, Olga G; Karajan, Bella P; Ivlev, Yuri F; Ivanova, Julia L; Skarlato, Sergei O; Popenko, Vladimir I

    2013-01-01

    We have earlier shown that the typical Didinium nasutum nucleolus is a complex convoluted branched domain, comprising a dense fibrillar component located at the periphery of the nucleolus and a granular component located in the central part. Here our main interest was to study quantitatively the spatial distribution of nucleolar chromatin structures in these convoluted nucleoli. There are no "classical" fibrillar centers in D.nasutum nucleoli. The spatial distribution of nucleolar chromatin bodies, which play the role of nucleolar organizers in the macronucleus of D.nasutum, was studied using 3D reconstructions based on serial ultrathin sections. The relative number of nucleolar chromatin bodies was determined in macronuclei of recently fed, starved D.nasutum cells and in resting cysts. This parameter is shown to correlate with the activity of the nucleolus. However, the relative number of nucleolar chromatin bodies in different regions of the same convoluted nucleolus is approximately the same. This finding suggests equal activity in different parts of the nucleolar domain and indicates the existence of some molecular mechanism enabling it to synchronize this activity in D. nasutum nucleoli. Our data show that D. nasutum nucleoli display bipartite structure. All nucleolar chromatin bodies are shown to be located outside of nucleoli, at the periphery of the fibrillar component.

  2. Epigenomic regulation of oncogenesis by chromatin remodeling.

    PubMed

    Kumar, R; Li, D-Q; Müller, S; Knapp, S

    2016-08-25

    Disruption of the intricate gene expression program represents one of major driving factors for the development, progression and maintenance of human cancer, and is often associated with acquired therapeutic resistance. At the molecular level, cancerous phenotypes are the outcome of cellular functions of critical genes, regulatory interactions of histones and chromatin remodeling complexes in response to dynamic and persistent upstream signals. A large body of genetic and biochemical evidence suggests that the chromatin remodelers integrate the extracellular and cytoplasmic signals to control gene activity. Consequently, widespread dysregulation of chromatin remodelers and the resulting inappropriate expression of regulatory genes, together, lead to oncogenesis. We summarize the recent developments and current state of the dysregulation of the chromatin remodeling components as the driving mechanism underlying the growth and progression of human tumors. Because chromatin remodelers, modifying enzymes and protein-protein interactions participate in interpreting the epigenetic code, selective chromatin remodelers and bromodomains have emerged as new frontiers for pharmacological intervention to develop future anti-cancer strategies to be used either as single-agent or in combination therapies with chemotherapeutics or radiotherapy.

  3. Progressive changes in chromatin structure and DNA damage response signals in bone marrow and peripheral blood during myelomagenesis.

    PubMed

    Gkotzamanidou, M; Terpos, E; Bamia, C; Kyrtopoulos, S A; Sfikakis, P P; Dimopoulos, M A; Souliotis, V L

    2014-05-01

    The molecular pathways implicated in multiple myeloma (MM) development are rather unknown. We studied epigenetic and DNA damage response (DDR) signals at selected model loci (N-ras, p53, d-globin) in bone marrow plasma cells and peripheral blood mononuclear cells (PBMCs) from patients with monoclonal gammopathy of undetermined significance (MGUS; n=20), smoldering/asymptomatic MM (SMM; n=29) and MM (n=18), as well as in healthy control-derived PBMCs (n=20). In both tissues analyzed, a progressive, significant increase in the looseness of local chromatin structure, gene expression levels and DNA repair efficiency from MGUS to SMM and finally to MM was observed (all P<0.002). Following ex vivo treatment with melphalan, a gradual suppression of the apoptotic pathway occurred in samples collected at different stages of myelomagenesis, with the severity and duration of the inhibition of RNA synthesis, p53 phosphorylation at serine15 and induction of apoptosis being higher in MGUS than SMM and lowest in MM patients (all P<0.0103). Interestingly, for all endpoints analyzed, a strong correlation between plasma cells and corresponding PBMCs was observed (all P<0.0003). We conclude that progressive changes in chromatin structure, transcriptional activity and DDR pathways during myelomagenesis occur in malignant plasma cells and that these changes are also reflected in PBMCs.

  4. Stress and the Emerging Roles of Chromatin Remodeling in Signal Integration and Stable Transmission of Reversible Phenotypes

    PubMed Central

    Weaver, Ian C. G.; Korgan, Austin C.; Lee, Kristen; Wheeler, Ryan V.; Hundert, Amos S.; Goguen, Donna

    2017-01-01

    The influence of early life experience and degree of parental-infant attachment on emotional development in children and adolescents has been comprehensively studied. Structural and mechanistic insight into the biological foundation and maintenance of mammalian defensive systems (metabolic, immune, nervous and behavioral) is slowly advancing through the emerging field of developmental molecular (epi)genetics. Initial evidence revealed that differential nurture early in life generates stable differences in offspring hypothalamic-pituitary-adrenal (HPA) regulation, in part, through chromatin remodeling and changes in DNA methylation of specific genes expressed in the brain, revealing physical, biochemical and molecular paths for the epidemiological concept of gene-environment interactions. Herein, a primary molecular mechanism underpinning the early developmental programming and lifelong maintenance of defensive (emotional) responses in the offspring is the alteration of chromatin domains of specific genomic regions from a condensed state (heterochromatin) to a transcriptionally accessible state (euchromatin). Conversely, DNA methylation promotes the formation of heterochromatin, which is essential for gene silencing, genomic integrity and chromosome segregation. Therefore, inter-individual differences in chromatin modifications and DNA methylation marks hold great potential for assessing the impact of both early life experience and effectiveness of intervention programs—from guided psychosocial strategies focused on changing behavior to pharmacological treatments that target chromatin remodeling and DNA methylation enzymes to dietary approaches that alter cellular pools of metabolic intermediates and methyl donors to affect nutrient bioavailability and metabolism. In this review article, we discuss the potential molecular mechanism(s) of gene regulation associated with chromatin modeling and programming of endocrine (e.g., HPA and metabolic or cardiovascular) and

  5. High-Resolution Whole-Genome Sequencing Reveals That Specific Chromatin Domains from Most Human Chromosomes Associate with Nucleoli

    PubMed Central

    van Koningsbruggen, Silvana; Gierliński, Marek; Schofield, Pietá; Martin, David; Barton, Geoffey J.; Ariyurek, Yavuz; den Dunnen, Johan T.

    2010-01-01

    The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope. PMID:20826608

  6. High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli.

    PubMed

    van Koningsbruggen, Silvana; Gierlinski, Marek; Schofield, Pietá; Martin, David; Barton, Geoffey J; Ariyurek, Yavuz; den Dunnen, Johan T; Lamond, Angus I

    2010-11-01

    The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

  7. Effects of DNA Methylation and Chromatin State on Rates of Molecular Evolution in Insects.

    PubMed

    Glastad, Karl M; Goodisman, Michael A D; Yi, Soojin V; Hunt, Brendan G

    2015-12-04

    Epigenetic information is widely appreciated for its role in gene regulation in eukaryotic organisms. However, epigenetic information can also influence genome evolution. Here, we investigate the effects of epigenetic information on gene sequence evolution in two disparate insects: the fly Drosophila melanogaster, which lacks substantial DNA methylation, and the ant Camponotus floridanus, which possesses a functional DNA methylation system. We found that DNA methylation was positively correlated with the synonymous substitution rate in C. floridanus, suggesting a key effect of DNA methylation on patterns of gene evolution. However, our data suggest the link between DNA methylation and elevated rates of synonymous substitution was explained, in large part, by the targeting of DNA methylation to genes with signatures of transcriptionally active chromatin, rather than the mutational effect of DNA methylation itself. This phenomenon may be explained by an elevated mutation rate for genes residing in transcriptionally active chromatin, or by increased structural constraints on genes in inactive chromatin. This result highlights the importance of chromatin structure as the primary epigenetic driver of genome evolution in insects. Overall, our study demonstrates how different epigenetic systems contribute to variation in the rates of coding sequence evolution. Copyright © 2016 Glastad et al.

  8. Sperm chromatin structure assay results after swim-up are related only to embryo quality but not to fertilization and pregnancy rates following IVF.

    PubMed

    Niu, Zhi-Hong; Shi, Hui-Juan; Zhang, Hui-Qin; Zhang, Ai-Jun; Sun, Yi-Juan; Feng, Yun

    2011-11-01

    The aim of this study was to investigate whether the sperm chromatin structure assay (SCSA) results after swim-up are related to fertilization rates, embryo quality and pregnancy rates following in vitro fertilization (IVF). A total of 223 couples undergoing IVF in our hospital from October 2008 to September 2009 were included in this study. Data on the IVF process and sperm chromatin structure assay results were collected. Fertilization rate, embryo quality and IVF success rates of different DNA fragmentation index (DFI) subgroups and high DNA stainability (HDS) subgroups were compared. There were no significant differences in fertilization rate, clinical pregnancy or delivery rates between the DFI and HDS subgroups. However, the group with abnormal DFI had a lower good embryo rate. So, we concluded that the SCSA variables, either DFI or HDS after swim-up preparation, were not valuable in predicting fertilization failure or pregnancy rate, but an abnormal DFI meant a lower good embryo rate following IVF.

  9. Atomic force microscopy of chromatin arrays reveal non-monotonic salt dependence of array compaction in solution

    PubMed Central

    Krzemien, Katarzyna M.; Beckers, Maximilian; Quack, Salina; Michaelis, Jens

    2017-01-01

    Compaction of DNA in chromatin is a hallmark of the eukaryotic cell and unravelling its structure is required for an understanding of DNA involving processes. Despite strong experimental efforts, many questions concerning the DNA packing are open. In particular, it is heavily debated whether an ordered structure referred to as the “30 nm fibre” exist in vivo. Scanning probe microscopy has become a cutting edge technology for the high-resolution imaging of DNA- protein complexes. Here, we perform high-resolution atomic force microscopy of non-cross-linked chromatin arrays in liquid, under different salt conditions. A statistical analysis of the data reveals that array compaction is salt dependent in a non-monotonic fashion. A simple physical model can qualitatively explain the observed findings due to the opposing effects of salt dependent stiffening of DNA, nucleosome stability and histone-histone interactions. While for different salt concentrations different compaction states are observed, our data do not provide support for the existence of regular chromatin fibres. Our studies add new insights into chromatin structure, and with that contribute to a further understanding of the DNA condensation. PMID:28296908

  10. Chromatin dynamics in plants.

    PubMed

    Fransz, Paul F; de Jong, J Hans

    2002-12-01

    Recent studies in yeast, animals and plants have provided major breakthroughs in unraveling the molecular mechanism of higher-order gene regulation. In conjunction with the DNA code, proteins that are involved in chromatin remodeling, histone modification and epigenetic imprinting form a large network of interactions that control the nuclear programming of cell identity. New insight into how chromatin conformations are regulated in plants sheds light on the relationships between chromosome function, cell differentiation and developmental patterns.

  11. Acetylation-Dependent Chromatin Reorganization by BRDT, a Testis-Specific Bromodomain-Containing Protein

    PubMed Central

    Pivot-Pajot, Christophe; Caron, Cécile; Govin, Jérôme; Vion, Alexandre; Rousseaux, Sophie; Khochbin, Saadi

    2003-01-01

    The association between histone acetylation and replacement observed during spermatogenesis prompted us to consider the testis as a source for potential factors capable of remodelling acetylated chromatin. A systematic search of data banks for open reading frames encoding testis-specific bromodomain-containing proteins focused our attention on BRDT, a testis-specific protein of unknown function containing two bromodomains. BRDT specifically binds hyperacetylated histone H4 tail depending on the integrity of both bromodomains. Moreover, in somatic cells, the ectopic expression of BRDT triggered a dramatic reorganization of the chromatin only after induction of histone hyperacetylation by trichostatin A (TSA). We then defined critical domains of BRDT involved in its activity. Both bromodomains of BRDT, as well as flanking regions, were found indispensable for its histone acetylation-dependent remodelling activity. Interestingly, we also observed that recombinant BRDT was capable of inducing reorganization of the chromatin of isolated nuclei in vitro only when the nuclei were from TSA-treated cells. This assay also allowed us to show that the action of BRDT was ATP independent, suggesting a structural role for the protein in the remodelling of acetylated chromatin. This is the first demonstration of a large-scale reorganization of acetylated chromatin induced by a specific factor. PMID:12861021

  12. Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chassé, Maggie H.; Muthurajan, Uma M.; Clark, Nicholas J.

    Poly (ADP-Ribose) Polymerase I (PARP-1) is a first responder to DNA damage and participates in the regulation of gene expression. The interaction of PARP-1 with chromatin and DNA is complex and involves at least two different modes of interaction. In its enzymatically inactive state, PARP-1 binds native chromatin with similar affinity as it binds free DNA ends. Automodification of PARP-1 affects interaction with chromatin and DNA to different extents. Here we describe a series of biochemical and biophysical techniques to quantify and dissect the different binding modes of PARP-1 with its various substrates. The techniques listed here allow for highmore » throughput and quantitative measurements of the interaction of different PARP-1 constructs (inactive and automodified) with chromatin and DNA damage models.« less

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

    PubMed

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

    2014-11-13

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

  14. Human linker histones: interplay between phosphorylation and O-β-GlcNAc to mediate chromatin structural modifications

    PubMed Central

    2011-01-01

    Eukaryotic chromatin is a combination of DNA and histone proteins. It is established fact that epigenetic mechanisms are associated with DNA and histones. Initial studies emphasize on core histones association with DNA, however later studies prove the importance of linker histone H1 epigenetic. There are many types of linker histone H1 found in mammals. These subtypes are cell specific and their amount in different types of cells varies as the cell functions. Many types of post-translational modifications which occur on different residues in each subtype of linker histone H1 induce conformational changes and allow the different subtypes of linker histone H1 to interact with chromatin at different stages during cell cycle which results in the regulation of transcription and gene expression. Proposed O-glycosylation of linker histone H1 promotes condensation of chromatin while phosphorylation of linker histone H1 is known to activate transcription and gene regulation by decondensation of chromatin. Interplay between phosphorylation and O-β-GlcNAc modification on Ser and Thr residues in each subtype of linker histone H1 in Homo sapiens during cell cycle may result in diverse functional regulation of proteins. This in silico study describes the potential phosphorylation, o-glycosylation and their possible interplay sites on conserved Ser/Thr residues in various subtypes of linker histone H1 in Homo sapiens. PMID:21749719

  15. Modulating chromatin structure and DNA accessibility by deacetylase inhibition enhances the anti-cancer activity of silver nanoparticles.

    PubMed

    Igaz, Nóra; Kovács, Dávid; Rázga, Zsolt; Kónya, Zoltán; Boros, Imre M; Kiricsi, Mónika

    2016-10-01

    Histone deacetylase (HDAC) inhibitors are considered as novel therapeutic agents inducing cell cycle arrest and apoptotic cell death in various cancer cells. Inhibition of deacetylase activity results in a relaxed chromatin structure thereby rendering the genetic material more vulnerable to DNA targeting agents that could be exploited by combinational cancer therapy. The unique potential of silver nanoparticles (AgNPs) in tumor therapy relies on the generation of reactive radicals which trigger oxidative stress, DNA damage and apoptosis in cancer cells. The revolutionary application of AgNPs as chemotherapeutical drugs seems very promising, nevertheless the exact molecular mechanisms of AgNP action in combination with other anti-cancer agents have yet to be elucidated in details before clinical administrations. As a step towards this we investigated the combinational effect of HDAC inhibition and AgNP administration in HeLa cervical cancer cells. We identified synergistic inhibition of cancer cell growth and migration upon combinational treatments. Here we report that the HDAC inhibitor Trichostatin A enhances the DNA targeting capacity and apoptosis inducing efficacy of AgNPs most probably due to its effect on chromatin condensation. These results point to the potential benefits of combinational application of HDAC inhibitors and AgNPs in novel cancer medication protocols. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Ionic strength and temperature induced conformational changes in mononucleosomes and oligonucleosomes. [Chromatin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schmitz, K.S.; Kent, J.C.; Parthasarathy, N.

    1980-10-01

    Chromatin is a nucleohistone complex which exhibits a repeat unit structure as inferred from nuclease digestion studies. The repeat unit, or nucleosome, is defined as approx. 200 base pairs of DNA wrapped about the surface of an octameric histone complex (two copies each of the histones H2A, H2B, H3, and H4). We report in this communication preliminary studies on the conformation of chromatin mononucleosomes and oligonucleosomes as a function of temperature and ionic strength. The methods used were conductivity, fluorescence of bound proflavine, and quasielastic light scattering.

  17. Chromatin organization regulates viral egress dynamics

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aho, Vesa; Myllys, Markko; Ruokolainen, Visa

    Various types of DNA viruses are known to elicit the formation of a large nuclear viral replication compartment and marginalization of the cell chromatin. We used three-dimensional soft x-ray tomography, confocal and electron microscopy, combined with numerical modelling of capsid diffusion to analyse the molecular organization of chromatin in herpes simplex virus 1 infection and its effect on the transport of progeny viral capsids to the nuclear envelope. Our data showed that the formation of the viral replication compartment at late infection resulted in the enrichment of heterochromatin in the nuclear periphery accompanied by the compaction of chromatin. Random walkmore » modelling of herpes simplex virus 1–sized particles in a three-dimensional soft x-ray tomography reconstruction of an infected cell nucleus demonstrated that the peripheral, compacted chromatin restricts viral capsid diffusion, but due to interchromatin channels capsids are able to reach the nuclear envelope, the site of their nuclear egress.« less

  18. Chromatin organization regulates viral egress dynamics

    DOE PAGES

    Aho, Vesa; Myllys, Markko; Ruokolainen, Visa; ...

    2017-06-16

    Various types of DNA viruses are known to elicit the formation of a large nuclear viral replication compartment and marginalization of the cell chromatin. We used three-dimensional soft x-ray tomography, confocal and electron microscopy, combined with numerical modelling of capsid diffusion to analyse the molecular organization of chromatin in herpes simplex virus 1 infection and its effect on the transport of progeny viral capsids to the nuclear envelope. Our data showed that the formation of the viral replication compartment at late infection resulted in the enrichment of heterochromatin in the nuclear periphery accompanied by the compaction of chromatin. Random walkmore » modelling of herpes simplex virus 1–sized particles in a three-dimensional soft x-ray tomography reconstruction of an infected cell nucleus demonstrated that the peripheral, compacted chromatin restricts viral capsid diffusion, but due to interchromatin channels capsids are able to reach the nuclear envelope, the site of their nuclear egress.« less

  19. Visualization of chromatin domains created by the gypsy insulator of Drosophila.

    PubMed

    Byrd, Keith; Corces, Victor G

    2003-08-18

    Insulators might regulate gene expression by establishing and maintaining the organization of the chromatin fiber within the nucleus. Biochemical fractionation and in situ high salt extraction of lysed cells show that two known protein components of the gypsy insulator are present in the nuclear matrix. Using FISH with DNA probes located between two endogenous Su(Hw) binding sites, we show that the intervening DNA is arranged in a loop, with the two insulators located at the base. Mutations in insulator proteins, subjecting the cells to a brief heat shock, or destruction of the nuclear matrix lead to disruption of the loop. Insertion of an additional gypsy insulator in the center of the loop results in the formation of paired loops through the attachment of the inserted sequences to the nuclear matrix. These results suggest that the gypsy insulator might establish higher-order domains of chromatin structure and regulate nuclear organization by tethering the DNA to the nuclear matrix and creating chromatin loops.

  20. Chromatin organisation and cancer prognosis: a pan-cancer study.

    PubMed

    Kleppe, Andreas; Albregtsen, Fritz; Vlatkovic, Ljiljana; Pradhan, Manohar; Nielsen, Birgitte; Hveem, Tarjei S; Askautrud, Hanne A; Kristensen, Gunnar B; Nesbakken, Arild; Trovik, Jone; Wæhre, Håkon; Tomlinson, Ian; Shepherd, Neil A; Novelli, Marco; Kerr, David J; Danielsen, Håvard E

    2018-03-01

    Chromatin organisation affects gene expression and regional mutation frequencies and contributes to carcinogenesis. Aberrant organisation of DNA has been correlated with cancer prognosis in analyses of the chromatin component of tumour cell nuclei using image texture analysis. As yet, the methodology has not been sufficiently validated to permit its clinical application. We aimed to define and validate a novel prognostic biomarker for the automatic detection of heterogeneous chromatin organisation. Machine learning algorithms analysed the chromatin organisation in 461 000 images of tumour cell nuclei stained for DNA from 390 patients (discovery cohort) treated for stage I or II colorectal cancer at the Aker University Hospital (Oslo, Norway). The resulting marker of chromatin heterogeneity, termed Nucleotyping, was subsequently independently validated in six patient cohorts: 442 patients with stage I or II colorectal cancer in the Gloucester Colorectal Cancer Study (UK); 391 patients with stage II colorectal cancer in the QUASAR 2 trial; 246 patients with stage I ovarian carcinoma; 354 patients with uterine sarcoma; 307 patients with prostate carcinoma; and 791 patients with endometrial carcinoma. The primary outcome was cancer-specific survival. In all patient cohorts, patients with chromatin heterogeneous tumours had worse cancer-specific survival than patients with chromatin homogeneous tumours (univariable analysis hazard ratio [HR] 1·7, 95% CI 1·2-2·5, in the discovery cohort; 1·8, 1·0-3·0, in the Gloucester validation cohort; 2·2, 1·1-4·5, in the QUASAR 2 validation cohort; 3·1, 1·9-5·0, in the ovarian carcinoma cohort; 2·5, 1·8-3·4, in the uterine sarcoma cohort; 2·3, 1·2-4·6, in the prostate carcinoma cohort; and 4·3, 2·8-6·8, in the endometrial carcinoma cohort). After adjusting for established prognostic patient characteristics in multivariable analyses, Nucleotyping was prognostic in all cohorts except for the prostate carcinoma

  1. A chromatin link to caste identity in the carpenter ant Camponotus floridanus

    PubMed Central

    Simola, Daniel F.; Ye, Chaoyang; Mutti, Navdeep S.; Dolezal, Kelly; Bonasio, Roberto; Liebig, Jürgen; Reinberg, Danny; Berger, Shelley L.

    2013-01-01

    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

  2. Sperm chromatin alterations in fertile and subfertile bulls.

    PubMed

    Souza, Elisson Terêncio; Silva, Cláudio Vieira; Travençolo, Bruno Augusto Nassif; Alves, Benner Geraldo; Beletti, Marcelo Emílio

    2018-06-01

    Alterations in sperm chromatin have been related with subfertility in several mammals. In this study, chromatin alteration types (Base, Basal half, Central axis, Dispersed, and Whole) were assessed by toluidine blue (TB) staining, 6-diamidino-2-fenilindole (DAPI) and anti-protamine 1 antibody (anti-PR1) labeling in sperm samples of fertile and subfertile bulls. Semen samples were obtained from bulls kept in Artificial Insemination Center (fertile bulls) or from bulls subjected to scrotal insulation (subfertile bulls). The percentage of chromatin alterations identified by TB was similar (P > 0.05) in semen samples of fertile and subfertile bulls. In contrast, a greater (P < 0.01) chromatin decondensation and heterogeneity were recorded in semen samples of subfertile bulls. In DAPI and anti-PR1 methods, the subfertile bulls samples had a higher (P < 0.05) percentage of alteration in the base as well as overall chromatin alterations (P < 0.05). Moreover, the chromatin alterations recorded with TB, DAPI, and anti-PR1 were compared in semen samples of fertile and subfertile bulls. In fertile bulls, the overall chromatin alterations were similar (P > 0.05) among the methods In contrast, semen samples of subfertile bulls had a higher (P < 0.05) percentage of overall chromatin alterations when labeled with DAPI. In conclusion, our findings shown that all dye tested had specific sperm stainability and can be feasible to monitor subfertility condition in bulls. Also, different chromatin alteration types in sperm samples of fertile and suberftile bulls were recorded. Copyright © 2018 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.

  3. Proteomics in chromatin biology and epigenetics: Elucidation of post-translational modifications of histone proteins by mass spectrometry.

    PubMed

    Sidoli, Simone; Cheng, Lei; Jensen, Ole N

    2012-06-27

    Histone proteins contribute to the maintenance and regulation of the dynamic chromatin structure, to gene activation, DNA repair and many other processes in the cell nucleus. Site-specific reversible and irreversible post-translational modifications of histone proteins mediate biological functions, including recruitment of transcription factors to specific DNA regions, assembly of epigenetic reader/writer/eraser complexes onto DNA, and modulation of DNA-protein interactions. Histones thereby regulate chromatin structure and function, propagate inheritance and provide memory functions in the cell. Dysfunctional chromatin structures and misregulation may lead to pathogenic states, including diabetes and cancer, and the mapping and quantification of multivalent post-translational modifications has therefore attracted significant interest. Mass spectrometry has quickly been accepted as a versatile tool to achieve insights into chromatin biology and epigenetics. High sensitivity and high mass accuracy and the ability to sequence post-translationally modified peptides and perform large-scale analyses make this technique very well suited for histone protein characterization. In this review we discuss a range of analytical methods and various mass spectrometry-based approaches for histone analysis, from sample preparation to data interpretation. Mass spectrometry-based proteomics is already an integrated and indispensable tool in modern chromatin biology, providing insights into the mechanisms and dynamics of nuclear and epigenetic processes. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

    PubMed Central

    Gartenberg, Marc R.; Smith, Jeffrey S.

    2016-01-01

    Transcriptional silencing in Saccharomyces cerevisiae occurs at several genomic sites including the silent mating-type loci, telomeres, and the ribosomal DNA (rDNA) tandem array. Epigenetic silencing at each of these domains is characterized by the absence of nearly all histone modifications, including most prominently the lack of histone H4 lysine 16 acetylation. In all cases, silencing requires Sir2, a highly-conserved NAD+-dependent histone deacetylase. At locations other than the rDNA, silencing also requires additional Sir proteins, Sir1, Sir3, and Sir4 that together form a repressive heterochromatin-like structure termed silent chromatin. The mechanisms of silent chromatin establishment, maintenance, and inheritance have been investigated extensively over the last 25 years, and these studies have revealed numerous paradigms for transcriptional repression, chromatin organization, and epigenetic gene regulation. Studies of Sir2-dependent silencing at the rDNA have also contributed to understanding the mechanisms for maintaining the stability of repetitive DNA and regulating replicative cell aging. The goal of this comprehensive review is to distill a wide array of biochemical, molecular genetic, cell biological, and genomics studies down to the “nuts and bolts” of silent chromatin and the processes that yield transcriptional silencing. PMID:27516616

  5. Role of ND10 nuclear bodies in the chromatin repression of HSV-1.

    PubMed

    Gu, Haidong; Zheng, Yi

    2016-04-05

    Herpes simplex virus (HSV) is a neurotropic virus that establishes lifelong latent infection in human ganglion sensory neurons. This unique life cycle necessitates an intimate relation between the host defenses and virus counteractions over the long course of infection. Two important aspects of host anti-viral defense, nuclear substructure restriction and epigenetic chromatin regulation, have been intensively studied in the recent years. Upon viral DNA entering the nucleus, components of discrete nuclear bodies termed nuclear domain 10 (ND10), converge at viral DNA and place restrictions on viral gene expression. Meanwhile the infected cell mobilizes its histones and histone-associated repressors to force the viral DNA into nucleosome-like structures and also represses viral transcription. Both anti-viral strategies are negated by various HSV countermeasures. One HSV gene transactivator, infected cell protein 0 (ICP0), is a key player in antagonizing both the ND10 restriction and chromatin repression. On one hand, ICP0 uses its E3 ubiquitin ligase activity to target major ND10 components for proteasome-dependent degradation and thereafter disrupts the ND10 nuclear bodies. On the other hand, ICP0 participates in de-repressing the HSV chromatin by changing histone composition or modification and therefore activates viral transcription. Involvement of a single viral protein in two seemingly different pathways suggests that there is coordination in host anti-viral defense mechanisms and also cooperation in viral counteraction strategies. In this review, we summarize recent advances in understanding the role of chromatin regulation and ND10 dynamics in both lytic and latent HSV infection. We focus on the new observations showing that ND10 nuclear bodies play a critical role in cellular chromatin regulation. We intend to find the connections between the two major anti-viral defense pathways, chromatin remodeling and ND10 structure, in order to achieve a better

  6. The ISW1 and CHD1 ATP-dependent chromatin remodelers compete to set nucleosome spacing in vivo.

    PubMed

    Ocampo, Josefina; Chereji, Răzvan V; Eriksson, Peter R; Clark, David J

    2016-06-02

    Adenosine triphosphate-dependent chromatin remodeling machines play a central role in gene regulation by manipulating chromatin structure. Most genes have a nucleosome-depleted region at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. In vitro, the three known yeast nucleosome spacing enzymes (CHD1, ISW1 and ISW2) form arrays with different spacing. We used genome-wide nucleosome sequencing to determine whether these enzymes space nucleosomes differently in vivo We find that CHD1 and ISW1 compete to set the spacing on most genes, such that CHD1 dominates genes with shorter spacing and ISW1 dominates genes with longer spacing. In contrast, ISW2 plays a minor role, limited to transcriptionally inactive genes. Heavily transcribed genes show weak phasing and extreme spacing, either very short or very long, and are depleted of linker histone (H1). Genes with longer spacing are enriched in H1, which directs chromatin folding. We propose that CHD1 directs short spacing, resulting in eviction of H1 and chromatin unfolding, whereas ISW1 directs longer spacing, allowing H1 to bind and condense the chromatin. Thus, competition between the two remodelers to set the spacing on each gene may result in a highly dynamic chromatin structure. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  7. Chromatin Remodeling and Plant Immunity.

    PubMed

    Chen, W; Zhu, Q; Liu, Y; Zhang, Q

    Chromatin remodeling, an important facet of the regulation of gene expression in eukaryotes, is performed by two major types of multisubunit complexes, covalent histone- or DNA-modifying complexes, and ATP-dependent chromosome remodeling complexes. Snf2 family DNA-dependent ATPases constitute the catalytic subunits of ATP-dependent chromosome remodeling complexes, which accounts for energy supply during chromatin remodeling. Increasing evidence indicates a critical role of chromatin remodeling in the establishment of long-lasting, even transgenerational immune memory in plants, which is supported by the findings that DNA methylation, histone deacetylation, and histone methylation can prime the promoters of immune-related genes required for disease defense. So what are the links between Snf2-mediated ATP-dependent chromosome remodeling and plant immunity, and what mechanisms might support its involvement in disease resistance? © 2017 Elsevier Inc. All rights reserved.

  8. Nuclear matrix - structure, function and pathogenesis.

    PubMed

    Wasąg, Piotr; Lenartowski, Robert

    2016-12-20

    The nuclear matrix (NM), or nuclear skeleton, is the non-chromatin, ribonucleoproteinaceous framework that is resistant to high ionic strength buffers, nonionic detergents, and nucleolytic enzymes. The NM fulfills a structural role in eukaryotic cells and is responsible for maintaining the shape of the nucleus and the spatial organization of chromatin. Moreover, the NM participates in several cellular processes, such as DNA replication/repair, gene expression, RNA transport, cell signaling and differentiation, cell cycle regulation, apoptosis and carcinogenesis. Short nucleotide sequences called scaffold/matrix attachment regions (S/MAR) anchor the chromatin loops to the NM proteins (NMP). The NMP composition is dynamic and depends on the cell type and differentiation stage or metabolic activity. Alterations in the NMP composition affect anchoring of the S/MARs and thus alter gene expression. This review aims to systematize information about the skeletal structure of the nucleus, with particular emphasis on the organization of the NM and its role in selected cellular processes. We also discuss several diseases that are caused by aberrant NM structure or dysfunction of individual NM elements.

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

    PubMed Central

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

    2015-01-01

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

  10. Radiation induced chromatin conformation changes analysed by fluorescent localization microscopy, statistical physics, and graph theory.

    PubMed

    Zhang, Yang; Máté, Gabriell; Müller, Patrick; Hillebrandt, Sabina; Krufczik, Matthias; Bach, Margund; Kaufmann, Rainer; Hausmann, Michael; Heermann, Dieter W

    2015-01-01

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

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

    PubMed

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

    2009-06-01

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

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

    PubMed

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

    2016-02-01

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

  13. Repressive Chromatin in Caenorhabditis elegans: Establishment, Composition, and Function

    PubMed Central

    Ahringer, Julie; Gasser, Susan M.

    2018-01-01

    Chromatin is organized and compacted in the nucleus through the association of histones and other proteins, which together control genomic activity. Two broad types of chromatin can be distinguished: euchromatin, which is generally transcriptionally active, and heterochromatin, which is repressed. Here we examine the current state of our understanding of repressed chromatin in Caenorhabditis elegans, focusing on roles of histone modifications associated with repression, such as methylation of histone H3 lysine 9 (H3K9me2/3) or the Polycomb Repressive Complex 2 (MES-2/3/6)-deposited modification H3K27me3, and on proteins that recognize these modifications. Proteins involved in chromatin repression are important for development, and have demonstrated roles in nuclear organization, repetitive element silencing, genome integrity, and the regulation of euchromatin. Additionally, chromatin factors participate in repression with small RNA pathways. Recent findings shed light on heterochromatin function and regulation in C. elegans, and should inform our understanding of repressed chromatin in other animals. PMID:29378810

  14. Models of chromatin spatial organisation in the cell nucleus

    NASA Astrophysics Data System (ADS)

    Nicodemi, Mario

    2014-03-01

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

  15. Open chromatin reveals the functional maize genome

    USDA-ARS?s Scientific Manuscript database

    Every cellular process mediated through nuclear DNA must contend with chromatin. As results from ENCODE show, open chromatin assays can efficiently integrate across diverse regulatory elements, revealing functional non-coding genome. In this study, we use a MNase hypersensitivity assay to discover o...

  16. Chromatin decompaction by the nucleosomal binding protein HMGN5 impairs nuclear sturdiness

    NASA Astrophysics Data System (ADS)

    Furusawa, Takashi; Rochman, Mark; Taher, Leila; Dimitriadis, Emilios K.; Nagashima, Kunio; Anderson, Stasia; Bustin, Michael

    2015-01-01

    In most metazoan nuclei, heterochromatin is located at the nuclear periphery in contact with the nuclear lamina, which provides mechanical stability to the nucleus. We show that in cultured cells, chromatin decompaction by the nucleosome binding protein HMGN5 decreases the sturdiness, elasticity and rigidity of the nucleus. Mice overexpressing HMGN5, either globally or only in the heart, are normal at birth but develop hypertrophic heart with large cardiomyoctyes, deformed nuclei and disrupted lamina and die of cardiac malfunction. Chromatin decompaction is seen in cardiomyocytes of newborn mice but misshaped nuclei with disrupted lamina are seen only in adult cardiomyocytes, suggesting that loss of heterochromatin diminishes the ability of the nucleus to withstand the mechanical forces of the contracting heart. Thus, heterochromatin enhances the ability of the nuclear lamina to maintain the sturdiness and shape of the eukaryotic nucleus; a structural role for chromatin that is distinct from its genetic functions.

  17. [Mechanisms of genoprotective action of a phytoecdysteroid drug(BTK-8L) in chromatin damage by tetrachloromethane].

    PubMed

    Gubskiĭ, Iu I; Levitskiĭ, E L; Kholodova, Iu D; Goriushko, A G; Primak, R G; Vistunova, I E; Sachenko, L G

    1993-01-01

    Hepatoprotective action of prophylactic injection of aqueous solution of preparation BTK-8L from plant ecdysteroids to experimental animals with the liver damage by tetrachloromethane was revealed. This effect at least partially was connected with the genoprotective action of the given preparation. As a result, normalization of free radical chromatin lipid peroxidation reaction, modified at the intoxication, as well as partial correction of physical and chemical properties of chromatin protein-lipid complex were those molecular mechanisms of genoprotective action of BTK-8L, which were manifested by the influence of the preparation on such indices which characterized the depth structure of the complex as microviscosity and energy transfer from the protein to the lipid probe. Investigation of the interaction of the preparation with chromatin fractions in vitro and comparison of this interaction with the analogous process in model systems allowed revealing determinative participation of chromatin proteins and lipids in the given process. The preparation interacted more intensively with the active chromatin fraction, which contained a more marked protein-lipid complex, as comparing to the repressed one. Injection of the preparation also normalized such indices as relation between the chromatine fractions and protein/DNA ratio in them. On the contrary, injection of the alcoholic solution of the preparation to experimental animals, aggravated genotoxic tetrachloromethane action.

  18. Rapid and reversible epigenome editing by endogenous chromatin regulators.

    PubMed

    Braun, Simon M G; Kirkland, Jacob G; Chory, Emma J; Husmann, Dylan; Calarco, Joseph P; Crabtree, Gerald R

    2017-09-15

    Understanding the causal link between epigenetic marks and gene regulation remains a central question in chromatin biology. To edit the epigenome we developed the FIRE-Cas9 system for rapid and reversible recruitment of endogenous chromatin regulators to specific genomic loci. We enhanced the dCas9-MS2 anchor for genome targeting with Fkbp/Frb dimerizing fusion proteins to allow chemical-induced proximity of a desired chromatin regulator. We find that mSWI/SNF (BAF) complex recruitment is sufficient to oppose Polycomb within minutes, leading to activation of bivalent gene transcription in mouse embryonic stem cells. Furthermore, Hp1/Suv39h1 heterochromatin complex recruitment to active promoters deposits H3K9me3 domains, resulting in gene silencing that can be reversed upon washout of the chemical dimerizer. This inducible recruitment strategy provides precise kinetic information to model epigenetic memory and plasticity. It is broadly applicable to mechanistic studies of chromatin in mammalian cells and is particularly suited to the analysis of endogenous multi-subunit chromatin regulator complexes.Understanding the link between epigenetic marks and gene regulation requires the development of new tools to directly manipulate chromatin. Here the authors demonstrate a Cas9-based system to recruit chromatin remodelers to loci of interest, allowing rapid, reversible manipulation of epigenetic states.

  19. In Situ Hi-C Library Preparation for Plants to Study Their Three-Dimensional Chromatin Interactions on a Genome-Wide Scale.

    PubMed

    Liu, Chang

    2017-01-01

    The spatial organization of the genome in the nucleus is critical for many cellular processes. It has been broadly accepted that the packing of chromatin inside the nucleus is not random, but structured at several hierarchical levels. The Hi-C method combines Chromatin Conformation Capture and high-throughput sequencing, which allows interrogating genome-wide chromatin interactions. Depending on the sequencing depth, chromatin packing patterns derived from Hi-C experiments can be viewed on a chromosomal scale or at a local genic level. Here, I describe a protocol of plant in situ Hi-C library preparation, which covers procedures starting from tissue fixation to library amplification.

  20. Formation of new chromatin domains determines pathogenicity of genomic duplications.

    PubMed

    Franke, Martin; Ibrahim, Daniel M; Andrey, Guillaume; Schwarzer, Wibke; Heinrich, Verena; Schöpflin, Robert; Kraft, Katerina; Kempfer, Rieke; Jerković, Ivana; Chan, Wing-Lee; Spielmann, Malte; Timmermann, Bernd; Wittler, Lars; Kurth, Ingo; Cambiaso, Paola; Zuffardi, Orsetta; Houge, Gunnar; Lambie, Lindsay; Brancati, Francesco; Pombo, Ana; Vingron, Martin; Spitz, Francois; Mundlos, Stefan

    2016-10-13

    Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes. However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood. Here we show by chromosome conformation capture (capture Hi-C and 4C-seq methods) that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology. Duplications of non-coding DNA within the mouse Sox9 TAD (intra-TAD) that cause female to male sex reversal in humans, showed increased contact of the duplicated regions within the TAD, but no change in the overall TAD structure. In contrast, overlapping duplications that extended over the next boundary into the neighbouring TAD (inter-TAD), resulted in the formation of a new chromatin domain (neo-TAD) that was isolated from the rest of the genome. As a consequence of this insulation, inter-TAD duplications had no phenotypic effect. However, incorporation of the next flanking gene, Kcnj2, in the neo-TAD resulted in ectopic contacts of Kcnj2 with the duplicated part of the Sox9 regulatory region, consecutive misexpression of Kcnj2, and a limb malformation phenotype. Our findings provide evidence that TADs are genomic regulatory units with a high degree of internal stability that can be sculptured by structural genomic variations. This process is important for the interpretation of copy number variations, as these variations are routinely detected in diagnostic tests for genetic disease and cancer. This finding also has relevance in an evolutionary setting because copy-number differences are thought to have a crucial role in the evolution of

  1. MacroH2A1 associates with nuclear lamina and maintains chromatin architecture in mouse liver cells.

    PubMed

    Fu, Yuhua; Lv, Pin; Yan, Guoquan; Fan, Hui; Cheng, Lu; Zhang, Feng; Dang, Yongjun; Wu, Hao; Wen, Bo

    2015-11-25

    In the interphase nucleus, chromatin is organized into three-dimensional conformation to coordinate genome functions. The lamina-chromatin association is important to facilitate higher-order chromatin in mammalian cells, but its biological significances and molecular mechanisms remain poorly understood. One obstacle is that the list of lamina-associated proteins remains limited, presumably due to the inherent insolubility of lamina proteins. In this report, we identified 182 proteins associated with lamin B1 (a constitutive component of lamina) in mouse hepatocytes, by adopting virus-based proximity-dependent biotin identification. These proteins are functionally related to biological processes such as chromatin organization. As an example, we validated the association between lamin B1 and core histone macroH2A1, a histone associated with repressive chromatin. Furthermore, we mapped Lamina-associated domains (LADs) in mouse liver cells and found that boundaries of LADs are enriched for macroH2A. More interestingly, knocking-down of macroH2A1 resulted in the release of heterochromatin foci marked by histone lysine 9 trimethylation (H3K9me3) and the decondensation of global chromatin structure. However, down-regulation of lamin B1 led to redistribution of macroH2A1. Taken together, our data indicated that macroH2A1 is associated with lamina and is required to maintain chromatin architecture in mouse liver cells.

  2. Chromatin and Epigenetics at the Forefront: Finding Clues among Peaks

    PubMed Central

    Shi, Yang

    2016-01-01

    The Keystone Symposium on Chromatin and Epigenetics, organized by Luciano Di Croce (Center for Genomic Regulation, Spain) and Yang Shi (Harvard Medical School, USA), took place 20 to 24 March 2016 at Whistler (British Columbia, Canada). The symposium brought together some of the most outstanding scientists studying how chromatin structure and epigenetic mechanisms regulate gene function in both development and disease. Junior scientists had the opportunity to interact with experienced researchers by presenting their work and discussing ideas and novel hypotheses. In order to foster interaction and networking, the scientific agenda was balanced with an extended social agenda. This meeting review describes several of the most provocative and exciting talks from the symposium, revealing how fast this research field is evolving and the profound impact it will have on human health. PMID:27402863

  3. Mutations in chromatin regulators functionally link Cornelia de Lange syndrome and clinically overlapping phenotypes.

    PubMed

    Parenti, Ilaria; Teresa-Rodrigo, María E; Pozojevic, Jelena; Ruiz Gil, Sara; Bader, Ingrid; Braunholz, Diana; Bramswig, Nuria C; Gervasini, Cristina; Larizza, Lidia; Pfeiffer, Lutz; Ozkinay, Ferda; Ramos, Feliciano; Reiz, Benedikt; Rittinger, Olaf; Strom, Tim M; Watrin, Erwan; Wendt, Kerstin; Wieczorek, Dagmar; Wollnik, Bernd; Baquero-Montoya, Carolina; Pié, Juan; Deardorff, Matthew A; Gillessen-Kaesbach, Gabriele; Kaiser, Frank J

    2017-03-01

    The coordinated tissue-specific regulation of gene expression is essential for the proper development of all organisms. Mutations in multiple transcriptional regulators cause a group of neurodevelopmental disorders termed "transcriptomopathies" that share core phenotypical features including growth retardation, developmental delay, intellectual disability and facial dysmorphism. Cornelia de Lange syndrome (CdLS) belongs to this class of disorders and is caused by mutations in different subunits or regulators of the cohesin complex. Herein, we report on the clinical and molecular characterization of seven patients with features overlapping with CdLS who were found to carry mutations in chromatin regulators previously associated to other neurodevelopmental disorders that are frequently considered in the differential diagnosis of CdLS. The identified mutations affect the methyltransferase-encoding genes KMT2A and SETD5 and different subunits of the SWI/SNF chromatin-remodeling complex. Complementary to this, a patient with Coffin-Siris syndrome was found to carry a missense substitution in NIPBL. Our findings indicate that mutations in a variety of chromatin-associated factors result in overlapping clinical phenotypes, underscoring the genetic heterogeneity that should be considered when assessing the clinical and molecular diagnosis of neurodevelopmental syndromes. It is clear that emerging molecular mechanisms of chromatin dysregulation are central to understanding the pathogenesis of these clinically overlapping genetic disorders.

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

    PubMed Central

    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. Peptide Epitalon activates chromatin at the old age.

    PubMed

    Khavinson, Vladimir Kh; Lezhava, Teimuraz A; Monaselidze, Jamlet R; Jokhadze, Tinatin A; Dvalishvili, Nana A; Bablishvili, Nino K; Trofimova, Svetlana V

    2003-10-01

    OBJECTIVES and design. We have studied the effect of synthetic peptide Epitalon on the activity of ribosomal genes, denaturation parameters of total heterochromatin, polymorphism of structural C-heterochromatin and the variability of facultative heterochromatin in cultured lymphocytes of persons aged 76-80 years. The obtained data demonstrate that Epitalon induces the activation of ribosomal genes, decondensation of pericentromeric structural heterochromatin and the release of genes repressed due to the age-related condensation of euchromatic chromosome regions. Epitalon has shown its ability to activate chromatin by modifying heterochromatin and heterochromatinized chromosome regions in the cells of older persons.

  6. A Bayesian mixture model for chromatin interaction data.

    PubMed

    Niu, Liang; Lin, Shili

    2015-02-01

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

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

    DOE PAGES

    Ugarte, Fernando; Sousae, Rebekah; Cinquin, Bertrand; ...

    2015-10-17

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ugarte, Fernando; Sousae, Rebekah; Cinquin, Bertrand

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

  9. Studies on sex-organ development. Changes in chromatin structure during spermatogenesis in maturing rooster testis as demonstrated by the initiation pattern of ribonucleic acid synthesis in vitro.

    PubMed Central

    Mezquita, C; Teng, C S

    1978-01-01

    To probe the structural change in the genome of the differentiating germ cell of the maturing rooster testis, the chromatin from nuclei at various stages of differentiation were transcribed with prokaryotic RNA polymerase from Escherichia coli or with eukaryotic RNA polymerase II from wheat germ. The transcription was performed under conditions of blockage of RNA chain reinitiation in vitro with rifampicin or rifampicin AF/013. With the E. coli enzyme, the changes in (1) the titration curve for the enzyme-chromatin interaction, (2) the number of initiation sites, (3) the rate of elongation of RNA chains, and (4) the kinetics of the formation of stable initiation complexes revealed the unmasking of DNA in elongated spermatids and the masking of DNA in spermatozoa. In both cases the stability of the DNA duplex in the initiation region for RNA synthesis greatly increased. In contrast with the E. coli enzyme, the wheat-germ RNA polymerase II was relatively inefficient at transcribing chromatin of elongated spermatids. Such behaviour can be predicted if unmasked double-stranded DNA is present in elongated spermatids. PMID:346018

  10. Flightless I (Drosophila) homolog facilitates chromatin accessibility of the estrogen receptor α target genes in MCF-7 breast cancer cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 complexmore » 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.« less

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

    PubMed

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

    2013-11-01

    Recently, we have investigated the endocannabinoid involvement in chromatin remodeling events occurring in male spermatids. Indeed, we have demonstrated that genetic inactivation of the cannabinoid receptor type 1 (Cnr1) negatively influences chromatin remodeling mechanisms, by reducing histone displacement and indices of sperm chromatin quality (chromatin condensation and DNA integrity). Conversely, Cnr1 knock-out (Cnr1(-/-)) male mice, treated with estrogens, replaced histones and rescued chromatin condensation as well as DNA integrity. In the present study, by exploiting Cnr1(+/+), Cnr(+/-) and Cnr1(-/-) epididymal sperm samples, we show that histone retention directly correlates with low values of sperm chromatin quality indices determining sperm nuclear size elongation. Moreover, we demonstrate that estrogens, by promoting histone displacement and chromatin condensation rescue, are able to efficiently reduce the greater nuclear length observed in Cnr1(-/-) sperm. As a consequence of our results, we suggest that nucleus length may be used as a morphological parameter useful to screen out spermatozoa with low chromatin quality. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

    PubMed Central

    Sun, Fei; Huang, Li

    2013-01-01

    Strand displacement by a DNA polymerase serves a key role in Okazaki fragment maturation, which involves displacement of the RNA primer of the preexisting Okazaki fragment into a flap structure, and subsequent flap removal and fragment ligation. We investigated the role of Sulfolobus chromatin proteins Sso7d and Cren7 in strand displacement by DNA polymerase B1 (PolB1) from the hyperthermophilic archaeon Sulfolobus solfataricus. PolB1 showed a robust strand displacement activity and was capable of synthesizing thousands of nucleotides on a DNA-primed 72-nt single-stranded circular DNA template. This activity was inhibited by both Sso7d and Cren7, which limited the flap length to 3–4 nt at saturating concentrations. However, neither protein inhibited RNA displacement on an RNA-primed single-stranded DNA minicircle by PolB1. Strand displacement remained sensitive to modulation by the chromatin proteins when PolB1 was in association with proliferating cell nuclear antigen. Inhibition of DNA instead of RNA strand displacement by the chromatin proteins is consistent with the finding that double-stranded DNA was more efficiently bound and stabilized than an RNA:DNA duplex by these proteins. Our results suggest that Sulfolobus chromatin proteins modulate strand displacement by PolB1, permitting efficient removal of the RNA primer while inhibiting excessive displacement of the newly synthesized DNA strand during Okazaki fragment maturation. PMID:23821667

  13. Chromatin assembly: Journey to the CENter of the chromosome

    PubMed Central

    Chen, Chin-Chi

    2016-01-01

    All eukaryotic genomes are packaged into basic units of DNA wrapped around histone proteins called nucleosomes. The ability of histones to specify a variety of epigenetic states at defined chromatin domains is essential for cell survival. The most distinctive type of chromatin is found at centromeres, which are marked by the centromere-specific histone H3 variant CENP-A. Many of the factors that regulate CENP-A chromatin have been identified; however, our understanding of the mechanisms of centromeric nucleosome assembly, maintenance, and reorganization remains limited. This review discusses recent insights into these processes and draws parallels between centromeric and noncentromeric chromatin assembly mechanisms. PMID:27377247

  14. From the chromatin interaction network to the organization of the human genome into replication N/U-domains

    NASA Astrophysics Data System (ADS)

    Boulos, Rasha E.; Julienne, Hanna; Baker, Antoine; Chen, Chun-Long; Petryk, Nataliya; Kahli, Malik; dʼAubenton-Carafa, Yves; Goldar, Arach; Jensen, Pablo; Hyrien, Olivier; Thermes, Claude; Arneodo, Alain; Audit, Benjamin

    2014-11-01

    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.

  15. Distinct modes of DNA accessibility in plant chromatin.

    PubMed

    Shu, Huan; Wildhaber, Thomas; Siretskiy, Alexey; Gruissem, Wilhelm; Hennig, Lars

    2012-01-01

    The accessibility of DNA to regulatory proteins is a major property of the chromatin environment that favours or hinders transcription. Recent studies in flies reported that H3K9me2-marked heterochromatin is accessible while H3K27me3-marked chromatin forms extensive domains of low accessibility. Here we show that plants regulate DNA accessibility differently. H3K9me2-marked heterochromatin is the least accessible in the Arabidopsis thaliana genome, and H3K27me3-marked chromatin also has low accessibility. We see that very long genes without H3K9me2 or H3K27me3 are often inaccessible and generated significantly lower amounts of antisense transcripts than other genes, suggesting that reduced accessibility is associated with reduced recognition of alternative promoters. Low accessibility of H3K9me2-marked heterochromatin and long genes depend on cytosine methylation, explaining why chromatin accessibility differs between plants and flies. Together, we conclude that restriction of DNA accessibility is a local property of chromatin and not necessarily a consequence of microscopically visible compaction.

  16. Chromatin De-Compaction By The Nucleosomal Binding Protein HMGN5 Impairs Nuclear Sturdiness

    PubMed Central

    Furusawa, Takashi; Rochman, Mark; Taher, Leila; Dimitriadis, Emilios K.; Nagashima, Kunio; Anderson, Stasia; Bustin, Michael

    2014-01-01

    In most metazoan nuclei, heterochromatin is located at the nuclear periphery in contact with the nuclear lamina, which provides mechanical stability to the nucleus. We show that in cultured cells, chromatin de-compaction by the nucleosome binding protein HMGN5 decreases the sturdiness, elasticity, and rigidity of the nucleus. Mice overexpressing HMGN5, either globally or only in the heart, are normal at birth but develop hypertrophic heart with large cardiomyoctyes, deformed nuclei and disrupted lamina, and die of cardiac malfunction. Chromatin de-compaction is seen in cardiomyocytes of newborn mice but misshaped nuclei with disrupted lamina are seen only in adult cardiomyocytes, suggesting that loss of heterochromatin diminishes the ability of the nucleus to withstand the mechanical forces of the contracting heart. Thus, heterochromatin enhances the ability of the nuclear lamina to maintain the sturdiness and shape of the eukaryotic nucleus; a structural role for chromatin that is distinct from its genetic functions. PMID:25609380

  17. The Correlation of Interphase Chromatin Structure with the Radiation-Induced Inter- and Intrachromosome Exchange Hotspots

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Mangala, Lingegowda S.; Purgason, Ashley M.; Hada, Megumi; Cucinotta, Francis A.; Wu, Honglu

    2011-01-01

    To investigate the relationship between chromosome aberrations induced by radiation and chromatin folding, we reconstructed three dimensional structure of chromosome 3 and measured the physical distances between different regions of the chromosome. Previously, we have investigated the location of breaks involved in inter- and intrachromosomal type exchange events in human chromosome 3, using the multicolor banding in situ hybridization (mBAND) technique. In human epithelial cells exposed to both low- and high-LET radiations in vitro, we reported that intra-chromosome exchanges occurred preferentially between a break in the 3p21 and one in the 3q11 regions, and the breaks involving in inter-chromosome exchanges occurred in two regions towards the telomeres of the chromosome. Exchanges were also observed between a break in 3p21 and one in 3q26, but few exchanges were observed between breaks in 3q11 and 3q26, even though the two regions are located on the same arm of the chromosome. In this study, human epithelial cells were fixed at G1 phase and the interphase cells were hybridized using the XCyte3 mBAND kit from MetaSystems. The z-section images of chromosome 3 were captured with a Leica and an LSM 510 Meta laser scanning confocal microscopes. A total of 100 chromosomes were analyzed. The reconstruction of three dimensional structure of interphase chromosome 3 with six different colored regions was achieved using the Imaris software. The relative distance between different regions was measured as well. We further analyzed fragile sites on the chromosome that have been identified in various types of cancers. The data showed that, in majority of the cells, the regions containing 3p21 and 3q11 are colocalized in the center of the chromosome, whereas, the regions towards the telomeres of the chromosome are either physically wrapping outside the chromosome center or with arms sticking out. Our results demonstrated that the distribution of breaks involved in radiation

  18. Acetyllysine-binding and function of bromodomain-containing proteins in chromatin.

    PubMed

    Dyson, M H; Rose, S; Mahadevan, L C

    2001-08-01

    Acetylated histones are generally associated with active chromatin. The bromodomain has recently been identified as a protein module capable of binding to acetylated lysine residues, and hence is able to mediate the recruitment of factors to acetylated chromatin. Functional studies of bromodomain-containing proteins indicate how this domain contributes to the activity of a number of nuclear factors including histone acetyltransferases and chromatin remodelling complexes. Here, we review the characteristics of acetyllysine-binding by bromodomains, discuss associated domains found in these proteins, and address the function of the bromodomain in the context of chromatin. Finally, the modulation of bromodomain binding by neighbouring post-translational modifications within histone tails might provide a mechanism through which combinations of covalent marks could exert control on chromatin function.

  19. Sleep duration is associated with sperm chromatin integrity among young men in Chongqing, China.

    PubMed

    Wang, Xiaogang; Chen, Qing; Zou, Peng; Liu, Taixiu; Mo, Min; Yang, Huan; Zhou, Niya; Sun, Lei; Chen, Hongqiang; Ling, Xi; Peng, Kaige; Ao, Lin; Yang, Huifang; Cao, Jia; Cui, Zhihong

    2017-10-09

    This study explores whether sleep duration is associated with sperm chromatin integrity. To do so, we conducted a three-phase panel study of 796 male volunteers from colleges in Chongqing (China) from 2013 to 2015. Sleep duration was measured using a modified Munich Chronotype Questionnaire. Sperm DNA integrity was examined via Sperm Chromatin Structure Assay and Comet assay. Setting 7-7.5 h day -1 of sleep duration as a reference, either longer or shorter sleep duration was associated negatively with high DNA stainability (HDS) (P = 0.009), which reflected the immaturity of sperm chromatin. The volunteers with > 9.0 h day -1 sleep and those with ≤ 6.5 h day -1 sleep had 40.7 and 30.3% lower HDS than did volunteers with 7-7.5 h day -1 sleep. No association was found between sleep duration and DNA fragmentation index or Comet assay parameters. This study suggests that sleep duration is associated with sperm chromatin integrity. Further studies are required to validate these findings and investigate the mechanism underlying this association. © 2017 European Sleep Research Society.

  20. Transcription upregulation via force-induced direct stretching of chromatin

    NASA Astrophysics Data System (ADS)

    Tajik, Arash; Zhang, Yuejin; Wei, Fuxiang; Sun, Jian; Jia, Qiong; Zhou, Wenwen; Singh, Rishi; Khanna, Nimish; Belmont, Andrew S.; Wang, Ning

    2016-12-01

    Mechanical forces play critical roles in the function of living cells. However, the underlying mechanisms of how forces influence nuclear events remain elusive. Here, we show that chromatin deformation as well as force-induced transcription of a green fluorescent protein (GFP)-tagged bacterial-chromosome dihydrofolate reductase (DHFR) transgene can be visualized in a living cell by using three-dimensional magnetic twisting cytometry to apply local stresses on the cell surface via an Arg-Gly-Asp-coated magnetic bead. Chromatin stretching depended on loading direction. DHFR transcription upregulation was sensitive to load direction and proportional to the magnitude of chromatin stretching. Disrupting filamentous actin or inhibiting actomyosin contraction abrogated or attenuated force-induced DHFR transcription, whereas activating endogenous contraction upregulated force-induced DHFR transcription. Our findings suggest that local stresses applied to integrins propagate from the tensed actin cytoskeleton to the LINC complex and then through lamina-chromatin interactions to directly stretch chromatin and upregulate transcription.

  1. Small angle x-ray scattering of chromatin. Radius and mass per unit length depend on linker length.

    PubMed Central

    Williams, S P; Langmore, J P

    1991-01-01

    Analyses of low angle x-ray scattering from chromatin, isolated by identical procedures but from different species, indicate that fiber diameter and number of nucleosomes per unit length increase with the amount of nucleosome linker DNA. Experiments were conducted at physiological ionic strength to obtain parameters reflecting the structure most likely present in living cells. Guinier analyses were performed on scattering from solutions of soluble chromatin from Necturus maculosus erythrocytes (linker length 48 bp), chicken erythrocytes (linker length 64 bp), and Thyone briareus sperm (linker length 87 bp). The results were extrapolated to infinite dilution to eliminate interparticle contributions to the scattering. Cross-sectional radii of gyration were found to be 10.9 +/- 0.5, 12.1 +/- 0.4, and 15.9 +/- 0.5 nm for Necturus, chicken, and Thyone chromatin, respectively, which are consistent with fiber diameters of 30.8, 34.2, and 45.0 nm. Mass per unit lengths were found to be 6.9 +/- 0.5, 8.3 +/- 0.6, and 11.8 +/- 1.4 nucleosomes per 10 nm for Necturus, chicken, and Thyone chromatin, respectively. The geometrical consequences of the experimental mass per unit lengths and radii of gyration are consistent with a conserved interaction among nucleosomes. Cross-linking agents were found to have little effect on fiber external geometry, but significant effect on internal structure. The absolute values of fiber diameter and mass per unit length, and their dependencies upon linker length agree with the predictions of the double-helical crossed-linker model. A compilation of all published x-ray scattering data from the last decade indicates that the relationship between chromatin structure and linker length is consistent with data obtained by other investigators. Images FIGURE 1 PMID:2049522

  2. Chromatin relaxation-mediated induction of p19INK4d increases the ability of cells to repair damaged DNA.

    PubMed

    Ogara, María F; Sirkin, Pablo F; Carcagno, Abel L; Marazita, Mariela C; Sonzogni, Silvina V; Ceruti, Julieta M; Cánepa, Eduardo T

    2013-01-01

    The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies.

  3. Chromatin Relaxation-Mediated Induction of p19INK4d Increases the Ability of Cells to Repair Damaged DNA

    PubMed Central

    Carcagno, Abel L.; Marazita, Mariela C.; Sonzogni, Silvina V.; Ceruti, Julieta M.; Cánepa, Eduardo T.

    2013-01-01

    The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies. PMID:23593412

  4. Time-resolved Global and Chromatin Proteomics during Herpes Simplex Virus Type 1 (HSV-1) Infection*

    PubMed Central

    Kulej, Katarzyna; Avgousti, Daphne C.; Sidoli, Simone; Herrmann, Christin; Della Fera, Ashley N.; Kim, Eui Tae; Garcia, Benjamin A.; Weitzman, Matthew D.

    2017-01-01

    Herpes simplex virus (HSV-1) lytic infection results in global changes to the host cell proteome and the proteins associated with host chromatin. We present a system level characterization of proteome dynamics during infection by performing a multi-dimensional analysis during HSV-1 lytic infection of human foreskin fibroblast (HFF) cells. Our study includes identification and quantification of the host and viral proteomes, phosphoproteomes, chromatin bound proteomes and post-translational modifications (PTMs) on cellular histones during infection. We analyzed proteomes across six time points of virus infection (0, 3, 6, 9, 12 and 15 h post-infection) and clustered trends in abundance using fuzzy c-means. Globally, we accurately quantified more than 4000 proteins, 200 differently modified histone peptides and 9000 phosphorylation sites on cellular proteins. In addition, we identified 67 viral proteins and quantified 571 phosphorylation events (465 with high confidence site localization) on viral proteins, which is currently the most comprehensive map of HSV-1 phosphoproteome. We investigated chromatin bound proteins by proteomic analysis of the high-salt chromatin fraction and identified 510 proteins that were significantly different in abundance during infection. We found 53 histone marks significantly regulated during virus infection, including a steady increase of histone H3 acetylation (H3K9ac and H3K14ac). Our data provide a resource of unprecedented depth for human and viral proteome dynamics during infection. Collectively, our results indicate that the proteome composition of the chromatin of HFF cells is highly affected during HSV-1 infection, and that phosphorylation events are abundant on viral proteins. We propose that our epi-proteomics approach will prove to be important in the characterization of other model infectious systems that involve changes to chromatin composition. PMID:28179408

  5. Time-resolved Global and Chromatin Proteomics during Herpes Simplex Virus Type 1 (HSV-1) Infection.

    PubMed

    Kulej, Katarzyna; Avgousti, Daphne C; Sidoli, Simone; Herrmann, Christin; Della Fera, Ashley N; Kim, Eui Tae; Garcia, Benjamin A; Weitzman, Matthew D

    2017-04-01

    Herpes simplex virus (HSV-1) lytic infection results in global changes to the host cell proteome and the proteins associated with host chromatin. We present a system level characterization of proteome dynamics during infection by performing a multi-dimensional analysis during HSV-1 lytic infection of human foreskin fibroblast (HFF) cells. Our study includes identification and quantification of the host and viral proteomes, phosphoproteomes, chromatin bound proteomes and post-translational modifications (PTMs) on cellular histones during infection. We analyzed proteomes across six time points of virus infection (0, 3, 6, 9, 12 and 15 h post-infection) and clustered trends in abundance using fuzzy c-means. Globally, we accurately quantified more than 4000 proteins, 200 differently modified histone peptides and 9000 phosphorylation sites on cellular proteins. In addition, we identified 67 viral proteins and quantified 571 phosphorylation events (465 with high confidence site localization) on viral proteins, which is currently the most comprehensive map of HSV-1 phosphoproteome. We investigated chromatin bound proteins by proteomic analysis of the high-salt chromatin fraction and identified 510 proteins that were significantly different in abundance during infection. We found 53 histone marks significantly regulated during virus infection, including a steady increase of histone H3 acetylation (H3K9ac and H3K14ac). Our data provide a resource of unprecedented depth for human and viral proteome dynamics during infection. Collectively, our results indicate that the proteome composition of the chromatin of HFF cells is highly affected during HSV-1 infection, and that phosphorylation events are abundant on viral proteins. We propose that our epi-proteomics approach will prove to be important in the characterization of other model infectious systems that involve changes to chromatin composition. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Toxic effects of lead and nickel nitrate on rat liver chromatin components.

    PubMed

    Rabbani-Chadegani Iii, Azra; Fani, Nesa; Abdossamadi, Sayeh; Shahmir, Nosrat

    2011-01-01

    The biological activity of heavy metals is related to their physicochemical interaction with biological receptors. In the present study, the effect of low concentrations of nickel nitrate and lead nitrate (<0.3 mM) on rat liver soluble chromatin and histone proteins was examined. The results showed that addition of various concentrations of metals to chromatin solution preceded the chromatin into aggregation and precipitation in a dose-dependant manner; however, the extent of absorbance changes at 260 and 400 nm was different between two metals. Gel electrophoresis of histone proteins and DNA of the supernatants obtained from the metal-treated chromatin and the controls revealed higher affinity of lead nitrate to chromatin compared to nickel nitrate. Also, the binding affinity of lead nitrate to histone proteins free in solution was higher than nickel. On the basis of the results, it is concluded that lead reacts with chromatin components even at very low concentrations and induce chromatin aggregation through histone-DNA cross-links. Whereas, nickel nitrate is less effective on chromatin at low concentrations, suggesting higher toxicity of lead nitrate on chromatin compared to nickel. Copyright © 2010 Wiley Periodicals, Inc.

  7. Anti-aging peptide bioregulators induce reactivation of chromatin.

    PubMed

    Lezhava, T; Monaselidze, J; Kadotani, T; Dvalishvili, N; Buadze, T

    2006-04-01

    The effect of synthetic peptide bioregulators (Epitalon, Livagen and Vilon) on structural and facultative heterochromatin of cultivated lymphocytes have been studied among old (75-88yr.) people. The data obtained indicate that epitalon, livagen and vilon: 1) activate synthetic processes, caused by reactivation of ribosomal genes as a result of deheterochromatinization (decondensation) of nucleolus organizer regions; 2) induce unrolling (deheterochromatinization) of total heterochromatin; 3) release genes repressed by heterochromatinization (condensation) of euchromatic regions forming facultative heterochromatin; 4) epitalon and livagen induce deheterochromatinization (decondensation) of pericentromeric structural heterochromatin of the chromosomes1 and 9. However, vilon does not induce deheterochromatinization of pericentromeric structural heterochromatin. These results indicate that peptide bioregulators Epitalon, Livagen and Vilon cause activation (deheterochromatinization) of chromatin in lymphocytes of old individuals.

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

    PubMed

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

    2015-06-01

    The presence or absence of specific transcription factors, chromatin remodeling machineries, chromatin modification enzymes, post-translational histone modifications and histone variants all play crucial roles in the regulation of pathogenicity genes. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) provides an important tool to study genome-wide protein-DNA interactions to help understand gene regulation in the context of native chromatin. ChIP-seq is a convenient in vivo technique to identify, map and characterize occupancy of specific DNA fragments with proteins against which specific antibodies exist or which can be epitope-tagged in vivo. We optimized existing ChIP protocols for use in the wheat pathogen Zymoseptoria tritici and closely related sister species. Here, we provide a detailed method, underscoring which aspects of the technique are organism-specific. Library preparation for Illumina sequencing is described, as this is currently the most widely used ChIP-seq method. One approach for the analysis and visualization of representative sequence is described; improved tools for these analyses are constantly being developed. Using ChIP-seq with antibodies against H3K4me2, which is considered a mark for euchromatin or H3K9me3 and H3K27me3, which are considered marks for heterochromatin, the overall distribution of euchromatin and heterochromatin in the genome of Z. tritici can be determined. Our ChIP-seq protocol was also successfully applied to Z. tritici strains with high levels of melanization or aberrant colony morphology, and to different species of the genus (Z. ardabiliae and Z. pseudotritici), suggesting that our technique is robust. The methods described here provide a powerful framework to study new aspects of chromatin biology and gene regulation in this prominent wheat pathogen. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function.

    PubMed

    Johnston, S D; Satake, N; Zee, Y; López-Fernández, C; Holt, W V; Gosálvez, J

    2012-06-01

    This study investigated whether cryopreservation-induced injury to koala spermatozoa could be explained using an experimental model that mimics the structural and physiological effects of osmotic flux. DNA labelling after in situ nick translation of thawed cryopreserved spermatozoa revealed a positive correlation (r=0.573; P<0.001; n=50) between the area of relaxed chromatin in the nucleus and the degree of nucleotide labelling. While the chromatin of some spermatozoa increased more than eight times its normal size, not all sperm nuclei with relaxed chromatin showed evidence of nucleotide incorporation. Preferential staining associated with sperm DNA fragmentation (SDF) was typically located in the peri-acrosomal and peripheral regions of the sperm head and at the base of the spermatozoa where it appear to be 'hot spots' of DNA damage following cryopreservation. Results of the comparative effects of anisotonic media and cryopreservation on the integrity of koala spermatozoa revealed that injury induced by exposure to osmotic flux, essentially imitated the results found following cryopreservation. Plasma membrane integrity, chromatin relaxation and SDF appeared particularly susceptible to extreme hypotonic environments. Mitochondrial membrane potential (MMP), while susceptible to extreme hypo- and hypertonic environments, showed an ability to rebound from hypertonic stress when returned to isotonic conditions. Koala spermatozoa exposed to 64 mOsm/kg media showed an equivalent, or more severe, degree of structural and physiological injury to that of frozen-thawed spermatozoa, supporting the hypothesis that cryoinjury is principally associated with a hypo-osmotic effect. A direct comparison of SDF of thawed cryopreserved spermatozoa and those exposed to a 64 mOsm/kg excursion showed a significant correlation (r=0.878; P<0.05; n=5); however, no correlation was found when the percentage of sperm with relaxed chromatin was compared. While a cryo-induced osmotic

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

    PubMed Central

    Ronan, Jehnna L.; Wu, Wei

    2014-01-01

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

  11. Prediction of Developmentally Competent Chromatin Conformation in Mouse Antral Oocytes.

    PubMed

    Daszkiewicz, Regina; Szymoniak, Magdalena; Gąsior, Łukasz; Polański, Zbigniew

    Mouse prophase oocytes isolated from antral follicles may possess two alternative types of chromatin configuration: NSN configuration represents more dispersed chromatin and is characteristic mainly for growing oocytes whereas SN configuration, attained upon oocyte growth, comprises more condensed chromatin with a significant fraction concentrated around the nucleolus. Importantly, fully grown oocytes isolated from antral follicles represent a non-homogenous population in which some oocytes posses NSN-type and others SN-type of chromatin conformation. From these two, only oocytes with SN configuration are able to complete full development upon fertilization. We show that among mouse oocytes isolated from antral follicles, those surrounded by cumulus cells were larger and more frequently possessed SN chromatin than oocytes lacking the complete cumulus cell layer. Females primed with PMSG gave a higher number of oocytes with a complete layer of cumulus cells and the frequency of oocytes with SN chromatin was also elevated. Within the whole population of isolated antral oocytes, we observed subtle variation in size which allowed fractionation of oocytes under a stereomicroscope into groups representing oocytes of slightly different sizes. The occurrence of SN chromatin configuration was highly dependent on the oocyte size and its frequency increased gradually in subsequent size groups reaching 95-100% in the group representing the largest oocytes. These findings demonstrate that the subtle differences in the size of antral oocytes allow prediction of the status of their chromatin, thus providing a simple, fast, non-invasive and non-expensive way to select good quality oocytes for ART purposes in mammals.

  12. Nucleosome occupancy as a novel chromatin parameter for replication origin functions

    PubMed Central

    Rodriguez, Jairo; Lee, Laura; Lynch, Bryony; Tsukiyama, Toshio

    2017-01-01

    Eukaryotic DNA replication initiates from multiple discrete sites in the genome, termed origins of replication (origins). Prior to S phase, multiple origins are poised to initiate replication by recruitment of the pre-replicative complex (pre-RC). For proper replication to occur, origin activation must be tightly regulated. At the population level, each origin has a distinct firing time and frequency of activation within S phase. Many studies have shown that chromatin can strongly influence initiation of DNA replication. However, the chromatin parameters that affect properties of origins have not been thoroughly established. We found that nucleosome occupancy in G1 varies greatly around origins across the S. cerevisiae genome, and nucleosome occupancy around origins significantly correlates with the activation time and efficiency of origins, as well as pre-RC formation. We further demonstrate that nucleosome occupancy around origins in G1 is established during transition from G2/M to G1 in a pre-RC-dependent manner. Importantly, the diminished cell-cycle changes in nucleosome occupancy around origins in the orc1-161 mutant are associated with an abnormal global origin usage profile, suggesting that proper establishment of nucleosome occupancy around origins is a critical step for regulation of global origin activities. Our work thus establishes nucleosome occupancy as a novel and key chromatin parameter for proper origin regulation. PMID:27895110

  13. Single-cell Hi-C bridges microscopy and genome-wide sequencing approaches to study 3D chromatin organization.

    PubMed

    Ulianov, Sergey V; Tachibana-Konwalski, Kikue; Razin, Sergey V

    2017-10-01

    Recent years have witnessed an explosion of the single-cell biochemical toolbox including chromosome conformation capture (3C)-based methods that provide novel insights into chromatin spatial organization in individual cells. The observations made with these techniques revealed that topologically associating domains emerge from cell population averages and do not exist as static structures in individual cells. Stochastic nature of the genome folding is likely to be biologically relevant and may reflect the ability of chromatin fibers to adopt a number of alternative configurations, some of which could be transiently stabilized and serve regulatory purposes. Single-cell Hi-C approaches provide an opportunity to analyze chromatin folding in rare cell types such as stem cells, tumor progenitors, oocytes, and totipotent cells, contributing to a deeper understanding of basic mechanisms in development and disease. Here, we review key findings of single-cell Hi-C and discuss possible biological reasons and consequences of the inferred dynamic chromatin spatial organization. © 2017 WILEY Periodicals, Inc.

  14. Pharmacologic Targeting of Chromatin Modulators As Therapeutics of Acute Myeloid Leukemia.

    PubMed

    Lu, Rui; Wang, Gang Greg

    2017-01-01

    Acute myeloid leukemia (AML), a common hematological cancer of myeloid lineage cells, generally exhibits poor prognosis in the clinic and demands new treatment options. Recently, direct sequencing of samples from human AMLs and pre-leukemic diseases has unveiled their mutational landscapes and significantly advanced the molecular understanding of AML pathogenesis. The newly identified recurrent mutations frequently "hit" genes encoding epigenetic modulators, a wide range of chromatin-modifying enzymes and regulatory factors involved in gene expression regulation, supporting aberration of chromatin structure and epigenetic modification as a main oncogenic mechanism and cancer-initiating event. Increasing body of evidence demonstrates that chromatin modification aberrations underlying the formation of blood cancer can be reversed by pharmacological targeting of the responsible epigenetic modulators, thus providing new mechanism-based treatment strategies. Here, we summarize recent advances in development of small-molecule inhibitors specific to chromatin factors and their potential applications in the treatment of genetically defined AMLs. These compounds selectively inhibit various subclasses of "epigenetic writers" (such as histone methyltransferases MLL/KMT2A, G9A/KMT1C, EZH2/KMT6A, DOT1L/KMT4, and PRMT1), "epigenetic readers" (such as BRD4 and plant homeodomain finger proteins), and "epigenetic erasers" (such as histone demethylases LSD1/KDM1A and JMJD2C/KDM4C). We also discuss about the molecular mechanisms underpinning therapeutic effect of these epigenetic compounds in AML and favor their potential usage for combinational therapy and treatment of pre-leukemia diseases.

  15. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) for Transcription Factors and Chromatin Factors in Arabidopsis thaliana Roots: From Material Collection to Data Analysis.

    PubMed

    Cortijo, Sandra; Charoensawan, Varodom; Roudier, François; Wigge, Philip A

    2018-01-01

    Chromatin immunoprecipitation combined with next-generation sequencing (ChIP-seq) is a powerful technique to investigate in vivo transcription factor (TF) binding to DNA, as well as chromatin marks. Here we provide a detailed protocol for all the key steps to perform ChIP-seq in Arabidopsis thaliana roots, also working on other A. thaliana tissues and in most non-ligneous plants. We detail all steps from material collection, fixation, chromatin preparation, immunoprecipitation, library preparation, and finally computational analysis based on a combination of publicly available tools.

  16. Role of chromatin in water stress responses in plants

    PubMed Central

    Han, Soon-Ki; Wagner, Doris

    2014-01-01

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

  17. Mechanisms and Consequences of Double-strand DNA Break Formation in Chromatin

    PubMed Central

    Cannan, Wendy J.; Pederson, David S.

    2016-01-01

    All organisms suffer double-strand breaks (DSBs) in their DNA as a result of exposure to ionizing radiation. DSBs can also form when replication forks encounter DNA lesions or repair intermediates. The processing and repair of DSBs can lead to mutations, loss of heterozygosity, and chromosome rearrangements that result in cell death or cancer. The most common pathway used to repair DSBs in metazoans (non-homologous DNA end joining) is more commonly mutagenic than the alternative pathway (homologous recombination mediated repair). Thus, factors that influence the choice of pathways used DSB repair can affect an individual’s mutation burden and risk of cancer. This review describes radiological, chemical and biological mechanisms that generate DSBs, and discusses the impact of such variables as DSB etiology, cell type, cell cycle, and chromatin structure on the yield, distribution, and processing of DSBs. The final section focuses on nucleosome-specific mechanisms that influence DSB production, and the possible relationship between higher order chromosome coiling and chromosome shattering (chromothripsis). PMID:26040249

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

    PubMed

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

    2014-09-29

    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.

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

    PubMed Central

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

    2015-01-01

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

  20. Direct screening for chromatin status on DNA barcodes in yeast delineates the regulome of H3K79 methylation by Dot1.

    PubMed

    Vlaming, Hanneke; Molenaar, Thom M; van Welsem, Tibor; Poramba-Liyanage, Deepani W; Smith, Desiree E; Velds, Arno; Hoekman, Liesbeth; Korthout, Tessy; Hendriks, Sjoerd; Altelaar, A F Maarten; van Leeuwen, Fred

    2016-12-06

    Given the frequent misregulation of chromatin in cancer, it is important to understand the cellular mechanisms that regulate chromatin structure. However, systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs. Here we describe a strategy, Epi-ID, to directly assess chromatin status in thousands of mutants. In Epi-ID, chromatin status on DNA barcodes is interrogated by chromatin immunoprecipitation followed by deep sequencing, allowing for quantitative comparison of many mutants in parallel. Screening of a barcoded yeast knock-out collection for regulators of histone H3K79 methylation by Dot1 identified all known regulators as well as novel players and processes. These include histone deposition, homologous recombination, and adenosine kinase, which influences the methionine cycle. Gcn5, the acetyltransferase within the SAGA complex, was found to regulate histone methylation and H2B ubiquitination. The concept of Epi-ID is widely applicable and can be readily applied to other chromatin features.

  1. Epigenetic regulation of open chromatin in pluripotent stem cells

    PubMed Central

    Kobayashi, Hiroshi; Kikyo, Nobuaki

    2014-01-01

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

  2. Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract

    PubMed Central

    Raut, Vishal V.; Pandey, Shashibhal M.; Sainis, Jayashree K.

    2011-01-01

    Background and Scope In eukaryotes, chromatin remodelling complexes are shown to be responsible for nucleosome mobility, leading to increased accessibility of DNA for DNA binding proteins. Although the existence of such complexes in plants has been surmised mainly at the genetic level from bioinformatics studies and analysis of mutants, the biochemical existence of such complexes has remained unexplored. Methods Histone H1-depleted donor chromatin was prepared by micrococcal nuclease digestion of wheat nuclei and fractionation by exclusion chromatography. Nuclear extract was partially purified by cellulose phosphate ion exchange chromatography. Histone octamer trans-transfer activity was analysed using the synthetic nucleosome positioning sequence in the absence and presence of ATP and its analogues. ATPase activity was measured as 32Pi released using liquid scintillation counting. Key Results ATP-dependent histone octamer trans-transfer activity, partially purified from wheat nuclei using cellulose phosphate, showed ATP-dependent octamer displacement in trans from the H1-depleted native donor chromatin of wheat to the labelled synthetic nucleosome positioning sequence. It also showed nucleosome-dependent ATPase activity. Substitution of ATP by ATP analogues, namely ATPγS, AMP-PNP and ADP abolished the octamer trans-transfer, indicating the requirement of ATP hydrolysis for this activity. Conclusions ATP-dependent histone octamer transfer in trans is a recognized activity of chromatin remodelling complexes required for chromatin structure dynamics in non-plant species. Our results suggested that wheat nuclei also possess a typical chromatin remodelling activity, similar to that in other eukaryotes. This is the first report on chromatin remodelling activity in vitro from plants. PMID:21896571

  3. Nucleolar chromatin organization at different activities of soybean root meristematic cell nucleoli.

    PubMed

    Stępiński, Dariusz

    2013-06-01

    Nucleolar chromatin, including nucleolus-associated chromatin as well as active and inactive condensed ribosomal DNA (rDNA) chromatin, derives mostly from secondary constrictions known as nucleolus organizer regions containing rDNA genes on nucleolus-forming chromosomes. This chromatin may occupy different nucleolar positions being in various condensation states which may imply different rDNA transcriptional competence. Sections of nucleoli originating from root meristematic cells of soybean seedlings grown at 25 °C (the control), then subjected to chilling stress (10 °C), and next transferred again to 25 °C (the recovery) were used to measure profile areas occupied by nucleolar condensed chromatin disclosed with sodium hydroxide methylation-acetylation plus uranyl acetate technique. The biggest total area of condensed chromatin was found in the nucleoli of chilled plants, while the smallest was found in those of recovered plants in relation to the amounts of chromatin in the control nucleoli. The condensed nucleolar chromatin, in the form of different-sized and different-shaped clumps, was mainly located in fibrillar centers. One can suppose that changes of condensed rDNA chromatin amounts might be a mechanism controlling the number of transcriptionally active rDNA genes as the nucleoli of plants grown under these experimental conditions show different transcriptional activity and morphology.

  4. Multiclassifier combinatorial proteomics of organelle shadows at the example of mitochondria in chromatin data.

    PubMed

    Kustatscher, Georg; Grabowski, Piotr; Rappsilber, Juri

    2016-02-01

    Subcellular localization is an important aspect of protein function, but the protein composition of many intracellular compartments is poorly characterized. For example, many nuclear bodies are challenging to isolate biochemically and thus remain inaccessible to proteomics. Here, we explore covariation in proteomics data as an alternative route to subcellular proteomes. Rather than targeting a structure of interest biochemically, we target it by machine learning. This becomes possible by taking data obtained for one organelle and searching it for traces of another organelle. As an extreme example and proof-of-concept we predict mitochondrial proteins based on their covariation in published interphase chromatin data. We detect about ⅓ of the known mitochondrial proteins in our chromatin data, presumably most as contaminants. However, these proteins are not present at random. We show covariation of mitochondrial proteins in chromatin proteomics data. We then exploit this covariation by multiclassifier combinatorial proteomics to define a list of mitochondrial proteins. This list agrees well with different databases on mitochondrial composition. This benchmark test raises the possibility that, in principle, covariation proteomics may also be applicable to structures for which no biochemical isolation procedures are available. © 2015 The Authors. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Chromatin Folding, Fragile Sites, and Chromosome Aberrations Induced by Low- and High- LET Radiation

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Cox, Bradley; Asaithamby, Aroumougame; Chen, David J.; Wu, Honglu

    2013-01-01

    We previously demonstrated non-random distributions of breaks involved in chromosome aberrations induced by low- and high-LET radiation. To investigate the factors contributing to the break point distribution in radiation-induced chromosome aberrations, human epithelial cells were fixed in 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 in separate colors. After the images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multimega base pair scale. Specific locations of the chromosome, in interphase, were also analyzed with bacterial artificial chromosome (BAC) probes. Both mBAND and BAC studies revealed non-random folding of chromatin in interphase, and suggested association of interphase chromatin folding to the radiation-induced chromosome aberration hotspots. We further investigated the distribution of genes, as well as the distribution of breaks found in tumor cells. Comparisons of these distributions to the radiation hotspots showed that some of the radiation hotspots coincide with the frequent breaks found in solid tumors and with the fragile sites for other environmental toxins. Our results suggest that multiple factors, including the chromatin structure and the gene distribution, can contribute to radiation-induced chromosome aberrations.

  6. Pathogen Infection and MORC Proteins Affect Chromatin Accessibility of Transposable Elements and Expression of Their Proximal Genes in Arabidopsis.

    PubMed

    Bordiya, Yogendra; Zheng, Yi; Nam, Ji-Chul; Bonnard, April C; Choi, Hyong Woo; Lee, Bum-Kyu; Kim, Jonghwan; Klessig, Daniel F; Fei, Zhangjun; Kang, Hong-Gu

    2016-09-01

    To assess the role of MORC1 in epigenetics in relation to plant immunity, genome-wide chromatin accessibility was compared between mock- or Pseudomonas syringae pv. tomato-inoculated wild type (WT) Arabidopsis, the morc1/2 double mutant, or both. Most changes in chromatin accessibility, scored by DNase I hypersensitive sites (DHSs), were located in the promoters of genes and transposable elements (TEs). Comparisons between morc1/2 and WT receiving the same treatment revealed differential DHSs (dDHSs) predominantly associated with heterochromatic TEs. By contrast, comparisons between mock- and P. syringae pv. tomato-inoculated plants from the same genotype showed dDHSs associated with biotic and abiotic stress-related genes; a smaller but significant population was in TEs. Moreover, many defense genes, including PR-1, PR-2, and PR-5, were proximal to P. syringae pv. tomato-induced, TE-associated dDHSs. A random subset of these defense genes showed moderately delayed or reduced expression or both in P. syringae pv. tomato-infected morc1/2 as compared with WT. MORC1 was physically bound to chromatin in a P. syringae pv. tomato infection-responsive manner at sites dispersed throughout the genome. Notably, silencing of TE-associated dDHSs proximal to these infection-induced, MORC1-interacting sites led to significant suppression of P. syringae pv. tomato-induced transcription of adjacent defense genes, including PR-1. These results provide evidence that MORC1 is associated with TEs and suggest that a subset of these TEs may help regulate their proximal defense genes.

  7. De Novo Chromosome Structure Prediction

    NASA Astrophysics Data System (ADS)

    di Pierro, Michele; Cheng, Ryan R.; Lieberman-Aiden, Erez; Wolynes, Peter G.; Onuchic, Jose'n.

    Chromatin consists of DNA and hundreds of proteins that interact with the genetic material. In vivo, chromatin folds into nonrandom structures. The physical mechanism leading to these characteristic conformations, however, remains poorly understood. We recently introduced MiChroM, a model that generates chromosome conformations by using the idea that chromatin can be subdivided into types based on its biochemical interactions. Here we extend and complete our previous finding by showing that structural chromatin types can be inferred from ChIP-Seq data. Chromatin types, which are distinct from DNA sequence, are partially epigenetically controlled and change during cell differentiation, thus constituting a link between epigenetics, chromosomal organization, and cell development. We show that, for GM12878 lymphoblastoid cells we are able to predict accurate chromosome structures with the only input of genomic data. The degree of accuracy achieved by our prediction supports the viability of the proposed physical mechanism of chromatin folding and makes the computational model a powerful tool for future investigations.

  8. The effect of human sperm chromatin maturity on ICSI outcomes.

    PubMed

    Gill, Kamil; Rosiak, Aleksandra; Gaczarzewicz, Dariusz; Jakubik, Joanna; Kurzawa, Rafal; Kazienko, Anna; Rymaszewska, Anna; Laszczynska, Maria; Grochans, Elzbieta; Piasecka, Malgorzata

    2018-03-29

    Because sperm chromatin may play a key role in reproductive success, we verify the associations between sperm chromatin abnormalities, embryo development and the ability to achieve pregnancy. The evaluation of sperm chromatin maturity using aniline blue (AB), chromomycin A3 (CMA3) and toluidine blue (TB) staining were carried out in group of males from infertile couples that underwent ICSI. Low levels of sperm chromatin abnormalities (< 16%) were found in most subjects (> 50%). A higher percentage of TB-positive sperm cells were discovered in the men from couples who achieved ≤ 50% fertilized oocytes compared to men who achieved > 50%. No significant differences were discovered by the applied tests between the men from couples who achieved ≤ 50% and those who achieved > 50% high-quality embryos on the 3rd or 5th day after fertilization, nor between the men from couples who achieved pregnancy and those who failed. The sperm chromatin maturity did not correlate with the ICSI results. However, the ROC analysis revealed a significant predictive value of TB-positive spermatozoa only for fertilization. Therefore, the TB assay can be considered as a useful test for the prediction of fertilization. Our findings suggest that the level of sperm chromatin abnormalities of the examined men was not clinically significant. No found associations between sperm chromatin maturity and embryo development and the ability to achieve pregnancy. We could not exclude the effects of the repairing processes in the fertilized oocyte. The use of complementary tests that verify the status of the sperm chromatin seems justified.

  9. Mapping protein-DNA and protein-protein interactions of ATP-dependent chromatin remodelers.

    PubMed

    Hota, Swetansu K; Dechassa, Mekonnen Lemma; Prasad, Punit; Bartholomew, Blaine

    2012-01-01

    Chromatin plays a key regulatory role in several DNA-dependent processes as it regulates DNA access to different protein factors. Several multisubunit protein complexes interact, modify, or mobilize nucleosomes: the basic unit of chromatin, from its original location in an ATP-dependent manner to facilitate processes, such as transcription, replication, repair, and recombination. Knowledge of the interactions of chromatin remodelers with nucleosomes is a crucial requirement to understand the mechanism of chromatin remodeling. Here, we describe several methods to analyze the interactions of multisubunit chromatin-remodeling enzymes with nucleosomes.

  10. Chromatin regulation at the frontier of synthetic biology.

    PubMed

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

    2015-03-01

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

  11. Chromatin regulation at the frontier of synthetic biology

    PubMed Central

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

    2016-01-01

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

  12. Protein and Genetic Composition of Four Chromatin Types in Drosophila melanogaster Cell Lines.

    PubMed

    Boldyreva, Lidiya V; Goncharov, Fyodor P; Demakova, Olga V; Zykova, Tatyana Yu; Levitsky, Victor G; Kolesnikov, Nikolay N; Pindyurin, Alexey V; Semeshin, Valeriy F; Zhimulev, Igor F

    2017-04-01

    Recently, we analyzed genome-wide protein binding data for the Drosophila cell lines S2, Kc, BG3 and Cl.8 (modENCODE Consortium) and identified a set of 12 proteins enriched in the regions corresponding to interbands of salivary gland polytene chromosomes. Using these data, we developed a bioinformatic pipeline that partitioned the Drosophila genome into four chromatin types that we hereby refer to as aquamarine, lazurite, malachite and ruby. Here, we describe the properties of these chromatin types across different cell lines. We show that aquamarine chromatin tends to harbor transcription start sites (TSSs) and 5' untranslated regions (5'UTRs) of the genes, is enriched in diverse "open" chromatin proteins, histone modifications, nucleosome remodeling complexes and transcription factors. It encompasses most of the tRNA genes and shows enrichment for non-coding RNAs and miRNA genes. Lazurite chromatin typically encompasses gene bodies. It is rich in proteins involved in transcription elongation. Frequency of both point mutations and natural deletion breakpoints is elevated within lazurite chromatin. Malachite chromatin shows higher frequency of insertions of natural transposons. Finally, ruby chromatin is enriched for proteins and histone modifications typical for the "closed" chromatin. Ruby chromatin has a relatively low frequency of point mutations and is essentially devoid of miRNA and tRNA genes. Aquamarine and ruby chromatin types are highly stable across cell lines and have contrasting properties. Lazurite and malachite chromatin types also display characteristic protein composition, as well as enrichment for specific genomic features. We found that two types of chromatin, aquamarine and ruby, retain their complementary protein patterns in four Drosophila cell lines.

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

    USGS Publications Warehouse

    Jenkins, Jill A.

    2013-01-01

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

  14. Mapping of protein- and chromatin-interactions at the nuclear lamina.

    PubMed

    Kubben, Nard; Voncken, Jan Willem; Misteli, Tom

    2010-01-01

    The nuclear envelope and the lamina define the nuclear periphery and are implicated in many nuclear processes including chromatin organization, transcription and DNA replication. Mutations in lamin A proteins, major components of the lamina, interfere with these functions and cause a set of phenotypically diverse diseases referred to as laminopathies. The phenotypic diversity of laminopathies is thought to be the result of alterations in specific protein- and chromatin interactions due to lamin A mutations. Systematic identification of lamin A-protein and -chromatin interactions will be critical to uncover the molecular etiology of laminopathies. Here we summarize and critically discuss recent technology to analyze lamina-protein and-chromatin interactions.

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

    PubMed Central

    Miles, Charles P.

    1962-01-01

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

  16. QuIN: A Web Server for Querying and Visualizing Chromatin Interaction Networks.

    PubMed

    Thibodeau, Asa; Márquez, Eladio J; Luo, Oscar; Ruan, Yijun; Menghi, Francesca; Shin, Dong-Guk; Stitzel, Michael L; Vera-Licona, Paola; Ucar, Duygu

    2016-06-01

    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/.

  17. QuIN: A Web Server for Querying and Visualizing Chromatin Interaction Networks

    PubMed Central

    Thibodeau, Asa; Márquez, Eladio J.; Luo, Oscar; Ruan, Yijun; Shin, Dong-Guk; Stitzel, Michael L.; Ucar, Duygu

    2016-01-01

    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. AVAILABILITY: 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/. PMID:27336171

  18. Modify the Histone to Win the Battle: Chromatin Dynamics in Plant–Pathogen Interactions

    PubMed Central

    Ramirez-Prado, Juan S.; Piquerez, Sophie J. M.; Bendahmane, Abdelhafid; Hirt, Heribert; Raynaud, Cécile; Benhamed, Moussa

    2018-01-01

    Relying on an immune system comes with a high energetic cost for plants. Defense responses in these organisms are therefore highly regulated and fine-tuned, permitting them to respond pertinently to the attack of a microbial pathogen. In recent years, the importance of the physical modification of chromatin, a highly organized structure composed of genomic DNA and its interacting proteins, has become evident in the research field of plant–pathogen interactions. Several processes, including DNA methylation, changes in histone density and variants, and various histone modifications, have been described as regulators of various developmental and defense responses. Herein, we review the state of the art in the epigenomic aspects of plant immunity, focusing on chromatin modifications, chromatin modifiers, and their physiological consequences. In addition, we explore the exciting field of understanding how plant pathogens have adapted to manipulate the plant epigenomic regulation in order to weaken their immune system and thrive in their host, as well as how histone modifications in eukaryotic pathogens are involved in the regulation of their virulence. PMID:29616066

  19. The Chromatin Remodeler BPTF Activates a Stemness Gene-Expression Program Essential for the Maintenance of Adult Hematopoietic Stem Cells.

    PubMed

    Xu, Bowen; Cai, Ling; Butler, Jason M; Chen, Dongliang; Lu, Xiongdong; Allison, David F; Lu, Rui; Rafii, Shahin; Parker, Joel S; Zheng, Deyou; Wang, Gang Greg

    2018-03-13

    Self-renewal and differentiation of adult stem cells are tightly regulated partly through configuration of chromatin structure by chromatin remodelers. Using knockout mice, we here demonstrate that bromodomain PHD finger transcription factor (BPTF), a component of the nucleosome remodeling factor (NURF) chromatin-remodeling complex, is essential for maintaining the population size of hematopoietic stem/progenitor cells (HSPCs), including long-term hematopoietic stem cells (HSCs). Bptf-deficient HSCs are defective in reconstituted hematopoiesis, and hematopoietic-specific knockout of Bptf caused profound defects including bone marrow failure and anemia. Genome-wide transcriptome profiling revealed that BPTF loss caused downregulation of HSC-specific gene-expression programs, which contain several master transcription factors (Meis1, Pbx1, Mn1, and Lmo2) required for HSC maintenance and self-renewal. Furthermore, we show that BPTF potentiates the chromatin accessibility of key HSC "stemness" genes. These results demonstrate an essential requirement of the chromatin remodeler BPTF and NURF for activation of "stemness" gene-expression programs and proper function of adult HSCs. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. Analysis of Chromatin Organisation

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

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

  1. Nucleosomal chromatin in the mature sperm of Drosophila melanogaster.

    PubMed

    Elnfati, Abdul Hakim; Iles, David; Miller, David

    2016-03-01

    During spermiogenesis in mammals and many other vertebrate classes, histone-containing nucleosomes are replaced by protamine toroids, which can repackage chromatin at a 10 to 20-fold higher density than in a typical somatic nucleus. However, recent evidence suggests that sperm of many species, including human and mouse retain a small compartment of nucleosomal chromatin, particularly near genes important for embryogenesis. As in mammals, spermiogenesis in the fruit fly, Drosophila melanogaster has also been shown to undergo a programmed substitution of nucleosomes with protamine-like proteins. Using chromatin immunoprecipitation (ChIP) and whole-genome tiling array hybridization (ChIP-chip), supported by immunocytochemical evidence, we show that in a manner analogous to nucleosomal chromatin retention in mammalian spermatozoa, distinct domains packaged by the canonical histones H2A, H2B, H3 and H4 are present in the fly sperm nucleus. We also find evidence for the retention of nucleosomes with specific histone H3 trimethylation marks characteristic of chromatin repression (H3K9me3, H3K27me3) and active transcription (H3K36me3). Raw and processed data from the experiments are available at GEO, accession GSE52165.

  2. Not just gene expression: 3D implications of chromatin modifications during sexual plant reproduction.

    PubMed

    Dukowic-Schulze, Stefanie; Liu, Chang; Chen, Changbin

    2018-01-01

    DNA methylation and histone modifications are epigenetic changes on a DNA molecule that alter the three-dimensional (3D) structure locally as well as globally, impacting chromatin looping and packaging on a larger scale. Epigenetic marks thus inform higher-order chromosome organization and placement in the nucleus. Conventional epigenetic marks are joined by chromatin modifiers like cohesins, condensins and membrane-anchoring complexes to support particularly 3D chromosome organization. The most popular consequences of epigenetic modifications are gene expression changes, but chromatin modifications have implications beyond this, particularly in actively dividing cells and during sexual reproduction. In this opinion paper, we will focus on epigenetic mechanisms and chromatin modifications during meiosis as part of plant sexual reproduction where 3D management of chromosomes and re-organization of chromatin are defining features and prime tasks in reproductive cells, not limited to modulating gene expression. Meiotic chromosome organization, pairing and synapsis of homologous chromosomes as well as distribution of meiotic double-strand breaks and resulting crossovers are presumably highly influenced by epigenetic mechanisms. Special mobile small RNAs have been described in anthers, where these so-called phasiRNAs seem to direct DNA methylation in meiotic cells. Intriguingly, many of the mentioned developmental processes make use of epigenetic changes and small RNAs in a manner other than gene expression changes. Widening our approaches and opening our mind to thinking three-dimensionally regarding epigenetics in plant development holds high promise for new discoveries and could give us a boost for further knowledge.

  3. Epigenetic chromatin silencing: bistability and front propagation

    NASA Astrophysics Data System (ADS)

    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. Promyelocytic extracellular chromatin exacerbates coagulation and fibrinolysis in acute promyelocytic leukemia

    PubMed Central

    Cao, Muhua; Li, Tao; He, Zhangxiu; Wang, Lixiu; Yang, Xiaoyan; Kou, Yan; Zou, Lili; Dong, Xue; Novakovic, Valerie A.; Bi, Yayan; Kou, Junjie; Yu, Bo; Fang, Shaohong; Wang, Jinghua; Zhou, Jin

    2017-01-01

    Despite routine treatment of unselected acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA), early death because of hemorrhage remains unacceptably common, and the mechanism underlying this complication remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, which involves release of extracellular chromatin. However, the role of promyelocytic extracellular chromatin in APL-associated coagulation remains unclear. Our objectives were to identify the novel role of ATRA-promoted extracellular chromatin in inducing a hypercoagulable and hyperfibrinolytic state in APL and to evaluate its interaction with fibrin and endothelial cells (ECs). Results from a series of coagulation assays have shown that promyelocytic extracellular chromatin increases thrombin and plasmin generation, causes a shortening of plasma clotting time of APL cells, and increases fibrin formation. DNase I but not anti-tissue factor antibody could inhibit these effects. Immunofluorescence staining showed that promyelocytic extracellular chromatin and phosphatidylserine on APL cells provide platforms for fibrin deposition and render clots more resistant to fibrinolysis. Additionally, coincubation assays revealed that promyelocytic extracellular chromatin is cytotoxic to ECs, converting them to a procoagulant phenotype. This cytotoxity was blocked by DNase I by 20% or activated protein C by 31%. Our current results thus delineate the pathogenic role of promyelocytic extracellular chromatin in APL coagulopathy. Furthermore, the remaining coagulation disturbance in high-risk APL patients after ATRA administration may be treatable by intrinsic pathway inhibition via accelerating extracellular chromatin degradation. PMID:28053193

  5. The activities of eukaryotic replication origins in chromatin.

    PubMed

    Weinreich, Michael; Palacios DeBeer, Madeleine A; Fox, Catherine A

    2004-03-15

    DNA replication initiates at chromosomal positions called replication origins. This review will focus on the activity, regulation and roles of replication origins in Saccharomyces cerevisiae. All eukaryotic cells, including S. cerevisiae, depend on the initiation (activity) of hundreds of replication origins during a single cell cycle for the duplication of their genomes. However, not all origins are identical. For example, there is a temporal order to origin activation with some origins firing early during the S-phase and some origins firing later. Recent studies provide evidence that posttranslational chromatin modifications, heterochromatin-binding proteins and nucleosome positioning can control the efficiency and/or timing of chromosomal origin activity in yeast. Many more origins exist than are necessary for efficient replication. The availability of excess replication origins leaves individual origins free to evolve distinct forms of regulation and/or roles in chromosomes beyond their fundamental role in DNA synthesis. We propose that some origins have acquired roles in controlling chromatin structure and/or gene expression. These roles are not linked obligatorily to replication origin activity per se, but instead exploit multi-subunit replication proteins with the potential to form context-dependent protein-protein interactions.

  6. Combgap Promotes Ovarian Niche Development and Chromatin Association of EcR-Binding Regions in BR-C.

    PubMed

    Hitrik, Anna; Popliker, Malka; Gancz, Dana; Mukamel, Zohar; Lifshitz, Aviezer; Schwartzman, Omer; Tanay, Amos; Gilboa, Lilach

    2016-11-01

    The development of niches for tissue-specific stem cells is an important aspect of stem cell biology. Determination of niche size and niche numbers during organogenesis involves precise control of gene expression. How this is achieved in the context of a complex chromatin landscape is largely unknown. Here we show that the nuclear protein Combgap (Cg) supports correct ovarian niche formation in Drosophila by controlling ecdysone-Receptor (EcR)- mediated transcription and long-range chromatin contacts in the broad locus (BR-C). Both cg and BR-C promote ovarian growth and the development of niches for germ line stem cells. BR-C levels were lower when Combgap was either reduced or over-expressed, indicating an intricate regulation of the BR-C locus by Combgap. Polytene chromosome stains showed that Cg co-localizes with EcR, the major regulator of BR-C, at the BR-C locus and that EcR binding to chromatin was sensitive to changes in Cg levels. Proximity ligation assay indicated that the two proteins could reside in the same complex. Finally, chromatin conformation analysis revealed that EcR-bound regions within BR-C, which span ~30 KBs, contacted each other. Significantly, these contacts were stabilized in an ecdysone- and Combgap-dependent manner. Together, these results highlight Combgap as a novel regulator of chromatin structure that promotes transcription of ecdysone target genes and ovarian niche formation.

  7. Combgap Promotes Ovarian Niche Development and Chromatin Association of EcR-Binding Regions in BR-C

    PubMed Central

    Gancz, Dana; Lifshitz, Aviezer; Tanay, Amos

    2016-01-01

    The development of niches for tissue-specific stem cells is an important aspect of stem cell biology. Determination of niche size and niche numbers during organogenesis involves precise control of gene expression. How this is achieved in the context of a complex chromatin landscape is largely unknown. Here we show that the nuclear protein Combgap (Cg) supports correct ovarian niche formation in Drosophila by controlling ecdysone-Receptor (EcR)- mediated transcription and long-range chromatin contacts in the broad locus (BR-C). Both cg and BR-C promote ovarian growth and the development of niches for germ line stem cells. BR-C levels were lower when Combgap was either reduced or over-expressed, indicating an intricate regulation of the BR-C locus by Combgap. Polytene chromosome stains showed that Cg co-localizes with EcR, the major regulator of BR-C, at the BR-C locus and that EcR binding to chromatin was sensitive to changes in Cg levels. Proximity ligation assay indicated that the two proteins could reside in the same complex. Finally, chromatin conformation analysis revealed that EcR-bound regions within BR-C, which span ~30 KBs, contacted each other. Significantly, these contacts were stabilized in an ecdysone- and Combgap-dependent manner. Together, these results highlight Combgap as a novel regulator of chromatin structure that promotes transcription of ecdysone target genes and ovarian niche formation. PMID:27846223

  8. Atomic force microscopy on chromosomes, chromatin and DNA: a review.

    PubMed

    Kalle, Wouter; Strappe, Padraig

    2012-12-01

    The purpose of this review is to discuss the achievements and progress that has been made in the use of atomic force microscopy in DNA related research in the last 25 years. For this review DNA related research is split up in chromosomal-, chromatin- and DNA focused research to achieve a logical flow from large- to smaller structures. The focus of this review is not only on the AFM as imaging tool but also on the AFM as measuring tool using force spectroscopy, as therein lays its greatest advantage and future. The amazing technological and experimental progress that has been made during the last 25 years is too extensive to fully cover in this review but some key developments and experiments have been described to give an overview of the evolution of AFM use from 'imaging tool' to 'measurement tool' on chromosomes, chromatin and DNA. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  9. Sperm nuclear protamines: A checkpoint to control sperm chromatin quality.

    PubMed

    Steger, Klaus; Balhorn, Rod

    2018-05-23

    Protamines are nuclear proteins which are specifically expressed in haploid male germ cells. Their replacement of histones and binding to DNA is followed by chromatin hypercondensation that protects DNA from negative influences by environmental factors. Mammalian sperm contain two types of protamines: PRM1 and PRM2. While the proportion of the two protamines is highly variable between different species, abnormal ratios within a species are known to be associated with male subfertility. Therefore, it is more than likely that correct protamine expression represents a kind of chromatin checkpoint during sperm development rendering protamines as suitable biomarkers for the estimation of sperm quality. This review presents an overview of our current knowledge on protamines comparing gene and protein structures between different mammalian species with particular consideration given to man, mouse and stallion. At last, recent insights into the possible role of inherited sperm histones for early embryo development are provided. © 2018 Blackwell Verlag GmbH.

  10. CTCF and cohesin regulate chromatin loop stability with distinct dynamics

    PubMed Central

    Hansen, Anders S; Pustova, Iryna; Cattoglio, Claudia; Tjian, Robert; Darzacq, Xavier

    2017-01-01

    Folding of mammalian genomes into spatial domains is critical for gene regulation. The insulator protein CTCF and cohesin control domain location by folding domains into loop structures, which are widely thought to be stable. Combining genomic and biochemical approaches we show that CTCF and cohesin co-occupy the same sites and physically interact as a biochemically stable complex. However, using single-molecule imaging we find that CTCF binds chromatin much more dynamically than cohesin (~1–2 min vs. ~22 min residence time). Moreover, after unbinding, CTCF quickly rebinds another cognate site unlike cohesin for which the search process is long (~1 min vs. ~33 min). Thus, CTCF and cohesin form a rapidly exchanging 'dynamic complex' rather than a typical stable complex. Since CTCF and cohesin are required for loop domain formation, our results suggest that chromatin loops are dynamic and frequently break and reform throughout the cell cycle. DOI: http://dx.doi.org/10.7554/eLife.25776.001 PMID:28467304

  11. Nuclear location of a chromatin insulator in Drosophila melanogaster.

    PubMed

    Xu, Qinghao; Li, Mo; Adams, Jessica; Cai, Haini N

    2004-03-01

    Chromatin-related functions are associated with spatial organization in the nucleus. We have investigated the relationship between the enhancer-blocking activity and subnuclear localization of the Drosophila melanogaster suHw insulator. Using fluorescent in situ hybridization, we observed that genomic loci containing the gypsy retrotransposon were distributed closer to the nuclear periphery than regions without the gypsy retrotransposon. However, transgenes containing a functional 340 bp suHw insulator did not exhibit such biased distribution towards the nuclear periphery, which suggests that the suHw insulator sequence is not responsible for the peripheral localization of the gypsy retrotransposon. Antibody stains showed that the two proteins essential for the suHw insulator activity, SUHW and MOD(MDG4), are not restricted to the nuclear periphery. The enhancer-blocking activity of suHw remained intact under the heat shock conditions, which was shown to disrupt the association of gypsy, SUHW and MOD(MDG4) with the nuclear periphery. Our results indicate that the suHw insulator can function in the nuclear interior, possibly through local interactions with chromatin components or other nuclear structures.

  12. Cytology of DNA Replication Reveals Dynamic Plasticity of Large-Scale Chromatin Fibers.

    PubMed

    Deng, Xiang; Zhironkina, Oxana A; Cherepanynets, Varvara D; Strelkova, Olga S; Kireev, Igor I; Belmont, Andrew S

    2016-09-26

    In higher eukaryotic interphase nuclei, the 100- to >1,000-fold linear compaction of chromatin is difficult to reconcile with its function as a template for transcription, replication, and repair. It is challenging to imagine how DNA and RNA polymerases with their associated molecular machinery would move along the DNA template without transient decondensation of observed large-scale chromatin "chromonema" fibers [1]. Transcription or "replication factory" models [2], in which polymerases remain fixed while DNA is reeled through, are similarly difficult to conceptualize without transient decondensation of these chromonema fibers. Here, we show how a dynamic plasticity of chromatin folding within large-scale chromatin fibers allows DNA replication to take place without significant changes in the global large-scale chromatin compaction or shape of these large-scale chromatin fibers. Time-lapse imaging of lac-operator-tagged chromosome regions shows no major change in the overall compaction of these chromosome regions during their DNA replication. Improved pulse-chase labeling of endogenous interphase chromosomes yields a model in which the global compaction and shape of large-Mbp chromatin domains remains largely invariant during DNA replication, with DNA within these domains undergoing significant movements and redistribution as they move into and then out of adjacent replication foci. In contrast to hierarchical folding models, this dynamic plasticity of large-scale chromatin organization explains how localized changes in DNA topology allow DNA replication to take place without an accompanying global unfolding of large-scale chromatin fibers while suggesting a possible mechanism for maintaining epigenetic programming of large-scale chromatin domains throughout DNA replication. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Dynamic Control of Chromosome Topology and Gene Expression by a Chromatin Modification.

    PubMed

    Bian, Qian; Anderson, Erika C; Brejc, Katjuša; Meyer, Barbara J

    2018-02-22

    The function of chromatin modification in establishing higher-order chromosome structure during gene regulation has been elusive. We dissected the machinery and mechanism underlying the enrichment of histone modification H4K20me1 on hermaphrodite X chromosomes during Caenorhabditis elegans dosage compensation and discovered a key role for H4K20me1 in regulating X-chromosome topology and chromosome-wide gene expression. Structural and functional analysis of the dosage compensation complex (DCC) subunit DPY-21 revealed a novel Jumonji C demethylase subfamily that converts H4K20me2 to H4K20me1 in worms and mammals. Inactivation of demethylase activity in vivo by genome editing eliminated H4K20me1 enrichment on X chromosomes of somatic cells, increased X-linked gene expression, reduced X-chromosome compaction, and disrupted X-chromosome conformation by diminishing the formation of topologically associated domains. H4K20me1 is also enriched on the inactive X of female mice, making our studies directly relevant to mammalian development. Unexpectedly, DPY-21 also associates specifically with autosomes of nematode germ cells in a DCC-independent manner to enrich H4K20me1 and trigger chromosome compaction. Thus, DPY-21 is an adaptable chromatin regulator. Its H4K20me2 demethylase activity can be harnessed during development for distinct biological functions by targeting it to diverse genomic locations through different mechanisms. In both somatic cells and germ cells, H4K20me1 enrichment modulates three-dimensional chromosome architecture, demonstrating the direct link between chromatin modification and higher-order chromosome structure. © 2017 Bian et al.; Published by Cold Spring Harbor Laboratory Press.

  14. Initial high-resolution microscopic mapping of active and inactive regulatory sequences proves non-random 3D arrangements in chromatin domain clusters.

    PubMed

    Cremer, Marion; Schmid, Volker J; Kraus, Felix; Markaki, Yolanda; Hellmann, Ines; Maiser, Andreas; Leonhardt, Heinrich; John, Sam; Stamatoyannopoulos, John; Cremer, Thomas

    2017-08-07

    The association of active transcription regulatory elements (TREs) with DNAse I hypersensitivity (DHS[+]) and an 'open' local chromatin configuration has long been known. However, the 3D topography of TREs within the nuclear landscape of individual cells in relation to their active or inactive status has remained elusive. Here, we explored the 3D nuclear topography of active and inactive TREs in the context of a recently proposed model for a functionally defined nuclear architecture, where an active and an inactive nuclear compartment (ANC-INC) form two spatially co-aligned and functionally interacting networks. Using 3D structured illumination microscopy, we performed 3D FISH with differently labeled DNA probe sets targeting either sites with DHS[+], apparently active TREs, or DHS[-] sites harboring inactive TREs. Using an in-house image analysis tool, DNA targets were quantitatively mapped on chromatin compaction shaped 3D nuclear landscapes. Our analyses present evidence for a radial 3D organization of chromatin domain clusters (CDCs) with layers of increasing chromatin compaction from the periphery to the CDC core. Segments harboring active TREs are significantly enriched at the decondensed periphery of CDCs with loops penetrating into interchromatin compartment channels, constituting the ANC. In contrast, segments lacking active TREs (DHS[-]) are enriched toward the compacted interior of CDCs (INC). Our results add further evidence in support of the ANC-INC network model. The different 3D topographies of DHS[+] and DHS[-] sites suggest positional changes of TREs between the ANC and INC depending on their functional state, which might provide additional protection against an inappropriate activation. Our finding of a structural organization of CDCs based on radially arranged layers of different chromatin compaction levels indicates a complex higher-order chromatin organization beyond a dichotomic classification of chromatin into an 'open,' active and 'closed

  15. Direct screening for chromatin status on DNA barcodes in yeast delineates the regulome of H3K79 methylation by Dot1

    PubMed Central

    Vlaming, Hanneke; Molenaar, Thom M; van Welsem, Tibor; Poramba-Liyanage, Deepani W; Smith, Desiree E; Velds, Arno; Hoekman, Liesbeth; Korthout, Tessy; Hendriks, Sjoerd; Maarten Altelaar, AF; van Leeuwen, Fred

    2016-01-01

    Given the frequent misregulation of chromatin in cancer, it is important to understand the cellular mechanisms that regulate chromatin structure. However, systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs. Here we describe a strategy, Epi-ID, to directly assess chromatin status in thousands of mutants. In Epi-ID, chromatin status on DNA barcodes is interrogated by chromatin immunoprecipitation followed by deep sequencing, allowing for quantitative comparison of many mutants in parallel. Screening of a barcoded yeast knock-out collection for regulators of histone H3K79 methylation by Dot1 identified all known regulators as well as novel players and processes. These include histone deposition, homologous recombination, and adenosine kinase, which influences the methionine cycle. Gcn5, the acetyltransferase within the SAGA complex, was found to regulate histone methylation and H2B ubiquitination. The concept of Epi-ID is widely applicable and can be readily applied to other chromatin features. DOI: http://dx.doi.org/10.7554/eLife.18919.001 PMID:27922451

  16. Chromatin organization and global regulation of Hox gene clusters

    PubMed Central

    Montavon, Thomas; Duboule, Denis

    2013-01-01

    During development, a properly coordinated expression of Hox genes, within their different genomic clusters is critical for patterning the body plans of many animals with a bilateral symmetry. The fascinating correspondence between the topological organization of Hox clusters and their transcriptional activation in space and time has served as a paradigm for understanding the relationships between genome structure and function. Here, we review some recent observations, which revealed highly dynamic changes in the structure of chromatin at Hox clusters, in parallel with their activation during embryonic development. We discuss the relevance of these findings for our understanding of large-scale gene regulation. PMID:23650639

  17. Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

    PubMed

    Kurat, Christoph F; Yeeles, Joseph T P; Patel, Harshil; Early, Anne; Diffley, John F X

    2017-01-05

    The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Conformation of chromatin oligomers. A new argument for a change with the hexanucleosome.

    PubMed

    Marion, C; Bezot, P; Hesse-Bezot, C; Roux, B; Bernengo, J C

    1981-11-01

    Quasielastic laser light scattering measurements have been made on chromatin oligomers to obtain information on the transition in their electrooptical properties, previously observed for the hexameric structures [Marion, C. and Roux, B. (1978) Nucleic Acids Res. 5, 4431-4449]. Translational diffusion coefficients were determined for mononucleosomes to octanucleosomes containing histone H1 over a range of ionic strength. At high ionic strength, oligomers show a linear dependence of the logarithm of diffusion coefficient upon the logarithm of number of nucleosomes. At low ionic strength a change occurs between hexamer and heptamer. Our results agree well with the recent sedimentation data of Osipova et al. [Eur. J. Biochem. (1980) 113, 183-188] and of Butler and Thomas [J. Mol. Biol. (1980) 140, 505-529] showing a change in stability with hexamer. Various models for the arrangements of nucleosomes in the superstructure of chromatin are discussed. All calculations clearly indicate a conformational change with the hexanucleosome and the results suggest that, at low ionic strength, the chromatin adopts a loosely helical structure of 28-nm diameter and 22-nm pitch. These results are also consistent with a discontinuity every sixth nucleosome, corresponding to a turn of the helix. This discontinuity may explain the recent electric dichroism data of Lee et al. [Biochemistry (1981) 20, 1438-1445]. The hexanucleosome structure which we have previously suggested, with the faces of nucleosomes arranged radially to the helical axis has been recently confirmed by Mc Ghee et al. [Cell (1980) 22, 87-96]. With an increase of ionic strength, the helix becomes more regular and compact with a slightly reduced outer diameter and a decreased pitch, the dimensions resembling those proposed for solenoid models.

  19. The molecular topography of silenced chromatin in Saccharomyces cerevisiae

    PubMed Central

    Thurtle, Deborah M.; Rine, Jasper

    2014-01-01

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

  20. Interplay between chromatin modulators and histone acetylation regulates the formation of accessible chromatin in the upstream regulatory region of fission yeast fbp1.

    PubMed

    Adachi, Akira; Senmatsu, Satoshi; Asada, Ryuta; Abe, Takuya; Hoffman, Charles S; Ohta, Kunihiro; Hirota, Kouji

    2018-05-03

    Numerous noncoding RNA transcripts are detected in eukaryotic cells. Noncoding RNAs transcribed across gene promoters are involved in the regulation of mRNA transcription via chromatin modulation. This function of noncoding RNA transcription was first demonstrated for the fission yeast fbp1 gene, where a cascade of noncoding RNA transcription events induces chromatin remodeling to facilitate transcription factor binding. We recently demonstrated that the noncoding RNAs from the fbp1 upstream region facilitate binding of the transcription activator Atf1 and thereby promote histone acetylation. Histone acetylation by histone acetyl transferases (HATs) and ATP-dependent chromatin remodelers (ADCRs) are implicated in chromatin remodeling, but the interplay between HATs and ADCRs in this process has not been fully elucidated. Here, we examine the roles played by two distinct ADCRs, Snf22 and Hrp3, and by the HAT Gcn5 in the transcriptional activation of fbp1. Snf22 and Hrp3 redundantly promote disassembly of chromatin in the fbp1 upstream region. Gcn5 critically contributes to nucleosome eviction in the absence of either Snf22 or Hrp3, presumably by recruiting Hrp3 in snf22∆ cells and Snf22 in hrp3∆ cells. Conversely, Gcn5-dependent histone H3 acetylation is impaired in snf22∆/hrp3∆ cells, suggesting that both redundant ADCRs induce recruitment of Gcn5 to the chromatin array in the fbp1 upstream region. These results reveal a previously unappreciated interplay between ADCRs and histone acetylation in which histone acetylation facilitates recruitment of ADCRs, while ADCRs are required for histone acetylation.

  1. Protective role of RAD50 on chromatin bridges during abnormal cytokinesis.

    PubMed

    Schröder-Heurich, Bianca; Wieland, Britta; Lavin, Martin F; Schindler, Detlev; Dörk, Thilo

    2014-03-01

    Faithful chromosome segregation is required for preserving genomic integrity. Failure of this process may entail chromatin bridges preventing normal cytokinesis. To test whether RAD50, a protein normally involved in DNA double-strand break repair, is involved in abnormal cytokinesis and formation of chromatin bridges, we used immunocytochemical and protein interaction assays. RAD50 localizes to chromatin bridges during aberrant cytokinesis and subsequent stages of the cell cycle, either decorating the entire bridge or focally accumulating at the midbody zone. Ionizing radiation led to an ∼4-fold increase in the rate of chromatin bridges in an ataxia telangiectatica mutated (ATM)-dependent manner in human RAD50-proficient fibroblasts but not in RAD50-deficient cells. Cells with a RAD50-positive chromatin bridge were able to continue cell cycling and to progress through S phase (44%), whereas RAD50 knockdown caused a deficiency in chromatin bridges as well as an ∼4-fold prolonged duration of mitosis. RAD50 colocalized and directly interacted with Aurora B kinase and phospho-histone H3, and Aurora B kinase inhibition led to a deficiency in RAD50-positive bridges. Based on these observations, we propose that RAD50 is a crucial factor for the stabilization and shielding of chromatin bridges. Our study provides evidence for a hitherto unknown role of RAD50 in abnormal cytokinesis.

  2. Circadian expression profiles of chromatin remodeling factor genes in Arabidopsis.

    PubMed

    Lee, Hong Gil; Lee, Kyounghee; Jang, Kiyoung; Seo, Pil Joon

    2015-01-01

    The circadian clock is a biological time keeper mechanism that regulates biological rhythms to a period of approximately 24 h. The circadian clock enables organisms to anticipate environmental cycles and coordinates internal cellular physiology with external environmental cues. In plants, correct matching of the clock with the environment confers fitness advantages to plant survival and reproduction. Therefore, circadian clock components are regulated at multiple layers to fine-tune the circadian oscillation. Epigenetic regulation provides an additional layer of circadian control. However, little is known about which chromatin remodeling factors are responsible for circadian control. In this work, we analyzed circadian expression of 109 chromatin remodeling factor genes and identified 17 genes that display circadian oscillation. In addition, we also found that a candidate interacts with a core clock component, supporting that clock activity is regulated in part by chromatin modification. As an initial attempt to elucidate the relationship between chromatin modification and circadian oscillation, we identified novel regulatory candidates that provide a platform for future investigations of chromatin regulation of the circadian clock.

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

    PubMed Central

    Kolade, Charles Oluwabukunmi

    2015-01-01

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

  4. Maize histone H2B-mCherry: a new fluorescent chromatin marker for somatic and meiotic chromosome research.

    PubMed

    Howe, Elizabeth S; Clemente, Thomas E; Bass, Hank W

    2012-06-01

    Cytological studies of fluorescent proteins are rapidly yielding insights into chromatin structure and dynamics. Here we describe the production and cytological characterization of new transgenic maize lines expressing a fluorescent histone fusion protein, H2B-mCherry. The transgene is expressed under the control of the maize ubiquitin1 promoter, including its first exon and intron. Polymerase chain reaction-based genotyping and root-tip microscopy showed that most of the lines carrying the transgene also expressed it, producing bright uniform staining of nuclei. Further, plants showing expression in root tips at the seedling stage also showed expression during meiosis, late in the life cycle. Detailed high-resolution three-dimensional imaging of cells and nuclei from various somatic and meiotic cell types showed that H2B-mCherry produced remarkably clear images of chromatin and chromosome fiber morphology, as seen in somatic, male meiotic prophase, and early microgametophyte cells. H2B-mCherry also yielded distinct nucleolus staining and was shown to be compatible with fluorescence in situ hybridization. We found several instances where H2B-mCherry was superior to DAPI as a generalized chromatin stain. Our study establishes these histone H2B-mCherry lines as new biological reagents for visualizing chromatin structure, chromosome morphology, and nuclear dynamics in fixed and living cells in a model plant genetic system.

  5. A multiplexed system for quantitative comparisons of chromatin landscapes

    PubMed Central

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

    2015-01-01

    Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of P300, EZH2 or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions and drug treatments. PMID:26687680

  6. Separate roles for chromatin and lamins in nuclear mechanics.

    PubMed

    Stephens, Andrew D; Banigan, Edward J; Marko, John F

    2018-01-01

    The cell nucleus houses, protects, and arranges the genome within the cell. Therefore, nuclear mechanics and morphology are important for dictating gene regulation, and these properties are perturbed in many human diseases, such as cancers and progerias. The field of nuclear mechanics has long been dominated by studies of the nuclear lamina, the intermediate filament shell residing just beneath the nuclear membrane. However, a growing body of work shows that chromatin and chromatin-related factors within the nucleus are an essential part of the mechanical response of the cell nucleus to forces. Recently, our group demonstrated that chromatin and the lamina provide distinct mechanical contributions to nuclear mechanical response. The lamina is indeed important for robust response to large, whole-nucleus stresses, but chromatin dominates the short-extension response. These findings offer a clarifying perspective on varied nuclear mechanics measurements and observations, and they suggest several new exciting possibilities for understanding nuclear morphology, organization, and mechanics.

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

    PubMed Central

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

    2013-01-01

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

  8. Histone hyperacetylation during meiosis interferes with large-scale chromatin remodeling, axial chromatid condensation and sister chromatid separation in the mammalian oocyte.

    PubMed

    Yang, Feikun; Baumann, Claudia; Viveiros, Maria M; De La Fuente, Rabindranath

    2012-01-01

    Histone acetylation regulates higher-order chromatin structure and function and is critical for the control of gene expression. Histone deacetylase inhibitors (HDACi) are currently under investigation as novel cancer therapeutic drugs. Here, we show that female germ cells are extremely susceptible to chromatin changes induced by HDACi. Our results indicate that exposure to trichostatin A (TSA) at nanomolar levels interferes with major chromatin remodeling events in the mammalian oocyte leading to chromosome instability. High resolution analysis of chromatin structure and live-cell imaging revealed a striking euchromatin decondensation associated with histone H4 hyperacetylation following exposure to 15 nM TSA in >90% of pre-ovulatory oocytes. Dynamic changes in large-scale chromatin structure were detected after 2 h of exposure and result in the formation of misaligned chromosomes in >75% (P<0.05) of in vitro matured oocytes showing chromosome lagging as well as abnormal sister chromatid separation at anaphase I. Abnormal axial chromatid condensation during meiosis results in the formation of elongated chromosomes exhibiting hyperacetylation of histone H4 at lysine 5 and lysine 16 at interstitial chromosome segments, but not pericentric heterochromatin, while highly decondensed bivalents exhibit prominent histone H3 phosphorylation at centromeric domains. Notably, no changes were observed in the chromosomal localization of the condensin protein SMC4. These results indicate that HDAC activity is required for proper chromosome condensation in the mammalian oocyte and that HDACi may induce abnormal chromosome segregation by interfering with both chromosome-microtubule interactions, as well as sister chromatid separation. Thus, HDACi, proposed for cancer therapy, may disrupt the epigenetic status of female germ cells, predisposing oocytes to aneuploidy at previously unrecognized low doses.

  9. Multifaceted Targeting of the Chromatin Mediates Gonadotropin-Releasing Hormone Effects on Gene Expression in the Gonadotrope.

    PubMed

    Melamed, Philippa; Haj, Majd; Yosefzon, Yahav; Rudnizky, Sergei; Wijeweera, Andrea; Pnueli, Lilach; Kaplan, Ariel

    2018-01-01

    Gonadotropin-releasing hormone (GnRH) stimulates the expression of multiple genes in the pituitary gonadotropes, most notably to induce synthesis of the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), but also to ensure the appropriate functioning of these cells at the center of the mammalian reproductive endocrine axis. Aside from the activation of gene-specific transcription factors, GnRH stimulates through its membrane-bound receptor, alterations in the chromatin that facilitate transcription of its target genes. These include changes in the histone and DNA modifications, nucleosome positioning, and chromatin packaging at the regulatory regions of each gene. The requirements for each of these events vary according to the DNA sequence which determines the basal chromatin packaging at the regulatory regions. Despite considerable progress in this field in recent years, we are only beginning to understand some of the complexities involved in the role and regulation of this chromatin structure, including new modifications, extensive cross talk, histone variants, and the actions of distal enhancers and non-coding RNAs. This short review aims to integrate the latest findings on GnRH-induced alterations in the chromatin of its target genes, which indicate multiple and diverse actions. Understanding these processes is illuminating not only in the context of the activation of these hormones during the reproductive life span but may also reveal how aberrant epigenetic regulation of these genes leads to sub-fertility.

  10. Multifaceted Targeting of the Chromatin Mediates Gonadotropin-Releasing Hormone Effects on Gene Expression in the Gonadotrope

    PubMed Central

    Melamed, Philippa; Haj, Majd; Yosefzon, Yahav; Rudnizky, Sergei; Wijeweera, Andrea; Pnueli, Lilach; Kaplan, Ariel

    2018-01-01

    Gonadotropin-releasing hormone (GnRH) stimulates the expression of multiple genes in the pituitary gonadotropes, most notably to induce synthesis of the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), but also to ensure the appropriate functioning of these cells at the center of the mammalian reproductive endocrine axis. Aside from the activation of gene-specific transcription factors, GnRH stimulates through its membrane-bound receptor, alterations in the chromatin that facilitate transcription of its target genes. These include changes in the histone and DNA modifications, nucleosome positioning, and chromatin packaging at the regulatory regions of each gene. The requirements for each of these events vary according to the DNA sequence which determines the basal chromatin packaging at the regulatory regions. Despite considerable progress in this field in recent years, we are only beginning to understand some of the complexities involved in the role and regulation of this chromatin structure, including new modifications, extensive cross talk, histone variants, and the actions of distal enhancers and non-coding RNAs. This short review aims to integrate the latest findings on GnRH-induced alterations in the chromatin of its target genes, which indicate multiple and diverse actions. Understanding these processes is illuminating not only in the context of the activation of these hormones during the reproductive life span but may also reveal how aberrant epigenetic regulation of these genes leads to sub-fertility. PMID:29535683

  11. RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation

    PubMed Central

    Zoabi, Muhammad; Nadar-Ponniah, Prathamesh T.; Khoury-Haddad, Hanan; Usaj, Marko; Budowski-Tal, Inbal; Haran, Tali; Henn, Arnon; Mandel-Gutfreund, Yael; Ayoub, Nabieh

    2014-01-01

    The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin. PMID:25378304

  12. Essential role of lncRNA binding for WDR5 maintenance of active chromatin and embryonic stem cell pluripotency

    PubMed Central

    Yang, Yul W; Flynn, Ryan A; Chen, Yong; Qu, Kun; Wan, Bingbing; Wang, Kevin C; Lei, Ming; Chang, Howard Y

    2014-01-01

    The WDR5 subunit of the MLL complex enforces active chromatin and can bind RNA; the relationship between these two activities is unclear. Here we identify a RNA binding pocket on WDR5, and discover a WDR5 mutant (F266A) that selectively abrogates RNA binding without affecting MLL complex assembly or catalytic activity. Complementation in ESCs shows that WDR5 F266A mutant is unable to accumulate on chromatin, and is defective in gene activation, maintenance of histone H3 lysine 4 trimethylation, and ESC self renewal. We identify a family of ESC messenger and lncRNAs that interact with wild type WDR5 but not F266A mutant, including several lncRNAs known to be important for ESC gene expression. These results suggest that specific RNAs are integral inputs into the WDR5-MLL complex for maintenance of the active chromatin state and embryonic stem cell fates. DOI: http://dx.doi.org/10.7554/eLife.02046.001 PMID:24521543

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

    PubMed Central

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

    2015-01-01

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

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

    DOE PAGES

    Myllys, Markko; Ruokolainen, Visa; Aho, Vesa; ...

    2016-06-28

    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. Here, 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 gapsmore » frequently contained viral nucleocapsids. Our 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.« less

  15. High-resolution mapping of transcription factor binding sites on native chromatin

    PubMed Central

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

    2014-01-01

    Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) is widely used for profiling of TF binding, but is limited by low resolution and poor specificity and sensitivity. We present a simple protocol that starts with micrococcal nuclease-digested uncross-linked chromatin and is followed by affinity purification of TFs and paired-end sequencing. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide highly accurate base-pair resolution maps that are not biased toward accessible chromatin, and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila melanogaster GAGA Factor and Pipsqueak. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions. PMID:24336359

  16. Hormone-induced modifications of the chromatin structure surrounding upstream regulatory regions conserved between the mouse and rabbit whey acidic protein genes.

    PubMed Central

    Millot, Benjamin; Montoliu, Lluís; Fontaine, Marie-Louise; Mata, Teresa; Devinoy, Eve

    2003-01-01

    The upstream regulatory regions of the mouse and rabbit whey acidic protein (WAP) genes have been used extensively to target the efficient expression of foreign genes into the mammary gland of transgenic animals. Therefore both regions have been studied to elucidate fully the mechanisms controlling WAP gene expression. Three DNase I-hypersensitive sites (HSS0, HSS1 and HSS2) have been described upstream of the rabbit WAP gene in the lactating mammary gland and correspond to important regulatory regions. These sites are surrounded by variable chromatin structures during mammary-gland development. In the present study, we describe the upstream sequence of the mouse WAP gene. Analysis of genomic sequences shows that the mouse WAP gene is situated between two widely expressed genes (Cpr2 and Ramp3). We show that the hypersensitive sites found upstream of the rabbit WAP gene are also detected in the mouse WAP gene. Further, they encompass functional signal transducer and activator of transcription 5-binding sites, as has been observed in the rabbit. A new hypersensitive site (HSS3), not specific to the mammary gland, was mapped 8 kb upstream of the rabbit WAP gene. Unlike the three HSSs described above, HSS3 is also detected in the liver, but similar to HSS1, it does not depend on lactogenic hormone treatments during cell culture. The region surrounding HSS3 encompasses a potential matrix attachment region, which is also conserved upstream of the mouse WAP gene and contains a functional transcription factor Ets-1 (E26 transformation-specific-1)-binding site. Finally, we demonstrate for the first time that variations in the chromatin structure are dependent on prolactin alone. PMID:12580766

  17. The genomic landscape of mantle cell lymphoma is related to the epigenetically determined chromatin state of normal B cells

    PubMed Central

    Zhang, Jenny; Jima, Dereje; Moffitt, Andrea B.; Liu, Qingquan; Czader, Magdalena; Hsi, Eric D.; Fedoriw, Yuri; Dunphy, Cherie H.; Richards, Kristy L.; Gill, Javed I.; Sun, Zhen; Love, Cassandra; Scotland, Paula; Lock, Eric; Levy, Shawn; Hsu, David S.; Dunson, David; Dave, Sandeep S.

    2014-01-01

    In this study, we define the genetic landscape of mantle cell lymphoma (MCL) through exome sequencing of 56 cases of MCL. We identified recurrent mutations in ATM, CCND1, MLL2, and TP53. We further identified a number of novel genes recurrently mutated in patients with MCL including RB1, WHSC1, POT1, and SMARCA4. We noted that MCLs have a distinct mutational profile compared with lymphomas from other B-cell stages. The ENCODE project has defined the chromatin structure of many cell types. However, a similar characterization of primary human mature B cells has been lacking. We defined, for the first time, the chromatin structure of primary human naïve, germinal center, and memory B cells through chromatin immunoprecipitation and sequencing for H3K4me1, H3K4me3, H3Ac, H3K36me3, H3K27me3, and PolII. We found that somatic mutations that occur more frequently in either MCLs or Burkitt lymphomas were associated with open chromatin in their respective B cells of origin, naïve B cells, and germinal center B cells. Our work thus elucidates the landscape of gene-coding mutations in MCL and the critical interplay between epigenetic alterations associated with B-cell differentiation and the acquisition of somatic mutations in cancer. PMID:24682267

  18. The Chromatin Remodeler Isw1 Prevents CAG Repeat Expansions During Transcription in Saccharomyces cerevisiae

    PubMed Central

    Koch, Melissa R.; House, Nealia C. M.; Cosetta, Casey M.; Jong, Robyn M.; Salomon, Christelle G.; Joyce, Cailin E.; Philips, Elliot A.; Su, Xiaofeng A.; Freudenreich, Catherine H.

    2018-01-01

    CAG/CTG trinucleotide repeats are unstable sequences that are difficult to replicate, repair, and transcribe due to their structure-forming nature. CAG repeats strongly position nucleosomes; however, little is known about the chromatin remodeling needed to prevent repeat instability. In a Saccharomyces cerevisiae model system with CAG repeats carried on a YAC, we discovered that the chromatin remodeler Isw1 is required to prevent CAG repeat expansions during transcription. CAG repeat expansions in the absence of Isw1 were dependent on both transcription-coupled repair (TCR) and base-excision repair (BER). Furthermore, isw1∆ mutants are sensitive to methyl methanesulfonate (MMS) and exhibit synergistic MMS sensitivity when combined with BER or TCR pathway mutants. We conclude that CAG expansions in the isw1∆ mutant occur during a transcription-coupled excision repair process that involves both TCR and BER pathways. We observed increased RNA polymerase II (RNAPII) occupancy at the CAG repeat when transcription of the repeat was induced, but RNAPII binding did not change in isw1∆ mutants, ruling out a role for Isw1 remodeling in RNAPII progression. However, nucleosome occupancy over a transcribed CAG tract was altered in isw1∆ mutants. Based on the known role of Isw1 in the reestablishment of nucleosomal spacing after transcription, we suggest that a defect in this function allows DNA structures to form within repetitive DNA tracts, resulting in inappropriate excision repair and repeat-length changes. These results establish a new function for Isw1 in directly maintaining the chromatin structure at the CAG repeat, thereby limiting expansions that can occur during transcription-coupled excision repair. PMID:29305386

  19. Statistical models for detecting differential chromatin interactions mediated by a protein.

    PubMed

    Niu, Liang; Li, Guoliang; Lin, Shili

    2014-01-01

    Chromatin interactions mediated by a protein of interest are of great scientific interest. Recent studies show that protein-mediated chromatin interactions can have different intensities in different types of cells or in different developmental stages of a cell. Such differences can be associated with a disease or with the development of a cell. Thus, it is of great importance to detect protein-mediated chromatin interactions with different intensities in different cells. A recent molecular technique, Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET), which uses formaldehyde cross-linking and paired-end sequencing, is able to detect genome-wide chromatin interactions mediated by a protein of interest. Here we proposed two models (One-Step Model and Two-Step Model) for two sample ChIA-PET count data (one biological replicate in each sample) to identify differential chromatin interactions mediated by a protein of interest. Both models incorporate the data dependency and the extent to which a fragment pair is related to a pair of DNA loci of interest to make accurate identifications. The One-Step Model makes use of the data more efficiently but is more computationally intensive. An extensive simulation study showed that the models can detect those differentially interacted chromatins and there is a good agreement between each classification result and the truth. Application of the method to a two-sample ChIA-PET data set illustrates its utility. The two models are implemented as an R package MDM (available at http://www.stat.osu.edu/~statgen/SOFTWARE/MDM).

  20. Statistical Models for Detecting Differential Chromatin Interactions Mediated by a Protein

    PubMed Central

    Niu, Liang; Li, Guoliang; Lin, Shili

    2014-01-01

    Chromatin interactions mediated by a protein of interest are of great scientific interest. Recent studies show that protein-mediated chromatin interactions can have different intensities in different types of cells or in different developmental stages of a cell. Such differences can be associated with a disease or with the development of a cell. Thus, it is of great importance to detect protein-mediated chromatin interactions with different intensities in different cells. A recent molecular technique, Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET), which uses formaldehyde cross-linking and paired-end sequencing, is able to detect genome-wide chromatin interactions mediated by a protein of interest. Here we proposed two models (One-Step Model and Two-Step Model) for two sample ChIA-PET count data (one biological replicate in each sample) to identify differential chromatin interactions mediated by a protein of interest. Both models incorporate the data dependency and the extent to which a fragment pair is related to a pair of DNA loci of interest to make accurate identifications. The One-Step Model makes use of the data more efficiently but is more computationally intensive. An extensive simulation study showed that the models can detect those differentially interacted chromatins and there is a good agreement between each classification result and the truth. Application of the method to a two-sample ChIA-PET data set illustrates its utility. The two models are implemented as an R package MDM (available at http://www.stat.osu.edu/~statgen/SOFTWARE/MDM). PMID:24835279

  1. The effect of magnesium ions on chromosome structure as observed by helium ion microscopy.

    PubMed

    Dwiranti, Astari; Hamano, Tohru; Takata, Hideaki; Nagano, Shoko; Guo, Hongxuan; Onishi, Keiko; Wako, Toshiyuki; Uchiyama, Susumu; Fukui, Kiichi

    2014-02-01

    One of the few conclusions known about chromosome structure is that Mg2+ is required for the organization of chromosomes. Scanning electron microscopy is a powerful tool for studying chromosome morphology, but being nonconductive, chromosomes require metal/carbon coating that may conceal information about the detailed surface structure of the sample. Helium ion microscopy (HIM), which has recently been developed, does not require sample coating due to its charge compensation system. Here we investigated the structure of isolated human chromosomes under different Mg2+ concentrations by HIM. High-contrast and resolution images from uncoated samples obtained by HIM enabled investigation on the effects of Mg2+ on chromosome structure. Chromatin fiber information was obtained more clearly with uncoated than coated chromosomes. Our results suggest that both overall features and detailed structure of chromatin are significantly affected by different Mg2+ concentrations. Chromosomes were more condensed and a globular structure of chromatin with 30 nm diameter was visualized with 5 mM Mg2+ treatment, while 0 mM Mg2+ resulted in a less compact and more fibrous structure 11 nm in diameter. We conclude that HIM is a powerful tool for investigating chromosomes and other biological samples without requiring metal/carbon coating.

  2. Knockdown Brm and Baf170, components of chromatin remodeling complex, facilitates reprogramming of somatic cells

    USDA-ARS?s Scientific Manuscript database

    The SWI/SNF (SWItch/Sucrose NonFermentable or BAF, Brg/Brahma-associated factors) complexes are epigenetic modifiers of chromatin structure and undergo progressive changes in subunit composition during cellular differentiation. For example, in embryonic stem cells (ESCs) esBAF contains Brg1 and Baf...

  3. Regulation of Chromatin Assembly and Cell Transformation by Formaldehyde Exposure in Human Cells

    PubMed Central

    Chen, Danqi; Fang, Lei; Mei, Shenglin; Li, Hongjie; Xu, Xia; Des Marais, Thomas L.; Lu, Kun; Liu, X. Shirley

    2017-01-01

    Background: Formaldehyde (FA) is an environmental and occupational chemical carcinogen. Recent studies have shown that exogenous FA causes only a modest increase in DNA adduct formation compared with the amount of adducts formed by endogenous FA, raising the possibility that epigenetic mechanisms may contribute to FA-mediated carcinogenicity. Objectives: We investigated the effects of FA exposure on histone modifications and chromatin assembly. We also examined the role of defective chromatin assembly in FA-mediated transcription and cell transformation. Methods: Cellular fractionation and Western blot analysis were used to measure the levels of histone modifications in human bronchial epithelial BEAS-2B cells and human nasal RPMI2650 cells in the presence of FA. Chromatin immunoprecipitation (ChIP) and micrococcal nuclease (MNase) digest assays were performed to examine the changes in chromatin assembly and accessibility after FA exposure. RNA sequencing (RNA-seq) and real-time polymerase chain reaction (PCR) were used to examine transcriptional dysregulation. Finally, anchorage-independent cell growth ability was tested by soft agar assay following FA exposure. Results: Exposure to FA dramatically decreased the acetylation of the N-terminal tails of cytosolic histones. These modifications are important for histone nuclear import and subsequent chromatin assembly. Histone proteins were depleted in both the chromatin fraction and at most of the genomic loci tested following FA exposure, suggesting that FA compromises chromatin assembly. Moreover, FA increased chromatin accessibility and altered the expression of hundreds of cancer-related genes. Knockdown of the histone H3.3 gene (an H3 variant), which mimics inhibition of chromatin assembly, facilitated FA-mediated anchorage-independent cell growth. Conclusions: We propose that the inhibition of chromatin assembly represents a novel mechanism of cell transformation induced by the environmental and occupational

  4. Chromatin configurations in the ferret germinal vesicle that reflect developmental competence for in vitro maturation.

    PubMed

    Sun, X; Li, Z; Yi, Y; Ding, W; Chen, J; Engelhardt, J F; Leno, G H

    2009-04-01

    In several mammalian species, the configuration of germinal vesicle (GV) chromatin correlates with the developmental competence of oocytes. Yet, no study has been published on the configuration of GV chromatin in ferret, nor is it known whether a specific configuration predicts meiotic competence in this species, in spite of the potential importance of ferret cloning to the study of human disease and to species conservation efforts. Here, we report on an analysis of the chromatin configuration in ferret GV oocytes and on how they correlate with meiotic development. Three distinct configurations were identified based on the degree of chromatin condensation: (1) fibrillar chromatin (FC), featuring strands of intertwined chromatin occupying most of the visible GV region; (2) intermediate condensed chromatin (ICC), characterized by dense, irregular chromatin masses throughout the GV; and (3) condensed chromatin (CC), which is highly compact and centered around the nucleolus. We also found that chromatin configuration was related to the extent of association with cumulus cells in cumulus-oocyte complexes; CC-configured oocytes were most often surrounded by a compact cumulus layer and also a compact corona but FC-configured oocytes were associated with neither. In addition, increasing chromatin condensation corresponded to an increase in oocyte diameter. Finally, following in vitro culture, significantly more CC-configured oocytes underwent maturation to meiotic metaphase II than did FC- or ICC-configured oocytes. We conclude that, in ferret, chromatin condensation is related to the sequential achievement of meiotic competencies during oocyte growth and differentiation, and thus can be used as a predictor of competence.

  5. Micromechanical-biochemical studies of mitotic chromosome elasticity and structure

    NASA Astrophysics Data System (ADS)

    Poirier, Michael Guy

    The structure of mitotic chromosomes was studied by combining micromechanical force measurements with microfluidic biochemical exposures. Our method is to use glass micropipettes attached to either end of a single chromosome to do mechanical experiments in the extracellular buffer. A third pipette can be used to locally 'spray' reactants so as to carry out dynamical mechanical-chemical experiments. The following elastic properties of mitotic chromosomes are found: Young's modulus, Y = 300 Pa; Poisson ratio, sigma = 0.1; Bending rigidity, B = 1 x 10 -22 J·m; Internal viscosity, eta' = 100 kg/m·sec; Volume fraction, ϕ = 0.7; Extensions of less than 3 times the relaxed length are linear and reversible; Extensions beyond 30 fold exhibit a force plateau at 15 nN and convert the chromosome to a disperse ghost-like state with little change in chromatin structure; Mitotic chromosomes are relatively isotropic; dsDNA cuts of at least every 3 kb cause the a mitotic chromosomes to fall apart; dsDNA cuts less frequently than every 50 kb do not affect mitotic chromosome structure. These results lead to the conclusion that mitotic chromosomes are a network crosslinked every 50 kb between which chromatin is fold by chromatin folding proteins, which are likely to be condensins.

  6. SSTY proteins colocalize with the postmeiotic sex chromatin and interact with regulators of its expression

    PubMed Central

    Comptour, Aurélie; Moretti, Charlotte; Serrentino, Maria-Elisabetta; Auer, Jana; Ialy-Radio, Côme; Ward, Monika A.; Touré, Aminata; Vaiman, Daniel; Cocquet, Julie

    2014-01-01

    In mammals, X- and Y-encoded genes are transcriptionally shut down during male meiosis, but the expression of many of them is (re)activated, after meiosis, in spermatids. Postmeiotic XY gene expression is timely regulated by active epigenetic marks, which are de novo incorporated in the sex chromatin of spermatids, and by repressive epigenetic marks inherited from meiosis; alteration in this process leads to male infertility. In the mouse, postmeiotic XY gene expression is known to depend on genetic information carried by the male specific region of the Y chromosome long arm (MSYq). The MSYq gene Sly has been shown to be a key regulator of postmeiotic sex chromosome gene expression and necessary for the maintenance/recruitment of repressive epigenetic marks on the sex chromatin, but studies suggest that another MSYq gene may be required. The best candidate to date is Ssty, an MSYq multicopy gene of unknown function. Here, we show that SSTY proteins are specifically expressed in round and elongating spermatids and colocalize with the postmeiotic sex chromatin. Moreover, SSTY proteins interact with SLY protein and its X-linked homolog SLX/SLXL1, and may be required for the localization of SLX/SLY proteins in the spermatid nucleus and sex chromatin. Our data suggest that SSTY is a second MSYq factor involved in the control of XY gene expression during sperm differentiation. As Slx/Slxl1 and Sly genes have been shown to be involved in the XY intragenomic conflict which affects the offspring sex-ratio, Ssty might constitute another actor of this conflict. PMID:24456183

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

    PubMed

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

    1998-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1998-11-01

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

  9. The TRPM7 chanzyme is cleaved to release a chromatin modifying kinase

    PubMed Central

    Krapivinsky, Grigory; Krapivinsky, Luba; Manasian, Yunona; Clapham, David E.

    2014-01-01

    SUMMARY TRPM7 is a ubiquitous ion channel and kinase, a unique ‘chanzyme’, required for proper early embryonic development. It conducts Zn2+, Mg2+, Ca2+ as well as monovalent cations, and contains a functional serine/threonine kinase at its carboxyl terminus. Here, we show that in normal tissues and cell lines, the kinase is proteolytically cleaved from the channel domain in a cell type-specific manner. These TRPM7 Cleaved Kinase fragments (M7CKs) translocate to the nucleus and bind multiple components of chromatin remodeling complexes, including Polycomb group proteins. In the nucleus, the kinase phosphorylates specific serines/threonines of histones. M7CK-dependent phosphorylation of H3Ser10 at promoters of TRPM7-dependent genes correlates with their activity. We also demonstrate that cytosolic free [Zn2+] is TRPM7-dependent and regulates M7CK binding to transcription factors containing zinc-finger domains. These findings suggest that TRPM7-mediated modulation of intracellular Zn2+ concentration couples ion channel signaling to epigenetic chromatin covalent modifications that affect gene expression patterns. PMID:24855944

  10. LEM-3 is a midbody-tethered DNA nuclease that resolves chromatin bridges during late mitosis.

    PubMed

    Hong, Ye; Sonneville, Remi; Wang, Bin; Scheidt, Viktor; Meier, Bettina; Woglar, Alexander; Demetriou, Sarah; Labib, Karim; Jantsch, Verena; Gartner, Anton

    2018-02-20

    Faithful chromosome segregation and genome maintenance requires the removal of all DNA bridges that physically link chromosomes before cells divide. Using C. elegans embryos we show that the LEM-3/Ankle1 nuclease defines a previously undescribed genome integrity mechanism by processing DNA bridges right before cells divide. LEM-3 acts at the midbody, the structure where abscission occurs at the end of cytokinesis. LEM-3 localization depends on factors needed for midbody assembly, and LEM-3 accumulation is increased and prolonged when chromatin bridges are trapped at the cleavage plane. LEM-3 locally processes chromatin bridges that arise from incomplete DNA replication, unresolved recombination intermediates, or the perturbance of chromosome structure. Proper LEM-3 midbody localization and function is regulated by AIR-2/Aurora B kinase. Strikingly, LEM-3 acts cooperatively with the BRC-1/BRCA1 homologous recombination factor to promote genome integrity. These findings provide a molecular basis for the suspected role of the LEM-3 orthologue Ankle1 in human breast cancer.

  11. Connecting the dots: chromatin and alternative splicing in EMT

    PubMed Central

    Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

    2015-01-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

  12. Connecting the dots: chromatin and alternative splicing in EMT.

    PubMed

    Warns, Jessica A; Davie, James R; Dhasarathy, Archana

    2016-02-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

  13. Chromatin loops as allosteric modulators of enhancer-promoter interactions.

    PubMed

    Doyle, Boryana; Fudenberg, Geoffrey; Imakaev, Maxim; Mirny, Leonid A

    2014-10-01

    The classic model of eukaryotic gene expression requires direct spatial contact between a distal enhancer and a proximal promoter. Recent Chromosome Conformation Capture (3C) studies show that enhancers and promoters are embedded in a complex network of looping interactions. Here we use a polymer model of chromatin fiber to investigate whether, and to what extent, looping interactions between elements in the vicinity of an enhancer-promoter pair can influence their contact frequency. Our equilibrium polymer simulations show that a chromatin loop, formed by elements flanking either an enhancer or a promoter, suppresses enhancer-promoter interactions, working as an insulator. A loop formed by elements located in the region between an enhancer and a promoter, on the contrary, facilitates their interactions. We find that different mechanisms underlie insulation and facilitation; insulation occurs due to steric exclusion by the loop, and is a global effect, while facilitation occurs due to an effective shortening of the enhancer-promoter genomic distance, and is a local effect. Consistently, we find that these effects manifest quite differently for in silico 3C and microscopy. Our results show that looping interactions that do not directly involve an enhancer-promoter pair can nevertheless significantly modulate their interactions. This phenomenon is analogous to allosteric regulation in proteins, where a conformational change triggered by binding of a regulatory molecule to one site affects the state of another site.

  14. Potential of chromatin modifying compounds for the treatment of Alzheimer's disease

    PubMed Central

    Karagiannis, Tom C.; Ververis, Katherine

    2012-01-01

    Alzheimer's disease is a very common progressive neurodegenerative disorder affecting the learning and memory centers in the brain. The hallmarks of disease are the accumulation of β-amyloid neuritic plaques and neurofibrillary tangles formed by abnormally phosphorylated tau protein. Alzheimer's disease is currently incurable and there is an intense interest in the development of new potential therapies. Chromatin modifying compounds such as sirtuin modulators and histone deacetylase inhibitors have been evaluated in models of Alzheimer's disease with some promising results. For example, the natural antioxidant and sirtuin 1 activator resveratrol has been shown to have beneficial effects in animal models of disease. Similarly, numerous histone deacetylase inhibitors including Trichostatin A, suberoylanilide hydroxamic acid, valproic acid and phenylbutyrate reduction have shown promising results in models of Alzheimer's disease. These beneficial effects include a reduction of β-amyloid production and stabilization of tau protein. In this review we provide an overview of the histone deacetylase enzymes, with a focus on enzymes that have been identified to have an important role in the pathobiology of Alzheimer's disease. Further, we discuss the potential for pharmacological intervention with chromatin modifying compounds that modulate histone deacetylase enzymes. PMID:22953035

  15. Potential of chromatin modifying compounds for the treatment of Alzheimer's disease.

    PubMed

    Karagiannis, Tom C; Ververis, Katherine

    2012-01-01

    Alzheimer's disease is a very common progressive neurodegenerative disorder affecting the learning and memory centers in the brain. The hallmarks of disease are the accumulation of β-amyloid neuritic plaques and neurofibrillary tangles formed by abnormally phosphorylated tau protein. Alzheimer's disease is currently incurable and there is an intense interest in the development of new potential therapies. Chromatin modifying compounds such as sirtuin modulators and histone deacetylase inhibitors have been evaluated in models of Alzheimer's disease with some promising results. For example, the natural antioxidant and sirtuin 1 activator resveratrol has been shown to have beneficial effects in animal models of disease. Similarly, numerous histone deacetylase inhibitors including Trichostatin A, suberoylanilide hydroxamic acid, valproic acid and phenylbutyrate reduction have shown promising results in models of Alzheimer's disease. These beneficial effects include a reduction of β-amyloid production and stabilization of tau protein. In this review we provide an overview of the histone deacetylase enzymes, with a focus on enzymes that have been identified to have an important role in the pathobiology of Alzheimer's disease. Further, we discuss the potential for pharmacological intervention with chromatin modifying compounds that modulate histone deacetylase enzymes.

  16. Chromatin-Remodeling-Factor ARID1B Represses Wnt/β-Catenin Signaling

    PubMed Central

    Vasileiou, Georgia; Ekici, Arif B.; Uebe, Steffen; Zweier, Christiane; Hoyer, Juliane; Engels, Hartmut; Behrens, Jürgen; Reis, André; Hadjihannas, Michel V.

    2015-01-01

    The link of chromatin remodeling to both neurodevelopment and cancer has recently been highlighted by the identification of mutations affecting BAF chromatin-remodeling components, such as ARID1B, in individuals with intellectual disability and cancer. However, the underlying molecular mechanism(s) remains unknown. Here, we show that ARID1B is a repressor of Wnt/β-catenin signaling. Through whole-transcriptome analysis, we find that in individuals with intellectual disability and ARID1B loss-of-function mutations, Wnt/β-catenin target genes are upregulated. Using cellular models of low and high Wnt/β-catenin activity, we demonstrate that knockdown of ARID1B activates Wnt/β-catenin target genes and Wnt/β-catenin-dependent transcriptional reporters in a β-catenin-dependent manner. Reciprocally, forced expression of ARID1B inhibits Wnt/β-catenin signaling downstream of the β-catenin destruction complex. Both endogenous and exogenous ARID1B associate with β-catenin and repress Wnt/β-catenin-mediated transcription through the BAF core subunit BRG1. Accordingly, mutations in ARID1B leading to partial or complete deletion of its BRG1-binding domain, as is often observed in intellectual disability and cancers, compromise association with β-catenin, and the resultant ARID1B mutant proteins fail to suppress Wnt/β-catenin signaling. Finally, knockdown of ARID1B in mouse neuroblastoma cells leads to neurite outgrowth through β-catenin. The data suggest that aberrations in chromatin-remodeling factors, such as ARID1B, might contribute to neurodevelopmental abnormalities and cancer through deregulation of developmental and oncogenic pathways, such as the Wnt/β-catenin signaling pathway. PMID:26340334

  17. Radial chromatin positioning is shaped by local gene density, not by gene expression

    PubMed Central

    2009-01-01

    G- and R-bands of metaphase chromosomes are characterized by profound differences in gene density, CG content, replication timing, and chromatin compaction. The preferential localization of gene-dense, transcriptionally active, and early replicating chromatin in the nuclear interior and of gene-poor, later replicating chromatin at the nuclear envelope has been demonstrated to be evolutionary-conserved in various cell types. Yet, the impact of different local chromatin features on the radial nuclear arrangement of chromatin is still not well understood. In particular, it is not known whether radial chromatin positioning is preferentially shaped by local gene density per se or by other related parameters such as replication timing or transcriptional activity. The interdependence of these distinct chromatin features on the linear deoxyribonucleic acid (DNA) sequence precludes a simple dissection of these parameters with respect to their importance for the reorganization of the linear DNA organization into the distinct radial chromatin arrangements observed in the nuclear space. To analyze this problem, we generated probe sets of pooled bacterial artificial chromosome (BAC) clones from HSA 11, 12, 18, and 19 representing R/G-band-assigned chromatin, segments with different gene density and gene loci with different expression levels. Using multicolor 3D flourescent in situ hybridization (FISH) and 3D image analysis, we determined their localization in the nucleus and their positions within or outside the corresponding chromosome territory (CT). For each BAC data on local gene density within 2- and 10-Mb windows, as well as GC (guanine and cytosine) content, replication timing and expression levels were determined. A correlation analysis of these parameters with nuclear positioning revealed regional gene density as the decisive parameter determining the radial positioning of chromatin in the nucleus in contrast to band assignment, replication timing, and transcriptional

  18. DNA packing in chromatine, a manifestation of the Bonnet transformation.

    PubMed

    Blum, Z; Lidin, S

    1988-08-01

    The packing of DNA is described using the formalism of differential geometry. Winding of the DNA double helix around the histone 2-5 octamer forming a nucleosome and the condensation of the so-formed bead-on-a-string chromatine aided by histone 1 is interpreted as two consecutive isometric, i.e. Bonnet, transformations. The DNA double helix can be approximated to a helicoid which can be transformed isometrically to a catenoid, an approximation of the nucleosome. Owing to the organization of the histone octamer the extended chromatine takes a helicoidal shape allowing a second Bonnet transformation to consummate the condensation into a chromatine fibre.

  19. Long-range looping of a locus control region drives tissue-specific chromatin packing within a multigene cluster

    PubMed Central

    Tsai, Yu-Cheng; Cooke, Nancy E.; Liebhaber, Stephen A.

    2016-01-01

    Abstract The relationships of higher order chromatin organization to mammalian gene expression remain incompletely defined. The human Growth Hormone (hGH) multigene cluster contains five gene paralogs. These genes are selectively activated in either the pituitary or the placenta by distinct components of a remote locus control region (LCR). Prior studies have revealed that appropriate activation of the placental genes is dependent not only on the actions of the LCR, but also on the multigene composition of the cluster itself. Here, we demonstrate that the hGH LCR ‘loops’ over a distance of 28 kb in primary placental nuclei to make specific contacts with the promoters of the two GH genes in the cluster. This long-range interaction sequesters the GH genes from the three hCS genes which co-assemble into a tightly packed ‘hCS chromatin hub’. Elimination of the long-range looping, via specific deletion of the placental LCR components, triggers a dramatic disruption of the hCS chromatin hub. These data reveal a higher-order structural pathway by which long-range looping from an LCR impacts on local chromatin architecture that is linked to tissue-specific gene regulation within a multigene cluster. PMID:26893355

  20. Contribution of transposable elements and distal enhancers to evolution of human-specific features of interphase chromatin architecture in embryonic stem cells.

    PubMed

    Glinsky, Gennadi V

    2018-03-01

    Transposable elements have made major evolutionary impacts on creation of primate-specific and human-specific genomic regulatory loci and species-specific genomic regulatory networks (GRNs). Molecular and genetic definitions of human-specific changes to GRNs contributing to development of unique to human phenotypes remain a highly significant challenge. Genome-wide proximity placement analysis of diverse families of human-specific genomic regulatory loci (HSGRL) identified topologically associating domains (TADs) that are significantly enriched for HSGRL and designated rapidly evolving in human TADs. Here, the analysis of HSGRL, hESC-enriched enhancers, super-enhancers (SEs), and specific sub-TAD structures termed super-enhancer domains (SEDs) has been performed. In the hESC genome, 331 of 504 (66%) of SED-harboring TADs contain HSGRL and 68% of SEDs co-localize with HSGRL, suggesting that emergence of HSGRL may have rewired SED-associated GRNs within specific TADs by inserting novel and/or erasing existing non-coding regulatory sequences. Consequently, markedly distinct features of the principal regulatory structures of interphase chromatin evolved in the hESC genome compared to mouse: the SED quantity is 3-fold higher and the median SED size is significantly larger. Concomitantly, the overall TAD quantity is increased by 42% while the median TAD size is significantly decreased (p = 9.11E-37) in the hESC genome. Present analyses illustrate a putative global role for transposable elements and HSGRL in shaping the human-specific features of the interphase chromatin organization and functions, which are facilitated by accelerated creation of novel transcription factor binding sites and new enhancers driven by targeted placement of HSGRL at defined genomic coordinates. A trend toward the convergence of TAD and SED architectures of interphase chromatin in the hESC genome may reflect changes of 3D-folding patterns of linear chromatin fibers designed to enhance both

  1. Advancing age increases sperm chromatin damage and impairs fertility in peroxiredoxin 6 null mice

    PubMed Central

    Ozkosem, Burak; Feinstein, Sheldon I.; Fisher, Aron B.; O’Flaherty, Cristian

    2015-01-01

    Due to socioeconomic factors, more couples are choosing to delay conception than ever. Increasing average maternal and paternal age in developed countries over the past 40 years has raised the question of how aging affects reproductive success of males and females. Since oxidative stress in the male reproductive tract increases with age, we investigated the impact of advanced paternal age on the integrity of sperm nucleus and reproductive success of males by using a Prdx6−/− mouse model. We compared sperm motility, cytoplasmic droplet retention sperm chromatin quality and reproductive outcomes of young (2-month-old), adult (8-month-old), and old (20-month-old) Prdx6−/− males with their age-matched wild type (WT) controls. Absence of PRDX6 caused age-dependent impairment of sperm motility and sperm maturation and increased sperm DNA fragmentation and oxidation as well as decreased sperm DNA compaction and protamination. Litter size, total number of litters and total number of pups per male were significantly lower in Prdx6−/− males compared to WT controls. These abnormal reproductive outcomes were severely affected by age in Prdx6−/− males. In conclusion, the advanced paternal age affects sperm chromatin integrity and fertility more severely in the absence of PRDX6, suggesting a protective role of PRDX6 in age-associated decline in the sperm quality and fertility in mice. PMID:25796034

  2. Advancing age increases sperm chromatin damage and impairs fertility in peroxiredoxin 6 null mice.

    PubMed

    Ozkosem, Burak; Feinstein, Sheldon I; Fisher, Aron B; O'Flaherty, Cristian

    2015-08-01

    Due to socioeconomic factors, more couples are choosing to delay conception than ever. Increasing average maternal and paternal age in developed countries over the past 40 years has raised the question of how aging affects reproductive success of males and females. Since oxidative stress in the male reproductive tract increases with age, we investigated the impact of advanced paternal age on the integrity of sperm nucleus and reproductive success of males by using a Prdx6(-/-) mouse model. We compared sperm motility, cytoplasmic droplet retention sperm chromatin quality and reproductive outcomes of young (2-month-old), adult (8-month-old), and old (20-month-old) Prdx6(-/-) males with their age-matched wild type (WT) controls. Absence of PRDX6 caused age-dependent impairment of sperm motility and sperm maturation and increased sperm DNA fragmentation and oxidation as well as decreased sperm DNA compaction and protamination. Litter size, total number of litters and total number of pups per male were significantly lower in Prdx6(-/-) males compared to WT controls. These abnormal reproductive outcomes were severely affected by age in Prdx6(-/-) males. In conclusion, the advanced paternal age affects sperm chromatin integrity and fertility more severely in the absence of PRDX6, suggesting a protective role of PRDX6 in age-associated decline in the sperm quality and fertility in mice. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  3. The Chd1 Chromatin Remodeler Shifts Nucleosomal DNA Bidirectionally as a Monomer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Qiu, Yupeng; Levendosky, Robert F.; Chakravarthy, Srinivas

    Chromatin remodelers catalyze dynamic packaging of the genome by carrying out nucleosome assembly/disassembly, histone exchange, and nucleosome repositioning. Remodeling results in evenly spaced nucleosomes, which requires probing both sides of the nucleosome, yet the way remodelers organize sliding activity to achieve this task is not understood. Here, we show that the monomeric Chd1 remodeler shifts DNA back and forth by dynamically alternating between different segments of the nucleosome. During sliding, Chd1 generates unstable remodeling intermediates that spontaneously relax to a pre-remodeled position. We demonstrate that nucleosome sliding is tightly controlled by two regulatory domains: the DNA-binding domain, which interferes withmore » sliding when its range is limited by a truncated linking segment, and the chromodomains, which play a key role in substrate discrimination. We propose that active interplay of the ATPase motor with the regulatory domains may promote dynamic nucleosome structures uniquely suited for histone exchange and chromatin reorganization during transcription.« less

  4. Tissue-Specific Regulation of Chromatin Insulator Function

    PubMed Central

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

    2012-01-01

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

  5. mTOR signaling regulates myotube hypertrophy by modulating protein synthesis, rDNA transcription, and chromatin remodeling.

    PubMed

    von Walden, Ferdinand; Liu, Chang; Aurigemma, Nicole; Nader, Gustavo A

    2016-10-01

    Ribosome production is an early event during skeletal muscle hypertrophy and precedes muscle protein accretion. Signaling via mTOR is crucial for ribosome production and hypertrophy; however, the mechanisms by which it regulates these processes remain to be identified. Herein, we investigated the activation of mTOR signaling in hypertrophying myotubes and determined that mTOR coordinates various aspects of gene expression important for ribosome production. First, inhibition of translation with cycloheximide had a more potent effect on protein synthesis than rapamycin indicating that mTOR function during hypertrophy is not on general, but rather on specific protein synthesis. Second, blocking Pol II transcription had a similar effect as Rapamycin and, unexpectedly, revealed the necessity of Pol II transcription for Pol I transcription, suggesting that mTOR may regulate ribosome production also by controlling Class II genes at the transcriptional level. Third, Pol I activity is essential for rDNA transcription and, surprisingly, for protein synthesis as selective Pol I inhibition blunted rDNA transcription, protein synthesis, and the hypertrophic response of myotubes. Finally, mTOR has nuclear localization in muscle, which is not sensitive to rapamycin. Inhibition of mTOR signaling by rapamycin disrupted mTOR-rDNA promoter interaction and resulted in altered histone marks indicative of repressed transcription and formation of higher-order chromatin structure. Thus mTOR signaling appears to regulate muscle hypertrophy by affecting protein synthesis, Class I and II gene expression, and chromatin remodeling. Copyright © 2016 the American Physiological Society.

  6. A Nucleotide-Driven Switch Regulates Flanking DNA Length Sensing by a Dimeric Chromatin Remodeler

    PubMed Central

    Leonard, John D.; Narlikar, Geeta J.

    2015-01-01

    SUMMARY The ATP-dependent chromatin assembly factor (ACF) is a dimeric motor that spaces nucleosomes to promote formation of silent chromatin. Two copies of its ATPase subunit SNF2h bind opposite sides of a nucleosome, but how these protomers avoid competition is unknown. SNF2h senses the length of DNA flanking a nucleosome via its HAND-SANT-SLIDE (HSS) domain, yet it is unclear how this interaction enhances remodeling. Using covalently connected SNF2h dimers we show that dimerization accelerates remodeling and that the HSS contributes to communication between protomers. We further identify a nucleotide-dependent conformational change in SNF2h. In one conformation the HSS binds flanking DNA, and in another conformation the HSS engages the nucleosome core. Based on these results, we propose a model in which DNA length sensing and translocation are performed by two distinct conformational states of SNF2h. Such separation of function suggests that these activities could be independently regulated to affect remodeling outcomes. PMID:25684208

  7. The Chromatin Remodeling Factor SMARCB1 Forms a Complex with Human Cytomegalovirus Proteins UL114 and UL44

    PubMed Central

    Ranneberg-Nilsen, Toril; Rollag, Halvor; Slettebakk, Ragnhild; Backe, Paul Hoff; Olsen, Øyvind; Luna, Luisa; Bjørås, Magnar

    2012-01-01

    Background Human cytomegalovirus (HCMV) uracil DNA glycosylase, UL114, is required for efficient viral DNA replication. Presumably, UL114 functions as a structural partner to other factors of the DNA-replication machinery and not as a DNA repair protein. UL114 binds UL44 (HCMV processivity factor) and UL54 (HCMV-DNA-polymerase). In the present study we have searched for cellular partners of UL114. Methodology/Principal Findings In a yeast two-hybrid screen SMARCB1, a factor of the SWI/SNF chromatin remodeling complex, was found to be an interacting partner of UL114. This interaction was confirmed in vitro by co-immunoprecipitation and pull-down. Immunofluorescence microscopy revealed that SMARCB1 along with BRG-1, BAF170 and BAF155, which are the core SWI/SNF components required for efficient chromatin remodeling, were present in virus replication foci 24–48 hours post infection (hpi). Furthermore a direct interaction was also demonstrated for SMARCB1 and UL44. Conclusions/Significance The core SWI/SNF factors required for efficient chromatin remodeling are present in the HCMV replication foci throughout infection. The proteins UL44 and UL114 interact with SMARCB1 and may participate in the recruitment of the SWI/SNF complex to the chromatinized virus DNA. Thus, the presence of the SWI/SNF chromatin remodeling complex in replication foci and its association with UL114 and with UL44 might imply its involvement in different DNA transactions. PMID:22479537

  8. Chromatin Pioneers | Center for Cancer Research

    Cancer.gov

    Taking advantage of their ability to explore provocative ideas, NCI investigators pioneered the study of chromatin to demonstrate its functional importance and lay the groundwork for understanding its role in cancer and other diseases.

  9. Evolution of histone 2A for chromatin compaction in eukaryotes

    PubMed Central

    Macadangdang, Benjamin R; Oberai, Amit; Spektor, Tanya; Campos, Oscar A; Sheng, Fang; Carey, Michael F; Vogelauer, Maria; Kurdistani, Siavash K

    2014-01-01

    During eukaryotic evolution, genome size has increased disproportionately to nuclear volume, necessitating greater degrees of chromatin compaction in higher eukaryotes, which have evolved several mechanisms for genome compaction. However, it is unknown whether histones themselves have evolved to regulate chromatin compaction. Analysis of histone sequences from 160 eukaryotes revealed that the H2A N-terminus has systematically acquired arginines as genomes expanded. Insertion of arginines into their evolutionarily conserved position in H2A of a small-genome organism increased linear compaction by as much as 40%, while their absence markedly diminished compaction in cells with large genomes. This effect was recapitulated in vitro with nucleosomal arrays using unmodified histones, indicating that the H2A N-terminus directly modulates the chromatin fiber likely through intra- and inter-nucleosomal arginine–DNA contacts to enable tighter nucleosomal packing. Our findings reveal a novel evolutionary mechanism for regulation of chromatin compaction and may explain the frequent mutations of the H2A N-terminus in cancer. DOI: http://dx.doi.org/10.7554/eLife.02792.001 PMID:24939988

  10. Functional sub-division of the Drosophila genome via chromatin looping

    PubMed Central

    Ahanger, Sajad H.; Shouche, Yogesh S.; Mishra, Rakesh K.

    2013-01-01

    Insulators help in organizing the eukaryotic genomes into physically and functionally autonomous regions through the formation of chromatin loops. Recent findings in Drosophila and vertebrates suggest that insulators anchor multiple loci through long-distance interactions which may be mechanistically linked to insulator function. Important to such processes in Drosophila is CP190, a common co-factor of insulator complexes. CP190 is also known to associate with the nuclear matrix, components of the RNAi machinery, active promoters and borders of the repressive chromatin domains. Although CP190 plays a pivotal role in insulator function in Drosophila, vertebrates lack a probable functional equivalent of CP190 and employ CTCF as the major factor to carry out insulator function/chromatin looping. In this review, we discuss the emerging role of CP190 in tethering genome, specifically in the perspective of insulator function in Drosophila. Future studies aiming genome-wide role of CP190 in chromatin looping is likely to give important insights into the mechanism of genome organization. PMID:23333867

  11. Measurement of Family Affective Structure.

    ERIC Educational Resources Information Center

    Lowman, Joseph

    1980-01-01

    Three studies demonstrate that the Inventory of Family Feelings, a measure of family affective structure, has high reliability and construct and concurrent validity. It is appropriate for affective comparisons by age, sex, and ordinal position of children and for measuring change after family or marital therapy, or after predictable stress…

  12. [The effect of structure of benzimidazoles on the character of forming intramolecular cross-links in DNA and chromatin].

    PubMed

    Mil', E M; Zhil'tsova, V M; Biniukov, V I; Zhizhina, G P; Stoliarova, L G; Kuznetsov, Iu P

    1994-01-01

    An investigation of a number of benzimidazole class preparations, being distinguished by a position of aminomethyl substitutes, has been carried out. It has been shown, that the non-substituted preparation BIO-10 does not form UV-cross-links in DNA and chromatine; BIO-40, having one substitute in the position 2, causes the formation of inter-molecular cross-links DNA-DNA. The preparation BIO-50, having 2 aminomethyl groups in the imidazole nucleus positions 2 and 6, forms cross-links DNA-DNA and DNA-protein in chromatine. The generation of radicals by the preparations BIO-10 and BIO-50 has been studied by the EPR-method by use of spin trap. It has been demonstrated, that BIO-10, unlike BIO-50, actively generates superoxide. A supposition has been made, that an UV-formation of superoxide-radical in the presence of BIO-10 might be a reason of DNA-macromolecule destruction.

  13. Drosophila transcription factor Tramtrack69 binds MEP1 to recruit the chromatin remodeler NuRD.

    PubMed

    Reddy, B Ashok; Bajpe, Prashanth Kumar; Bassett, Andrew; Moshkin, Yuri M; Kozhevnikova, Elena; Bezstarosti, Karel; Demmers, Jeroen A A; Travers, Andrew A; Verrijzer, C Peter

    2010-11-01

    ATP-dependent chromatin-remodeling complexes (remodelers) are essential regulators of chromatin structure and gene transcription. How remodelers can act in a gene-selective manner has remained enigmatic. A yeast two-hybrid screen for proteins binding the Drosophila transcription factor Tramtrack69 (TTK69) identified MEP1. Proteomic characterization revealed that MEP1 is a tightly associated subunit of the NuRD remodeler, harboring the Mi2 enzymatic core ATPase. In addition, we identified the fly homolog of human Deleted in oral cancer 1 (DOC1), also known as CDK2-associated protein 1 (CDK2AP1), as a bona fide NuRD subunit. Biochemical and genetic assays supported the functional association between MEP1, Mi2, and TTK69. Genomewide expression analysis established that TTK69, MEP1, and Mi2 cooperate closely to control transcription. The TTK69 transcriptome profile correlates poorly with remodelers other than NuRD, emphasizing the selectivity of remodeler action. On the genes examined, TTK69 is able to bind chromatin in the absence of NuRD, but targeting of NuRD is dependent on TTK69. Thus, there appears to be a hierarchical relationship in which transcription factor binding precedes remodeler recruitment.

  14. Quantitative Proteomic Analysis of Replicative and Nonreplicative Forms Reveals Important Insights into Chromatin Biology of Trypanosoma cruzi*

    PubMed Central

    Leandro de Jesus, Teresa Cristina; Calderano, Simone Guedes; Vitorino, Francisca Nathalia de Luna; Llanos, Ricardo Pariona; Lopes, Mariana de Camargo; de Araújo, Christiane Bezerra; Thiemann, Otavio Henrique; Reis, Marcelo da Silva; Elias, Maria Carolina

    2017-01-01

    Chromatin associated proteins are key regulators of many important processes in the cell. Trypanosoma cruzi, a protozoa flagellate that causes Chagas disease, alternates between replicative and nonreplicative forms accompanied by a shift on global transcription levels and by changes in its chromatin architecture. Here, we investigated the T. cruzi chromatin proteome using three different protocols and compared it between replicative (epimastigote) and nonreplicative (trypomastigote) forms by high-resolution mass spectrometry. More than 2000 proteins were identified and quantified both in chromatin and nonchromatin extracts. Besides histones and other known nuclear proteins, trypanosomes chromatin also contains metabolic (mainly from carbohydrate pathway), cytoskeleton and many other proteins with unknown functions. Strikingly, the two parasite forms differ greatly regarding their chromatin-associated factors composition and amount. Although the nucleosome content is the same for both life forms (as seen by MNase digestion), the remaining proteins were much less detected in nonreplicative forms, suggesting that they have a naked chromatin. Proteins associated to DNA proliferation, such as PCNA, RPA, and DNA topoisomerases were exclusively found in the chromatin of replicative stages. On the other hand, the nonreplicative stages have an enrichment of a histone H2B variant. Furthermore, almost 20% of replicative stages chromatin-associated proteins are expressed in nonreplicative forms, but located at nonchromatin space. We identified different classes of proteins including phosphatases and a Ran-binding protein, that may shuttle between chromatin and nonchromatin space during differentiation. Seven proteins, including those with unknown functions, were selected for further validation. We confirmed their location in chromatin and their differential expression, using Western blotting assays and chromatin immunoprecipitation (ChIP). Our results indicate that the

  15. Quantitative Proteomic Analysis of Replicative and Nonreplicative Forms Reveals Important Insights into Chromatin Biology of Trypanosoma cruzi.

    PubMed

    Leandro de Jesus, Teresa Cristina; Calderano, Simone Guedes; Vitorino, Francisca Nathalia de Luna; Llanos, Ricardo Pariona; Lopes, Mariana de Camargo; de Araújo, Christiane Bezerra; Thiemann, Otavio Henrique; Reis, Marcelo da Silva; Elias, Maria Carolina; Chagas da Cunha, Julia Pinheiro

    2017-01-01

    Chromatin associated proteins are key regulators of many important processes in the cell. Trypanosoma cruzi, a protozoa flagellate that causes Chagas disease, alternates between replicative and nonreplicative forms accompanied by a shift on global transcription levels and by changes in its chromatin architecture. Here, we investigated the T. cruzi chromatin proteome using three different protocols and compared it between replicative (epimastigote) and nonreplicative (trypomastigote) forms by high-resolution mass spectrometry. More than 2000 proteins were identified and quantified both in chromatin and nonchromatin extracts. Besides histones and other known nuclear proteins, trypanosomes chromatin also contains metabolic (mainly from carbohydrate pathway), cytoskeleton and many other proteins with unknown functions. Strikingly, the two parasite forms differ greatly regarding their chromatin-associated factors composition and amount. Although the nucleosome content is the same for both life forms (as seen by MNase digestion), the remaining proteins were much less detected in nonreplicative forms, suggesting that they have a naked chromatin. Proteins associated to DNA proliferation, such as PCNA, RPA, and DNA topoisomerases were exclusively found in the chromatin of replicative stages. On the other hand, the nonreplicative stages have an enrichment of a histone H2B variant. Furthermore, almost 20% of replicative stages chromatin-associated proteins are expressed in nonreplicative forms, but located at nonchromatin space. We identified different classes of proteins including phosphatases and a Ran-binding protein, that may shuttle between chromatin and nonchromatin space during differentiation. Seven proteins, including those with unknown functions, were selected for further validation. We confirmed their location in chromatin and their differential expression, using Western blotting assays and chromatin immunoprecipitation (ChIP). Our results indicate that the

  16. High-frequency promoter firing links THO complex function to heavy chromatin formation.

    PubMed

    Mouaikel, John; Causse, Sébastien Z; Rougemaille, Mathieu; Daubenton-Carafa, Yves; Blugeon, Corinne; Lemoine, Sophie; Devaux, Frédéric; Darzacq, Xavier; Libri, Domenico

    2013-11-27

    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 in situ 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 occupancy genome wide. We propose that the THO complex is required for tuning the dynamic of gene-nuclear pore association and mRNP release to the same high pace of transcription initiation. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  17. New insights into the mechanisms of mammalian erythroid chromatin condensation and enucleation.

    PubMed

    Ji, Peng

    2015-01-01

    A unique feature in mammalian erythropoiesis is the dramatic chromatin condensation followed by enucleation. This step-by-step process starts at the beginning of terminal erythropoiesis after the hematopoietic stem cells are committed to erythroid lineage. Although this phenomenon is known for decades, the mechanisms of chromatin condensation and enucleation remain elusive. Recent advances in cell and molecular biology have started to reveal the molecular pathways in the regulation of chromatin condensation, the establishment of nuclear polarity prior enucleation, and the rearrangement of actin cytoskeleton in enucleation. However, many challenging questions, especially whether and how the apoptotic mechanisms are involved in chromatin condensation and how to dissect the functions of many actin cytoskeleton proteins in cytokinesis and enucleation, remain to be answered. Here I review our current understanding of mammalian erythroid chromatin condensation and enucleation during terminal differentiation with a focus on more recent studies. I conclude with my perspective of future works in this rising topic in developmental and cell biology. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Chromatin remodeller SMARCA4 recruits topoisomerase 1 and suppresses transcription-associated genomic instability.

    PubMed

    Husain, Afzal; Begum, Nasim A; Taniguchi, Takako; Taniguchi, Hisaaki; Kobayashi, Maki; Honjo, Tasuku

    2016-02-04

    Topoisomerase 1, an enzyme that relieves superhelical tension, is implicated in transcription-associated mutagenesis and genome instability-associated with neurodegenerative diseases as well as activation-induced cytidine deaminase. From proteomic analysis of TOP1-associated proteins, we identify SMARCA4, an ATP-dependent chromatin remodeller; FACT, a histone chaperone; and H3K4me3, a transcriptionally active chromatin marker. Here we show that SMARCA4 knockdown in a B-cell line decreases TOP1 recruitment to chromatin, and leads to increases in Igh/c-Myc chromosomal translocations, variable and switch region mutations and negative superhelicity, all of which are also observed in response to TOP1 knockdown. In contrast, FACT knockdown inhibits association of TOP1 with H3K4me3, and severely reduces DNA cleavage and Igh/c-Myc translocations, without significant effect on TOP1 recruitment to chromatin. We thus propose that SMARCA4 is involved in the TOP1 recruitment to general chromatin, whereas FACT is required for TOP1 binding to H3K4me3 at non-B DNA containing chromatin for the site-specific cleavage.

  19. Fission yeast Tup1-like repressors repress chromatin remodeling at the fbp1+ promoter and the ade6-M26 recombination hotspot.

    PubMed Central

    Hirota, Kouji; Hoffman, Charles S; Shibata, Takehiko; Ohta, Kunihiro

    2003-01-01

    Chromatin remodeling plays crucial roles in the regulation of gene expression and recombination. Transcription of the fission yeast fbp1(+) gene and recombination at the meiotic recombination hotspot ade6-M26 (M26) are both regulated by cAMP responsive element (CRE)-like sequences and the CREB/ATF-type transcription factor Atf1*Pcr1. The Tup11 and Tup12 proteins, the fission yeast counterparts of the Saccharomyces cerevisiae Tup1 corepressor, are involved in glucose repression of the fbp1(+) transcription. We have analyzed roles of the Tup1-like corepressors in chromatin regulation around the fbp1(+) promoter and the M26 hotspot. We found that the chromatin structure around two regulatory elements for fbp1(+) was remodeled under derepressed conditions in concert with the robust activation of fbp1(+) transcription. Strains with tup11delta tup12delta double deletions grown in repressed conditions exhibited the chromatin state associated with wild-type cells grown in derepressed conditions. Interestingly, deletion of rst2(+), encoding a transcription factor controlled by the cAMP-dependent kinase, alleviated the tup11delta tup12delta defects in chromatin regulation but not in transcription repression. The chromatin at the M26 site in mitotic cultures of a tup11delta tup12delta mutant resembled that of wild-type meiotic cells. These observations suggest that these fission yeast Tup1-like corepressors repress chromatin remodeling at CRE-related sequences and that Rst2 antagonizes this function. PMID:14573465

  20. Heavy ion-induced lesions in DNA: A theoretical model for the initial induction of DNA strand breaks and chromatin breaks

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schmidt, J.B.

    1993-01-01

    A theoretical model has been developed and used to calculate yields and spatial distributions of DNA strand breaks resulting from the interactions of heavy ions with chromatin in aqueous systems. The three dimensional spatial distribution of ionizing events has been modeled for charged particles as a function of charge and velocity. Chromatin has been modeled as a 30 nm diameter solenoid of nucleosomal DNA. The Monte Carlo methods used by Chatterjee et al. have been applied to DNA in a chromatin conformation. Refinements to their methods include: a combined treatment of primary and low energy (<2 keV) secondary electron interactions,more » an improved low energy delta ray model, and the combined simulation of direct energy deposition on the DNA and attack by diffusing hydroxyl radicals. Individual particle tracks are treated independently, which is assumed to be applicable to low fluence irradiations in which multiple particle effects are negligible. Single strand break cross section [open quotes]hooks[close quotes] seen in experiments at very high LET appear to be due to the collapsing radial extent of the track, as predicted in the [open quotes]deep sieve[close quotes] hypothesis proposed by Tobias et al. Spatial distributions of lesions produced by particles have been found to depend on chromatin structure. In the future, heavy ions may be used as a tool to probe the organization of DNA in chromatin. A Neyman A-binomial variation of the [open quotes]cluster model[close quotes] for the distribution of chromatin breaks per irradiated cell has been theoretically tested. The model includes a treatment of the chromatin fragment detection technique's resolution, which places a limitation on the minimum size of fragments which can be detected. The model appears to fit some of the experimental data reasonably well. However, further experimental and theoretical refinements are desirable.« less

  1. Phosphorylated SAP155, the spliceosomal component, is localized to chromatin in postnatal mouse testes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Eto, Ko, E-mail: etoko@gpo.kumamoto-u.ac.jp; Sonoda, Yoshiyuki; Jin, Yuji

    SAP155 is an essential component of the spliceosome and its phosphorylation is required for splicing catalysis, but little is known concerning its expression and regulation during spermatogenesis in postnatal mouse testes. We report that SAP155 is ubiquitously expressed in nuclei of germ and Sertoli cells within the seminiferous tubules of 6- and 35-day postpartum (dpp) testes. Analyses by fractionation of testes revealed that (1) phosphorylated SAP155 was found in the fraction containing nuclear structures at 6 dpp in amounts much larger than that at other ages; (2) non-phosphorylated SAP155 was detected in the fraction containing nucleoplasm; and (3) phosphorylated SAP155more » was preferentially associated with chromatin. Our findings suggest that the active spliceosome, containing phosphorylated SAP155, performs pre-mRNA splicing on chromatin concomitant with transcription during testicular development.« less

  2. Structural basis for ATP-dependent chromatin remodelling by the INO80 complex.

    PubMed

    Eustermann, Sebastian; Schall, Kevin; Kostrewa, Dirk; Lakomek, Kristina; Strauss, Mike; Moldt, Manuela; Hopfner, Karl-Peter

    2018-04-01

    In the eukaryotic nucleus, DNA is packaged in the form of nucleosomes, each of which comprises about 147 base pairs of DNA wrapped around a histone protein octamer. The position and histone composition of nucleosomes is governed by ATP-dependent chromatin remodellers 1-3 such as the 15-subunit INO80 complex 4 . INO80 regulates gene expression, DNA repair and replication by sliding nucleosomes, the exchange of histone H2A.Z with H2A, and the positioning of + 1 and -1 nucleosomes at promoter DNA 5-8 . The structures and mechanisms of these remodelling reactions are currently unknown. Here we report the cryo-electron microscopy structure of the evolutionarily conserved core of the INO80 complex from the fungus Chaetomium thermophilum bound to a nucleosome, at a global resolution of 4.3 Å and with major parts at 3.7 Å. The INO80 core cradles one entire gyre of the nucleosome through multivalent DNA and histone contacts. An Rvb1/Rvb2 AAA + ATPase heterohexamer is an assembly scaffold for the complex and acts as a 'stator' for the motor and nucleosome-gripping subunits. The Swi2/Snf2 ATPase motor binds to nucleosomal DNA at superhelical location -6, unwraps approximately 15 base pairs, disrupts the H2A-DNA contacts and is poised to pump entry DNA into the nucleosome. Arp5 and Ies6 bind superhelical locations -2 and -3 to act as a counter grip for the motor, on the other side of the H2A-H2B dimer. The Arp5 insertion domain forms a grappler element that binds the nucleosome dyad, connects the Arp5 actin-fold and entry DNA over a distance of about 90 Å and packs against histone H2A-H2B near the 'acidic patch'. Our structure together with biochemical data 8 suggests a unified mechanism for nucleosome sliding and histone editing by INO80. The motor is part of a macromolecular ratchet, persistently pumping entry DNA across the H2A-H2B dimer against the Arp5 grip until a large nucleosome translocation step occurs. The transient exposure of H2A-H2B by motor activity

  3. Chromatin-Remodeling-Factor ARID1B Represses Wnt/β-Catenin Signaling.

    PubMed

    Vasileiou, Georgia; Ekici, Arif B; Uebe, Steffen; Zweier, Christiane; Hoyer, Juliane; Engels, Hartmut; Behrens, Jürgen; Reis, André; Hadjihannas, Michel V

    2015-09-03

    The link of chromatin remodeling to both neurodevelopment and cancer has recently been highlighted by the identification of mutations affecting BAF chromatin-remodeling components, such as ARID1B, in individuals with intellectual disability and cancer. However, the underlying molecular mechanism(s) remains unknown. Here, we show that ARID1B is a repressor of Wnt/β-catenin signaling. Through whole-transcriptome analysis, we find that in individuals with intellectual disability and ARID1B loss-of-function mutations, Wnt/β-catenin target genes are upregulated. Using cellular models of low and high Wnt/β-catenin activity, we demonstrate that knockdown of ARID1B activates Wnt/β-catenin target genes and Wnt/β-catenin-dependent transcriptional reporters in a β-catenin-dependent manner. Reciprocally, forced expression of ARID1B inhibits Wnt/β-catenin signaling downstream of the β-catenin destruction complex. Both endogenous and exogenous ARID1B associate with β-catenin and repress Wnt/β-catenin-mediated transcription through the BAF core subunit BRG1. Accordingly, mutations in ARID1B leading to partial or complete deletion of its BRG1-binding domain, as is often observed in intellectual disability and cancers, compromise association with β-catenin, and the resultant ARID1B mutant proteins fail to suppress Wnt/β-catenin signaling. Finally, knockdown of ARID1B in mouse neuroblastoma cells leads to neurite outgrowth through β-catenin. The data suggest that aberrations in chromatin-remodeling factors, such as ARID1B, might contribute to neurodevelopmental abnormalities and cancer through deregulation of developmental and oncogenic pathways, such as the Wnt/β-catenin signaling pathway. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  4. Human tRNA genes function as chromatin insulators

    PubMed Central

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

    2012-01-01

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

  5. Genome-wide chromatin state transitions associated with developmental and environmental cues.

    PubMed

    Zhu, Jiang; Adli, Mazhar; Zou, James Y; Verstappen, Griet; Coyne, Michael; Zhang, Xiaolan; Durham, Timothy; Miri, Mohammad; Deshpande, Vikram; De Jager, Philip L; Bennett, David A; Houmard, Joseph A; Muoio, Deborah M; Onder, Tamer T; Camahort, Ray; Cowan, Chad A; Meissner, Alexander; Epstein, Charles B; Shoresh, Noam; Bernstein, Bradley E

    2013-01-31

    Differences in chromatin organization are key to the multiplicity of cell states that arise from a single genetic background, yet the landscapes of in vivo tissues remain largely uncharted. Here, we mapped chromatin genome-wide in a large and diverse collection of human tissues and stem cells. The maps yield unprecedented annotations of functional genomic elements and their regulation across developmental stages, lineages, and cellular environments. They also reveal global features of the epigenome, related to nuclear architecture, that also vary across cellular phenotypes. Specifically, developmental specification is accompanied by progressive chromatin restriction as the default state transitions from dynamic remodeling to generalized compaction. Exposure to serum in vitro triggers a distinct transition that involves de novo establishment of domains with features of constitutive heterochromatin. We describe how these global chromatin state transitions relate to chromosome and nuclear architecture, and discuss their implications for lineage fidelity, cellular senescence, and reprogramming. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation

    PubMed Central

    Gomez-Godinez, Veronica; Wu, Tao; Sherman, Adria J.; Lee, Christopher S.; Liaw, Lih-Huei; Zhongsheng, You; Yokomori, Kyoko; Berns, Michael W.

    2010-01-01

    In this study the femtosecond near-IR and nanosecond green lasers are used to induce alterations in mitotic chromosomes. The subsequent double-strand break responses are studied. We show that both lasers are capable of creating comparable chromosomal alterations and that a phase paling observed within 1–2 s of laser exposure is associated with an alteration of chromatin as confirmed by serial section electron microscopy, DAPI, γH2AX and phospho-H3 staining. Additionally, the accumulation of dark material observed using phase contrast light microscopy (indicative of a change in refractive index of the chromatin) ∼34 s post-laser exposure corresponds spatially to the accumulation of Nbs1, Ku and ubiquitin. This study demonstrates that chromosomes selectively altered in mitosis initiate the DNA damage response within 30 s and that the accumulation of proteins are visually represented by phase-dark material at the irradiation site, allowing us to determine the fate of the damage as cells enter G1. These results occur with two widely different laser systems, making this approach to study DNA damage responses in the mitotic phase generally available to many different labs. Additionally, we present a summary of most of the published laser studies on chromosomes in order to provide a general guide of the lasers and operating parameters used by other laboratories. PMID:20923785

  7. The histone chaperone TAF-I/SET/INHAT is required for transcription in vitro of chromatin templates.

    PubMed

    Gamble, Matthew J; Erdjument-Bromage, Hediye; Tempst, Paul; Freedman, Leonard P; Fisher, Robert P

    2005-01-01

    To uncover factors required for transcription by RNA polymerase II on chromatin, we fractionated a mammalian cell nuclear extract. We identified the histone chaperone TAF-I (also known as INHAT [inhibitor of histone acetyltransferase]), which was previously proposed to repress transcription, as a potent activator of chromatin transcription responsive to the vitamin D3 receptor or to Gal4-VP16. TAF-I associates with chromatin in vitro and can substitute for the related protein NAP-1 in assembling chromatin onto cloned DNA templates in cooperation with the remodeling enzyme ATP-dependent chromatin assembly factor (ACF). The chromatin assembly and transcriptional activation functions are distinct, however, and can be dissociated temporally. Efficient transcription of chromatin assembled with TAF-I still requires the presence of TAF-I during the polymerization reaction. Conversely, TAF-I cannot stimulate transcript elongation when added after the other factors necessary for assembly of a preinitiation complex on naked DNA. Thus, TAF-I is required to facilitate transcription at a step after chromatin assembly but before transcript elongation.

  8. Chromatibody, a novel non-invasive molecular tool to explore and manipulate chromatin in living cells

    PubMed Central

    Jullien, Denis; Vignard, Julien; Fedor, Yoann; Béry, Nicolas; Olichon, Aurélien; Crozatier, Michèle; Erard, Monique; Cassard, Hervé; Ducommun, Bernard; Salles, Bernard

    2016-01-01

    ABSTRACT Chromatin function is involved in many cellular processes, its visualization or modification being essential in many developmental or cellular studies. Here, we present the characterization of chromatibody, a chromatin-binding single-domain, and explore its use in living cells. This non-intercalating tool specifically binds the heterodimer of H2A–H2B histones and displays a versatile reactivity, specifically labeling chromatin from yeast to mammals. We show that this genetically encoded probe, when fused to fluorescent proteins, allows non-invasive real-time chromatin imaging. Chromatibody is a dynamic chromatin probe that can be modulated. Finally, chromatibody is an efficient tool to target an enzymatic activity to the nucleosome, such as the DNA damage-dependent H2A ubiquitylation, which can modify this epigenetic mark at the scale of the genome and result in DNA damage signaling and repair defects. Taken together, these results identify chromatibody as a universal non-invasive tool for either in vivo chromatin imaging or to manipulate the chromatin landscape. PMID:27206857

  9. Brd2 gene disruption causes ‘metabolically healthy’ obesity: Epigenetic and chromatin-based mechanisms that uncouple obesity from Type 2 diabetes

    PubMed Central

    Wang, Fangnian; Deeney, Jude T.; Denis, Gerald V.

    2014-01-01

    Disturbed body energy balance can lead to obesity and obesity-driven diseases such as Type 2 diabetes, which have reached an epidemic level. Evidence indicates that obesity induced inflammation is a major cause of insulin resistance and Type 2 diabetes. Environmental factors, such as nutrients, affect body energy balance through epigenetic or chromatin-based mechanisms. As a bromodomain and external domain family transcription regulator, Brd2 regulates expression of many genes through interpretation of chromatin codes, and participates in the regulation of body energy balance and immune function. In the severely obese state, Brd2 knockdown in mice prevented obesity-induced inflammatory responses, protected animals from Type 2 diabetes, and thus uncoupled obesity from diabetes. Brd2 provides an important model for investigation of the function of transcription regulators and the development of obesity and diabetes; it also provides a possible target to treat obesity and diabetes through modulation of the function of a chromatin code reader. PMID:23374712

  10. Using Chromatin Immunoprecipitation in Toxicology: A Step-by-Step Guide to Increasing Efficiency, Reducing Variability, and Expanding Applications

    EPA Science Inventory

    Histone modifications work in concert with DNA methylation to regulate cellular structure, function, and the response to environmental stimuli. More than 130 unique histone modifications have been described to date and chromatin immunoprecipitation (ChIP) allows for the explorat...

  11. Coordinating Regulation of Gene Expression in Cardiovascular Disease: Interactions between Chromatin Modifiers and Transcription Factors

    PubMed Central

    Bauer, Ashley J.; Martin, Kathleen A.

    2017-01-01

    Cardiovascular disease is a leading cause of death with increasing economic burden. The pathogenesis of cardiovascular diseases is complex, but can arise from genetic and/or environmental risk factors. This can lead to dysregulated gene expression in numerous cell types including cardiomyocytes, endothelial cells, vascular smooth muscle cells, and inflammatory cells. While initial studies addressed transcriptional control of gene expression, epigenetics has been increasingly appreciated to also play an important role in this process through alterations in chromatin structure and gene accessibility. Chromatin-modifying proteins including enzymes that modulate DNA methylation, histone methylation, and histone acetylation can influence gene expression in numerous ways. These chromatin modifiers and their marks can promote or prevent transcription factor recruitment to regulatory regions of genes through modifications to DNA, histones, or the transcription factors themselves. This review will focus on the emerging question of how epigenetic modifiers and transcription factors interact to coordinately regulate gene expression in cardiovascular disease. While most studies have addressed the roles of either epigenetic or transcriptional control, our understanding of the integration of these processes is only just beginning. Interrogating these interactions is challenging, and improved technical approaches will be needed to fully dissect the temporal and spatial relationships between transcription factors, chromatin modifiers, and gene expression in cardiovascular disease. We summarize the current state of the field and provide perspectives on limitations and future directions. Through studies of epigenetic and transcriptional interactions, we can advance our understanding of the basic mechanisms of cardiovascular disease pathogenesis to develop novel therapeutics. PMID:28428957

  12. metaseq: a Python package for integrative genome-wide analysis reveals relationships between chromatin insulators and associated nuclear mRNA.

    PubMed

    Dale, Ryan K; Matzat, Leah H; Lei, Elissa P

    2014-08-01

    Here we introduce metaseq, a software library written in Python, which enables loading multiple genomic data formats into standard Python data structures and allows flexible, customized manipulation and visualization of data from high-throughput sequencing studies. We demonstrate its practical use by analyzing multiple datasets related to chromatin insulators, which are DNA-protein complexes proposed to organize the genome into distinct transcriptional domains. Recent studies in Drosophila and mammals have implicated RNA in the regulation of chromatin insulator activities. Moreover, the Drosophila RNA-binding protein Shep has been shown to antagonize gypsy insulator activity in a tissue-specific manner, but the precise role of RNA in this process remains unclear. Better understanding of chromatin insulator regulation requires integration of multiple datasets, including those from chromatin-binding, RNA-binding, and gene expression experiments. We use metaseq to integrate RIP- and ChIP-seq data for Shep and the core gypsy insulator protein Su(Hw) in two different cell types, along with publicly available ChIP-chip and RNA-seq data. Based on the metaseq-enabled analysis presented here, we propose a model where Shep associates with chromatin cotranscriptionally, then is recruited to insulator complexes in trans where it plays a negative role in insulator activity. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  13. SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

    PubMed Central

    Mund, Andreas; Schubert, Tobias; Staege, Hannah; Kinkley, Sarah; Reumann, Kerstin; Kriegs, Malte; Fritsch, Lauriane; Battisti, Valentine; Ait-Si-Ali, Slimane; Hoffbeck, Anne-Sophie; Soutoglou, Evi; Will, Hans

    2012-01-01

    Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure. PMID:23034801

  14. Comprehensive single cell-resolution analysis of the role of chromatin regulators in early C. elegans embryogenesis.

    PubMed

    Krüger, Angela V; Jelier, Rob; Dzyubachyk, Oleh; Zimmerman, Timo; Meijering, Erik; Lehner, Ben

    2015-02-15

    Chromatin regulators are widely expressed proteins with diverse roles in gene expression, nuclear organization, cell cycle regulation, pluripotency, physiology and development, and are frequently mutated in human diseases such as cancer. Their inhibition often results in pleiotropic effects that are difficult to study using conventional approaches. We have developed a semi-automated nuclear tracking algorithm to quantify the divisions, movements and positions of all nuclei during the early development of Caenorhabditis elegans and have used it to systematically study the effects of inhibiting chromatin regulators. The resulting high dimensional datasets revealed that inhibition of multiple regulators, including F55A3.3 (encoding FACT subunit SUPT16H), lin-53 (RBBP4/7), rba-1 (RBBP4/7), set-16 (MLL2/3), hda-1 (HDAC1/2), swsn-7 (ARID2), and let-526 (ARID1A/1B) affected cell cycle progression and caused chromosome segregation defects. In contrast, inhibition of cir-1 (CIR1) accelerated cell division timing in specific cells of the AB lineage. The inhibition of RNA polymerase II also accelerated these division timings, suggesting that normal gene expression is required to delay cell cycle progression in multiple lineages in the early embryo. Quantitative analyses of the dataset suggested the existence of at least two functionally distinct SWI/SNF chromatin remodeling complex activities in the early embryo, and identified a redundant requirement for the egl-27 and lin-40 MTA orthologs in the development of endoderm and mesoderm lineages. Moreover, our dataset also revealed a characteristic rearrangement of chromatin to the nuclear periphery upon the inhibition of multiple general regulators of gene expression. Our systematic, comprehensive and quantitative datasets illustrate the power of single cell-resolution quantitative tracking and high dimensional phenotyping to investigate gene function. Furthermore, the results provide an overview of the functions of essential

  15. Quantifying transient binding of ISWI chromatin remodelers in living cells by pixel-wise photobleaching profile evolution analysis.

    PubMed

    Erdel, Fabian; Rippe, Karsten

    2012-11-20

    Interactions between nuclear proteins and chromatin frequently occur on the time scale of seconds and below. These transient binding events are important for the fast identification of target sites as concluded from our previous analysis of the human chromatin remodelers Snf2H and Snf2L from the imitation switch (ISWI) family. Both ATP-driven molecular motor proteins are able to translocate nucleosomes along the DNA and appear to exert this activity only on a small number of nucleosomes to which they bind more tightly. For mechanistic studies, one needs to distinguish such translocation reactions or other long-lived interactions associated with conformational changes and/or ATP hydrolysis from nonproductive chromatin sampling during target search. These processes can be separated by measuring the duration of nucleosome binding with subsecond time resolution. To reach this goal, we have developed a fluorescence bleaching technique termed pixel-wise photobleaching profile evolution analysis (3PEA). It exploits the inherent time structure of confocal microscopy images and yields millisecond resolution. 3PEA represents a generally applicable approach to quantitate transient chromatin interactions in the 2- to 500-ms time regime within only ∼1 s needed for a measurement. The green autofluorescent protein (GFP)-tagged Snf2H and Snf2L and the inactive Snf2L+13 splice variant were studied by 3PEA in comparison to the isolated GFP or red autofluorescent protein and a GFP pentamer. Our results reveal that the residence time for transient chromatin binding of Snf2H and Snf2L is <2 ms, and strongly support the view that ISWI-type remodelers are only rarely active in unperturbed cells during G1 phase.

  16. Nitric oxide deficiency determines global chromatin changes in Duchenne muscular dystrophy.

    PubMed

    Colussi, Claudia; Gurtner, Aymone; Rosati, Jessica; Illi, Barbara; Ragone, Gianluca; Piaggio, Giulia; Moggio, Maurizio; Lamperti, Costanza; D'Angelo, Grazia; Clementi, Emilio; Minetti, Giulia; Mozzetta, Chiara; Antonini, Annalisa; Capogrossi, Maurizio C; Puri, Pier Lorenzo; Gaetano, Carlo

    2009-07-01

    The present study provides evidence that abnormal patterns of global histone modification are present in the skeletal muscle nuclei of mdx mice and Duchenne muscular dystrophy (DMD) patients. A combination of specific histone H3 modifications, including Ser-10 phosphorylation, acetylation of Lys 9 and 14, and Lys 79 methylation, were found enriched in muscle biopsies from human patients affected by DMD and in late-term fetuses, early postnatal pups, or adult mdx mice. In this context, chromatin immunoprecipitation experiments showed an enrichment of these modifications at the loci of genes involved in proliferation or inflammation, suggesting a regulatory effect on gene expression. Remarkably, the reexpression of dystrophin induced by gentamicin treatment or the administration of nitric oxide (NO) donors reversed the abnormal pattern of H3 histone modifications. These findings suggest an unanticipated link between the dystrophin-activated NO signaling and the remodeling of chromatin. In this context, the regulation of class IIa histone deacetylases (HDACs) 4 and 5 was found altered as a consequence of the reduced NO-dependent protein phosphatase 2A activity, indicating that both NO and class IIa HDACs are important for satellite cell differentiation and gene expression in mdx mice. In conclusion, this work provides the first evidence of a role for NO as an epigenetic regulator in DMD.

  17. Concerted action of the PHD, chromo and motor domains regulates the human chromatin remodelling ATPase CHD4.

    PubMed

    Morra, Rosa; Lee, Benjamin M; Shaw, Heather; Tuma, Roman; Mancini, Erika J

    2012-07-30

    CHD4, the core subunit of the Nucleosome Remodelling and Deacetylase (NuRD) complex, is a chromatin remodelling ATPase that, in addition to a helicase domain, harbors tandem plant homeo finger and chromo domains. By using a panel of domain constructs we dissect their roles and demonstrate that DNA binding, histone binding and ATPase activities are allosterically regulated. Molecular shape reconstruction from small-angle X-ray scattering reveals extensive domain-domain interactions, which provide a structural explanation for the regulation of CHD4 activities by intramolecular domain communication. Our results demonstrate functional interdependency between domains within a chromatin remodeller. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  18. The Usefulness of Selected Physicochemical Indices, Cell Membrane Integrity and Sperm Chromatin Structure in Assessments of Boar Semen Sensitivity

    PubMed Central

    Wysokińska, A.; Kondracki, S.; Iwanina, M.

    2015-01-01

    The present work describes experiments undertaken to evaluate the usefulness of selected physicochemical indices of semen, cell membrane integrity and sperm chromatin structure for the assessment of boar semen sensitivity to processes connected with pre-insemination procedures. The experiments were carried out on 30 boars: including 15 regarded as providers of sensitive semen and 15 regarded as providers of semen that is little sensitive to laboratory processing. The selection of boars for both groups was based on sperm morphology analyses, assuming secondary morphological change incidence in spermatozoa as the criterion. Two ejaculates were manually collected from each boar at an interval of 3 to 4 months. The following analyses were carried out for each ejaculate: sperm motility assessment, sperm pH measurement, sperm morphology assessment, sperm chromatin structure evaluation and cell membrane integrity assessment. The analyses were performed three times. Semen storage did not cause an increase in the incidence of secondary morphological changes in the group of boars considered to provide sperm of low sensitivity. On the other hand, with continued storage there was a marked increase in the incidence of spermatozoa with secondary morphological changes in the group of boars regarded as producing more sensitive semen. Ejaculates of group I boars evaluated directly after collection had an approximately 6% smaller share of spermatozoa with undamaged cell membranes than the ejaculates of boars in group II (p≤0.05). In the process of time the percentage of spermatozoa with undamaged cell membranes decreased. The sperm of group I boars was characterised with a lower sperm motility than the semen of group II boars. After 1 hour of storing diluted semen, the sperm motility of boars producing highly sensitive semen was already 4% lower (p≤0.05), and after 24 hours of storage it was 6.33% lower than that of the boars that produced semen with a low sensitivity. Factors

  19. Layer-specific chromatin accessibility landscapes reveal regulatory networks in adult mouse visual cortex

    PubMed Central

    Gray, Lucas T; Yao, Zizhen; Nguyen, Thuc Nghi; Kim, Tae Kyung; Zeng, Hongkui; Tasic, Bosiljka

    2017-01-01

    Mammalian cortex is a laminar structure, with each layer composed of a characteristic set of cell types with different morphological, electrophysiological, and connectional properties. Here, we define chromatin accessibility landscapes of major, layer-specific excitatory classes of neurons, and compare them to each other and to inhibitory cortical neurons using the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq). We identify a large number of layer-specific accessible sites, and significant association with genes that are expressed in specific cortical layers. Integration of these data with layer-specific transcriptomic profiles and transcription factor binding motifs enabled us to construct a regulatory network revealing potential key layer-specific regulators, including Cux1/2, Foxp2, Nfia, Pou3f2, and Rorb. This dataset is a valuable resource for identifying candidate layer-specific cis-regulatory elements in adult mouse cortex. DOI: http://dx.doi.org/10.7554/eLife.21883.001 PMID:28112643

  20. A high-resolution map of the three-dimensional chromatin interactome in human cells.

    PubMed

    Jin, Fulai; Li, Yan; Dixon, Jesse R; Selvaraj, Siddarth; Ye, Zhen; Lee, Ah Young; Yen, Chia-An; Schmitt, Anthony D; Espinoza, Celso A; Ren, Bing

    2013-11-14

    A large number of cis-regulatory sequences have been annotated in the human genome, but defining their target genes remains a challenge. One strategy is to identify the long-range looping interactions at these elements with the use of chromosome conformation capture (3C)-based techniques. However, previous studies lack either the resolution or coverage to permit a whole-genome, unbiased view of chromatin interactions. Here we report a comprehensive chromatin interaction map generated in human fibroblasts using a genome-wide 3C analysis method (Hi-C). We determined over one million long-range chromatin interactions at 5-10-kb resolution, and uncovered general principles of chromatin organization at different types of genomic features. We also characterized the dynamics of promoter-enhancer contacts after TNF-α signalling in these cells. Unexpectedly, we found that TNF-α-responsive enhancers are already in contact with their target promoters before signalling. Such pre-existing chromatin looping, which also exists in other cell types with different extracellular signalling, is a strong predictor of gene induction. Our observations suggest that the three-dimensional chromatin landscape, once established in a particular cell type, is relatively stable and could influence the selection or activation of target genes by a ubiquitous transcription activator in a cell-specific manner.

  1. Vitamin D receptor (VDR) promoter targeting through a novel chromatin remodeling complex.

    PubMed

    Kato, Shigeaki; Fujiki, Ryoji; Kitagawa, Hirochika

    2004-05-01

    We have purified nuclear complexes for Vitamin D receptor (VDR), and identified one of them as a novel ATP-dependent chromatine remodeling containing Williams syndrome transcription factor (WSTF), that is supposed to be responsible for Williams syndrome. This complex (WSTF including nucleosome assembly complex (WINAC)) exhibited an ATP-dependent chromatin remodeling activity in vitro. Transient expression assays revealed that WINAC potentiates ligand-induced function of VDR in gene activation and repression. Thus, this study describes a molecular basis of the VDR function on chromosomal DNA through chromatine remodeling.

  2. Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

    PubMed Central

    Jukam, David; Teran, Nicole A; Risca, Viviana I; Smith, Owen K; Johnson, Whitney L; Skotheim, Jan M; Greenleaf, William James

    2018-01-01

    RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here, we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. Using Drosophila cells, we show that ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing. PMID:29648534

  3. Chromatin insulator elements: establishing barriers to set heterochromatin boundaries.

    PubMed

    Barkess, Gráinne; West, Adam G

    2012-02-01

    Epigenomic profiling has revealed that substantial portions of genomes in higher eukaryotes are organized into extensive domains of transcriptionally repressive chromatin. The boundaries of repressive chromatin domains can be fixed by DNA elements known as barrier insulators, to both shield neighboring gene expression and to maintain the integrity of chromosomal silencing. Here, we examine the current progress in identifying vertebrate barrier elements and their binding factors. We overview the design of the reporter assays used to define enhancer-blocking and barrier insulators. We look at the mechanisms vertebrate barrier proteins, such as USF1 and VEZF1, employ to counteract Polycomb- and heterochromatin-associated repression. We also undertake a critical analysis of whether CTCF could also act as a barrier protein. There is good evidence that barrier elements in vertebrates can form repressive chromatin domain boundaries. Future studies will determine whether barriers are frequently used to define repressive domain boundaries in vertebrates.

  4. Chromatin Regulation and the Histone Code in HIV Latency
.

    PubMed

    Turner, Anne-Marie W; Margolis, David M

    2017-06-01

    The formation of a latent reservoir of Human Immunodeficiency Virus (HIV) infection hidden from immune clearance remains a significant obstacle to approaches to eradicate HIV infection. Towards an understanding of the mechanisms of HIV persistence, there is a growing body of work implicating epigenetic regulation of chromatin in establishment and maintenance of this latent reservoir. Here we discuss recent advances in the field of chromatin regulation, specifically in our understanding of the histone code, and how these discoveries relate to our current knowledge of the chromatin mechanisms linked to HIV transcriptional repression and the reversal of latency. We also examine mechanisms unexplored in the context of HIV latency and briefly discuss current therapies aimed at the induction of proviral expression within latently infected cells. We aim to emphasize that a greater understanding of the epigenetic mechanisms which govern HIV latency could lead to new therapeutic targets for latency reversal and clearance cure strategies.

  5. Chromatin Regulation and the Histone Code in HIV Latency


    PubMed Central

    Turner, Anne-Marie W.; Margolis, David M.

    2017-01-01

    The formation of a latent reservoir of Human Immunodeficiency Virus (HIV) infection hidden from immune clearance remains a significant obstacle to approaches to eradicate HIV infection. Towards an understanding of the mechanisms of HIV persistence, there is a growing body of work implicating epigenetic regulation of chromatin in establishment and maintenance of this latent reservoir. Here we discuss recent advances in the field of chromatin regulation, specifically in our understanding of the histone code, and how these discoveries relate to our current knowledge of the chromatin mechanisms linked to HIV transcriptional repression and the reversal of latency. We also examine mechanisms unexplored in the context of HIV latency and briefly discuss current therapies aimed at the induction of proviral expression within latently infected cells. We aim to emphasize that a greater understanding of the epigenetic mechanisms which govern HIV latency could lead to new therapeutic targets for latency reversal and clearance cure strategies. PMID:28656010

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

    PubMed

    Garai, Ashok; Mani, Jesrael; Chowdhury, Debashish

    2012-04-01

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

  7. [Comparative investigation of the non-histone proteins of chromatin from pigeon erythroblasts and erythrocytes].

    PubMed

    Fedina, A B; Gazarian, G G

    1976-01-01

    Chromosomal non-histone proteins are obtained from nuclei of two types of pigeon erythroid cells: erythroblasts (cells active in RNA synthesis) and erythrocytes (cells with repressed RNA synthesis). They are well soluble in solutions of low ionic strength. Electrophoretic separation of the obtained non-histone proteins in polyacrylamide gels with urea and SDS shows the presence of qualitative differences in the pattern of non-histone proteins of chromatine from erythroblasts and erythrocytes. By electrophoresis in urea some protein bands of non-histone proteins of chromatine from erythroblasts were found which disappear with the aging of cells. At the same time two protein fractions were observed in chromatine from erythrocytes which were absent in that of erythroblasts. Disappearance of some high molecular weight protein fractions from erythrocyte chromatine as compared to erythroblasts was observed by separation of the non-histone proteins in the presence of SDS. These fractions of the non-histone proteins disappearing during aging of cells are well extractable from erythroblast chromatine by 0.35 M NaCl solution. In the in vitro system with E. coli RNA polymerase addition of non-histone proteins of chromatine from erythroblasts to chromatine from erythrocytes increases RNA synthesis 2--3 times. At the same time addition of non-histone proteins from erythrocytes is either without any influence on this process or somewhat inhibiting.

  8. Tissue-Specific Chromatin Modifications at a Multigene Locus Generate Asymmetric Transcriptional Interactions

    PubMed Central

    Yoo, Eung Jae; Cajiao, Isabela; Kim, Jeong-Seon; Kimura, Atsushi P.; Zhang, Aiwen; Cooke, Nancy E.; Liebhaber, Stephen A.

    2006-01-01

    Random assortment within mammalian genomes juxtaposes genes with distinct expression profiles. This organization, along with the prevalence of long-range regulatory controls, generates a potential for aberrant transcriptional interactions. The human CD79b/GH locus contains six tightly linked genes with three mutually exclusive tissue specificities and interdigitated control elements. One consequence of this compact organization is that the pituitarycell-specific transcriptional events that activate hGH-N also trigger ectopic activation of CD79b. However, the B-cell-specific events that activate CD79b do not trigger reciprocal activation of hGH-N. Here we utilized DNase I hypersensitive site mapping, chromatin immunoprecipitation, and transgenic models to explore the basis for this asymmetric relationship. The results reveal tissue-specific patterns of chromatin structures and transcriptional controls at the CD79b/GH locus in B cells distinct from those in the pituitary gland and placenta. These three unique transcriptional environments suggest a set of corresponding gene expression pathways and transcriptional interactions that are likely to be found juxtaposed at multiple sites within the eukaryotic genome. PMID:16847312

  9. Mechanically Induced Chromatin Condensation Requires Cellular Contractility in Mesenchymal Stem Cells.

    PubMed

    Heo, Su-Jin; Han, Woojin M; Szczesny, Spencer E; Cosgrove, Brian D; Elliott, Dawn M; Lee, David A; Duncan, Randall L; Mauck, Robert L

    2016-08-23

    Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in chromatin condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-protein coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in chromatin condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced chromatin condensation. With loading, ATP release occurred very rapidly (within the first 10-20 s), whereas changes in chromatin occurred at a later time point (∼10 min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that chromatin condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/bone morphogenetic proteins signaling pathway contributes to the establishment of baseline contractility in MSCs. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. FIND: difFerential chromatin INteractions Detection using a spatial Poisson process

    PubMed Central

    Chen, Yang; Zhang, Michael Q.

    2018-01-01

    Polymer-based simulations and experimental studies indicate the existence of a spatial dependency between the adjacent DNA fibers involved in the formation of chromatin loops. However, the existing strategies for detecting differential chromatin interactions assume that the interacting segments are spatially independent from the other segments nearby. To resolve this issue, we developed a new computational method, FIND, which considers the local spatial dependency between interacting loci. FIND uses a spatial Poisson process to detect differential chromatin interactions that show a significant difference in their interaction frequency and the interaction frequency of their neighbors. Simulation and biological data analysis show that FIND outperforms the widely used count-based methods and has a better signal-to-noise ratio. PMID:29440282

  11. Sperm DNA fragmentation affects epigenetic feature in human male pronucleus.

    PubMed

    Rajabi, H; Mohseni-Kouchesfehani, H; Eslami-Arshaghi, T; Salehi, M

    2018-02-01

    To evaluate whether the sperm DNA fragmentation affects male pronucleus epigenetic factors, semen analysis was performed and DNA fragmentation was assessed by the method of sperm chromatin structure assay (SCSA). Human-mouse interspecies fertilisation was used to create human male pronucleus. Male pronucleus DNA methylation and H4K12 acetylation were evaluated by immunostaining. Results showed a significant positive correlation between the level of sperm DNA fragmentation and DNA methylation in male pronuclei. In other words, an increase in DNA damage caused an upsurge in DNA methylation. In the case of H4K12 acetylation, no correlation was detected between DNA damage and the level of histone acetylation in the normal group, but results for the group in which male pronuclei were derived from sperm cells with DNA fragmentation, increased DNA damage led to a decreased acetylation level. Sperm DNA fragmentation interferes with the active demethylation process and disrupts the insertion of histones into the male chromatin in the male pronucleus, following fertilisation. © 2017 Blackwell Verlag GmbH.

  12. Structure of interphase chromosomes in the nuclei of Drosophila cells.

    PubMed

    Banfalvi, Gaspar

    2006-10-01

    Fluorescent images of interphase chromatin structures and chromosome structures isolated from reversibly permeable Drosophila cells were analyzed. Decondensed chromatin in early S phase (2.0-2.5 C-value) consisted of a veil-like fibrillary network. Fibrillar chromatin formed rodlets later in the early S phase (2.5-2.75 C). Drosophila chromosomes contain several smaller subunits called rodlets. Fibrillar chromatin turned to chromatin ribbon and the early mid-S-phase globular chromosomes (2.75-3.0 C), then to opened fibrous globular forms later in the mid-S-phase (3.0-3.25 C), to late-S-phase supercoiled ribbons (3.25-3.5 C), end-S-phase elongated prechromosomes (3.5-3.75 C), bent and linear chromosomes (3.75-4.0 C). Early-S phase chromatin fibrils in the nuclei of Drosophila cells are thinner than the veil-like structures in mammalian cells. The connectivity of chromosomes shows linear arrangement (3, 1, 2, 4), with larger chromosomes (1 and 2) inside and smaller chromosomes (3, 4) at the two ends in the chromosomal chain.

  13. Citrullination regulates pluripotency and histone H1 binding to chromatin

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  14. Open chromatin structures regulate the efficiencies of pre-RC formation and replication initiation in Epstein-Barr virus

    PubMed Central

    Papior, Peer; Arteaga-Salas, José M.; Günther, Thomas; Grundhoff, Adam

    2012-01-01

    Whether or not metazoan replication initiates at random or specific but flexible sites is an unsolved question. The lack of sequence specificity in origin recognition complex (ORC) DNA binding complicates genome-scale chromatin immunoprecipitation (ChIP)-based studies. Epstein-Barr virus (EBV) persists as chromatinized minichromosomes that are replicated by the host replication machinery. We used EBV to investigate the link between zones of pre-replication complex (pre-RC) assembly, replication initiation, and micrococcal nuclease (MNase) sensitivity at different cell cycle stages in a genome-wide fashion. The dyad symmetry element (DS) of EBV’s latent origin, a well-established and very efficient pre-RC assembly region, served as an internal control. We identified 64 pre-RC zones that correlate spatially with 57 short nascent strand (SNS) zones. MNase experiments revealed that pre-RC and SNS zones were linked to regions of increased MNase sensitivity, which is a marker of origin strength. Interestingly, although spatially correlated, pre-RC and SNS zones were characterized by different features. We propose that pre-RCs are formed at flexible but distinct sites, from which only a few are activated per single genome and cell cycle. PMID:22891264

  15. Condensation of chromatin in transcriptional regions of an inactivated plant transgene: evidence for an active role of transcription in gene silencing.

    PubMed

    van Blokland, R; ten Lohuis, M; Meyer, P

    1997-12-01

    The chromatin structures of two epigenetic alleles of a transgene were investigated by measuring the local accessibility of transgene chromatin to endonucleases. The two epialleles represented the active, hypomethylated state of a transgene in line 17-I of Petunia hybrida, and a transcriptionally inactive, hypermethylated derivative of the same transgene in line 17-IV. In nuclear preparations the inactive epiallele was significantly less sensitive to DNasel digestion and nuclease S7 digestion than the transcriptionally active epiallele, whereas no significant differences in accessibility were observed between naked DNA samples of the two epialleles. Our data suggest that a condensed chromatin structure is specifically imposed on transcribed regions of the construct in line 17-IV. In contrast, in both epialleles the plasmid region of the transgene, which is not transcriptionally active in plants, retains the same accessibility to endonucleases as the chromosomal integration site. These data suggest that transcriptional inactivation is linked to the process of transcription, and imply that control of transgene expression via the use of inducible or tissue-specific promoters might prevent transgene silencing and conserve the active state of transgenes during sexual propagation.

  16. Distinct polymer physics principles govern chromatin dynamics in mouse and Drosophila topological domains.

    PubMed

    Ea, Vuthy; Sexton, Tom; Gostan, Thierry; Herviou, Laurie; Baudement, Marie-Odile; Zhang, Yunzhe; Berlivet, Soizik; Le Lay-Taha, Marie-Noëlle; Cathala, Guy; Lesne, Annick; Victor, Jean-Marc; Fan, Yuhong; Cavalli, Giacomo; Forné, Thierry

    2015-08-15

    In higher eukaryotes, the genome is partitioned into large "Topologically Associating Domains" (TADs) in which the chromatin displays favoured long-range contacts. While a crumpled/fractal globule organization has received experimental supports at higher-order levels, the organization principles that govern chromatin dynamics within these TADs remain unclear. Using simple polymer models, we previously showed that, in mouse liver cells, gene-rich domains tend to adopt a statistical helix shape when no significant locus-specific interaction takes place. Here, we use data from diverse 3C-derived methods to explore chromatin dynamics within mouse and Drosophila TADs. In mouse Embryonic Stem Cells (mESC), that possess large TADs (median size of 840 kb), we show that the statistical helix model, but not globule models, is relevant not only in gene-rich TADs, but also in gene-poor and gene-desert TADs. Interestingly, this statistical helix organization is considerably relaxed in mESC compared to liver cells, indicating that the impact of the constraints responsible for this organization is weaker in pluripotent cells. Finally, depletion of histone H1 in mESC alters local chromatin flexibility but not the statistical helix organization. In Drosophila, which possesses TADs of smaller sizes (median size of 70 kb), we show that, while chromatin compaction and flexibility are finely tuned according to the epigenetic landscape, chromatin dynamics within TADs is generally compatible with an unconstrained polymer configuration. Models issued from polymer physics can accurately describe the organization principles governing chromatin dynamics in both mouse and Drosophila TADs. However, constraints applied on this dynamics within mammalian TADs have a peculiar impact resulting in a statistical helix organization.

  17. High temporal-resolution view of transcription and chromatin states across distinct metabolic states in budding yeast

    PubMed Central

    Kuang, Zheng; Cai, Ling; Zhang, Xuekui; Ji, Hongkai; Tu, Benjamin P.; Boeke, Jef D.

    2014-01-01

    Under continuous, glucose-limited conditions, budding yeast exhibit robust metabolic cycles associated with major oscillations of gene expression. How such fluctuations are linked to changes in chromatin status is not well understood. Here we examine the correlated genome-wide transcription and chromatin states across the yeast metabolic cycle at unprecedented temporal resolution, revealing a “just-in-time supply chain” by which components from specific cellular processes such as ribosome biogenesis become available in a highly coordinated manner. We identify distinct chromatin and splicing patterns associated with different gene categories and determine the relative timing of chromatin modifications to maximal transcription. There is unexpected variation in the chromatin modification and expression relationship, with histone acetylation peaks occurring with varying timing and “sharpness” relative to RNA expression both within and between cycle phases. Chromatin modifier occupancy reveals subtly distinct spatial and temporal patterns compared to the modifications themselves. PMID:25173176

  18. Analysis of neonatal brain lacking ATRX or MeCP2 reveals changes in nucleosome density, CTCF binding and chromatin looping

    PubMed Central

    Kernohan, Kristin D.; Vernimmen, Douglas; Gloor, Gregory B.; Bérubé, Nathalie G.

    2014-01-01

    ATRX and MeCP2 belong to an expanding group of chromatin-associated proteins implicated in human neurodevelopmental disorders, although their gene-regulatory activities are not fully resolved. Loss of ATRX prevents full repression of an imprinted gene network in the postnatal brain and in this study we address the mechanistic aspects of this regulation. We show that ATRX binds many imprinted domains individually but that transient co-localization between imprinted domains in the nuclei of neurons does not require ATRX. We demonstrate that MeCP2 is required for ATRX recruitment and that deficiency of either ATRX or MeCP2 causes decreased frequency of long-range chromatin interactions associated with altered nucleosome density at CTCF-binding sites and reduced CTCF occupancy. These findings indicate that MeCP2 and ATRX regulate gene expression at a subset of imprinted domains by maintaining a nucleosome configuration conducive to CTCF binding and to the maintenance of higher order chromatin structure. PMID:24990380

  19. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators

    PubMed Central

    Polstein, Lauren R.; Perez-Pinera, Pablo; Kocak, D. Dewran; Vockley, Christopher M.; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E.; Reddy, Timothy E.; Gersbach, Charles A.

    2015-01-01

    Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. PMID:26025803

  20. 3D chromatin conformation correlates with replication timing and is conserved in resting cells

    PubMed Central

    Moindrot, Benoit; Audit, Benjamin; Klous, Petra; Baker, Antoine; Thermes, Claude; de Laat, Wouter; Bouvet, Philippe; Mongelard, Fabien; Arneodo, Alain

    2012-01-01

    Although chromatin folding is known to be of functional importance to control the gene expression program, less is known regarding its interplay with DNA replication. Here, using Circular Chromatin Conformation Capture combined with high-throughput sequencing, we identified megabase-sized self-interacting domains in the nucleus of a human lymphoblastoid cell line, as well as in cycling and resting peripheral blood mononuclear cells (PBMC). Strikingly, the boundaries of those domains coincide with early-initiation zones in every cell types. Preferential interactions have been observed between the consecutive early-initiation zones, but also between those separated by several tens of megabases. Thus, the 3D conformation of chromatin is strongly correlated with the replication timing along the whole chromosome. We furthermore provide direct clues that, in addition to the timing value per se, the shape of the timing profile at a given locus defines its set of genomic contacts. As this timing-related scheme of chromatin organization exists in lymphoblastoid cells, resting and cycling PBMC, this indicates that it is maintained several weeks or months after the previous S-phase. Lastly, our work highlights that the major chromatin changes accompanying PBMC entry into cell cycle occur while keeping largely unchanged the long-range chromatin contacts. PMID:22879376