WorldWideScience

Sample records for centromeric chromatin assembly

  1. Premitotic assembly of human CENPs -T and -W switches centromeric chromatin to a mitotic state.

    Directory of Open Access Journals (Sweden)

    Lisa Prendergast

    2011-06-01

    Full Text Available Centromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They are distinct genetic loci in that their identity in most organisms is determined not by the DNA sequences they are associated with, but through specific chromatin composition and context. The core nucleosomal protein CENP-A/cenH3 plays a primary role in centromere determination in all species and directs assembly of a large complex of associated proteins in vertebrates. While CENP-A itself is stably transmitted from one generation to the next, the nature of the template for centromere replication and its relationship to kinetochore function are as yet poorly understood. Here, we investigate the assembly and inheritance of a histone fold complex of the centromere, the CENP-T/W complex, which is integrated with centromeric chromatin in association with canonical histone H3 nucleosomes. We have investigated the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENPs -T and -W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations, properties reciprocal to those measured for CENP-A. We propose that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. Segregation of the assembly mechanisms for the two allows the cell to switch between chromatin configurations that reciprocally support the replication of the centromere and its conversion to a mitotic state on postreplicative chromatin.

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

    Science.gov (United States)

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

    2012-07-01

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

  3. Chromatin ring formation at plant centromeres

    Directory of Open Access Journals (Sweden)

    Veit eSchubert

    2016-02-01

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

  4. The cell cycle timing of centromeric chromatin assembly in Drosophila meiosis is distinct from mitosis yet requires CAL1 and CENP-C.

    Science.gov (United States)

    Dunleavy, Elaine M; Beier, Nicole L; Gorgescu, Walter; Tang, Jonathan; Costes, Sylvain V; Karpen, Gary H

    2012-01-01

    CENP-A (CID in flies) is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, we do not understand the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. Here we investigate the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila melanogaster, using fixed and live imaging combined with genetic approaches. We find that CID assembly initiates at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, we observe a novel decrease in CID levels after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. We also demonstrate that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, we show that the centromere proteins CAL1 and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. We conclude that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote.

  5. The cell cycle timing of centromeric chromatin assembly in Drosophila meiosis is distinct from mitosis yet requires CAL1 and CENP-C.

    Directory of Open Access Journals (Sweden)

    Elaine M Dunleavy

    Full Text Available CENP-A (CID in flies is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, we do not understand the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. Here we investigate the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila melanogaster, using fixed and live imaging combined with genetic approaches. We find that CID assembly initiates at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, we observe a novel decrease in CID levels after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. We also demonstrate that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, we show that the centromere proteins CAL1 and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. We conclude that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote.

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

    Directory of Open Access Journals (Sweden)

    Mickaël Durand-Dubief

    2012-09-01

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

  7. Chromatin assembly using Drosophila systems.

    Science.gov (United States)

    Fyodorov, Dmitry V; Levenstein, Mark E

    2002-05-01

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

  8. Assembly of Drosophila centromeric nucleosomes requires CID dimerization.

    Science.gov (United States)

    Zhang, Weiguo; Colmenares, Serafin U; Karpen, Gary H

    2012-01-27

    Centromeres are essential chromosomal regions required for kinetochore assembly and chromosome segregation. The composition and organization of centromeric nucleosomes containing the essential histone H3 variant CENP-A (CID in Drosophila) is a fundamental, unresolved issue. Using immunoprecipitation of CID mononucleosomes and cysteine crosslinking, we demonstrate that centromeric nucleosomes contain CID dimers in vivo. Furthermore, CID dimerization and centromeric targeting require a residue implicated in formation of the four-helix bundle, which mediates intranucleosomal H3 dimerization and nucleosome integrity. Taken together, our findings suggest that CID nucleosomes are octameric in vivo and that CID dimerization is essential for correct centromere assembly.

  9. Dynamic epigenetic states of maize centromeres

    Directory of Open Access Journals (Sweden)

    Yalin eLiu

    2015-10-01

    Full Text Available The centromere is a specialized chromosomal region identified as the major constriction, upon which the kinetochore complex is formed, ensuring accurate chromosome orientation and segregation during cell division. The rapid evolution of centromere DNA sequence and the conserved centromere function are two contradictory aspects of centromere biology. Indeed, the sole presence of genetic sequence is not sufficient for centromere formation. Various dicentric chromosomes with one inactive centromere have been recognized. It has also been found that de novo centromere formation is common on fragments in which centromeric DNA sequences are lost. Epigenetic factors play important roles in centromeric chromatin assembly and maintenance. Nondisjunction of the supernumerary B chromosome early prophase of meiosis I requires an active centromere. This review discusses recent studies in maize about genetic and epigenetic elements regulating formation and maintenance of centromere chromatin, as well as centromere behavior in meiosis.

  10. Rad51-Rad52 mediated maintenance of centromeric chromatin in Candida albicans.

    Directory of Open Access Journals (Sweden)

    Sreyoshi Mitra

    2014-04-01

    Full Text Available Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI proximal to an early replicating centromere (CEN7 in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-ACaCse4 levels at centromeres, as determined by chromatin immunoprecipitation (ChIP experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-ACaCse4 in vivo. Thus, the HR proteins Rad51 and Rad52

  11. Chromatin determinants of the inner-centromere rely on replication factors with functions that impart cohesion.

    Science.gov (United States)

    Abe, Takuya; Kawasumi, Ryotaro; Arakawa, Hiroshi; Hori, Tetsuya; Shirahige, Katsuhiko; Losada, Ana; Fukagawa, Tatsuo; Branzei, Dana

    2016-10-18

    Replication fork-associated factors promote genome integrity and protect against cancer. Mutations in the DDX11 helicase and the ESCO2 acetyltransferase also cause related developmental disorders classified as cohesinopathies. Here we generated vertebrate model cell lines of these disorders and cohesinopathies-related genes. We found that vertebrate DDX11 and Tim-Tipin are individually needed to compensate for ESCO2 loss in chromosome segregation, with DDX11 also playing complementary roles with ESCO2 in centromeric cohesion. Our study reveals that overt centromeric cohesion loss does not necessarily precede chromosome missegregation, while both these problems correlate with, and possibly originate from, inner-centromere defects involving reduced phosphorylation of histone H3T3 (pH3T3) in the region. Interestingly, the mitotic pH3T3 mark was defective in all analyzed replication-related mutants with functions in cohesion. The results pinpoint mitotic pH3T3 as a postreplicative chromatin mark that is sensitive to replication stress and conducts with different kinetics to robust centromeric cohesion and correct chromosome segregation.

  12. Switching the centromeres on and off: epigenetic chromatin alterations provide plasticity in centromere activity stabilizing aberrant dicentric chromosomes.

    Science.gov (United States)

    Sato, Hiroshi; Saitoh, Shigeaki

    2013-12-01

    The kinetochore, which forms on a specific chromosomal locus called the centromere, mediates interactions between the chromosome and the spindle during mitosis and meiosis. Abnormal chromosome rearrangements and/or neocentromere formation can cause the presence of multiple centromeres on a single chromosome, which results in chromosome breakage or cell cycle arrest. Analyses of artificial dicentric chromosomes suggested that the activity of the centromere is regulated epigenetically; on some stably maintained dicentric chromosomes, one of the centromeres no longer functions as a platform for kinetochore formation, although the DNA sequence remains intact. Such epigenetic centromere inactivation occurs in cells of various eukaryotes harbouring 'regional centromeres', such as those of maize, fission yeast and humans, suggesting that the position of the active centromere is determined by epigenetic markers on a chromosome rather than the nucleotide sequence. Our recent findings in fission yeast revealed that epigenetic centromere inactivation consists of two steps: disassembly of the kinetochore initiates inactivation and subsequent heterochromatinization prevents revival of the inactivated centromere. Kinetochore disassembly followed by heterochromatinization is also observed in normal senescent human cells. Thus epigenetic centromere inactivation may not only stabilize abnormally generated dicentric chromosomes, but also be part of an intrinsic mechanism regulating cell proliferation.

  13. Posttranslational modification of CENP-A influences the conformation of centromeric chromatin.

    Science.gov (United States)

    Bailey, Aaron O; Panchenko, Tanya; Sathyan, Kizhakke M; Petkowski, Janusz J; Pai, Pei-Jing; Bai, Dina L; Russell, David H; Macara, Ian G; Shabanowitz, Jeffrey; Hunt, Donald F; Black, Ben E; Foltz, Daniel R

    2013-07-16

    Centromeres are chromosomal loci required for accurate segregation of sister chromatids during mitosis. The location of the centromere on the chromosome is not dependent on DNA sequence, but rather it is epigenetically specified by the histone H3 variant centromere protein A (CENP-A). The N-terminal tail of CENP-A is highly divergent from other H3 variants. Canonical histone N termini are hotspots of conserved posttranslational modification; however, no broadly conserved modifications of the vertebrate CENP-A tail have been previously observed. Here, we report three posttranslational modifications on human CENP-A N termini using high-resolution MS: trimethylation of Gly1 and phosphorylation of Ser16 and Ser18. Our results demonstrate that CENP-A is subjected to constitutive initiating methionine removal, similar to other H3 variants. The nascent N-terminal residue Gly1 becomes trimethylated on the α-amino group. We demonstrate that the N-terminal RCC1 methyltransferase is capable of modifying the CENP-A N terminus. Methylation occurs in the prenucleosomal form and marks the majority of CENP-A nucleosomes. Serine 16 and 18 become phosphorylated in prenucleosomal CENP-A and are phosphorylated on asynchronous and mitotic nucleosomal CENP-A and are important for chromosome segregation during mitosis. The double phosphorylation motif forms a salt-bridged secondary structure and causes CENP-A N-terminal tails to form intramolecular associations. Analytical ultracentrifugation of phospho-mimetic CENP-A nucleosome arrays demonstrates that phosphorylation results in greater intranucleosome associations and counteracts the hyperoligomerized state exhibited by unmodified CENP-A nucleosome arrays. Our studies have revealed that the major modifications on the N-terminal tail of CENP-A alter the physical properties of the chromatin fiber at the centromere.

  14. A CENP-S/X complex assembles at the centromere in S and G2 phases of the human cell cycle.

    Science.gov (United States)

    Dornblut, Carsten; Quinn, Nadine; Monajambashi, Shamci; Prendergast, Lisa; van Vuuren, Chelly; Münch, Sandra; Deng, Wen; Leonhardt, Heinrich; Cardoso, M Cristina; Hoischen, Christian; Diekmann, Stephan; Sullivan, Kevin F

    2014-02-12

    The functional identity of centromeres arises from a set of specific nucleoprotein particle subunits of the centromeric chromatin fibre. These include CENP-A and histone H3 nucleosomes and a novel nucleosome-like complex of CENPs -T, -W, -S and -X. Fluorescence cross-correlation spectroscopy and Förster resonance energy transfer (FRET) revealed that human CENP-S and -X exist principally in complex in soluble form and retain proximity when assembled at centromeres. Conditional labelling experiments show that they both assemble de novo during S phase and G2, increasing approximately three- to fourfold in abundance at centromeres. Fluorescence recovery after photobleaching (FRAP) measurements documented steady-state exchange between soluble and assembled pools, with CENP-X exchanging approximately 10 times faster than CENP-S (t1/2 ∼ 10 min versus 120 min). CENP-S binding to sites of DNA damage was quite distinct, with a FRAP half-time of approximately 160 s. Fluorescent two-hybrid analysis identified CENP-T as a uniquely strong CENP-S binding protein and this association was confirmed by FRET, revealing a centromere-bound complex containing CENP-S, CENP-X and CENP-T in proximity to histone H3 but not CENP-A. We propose that deposition of the CENP-T/W/S/X particle reveals a kinetochore-specific chromatin assembly pathway that functions to switch centromeric chromatin to a mitosis-competent state after DNA replication. Centromeres shuttle between CENP-A-rich, replication-competent and H3-CENP-T/W/S/X-rich mitosis-competent compositions in the cell cycle.

  15. DNA Topoisomerase II Is a Determinant of the Tensile Properties of Yeast Centromeric Chromatin and the Tension Checkpoint

    OpenAIRE

    Warsi, Tariq H.; Navarro, Michelle S.; Bachant, Jeff

    2008-01-01

    Centromeric (CEN) chromatin is placed under mechanical tension and stretches as kinetochores biorient on the mitotic spindle. This deformation could conceivably provide a readout of biorientation to error correction mechanisms that monitor kinetochore–spindle interactions, but whether CEN chromatin acts in a tensiometer capacity is unresolved. Here, we report observations linking yeast Topoisomerase II (Top2) to both CEN mechanics and assessment of interkinetochore tension. First, in top2-4 a...

  16. Escape from Mitotic Arrest: An Unexpected Connection Between Microtubule Dynamics and Epigenetic Regulation of Centromeric Chromatin in Schizosaccharomyces pombe.

    Science.gov (United States)

    George, Anuja A; Walworth, Nancy C

    2015-12-01

    Accurate chromosome segregation is necessary to ensure genomic integrity. Segregation depends on the proper functioning of the centromere, kinetochore, and mitotic spindle microtubules and is monitored by the spindle assembly checkpoint (SAC). In the fission yeast Schizosaccharomyces pombe, defects in Dis1, a microtubule-associated protein that influences microtubule dynamics, lead to mitotic arrest as a result of an active SAC and consequent failure to grow at low temperature. In a mutant dis1 background (dis1-288), loss of function of Msc1, a fission yeast homolog of the KDM5 family of proteins, suppresses the growth defect and promotes normal mitosis. Genetic analysis implicates a histone deacetylase (HDAC)-linked pathway in suppression because HDAC mutants clr6-1, clr3∆, and sir2∆, though not hos2∆, also promote normal mitosis in the dis1-288 mutant. Suppression of the dis phenotype through loss of msc1 function requires the spindle checkpoint protein Mad2 and is limited by the presence of the heterochromatin-associated HP1 protein homolog Swi6. We speculate that alterations in histone acetylation promote a centromeric chromatin environment that compensates for compromised dis1 function by allowing for successful kinetochore-microtubule interactions that can satisfy the SAC. In cells arrested in mitosis by mutation of dis1, loss of function of epigenetic determinants such as Msc1 or specific HDACs can promote cell survival. Because the KDM5 family of proteins has been implicated in human cancers, an appreciation of the potential role of this family of proteins in chromosome segregation is warranted.

  17. Structure, Function, and Evolution of Rice Centromeres

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Jiming

    2010-02-04

    The centromere is the most characteristic landmark of eukaryotic chromosomes. Centromeres function as the site for kinetochore assembly and spindle attachment, allowing for the faithful pairing and segregation of sister chromatids during cell division. Characterization of centromeric DNA is not only essential to understand the structure and organization of plant genomes, but it is also a critical step in the development of plant artificial chromosomes. The centromeres of most model eukaryotic species, consist predominantly of long arrays of satellite DNA. Determining the precise DNA boundary of a centromere has proven to be a difficult task in multicellular eukaryotes. We have successfully cloned and sequenced the centromere of rice chromosome 8 (Cen8), representing the first fully sequenced centromere from any multicellular eukaryotes. The functional core of Cen8 spans ~800 kb of DNA, which was determined by chromatin immunoprecipitation (ChIP) using an antibody against the rice centromere-specific H3 histone. We discovered 16 actively transcribed genes distributed throughout the Cen8 region. In addition to Cen8, we have characterized eight additional rice centromeres using the next generation sequencing technology. We discovered four subfamilies of the CRR retrotransposon that is highly enriched in rice centromeres. CRR elements are constitutively transcribed and different CRR subfamilies are differentially processed by RNAi. These results suggest that different CRR subfamilies may play different roles in the RNAi-mediated pathway for formation and maintenance of centromeric chromatin.

  18. Nucleolar activity and CENP-C regulate CENP-A and CAL1 availability for centromere assembly in meiosis.

    Science.gov (United States)

    Kwenda, Lucretia; Collins, Caitriona M; Dattoli, Anna A; Dunleavy, Elaine M

    2016-04-15

    The centromere-specific histone CENP-A is the key epigenetic determinant of centromere identity. Whereas most histones are removed from mature sperm, CENP-A is retained to mark paternal centromeres. In Drosophila males we show that the centromere assembly factors CAL1 and CENP-C are required for meiotic chromosome segregation, CENP-A assembly and maintenance on sperm, as well as fertility. In meiosis, CENP-A accumulates with CAL1 in nucleoli. Furthermore, we show that CENP-C normally limits the release of CAL1 and CENP-A from nucleoli for proper centromere assembly in meiotic prophase I. Finally, we show that RNA polymerase I transcription is required for efficient CENP-A assembly in meiosis, as well as centromere tethering to nucleoli.

  19. Centromere domain organization and histone modifications

    Directory of Open Access Journals (Sweden)

    P. Bjerling

    2002-05-01

    Full Text Available Centromere function requires the proper coordination of several subfunctions, such as kinetochore assembly, sister chromatid cohesion, binding of kinetochore microtubules, orientation of sister kinetochores to opposite spindle poles, and their movement towards the spindle poles. Centromere structure appears to be organized in different, separable domains in order to accomplish these functions. Despite the conserved nature of centromere functions, the molecular genetic definition of the DNA sequences that form a centromere in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, in the fruit fly Drosophila melanogaster, and in humans has revealed little conservation at the level of centromere DNA sequences. Also at the protein level few centromere proteins are conserved in all of these four organisms and many are unique to the different organisms. The recent analysis of the centromere structure in the yeast S. pombe by electron microscopy and detailed immunofluorescence microscopy of Drosophila centromeres have brought to light striking similarities at the overall structural level between these centromeres and the human centromere. The structural organization of the centromere is generally multilayered with a heterochromatin domain and a central core/inner plate region, which harbors the outer plate structures of the kinetochore. It is becoming increasingly clear that the key factors for assembly and function of the centromere structure are the specialized histones and modified histones which are present in the centromeric heterochromatin and in the chromatin of the central core. Thus, despite the differences in the DNA sequences and the proteins that define a centromere, there is an overall structural similarity between centromeres in evolutionarily diverse eukaryotes.

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

    Science.gov (United States)

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

    2016-09-01

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

  1. DNA topoisomerase II is a determinant of the tensile properties of yeast centromeric chromatin and the tension checkpoint.

    Science.gov (United States)

    Warsi, Tariq H; Navarro, Michelle S; Bachant, Jeff

    2008-10-01

    Centromeric (CEN) chromatin is placed under mechanical tension and stretches as kinetochores biorient on the mitotic spindle. This deformation could conceivably provide a readout of biorientation to error correction mechanisms that monitor kinetochore-spindle interactions, but whether CEN chromatin acts in a tensiometer capacity is unresolved. Here, we report observations linking yeast Topoisomerase II (Top2) to both CEN mechanics and assessment of interkinetochore tension. First, in top2-4 and sumoylation-resistant top2-SNM mutants CEN chromatin stretches extensively during biorientation, resulting in increased sister kinetochore separation and preanaphase spindle extension. Our data indicate increased CEN stretching corresponds with alterations to CEN topology induced in response to tension. Second, Top2 potentiates aspects of the tension checkpoint. Mutations affecting the Mtw1 kinetochore protein activate Ipl1 kinase to detach kinetochores and induce spindle checkpoint arrest. In mtw1top2-4 and mtw1top2-SNM mutants, however, kinetochores are resistant to detachment and checkpoint arrest is attenuated. For top2-SNM cells, CEN stretching and checkpoint attenuation occur even in the absence of catenation linking sister chromatids. In sum, Top2 seems to play a novel role in CEN compaction that is distinct from decatenation. Perturbations to this function may allow weakened kinetochores to stretch CENs in a manner that mimics tension or evades Ipl1 surveillance.

  2. Chromosomes. CENP-C reshapes and stabilizes CENP-A nucleosomes at the centromere.

    Science.gov (United States)

    Falk, Samantha J; Guo, Lucie Y; Sekulic, Nikolina; Smoak, Evan M; Mani, Tomoyasu; Logsdon, Glennis A; Gupta, Kushol; Jansen, Lars E T; Van Duyne, Gregory D; Vinogradov, Sergei A; Lampson, Michael A; Black, Ben E

    2015-05-01

    Inheritance of each chromosome depends upon its centromere. A histone H3 variant, centromere protein A (CENP-A), is essential for epigenetically marking centromere location. We find that CENP-A is quantitatively retained at the centromere upon which it is initially assembled. CENP-C binds to CENP-A nucleosomes and is a prime candidate to stabilize centromeric chromatin. Using purified components, we find that CENP-C reshapes the octameric histone core of CENP-A nucleosomes, rigidifies both surface and internal nucleosome structure, and modulates terminal DNA to match the loose wrap that is found on native CENP-A nucleosomes at functional human centromeres. Thus, CENP-C affects nucleosome shape and dynamics in a manner analogous to allosteric regulation of enzymes. CENP-C depletion leads to rapid removal of CENP-A from centromeres, indicating their collaboration in maintaining centromere identity.

  3. Chromatin Assembly in a Yeast Whole-Cell Extract

    Science.gov (United States)

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

    1997-08-01

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

  4. Centromere binding specificity in assembly of the F plasmid partition complex

    OpenAIRE

    2011-01-01

    The segregation of plasmid F of Escherichia coli is highly reliable. The Sop partition locus, responsible for this stable maintenance, is composed of two genes, sopA and sopB and a centromere, sopC, consisting of 12 direct repeats of 43 bp. Each repeat carries a 16-bp inverted repeat motif to which SopB binds to form a nucleoprotein assembly called the partition complex. A database search for sequences closely related to sopC revealed unexpected features that appeared highly conserved. We hav...

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

    Science.gov (United States)

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

    2015-11-20

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

  6. Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions

    Science.gov (United States)

    Burton, Joshua N.; Adey, Andrew; Patwardhan, Rupali P.; Qiu, Ruolan; Kitzman, Jacob O.; Shendure, Jay

    2014-01-01

    Genomes assembled de novo from short reads are highly fragmented relative to the finished chromosomes of H. sapiens and key model organisms generated by the Human Genome Project. To address this, we need scalable, cost-effective methods enabling chromosome-scale contiguity. Here we show that genome-wide chromatin interaction datasets, such as those generated by Hi-C, are a rich source of long-range information for assigning, ordering and orienting genomic sequences to chromosomes, including across centromeres. To exploit this, we developed an algorithm that uses Hi-C data for ultra-long-range scaffolding of de novo genome assemblies. We demonstrate the approach by combining shotgun fragment and short jump mate-pair sequences with Hi-C data to generate chromosome-scale de novo assemblies of the human, mouse and Drosophila genomes, achieving – for human – 98% accuracy in assigning scaffolds to chromosome groups and 99% accuracy in ordering and orienting scaffolds within chromosome groups. Hi-C data can also be used to validate chromosomal translocations in cancer genomes. PMID:24185095

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

    Science.gov (United States)

    Keck, Kristin M; Pemberton, Lucy F

    2013-01-01

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

  8. Structure and function of centromeric and pericentromeric heterochromatin in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Lauriane eSimon

    2015-11-01

    Full Text Available The centromere is a specific chromosomal region where the kinetochore assembles to ensure the faithful segregation of sister chromatids during mitosis and meiosis. Centromeres are defined by a local enrichment of the specific histone variant CenH3 mostly at repetitive satellite sequences. A larger pericentromeric region containing repetitive sequences and transposable elements surrounds the centromere that adopts a particular chromatin state characterized by specific histone variants and post-translational modifications and forms a transcriptionally repressive chromosomal environment. In the model organism Arabidopsis thaliana centromeric and pericentromeric domains form conspicuous heterochromatin clusters called chromocenters in interphase. Here we discuss, using Arabidopsis as example, recent insight into mechanisms involved in maintenance and establishment of centromeric and pericentromeric chromatin signatures as well as in chromocenter formation.

  9. High-resolution mapping and transcriptional activity analysis of chicken centromere sequences on giant lampbrush chromosomes.

    Science.gov (United States)

    Krasikova, Alla; Fukagawa, Tatsuo; Zlotina, Anna

    2012-12-01

    Exploration into morphofunctional organisation of centromere DNA sequences is important for understanding the mechanisms of kinetochore specification and assembly. In-depth epigenetic analysis of DNA fragments associated with centromeric nucleosome proteins has demonstrated unique features of centromere organisation in chicken karyotype: there are both mature centromeres, which comprise chromosome-specific homogeneous arrays of tandem repeats, and recently evolved primitive centromeres, which consist of non-tandemly organised DNA sequences. In this work, we describe the arrangement and transcriptional activity of chicken centromere repeats for Cen1, Cen2, Cen3, Cen4, Cen7, Cen8, and Cen11 and non-repetitive centromere sequences of chromosomes 5, 27, and Z using highly elongated lampbrush chromosomes, which are characteristic of the diplotene stage of oogenesis. The degree of chromatin packaging and fine spatial organisations of tandemly repetitive and non-tandemly repetitive centromeric sequences significantly differ at the lampbrush stage. Using DNA/RNA FISH, we have demonstrated that during the lampbrush stage, DNA sequences are transcribed within the centromere regions of chromosomes that lack centromere-specific tandem repeats. In contrast, chromosome-specific centromeric repeats Cen1, Cen2, Cen3, Cen4, Cen7, Cen8, and Cen11 do not demonstrate any transcriptional activity during the lampbrush stage. In addition, we found that CNM repeat cluster localises adjacent to non-repetitive centromeric sequences in chicken microchromosome 27 indicating that centromere region in this chromosome is repeat-rich. Cross-species FISH allowed localisation of the sequences homologous to centromeric DNA of chicken chromosomes 5 and 27 in centromere regions of quail orthologous chromosomes.

  10. Overexpression of LSD1 contributes to human carcinogenesis through chromatin regulation in various cancers.

    Science.gov (United States)

    Hayami, Shinya; Kelly, John D; Cho, Hyun-Soo; Yoshimatsu, Masanori; Unoki, Motoko; Tsunoda, Tatsuhiko; Field, Helen I; Neal, David E; Yamaue, Hiroki; Ponder, Bruce A J; Nakamura, Yusuke; Hamamoto, Ryuji

    2011-02-01

    A number of histone demethylases have been identified and biochemically characterized, but the pathological roles of their dysfunction in human disease like cancer have not been well understood. Here, we demonstrate important roles of lysine-specific demethylase 1 (LSD1) in human carcinogenesis. Expression levels of LSD1 are significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (p human embryonic kidney fibroblast cells. Expression profile analysis showed that LSD1 could affect the expression of genes involved in various chromatin-modifying pathways such as chromatin remodeling at centromere, centromeric heterochromatin formation and chromatin assembly, indicating its essential roles in carcinogenesis through chromatin modification.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  13. Identification of Drosophila centromere associated proteins by quantitative affinity purification-mass spectrometry

    Science.gov (United States)

    Barth, Teresa K.; Schade, Georg O.M.; Schmidt, Andreas; Vetter, Irene; Wirth, Marc; Heun, Patrick; Imhof, Axel; Thomae, Andreas W.

    2015-01-01

    Centromeres of higher eukaryotes are epigenetically defined by the centromere specific histone H3 variant CENP-ACID. CENP-ACID builds the foundation for the assembly of a large network of proteins. In contrast to mammalian systems, the protein composition of Drosophila centromeres has not been comprehensively investigated. Here we describe the proteome of Drosophila melanogaster centromeres as analyzed by quantitative affinity purification-mass spectrometry (AP-MS). The AP-MS input chromatin material was prepared from D. melanogaster cell lines expressing CENP-ACID or H3.3 fused to EGFP as baits. Centromere chromatin enriched proteins were identified based on their relative abundance in CENP-ACID–GFP compared to H3.3-GFP or mock affinity-purifications. The analysis yielded 86 proteins specifically enriched in centromere chromatin preparations. The data accompanying the manuscript on this approach (Barth et al., 2015, Proteomics 14:2167-78, DOI: 10.1002/pmic.201400052) has been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000758. PMID:26306323

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Davide F V Corona

    2007-09-01

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

  16. Data on the kinetics of in vitro assembled chromatin

    Directory of Open Access Journals (Sweden)

    Moritz Carl Völker-Albert

    2016-09-01

    The data accompanying the manuscript on this approach, Völker-Albert et al., 2016 “A quantitative proteomic analysis of in vitro assembled chromatin” [1], has been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org via the PRIDE partner repository with the dataset identifier submission number PRIDE: PXD002537 and PRIDE: PXD003445.

  17. Chromatin remodeling complexes in the assembly of long noncoding RNA-dependent nuclear bodies.

    Science.gov (United States)

    Kawaguchi, Tetsuya; Hirose, Tetsuro

    2015-01-01

    Paraspeckles are subnuclear structures that assemble on nuclear paraspeckle assembly transcript 1 (NEAT1) long noncoding (lnc)RNA. Paraspeckle formation requires appropriate NEAT1 biogenesis and subsequent assembly with multiple prion-like domain (PLD) containing RNA-binding proteins. We found that SWI/SNF chromatin remodeling complexes function as paraspeckle components that interact with paraspeckle proteins (PSPs) and NEAT1. SWI/SNF complexes play an essential role in paraspeckle formation that does not require their ATP-dependent chromatin remodeling activity. Instead, SWI/SNF complexes facilitate organization of the PSP interaction network required for intact paraspeckle assembly. SWI/SNF complexes may collectively bind multiple PSPs to recruit them onto NEAT1. SWI/SNF complexes are also required for Sat III (Satellite III) lncRNA-dependent formation of nuclear stress bodies under heat shock conditions. Organization of the lncRNA-dependent omega speckle in Drosophila also depends on the chromatin remodeling complex. These findings raise the possibility that a common mechanism controls the formation of lncRNA-dependent nuclear body architecture.

  18. Replication-coupled chromatin assembly of newly synthesized histones: distinct functions for the histone tail domains.

    Science.gov (United States)

    Ejlassi-Lassallette, Aïda; Thiriet, Christophe

    2012-02-01

    The maintenance of the genome during replication requires the assembly of nucleosomes with newly synthesized histones. Achieving the deposition of newly synthesized histones in chromatin implies their transport from the cytoplasm to the nucleus at the replication sites. Several lines of evidence have revealed critical functions of the histone tail domains in these conserved cellular processes. In this review, we discuss the role of the amino termini of the nucleosome building blocks, H2A/H2B and H3/H4, in different model systems. The experimental data showed that H2A/H2B tails and H3/H4 tails display distinct functions in nuclear import and chromatin assembly. Furthermore, we describe recent studies exploiting the unique properties of the slime mold, Physarum polycephalum , that have advanced understanding of the function of the highly conserved replication-dependent diacetylation of H4.

  19. Chromatin-bound NLS proteins recruit membrane vesicles and nucleoporins for nuclear envelope assembly via importin-α/β

    Institute of Scientific and Technical Information of China (English)

    Quanlong Lu; Zhigang Lu; Qinying Liu; Li Guo; He Ren; Jingyan Fu; Qing Jiang; Paul R Clarke; Chuanmao Zhang

    2012-01-01

    The mechanism for nuclear envelope (NE) assembly is not fully understood.Importin-β and the small GTPase Ran have been implicated in the spatial regulation of NE assembly process.Here we report that chromatin-bound NLS (nuclear localization sequence) proteins provide docking sites for the NE precursor membrane vesicles and nucleoporins via importin-α and -β during NE assembly in Xenopus egg extracts.We show that along with the fast recruitment of the abundant NLS proteins such as nucleoplasmin and histones to the demembranated sperm chromatin in the extracts,importin-α binds the chromatin NLS proteins rapidly.Meanwhile,importin-β binds cytoplasmic NE precursor membrane vesicles and nucleoporins.Through interacting with importin-α on the chromatin NLS proteins,importin-β targets the membrane vesicles and nucleoporins to the chromatin surface.Once encountering RanGTP on the chromatin generated by RCC1,importin-β preferentially binds Ran-GTP and releases the membrane vesicles and nucleoporins for NE assembly.NE assembly is disrupted by blocking the interaction between importin-α and NLS proteins with excess soluble NLS proteins or by depletion of importin-β from the extract.Our findings reveal a novel molecular mechanism for NE assembly in Xenopus egg extracts.

  20. Synaptonemal complex components persist at centromeres and are required for homologous centromere pairing in mouse spermatocytes.

    Directory of Open Access Journals (Sweden)

    C Gaston Bisig

    2012-06-01

    Full Text Available Recent studies in simple model organisms have shown that centromere pairing is important for ensuring high-fidelity meiotic chromosome segregation. However, this process and the mechanisms regulating it in higher eukaryotes are unknown. Here we present the first detailed study of meiotic centromere pairing in mouse spermatogenesis and link it with key events of the G2/metaphase I transition. In mouse we observed no evidence of the persistent coupling of centromeres that has been observed in several model organisms. We do however find that telomeres associate in non-homologous pairs or small groups in B type spermatogonia and pre-leptotene spermatocytes, and this association is disrupted by deletion of the synaptonemal complex component SYCP3. Intriguingly, we found that, in mid prophase, chromosome synapsis is not initiated at centromeres, and centromeric regions are the last to pair in the zygotene-pachytene transition. In late prophase, we first identified the proteins that reside at paired centromeres. We found that components of the central and lateral element and transverse filaments of the synaptonemal complex are retained at paired centromeres after disassembly of the synaptonemal complex along diplotene chromosome arms. The absence of SYCP1 prevents centromere pairing in knockout mouse spermatocytes. The localization dynamics of SYCP1 and SYCP3 suggest that they play different roles in promoting homologous centromere pairing. SYCP1 remains only at paired centromeres coincident with the time at which some kinetochore proteins begin loading at centromeres, consistent with a role in assembly of meiosis-specific kinetochores. After removal of SYCP1 from centromeres, SYCP3 then accumulates at paired centromeres where it may promote bi-orientation of homologous centromeres. We propose that, in addition to their roles as synaptonemal complex components, SYCP1 and SYCP3 act at the centromeres to promote the establishment and/or maintenance of

  1. Testing Whether Defective Chromatin Assembly in S-Phase Contributes to Breast Cancer

    Science.gov (United States)

    2005-10-01

    TACTTGTACC) was designed using the approach described by Dr. les (Phair and Misteli, 2000) and Cajal bodies (Dundr et Greg Hannon. al., 2004) and with...machin- K.B., Meier, U.T., Neugebauer, K.M., Matera, A.G., and Misteli, T. ery. Genes Dev. 16, 2621-2626. (2004). In vivo kinetics of Cajal body components...years of starting my lab has been crucial. Body . The initial goal of this application was to investigate whether defective chromatin assembly in S-phase

  2. Euchromatic subdomains in rice centromeres are associated with genes and transcription.

    Science.gov (United States)

    Wu, Yufeng; Kikuchi, Shinji; Yan, Huihuang; Zhang, Wenli; Rosenbaum, Heidi; Iniguez, A Leonardo; Jiang, Jiming

    2011-11-01

    The presence of the centromere-specific histone H3 variant, CENH3, defines centromeric (CEN) chromatin, but poorly understood epigenetic mechanisms determine its establishment and maintenance. CEN chromatin is embedded within pericentromeric heterochromatin in most higher eukaryotes, but, interestingly, it can show euchromatic characteristics; for example, the euchromatic histone modification mark dimethylated H3 Lys 4 (H3K4me2) is uniquely associated with animal centromeres. To examine the histone marks and chromatin properties of plant centromeres, we developed a genomic tiling array for four fully sequenced rice (Oryza sativa) centromeres and used chromatin immunoprecipitation-chip to study the patterns of four euchromatic histone modification marks: H3K4me2, trimethylated H3 Lys 4, trimethylated H3 Lys 36, and acetylated H3 Lys 4, 9. The vast majority of the four histone marks were associated with genes located in the H3 subdomains within the centromere cores. We demonstrate that H3K4me2 is not a ubiquitous component of rice CEN chromatin, and the euchromatic characteristics of rice CEN chromatin are hallmarks of the transcribed sequences embedded in the centromeric H3 subdomains. We propose that the transcribed sequences located in rice centromeres may provide a barrier preventing loading of CENH3 into the H3 subdomains. The separation of CENH3 and H3 subdomains in the centromere core may be favorable for the formation of three-dimensional centromere structure and for rice centromere function.

  3. Strong nucleosomes of A. thaliana concentrate in centromere regions.

    Science.gov (United States)

    Salih, Bilal; Trifonov, Edward N

    2015-01-01

    Earlier identified strongest nucleosome DNA sequences of A. thaliana, those with visible 10-11 base sequence periodicity, are mapped along chromosomes. Resulting positional distributions reveal distinct maxima, one per chromosome, located in the centromere regions. Sequence-directed nucleosome mapping demonstrates that the strong nucleosomes (SNs) make tight arrays, several 'parallel' nucleosomes each, suggesting a columnar chromatin structure. The SNs represent a new class of centromeric nucleosomes, presumably, participating in synapsis of chromatids and securing the centromere architecture.

  4. Atypical centromeres in plants – what they can tell us

    Directory of Open Access Journals (Sweden)

    Maria eCuacos

    2015-10-01

    Full Text Available The centromere, visible as the primary constriction of condensed metaphase chromosomes, is a defined chromosomal locus essential for genome stability. It mediates transient assembly of a multi-protein complex, the kinetochore, which enables interaction with spindle fibers and thus faithful segregation of the genetic information during nuclear divisions. Centromeric DNA varies in extent and sequence composition among organisms, but a common feature of almost all active eukaryotic centromeres is the presence of the centromeric histone H3 variant cenH3 (a.k.a. CENP-A.These typical centromere features apply to most studied species. However, a number of species display atypical centromeres, such as holocentromeres (centromere extension along almost the entire chromatid length or neocentromeres (ectopic centromere activity.In this review, we provide an overview of different atypical centromere types found in plants including holocentromeres, de novo formed centromeres and terminal neocentromeres as well as di-, tri- and metapolycentromeres (more than one centromere per chromosomes. We discuss their specific and common features and compare them to centromere types found in other eukaryotic species. We also highlight new insights into centromere biology gained in plants with atypical centromeres such as distinct mechanisms to define a holocentromere, specific adaptations in species with holocentromeres during meiosis or various scenarios leading to neocentromere formation.

  5. Domain architectures of the Scm3p protein provide insights into centromere function and evolution

    OpenAIRE

    Aravind, L.; Lakshminarayan, M. Iyer; Wu, Carl

    2007-01-01

    Recently, Scm3p has been shown to be a nonhistone component of centromeric chromatin that binds stoichiometrically to CenH3–H4 histones, and to be required for the assembly of kinetochores in S. cerevisiae. Scm3p is conserved across fungi, and displays a remarkable variation in protein size, ranging from ~200 amino acids in Saccharomyces cerevisiae to ~1300 amino acids in Neurospora crassa. This is primarily due a variable C-terminal segment that is linked to a conserved N-terminal, CenH3-int...

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

    OpenAIRE

    2002-01-01

    The chromatin loop model predicts that genes within the same chromatin domain exhibit coordinated regulation. We here present the first direct experimental support for this model in plants. Two reporter genes, the E. coli beta-glucuronidase gene and the firefly luciferase gene, driven by different promoters, were placed between copies of the chicken lysozyme A element, a member of the matrix-associated region (MAR) group of chromatin boundary elements, and introduced in tobacco (Nicotiana tab...

  7. The microtubule aster formation and its role in nuclear envelope assembly around the sperm chromatin in Xenopus egg extracts

    Institute of Scientific and Technical Information of China (English)

    YANG Ning; CHEN Zhongcai; LU Ping; ZHANG Chuanmao; ZHAI Zhonghe; TANG Xiaowei

    2003-01-01

    Nuclear envelope is a dynamic structure in the cell cycle. At the beginning of mitosis, nuclear envelope breaks down and its components disperse into the cytoplasm. At the end of mitosis, nuclear envelope reassembles using the dispersed components. Searching for the mechanisms of the nuclear disassembly and reassembly has for a long time been one of the key projects for cell biologists. In this report we show that microtubules take a role in the nuclear envelope assembly around the sperm chromatin in Xenopus egg extracts. Microtubule cytoskeleton has been demonstrated to take roles in the transport of intracellular membranes such as Golgi and ER vesicles. We found that the nuclear envelope assembly needs functional microtubules. At the beginning of the nuclear assembly, microtubules nucleated to form a microtubule aster around the centrosome at the base of the sperm head. Using the microtubule drug colchicine to disrupt the microtubule nucleation, nuclear envelope reassembly was seriously inhibited. If the microtubules were stabilized by taxol, another microtubule drug, the nuclear envelope reassembly was also interfered, although a significantly large aster formed around the chromatin. Based on these observations, we propose that microtubules play an important role in the nuclear envelope reassembly maybe by transporting the nuclear envelope precursors to the chromatin surfaces.

  8. Identification of novel Drosophila centromere-associated proteins.

    Science.gov (United States)

    Barth, Teresa K; Schade, Georg O M; Schmidt, Andreas; Vetter, Irene; Wirth, Marc; Heun, Patrick; Thomae, Andreas W; Imhof, Axel

    2014-10-01

    Centromeres are chromosomal regions crucial for correct chromosome segregation during mitosis and meiosis. They are epigenetically defined by centromeric proteins such as the centromere-specific histone H3-variant centromere protein A (CENP-A). In humans, 16 additional proteins have been described to be constitutively associated with centromeres throughout the cell cycle, known as the constitutive centromere-associated network (CCAN). In contrast, only one additional constitutive centromeric protein is known in Drosophila melanogaster (D.mel), the conserved CCAN member CENP-C. To gain further insights into D.mel centromere composition and biology, we analyzed affinity-purified chromatin prepared from D.mel cell lines expressing green fluorescent protein tagged histone three variants by MS. In addition to already-known centromeric proteins, we identified novel factors that were repeatedly enriched in affinity purification-MS experiments. We analyzed the cellular localization of selected candidates by immunocytochemistry and confirmed localization to the centromere and other genomic regions for ten factors. Furthermore, RNA interference mediated depletion of CG2051, CG14480, and hyperplastic discs, three of our strongest candidates, leads to elevated mitotic defects. Knockdowns of these candidates neither impair the localization of several known kinetochore proteins nor CENP-A(CID) loading, suggesting their involvement in alternative pathways that contribute to proper centromere function. In summary, we provide a comprehensive analysis of the proteomic composition of Drosophila centromeres. All MS data have been deposited in the ProteomeXchange with identifier PXD000758 (http://proteomecentral.proteomexchange.org/dataset/PXD000758).

  9. The differential mobilization of histones H3.1 and H3.3 by herpes simplex virus 1 relates histone dynamics to the assembly of viral chromatin.

    Science.gov (United States)

    Conn, Kristen L; Hendzel, Michael J; Schang, Luis M

    2013-01-01

    During lytic infections, HSV-1 genomes are assembled into unstable nucleosomes. The histones required for HSV-1 chromatin assembly, however, are in the cellular chromatin. We have shown that linker (H1) and core (H2B and H4) histones are mobilized during HSV-1 infection, and proposed that the mobilized histones are available for assembly into viral chromatin. However, the actual relevance of histone mobilization remained unknown. We now show that canonical H3.1 and variant H3.3 are also mobilized during HSV-1 infection. Mobilization required no HSV-1 protein expression, although immediate early or early proteins enhanced it. We used the previously known differential association of H3.3 and H3.1 with HSV-1 DNA to test the relevance of histone mobilization. H3.3 binds to HSV-1 genomes first, whereas H3.1 only binds after HSV-1 DNA replication initiates. Consistently, H3.3 and H3.1 were differentially mobilized. H3.1 mobilization decreased with HSV-1 DNA replication, whereas H3.3 mobilization was largely unaffected by it. These results support a model in which previously mobilized H3.1 is immobilized by assembly into viral chromatin during HSV-1 DNA replication, whereas H3.3 is mobilized and assembled into HSV-1 chromatin throughout infection. The differential mobilizations of H3.3 and H3.1 are consistent with their differential assembly into viral chromatin. These data therefore relate nuclear histone dynamics to the composition of viral chromatin and provide the first evidence that histone mobilization relates to viral chromatin assembly.

  10. Centromere architecture breakdown induced by the viral E3 ubiquitin ligase ICP0 protein of herpes simplex virus type 1.

    Directory of Open Access Journals (Sweden)

    Sylvain Gross

    Full Text Available The viral E3 ubiquitin ligase ICP0 protein has the unique property to temporarily localize at interphase and mitotic centromeres early after infection of cells by the herpes simplex virus type 1 (HSV-1. As a consequence ICP0 induces the proteasomal degradation of several centromeric proteins (CENPs, namely CENP-A, the centromeric histone H3 variant, CENP-B and CENP-C. Following ICP0-induced centromere modification cells trigger a specific response to centromeres called interphase Centromere Damage Response (iCDR. The biological significance of the iCDR is unknown; so is the degree of centromere structural damage induced by ICP0. Interphase centromeres are complex structures made of proximal and distal protein layers closely associated to CENP-A-containing centromeric chromatin. Using several cell lines constitutively expressing GFP-tagged CENPs, we investigated the extent of the centromere destabilization induced by ICP0. We show that ICP0 provokes the disappearance from centromeres, and the proteasomal degradation of several CENPs from the NAC (CENP-A nucleosome associated and CAD (CENP-A Distal complexes. We then investigated the nucleosomal occupancy of the centromeric chromatin in ICP0-expressing cells by micrococcal nuclease (MNase digestion analysis. ICP0 expression either following infection or in cell lines constitutively expressing ICP0 provokes significant modifications of the centromeric chromatin structure resulting in higher MNase accessibility. Finally, using human artificial chromosomes (HACs, we established that ICP0-induced iCDR could also target exogenous centromeres. These results demonstrate that, in addition to the protein complexes, ICP0 also destabilizes the centromeric chromatin resulting in the complete breakdown of the centromere architecture, which consequently induces iCDR.

  11. ATRX in chromatin assembly and genome architecture during development and disease.

    Science.gov (United States)

    Bérubé, Nathalie G

    2011-10-01

    The regulation of genome architecture is essential for a variety of fundamental cellular phenomena that underlie the complex orchestration of mammalian development. The ATP-dependent chromatin remodeling protein ATRX is emerging as a key regulatory component of nucleosomal dynamics and higher order chromatin conformation. Here we provide an overview of the role of ATRX at chromatin and during development, and discuss recent studies exposing a repertoire of ATRX functions at heterochromatin, in gene regulation, and during mitosis and meiosis. Exciting new progress on several fronts suggest that ATRX operates in histone variant deposition and in the modulation of higher order chromatin structure. Not surprisingly, dysfunction or absence of ATRX protein has devastating consequences on embryonic development and leads to human disease.

  12. Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA.

    Science.gov (United States)

    Sirbu, Bianca M; Couch, Frank B; Cortez, David

    2012-03-01

    Understanding the processes of DNA replication, chromatin assembly and maturation, and the replication stress response requires the ability to monitor protein dynamics at active and damaged replication forks. Detecting protein accumulation at replication forks or damaged sites has primarily relied on immunofluorescence imaging, which is limited in resolution and antibody sensitivity. Here we describe a procedure to isolate proteins on nascent DNA (iPOND) that permits a high-resolution spatiotemporal analysis of proteins at replication forks or on chromatin following DNA replication in cultured cells. iPOND relies on labeling of nascent DNA with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). Biotin conjugation to EdU-labeled DNA using click chemistry facilitates a single-step streptavidin purification of proteins bound to the nascent DNA. iPOND permits an interrogation of any cellular process linked to DNA synthesis using a 3- to 4-d protocol.

  13. The Past, Present, and Future of Human Centromere Genomics

    Directory of Open Access Journals (Sweden)

    Megan E. Aldrup-MacDonald

    2014-01-01

    Full Text Available The centromere is the chromosomal locus essential for chromosome inheritance and genome stability. Human centromeres are located at repetitive alpha satellite DNA arrays that compose approximately 5% of the genome. Contiguous alpha satellite DNA sequence is absent from the assembled reference genome, limiting current understanding of centromere organization and function. Here, we review the progress in centromere genomics spanning the discovery of the sequence to its molecular characterization and the work done during the Human Genome Project era to elucidate alpha satellite structure and sequence variation. We discuss exciting recent advances in alpha satellite sequence assembly that have provided important insight into the abundance and complex organization of this sequence on human chromosomes. In light of these new findings, we offer perspectives for future studies of human centromere assembly and function.

  14. Three wise centromere functions: see no error, hear no break, speak no delay.

    OpenAIRE

    Tanaka, Tomoyuki U; Clayton, Lesley; Natsume, Toyoaki

    2013-01-01

    The main function of the centromere is to promote kinetochore assembly for spindle microtubule attachment. Two additional functions of the centromere, however, are becoming increasingly clear: facilitation of robust sister-chromatid cohesion at pericentromeres and advancement of replication of centromeric regions. The combination of these three centromere functions ensures correct chromosome segregation during mitosis. Here, we review the mechanisms of the kinetochore–microtubule interaction,...

  15. 植物着丝粒结构及进化的研究进展%Research Progress on Structure and Evolution of Plant Centromeres

    Institute of Scientific and Technical Information of China (English)

    刘青

    2015-01-01

    植物着丝粒是染色体重要结构域,介导动粒装配。不同物种间着丝粒重复序列快速趋异进化,着丝粒功能保守,确保有丝分裂和减数分裂过程中染色体正确分离和准确传递。伴随染色质免疫共沉淀技术(Chromatin immunoprecipitation, ChIP)、ChIP与高密度芯片相结合技术(ChIP-chip)、ChIP与高通量测序相结合技术(ChIP-seq)的应用,植物着丝粒研究获得里程碑式进展:某些模式植物着丝粒DNA序列、蛋白质结构、功能获得大量新认识;着丝粒基本蛋白质组蛋白H3被用来界定着丝粒大小和边界;某些非着丝粒区域被激活为新着丝粒,在世代传递中保持稳定性。本文对植物着丝粒结构、功能、进化研究进行了综述,并探讨了植物着丝粒研究存在的问题。%The plant centromere is the most important chromosome domain mediating the assembly of kinetochore. The rapid divergent evolution of centromeric repeat sequences and function conservation of centromeres among different species ensure correct segregation and faithful transmission of chromosome in mitosis and meiosis. Along with the development of chromatin immunoprecipitation (ChIP), ChIP-chip, and ChIP-sequencing (ChIP-seq) technologies, three milestone discoveries have achieved in plant centromere research since the last 20 years, such as a lot of new knowledge on the structure, function, and evolution of centromeres from model plants, the fundamental kinetochore protein CENH3 used to delimiting the size and boundaries of centromere, the neocentromeres activated from non-centromeric regions stably transmitted to subsequent generations. The research progress on structure, function, and evolution of plant centromeres are reviewed and the remaining questions of plant centromere studies are discussed.

  16. Tissue Microarray-Based Evaluation of Chromatin Assembly Factor-1 (CAF-1/p60 as Tumour Prognostic Marker

    Directory of Open Access Journals (Sweden)

    Stefania Staibano

    2012-09-01

    Full Text Available In this study we aimed to confirm the emerging role of Chromatin Assembly Factor 1 (CAF-1 p60 as a new proliferation and prognostic marker for cancer and to test the usefulness of the tissue microarray technique (TMA for CAF-1 p60 rapid screening in several human malignancies. CAF-1 is a histone chaperone, regulating chromatin dynamics during DNA replication and repair in eukaryotics. TMA is a powerful high-throughput methodology in the study of cancer, allowing simultaneous assessment of different biomarkers within large numbers of tissue specimens. We generated TMA taking 3 mm diameter-core biopsies from oral squamous cell carcinoma, prostate cancer, salivary gland tumours and skin melanoma specimens, which had been previously tested for CAF-1 p60 on routine tissue sections. We also analysed, for the first time, 30 larynx and 30 skin squamous cell carcinomas. CAF-1 p60 resulted over-expressed in both the tissue sections and the TMA specimens, with the highest levels of expression in tumours which were more aggressive and metastasizing. Notably, a high degree of agreement was found between the CAF-1 p60 assessment on TMAs and on routine tissue sections. Our findings confirm the prognostic role of CAF-1 p60 and indicate TMA as a really advantageous method for CAF-1 p60 immunohistochemical screening, allowing savings on both tissue quantity and operator-time.

  17. A dual inhibitory mechanism sufficient to maintain cell cycle restricted CENP-A assembly

    Science.gov (United States)

    Stankovic, Ana; Guo, Lucie Y.; Mata, João F.; Bodor, Dani L.; Cao, Xing-Jun; Bailey, Aaron O.; Shabanowitz, Jeffrey; Hunt, Donald F.; Garcia, Benjamin A.; Black, Ben E.; Jansen, Lars E.T

    2017-01-01

    Summary Chromatin featuring the H3 variant CENP-A at the centromere is critical for its mitotic function and epigenetic maintenance. Assembly of centromeric chromatin is restricted to G1 phase through inhibitory action of Cdk1/2 kinases in other phases of the cell cycle. Here, we identify the two key targets sufficient to maintain cell cycle control of CENP-A assembly. We uncovered a single phosphorylation site in the licensing factor M18BP1 and a cyclin A binding site in the CENP-A chaperone, HJURP, mediating specific inhibitory phosphorylation. Simultaneous expression of mutant proteins lacking these residues, results in complete uncoupling from the cell cycle. Consequently, CENP-A assembly is fully recapitulated under high Cdk activities, indistinguishable from G1 assembly. We find that Cdk-mediated inhibition is exerted by sequestering active factors away from the centromere. Finally, we show that displacement of M18BP1 from the centromere is critical for the assembly mechanism of CENP-A. PMID:28017591

  18. Three wise centromere functions: see no error, hear no break, speak no delay.

    Science.gov (United States)

    Tanaka, Tomoyuki U; Clayton, Lesley; Natsume, Toyoaki

    2013-12-01

    The main function of the centromere is to promote kinetochore assembly for spindle microtubule attachment. Two additional functions of the centromere, however, are becoming increasingly clear: facilitation of robust sister-chromatid cohesion at pericentromeres and advancement of replication of centromeric regions. The combination of these three centromere functions ensures correct chromosome segregation during mitosis. Here, we review the mechanisms of the kinetochore-microtubule interaction, focusing on sister-kinetochore bi-orientation (or chromosome bi-orientation). We also discuss the biological importance of robust pericentromeric cohesion and early centromere replication, as well as the mechanisms orchestrating these two functions at the microtubule attachment site.

  19. Chromatin Structure and Function

    CERN Document Server

    Wolffe, Alan P

    1999-01-01

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

  20. Chromatin replication and epigenome maintenance

    DEFF Research Database (Denmark)

    Alabert, Constance; Groth, Anja

    2012-01-01

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

  1. Chromatin replication and epigenome maintenance

    DEFF Research Database (Denmark)

    Alabert, Constance; Groth, Anja

    2012-01-01

    Stability and function of eukaryotic genomes are closely linked to chromatin structure and organization. During cell division the entire genome must be accurately replicated and the chromatin landscape reproduced on new DNA. Chromatin and nuclear structure influence where and when DNA replication...... initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA...

  2. Tetrameric structure of centromeric nucleosomes in interphase Drosophila cells.

    Directory of Open Access Journals (Sweden)

    Yamini Dalal

    2007-08-01

    Full Text Available Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3. However, the mechanistic basis for centromere maintenance is unknown. We investigated biochemical properties of CenH3 nucleosomes from Drosophila melanogaster cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical "beads-on-a-string" appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity.

  3. CENP-A chromatin disassembly in stressed and senescent murine cells

    Science.gov (United States)

    Hédouin, Sabrine; Grillo, Giacomo; Ivkovic, Ivana; Velasco, Guillaume; Francastel, Claire

    2017-01-01

    Centromeres are chromosomal domains essential for genomic stability. We report here the remarkable transcriptional and epigenetic perturbations at murine centromeres in genotoxic stress conditions. A strong and selective transcriptional activation of centromeric repeats is detected within hours. This is followed by disorganization of centromeres with striking delocalization of nucleosomal CENP-A, the key determinant of centromere identity and function, in a mechanism requiring active transcription of centromeric repeats, the DNA Damage Response (DDR) effector ATM and chromatin remodelers/histone chaperones. In the absence of p53 checkpoint, activated transcription of centromeric repeats and CENP-A delocalization do not occur and cells accumulate micronuclei indicative of genomic instability. In addition, activated transcription and loss of centromeres identity are features of permanently arrested senescent cells with persistent DDR activation. Together, these findings bring out cooperation between DDR effectors and loss of centromere integrity as a safeguard mechanism to prevent genomic instability in context of persistent DNA damage signalling. PMID:28186195

  4. Organization and evolution of Gorilla centromeric DNA from old strategies to new approaches

    OpenAIRE

    Catacchio, C. R.; Ragone, R.; Chiatante, G.; M. Ventura

    2015-01-01

    The centromere/kinetochore interaction is responsible for the pairing and segregation of replicated chromosomes in eukaryotes. Centromere DNA is portrayed as scarcely conserved, repetitive in nature, quickly evolving and protein-binding competent. Among primates, the major class of centromeric DNA is the pancentromeric α-satellite, made of arrays of 171 bp monomers, repeated in a head-to-tail pattern. α-satellite sequences can either form tandem heterogeneous monomeric arrays or assemble in h...

  5. A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli

    OpenAIRE

    2014-01-01

    Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element–containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishe...

  6. Reprogramming chromatin

    DEFF Research Database (Denmark)

    Ehrensberger, Andreas Hasso; Svejstrup, Jesper Qualmann

    2012-01-01

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

  7. The nucleoplasmin homolog NLP mediates centromere clustering and anchoring to the nucleolus.

    Science.gov (United States)

    Padeken, Jan; Mendiburo, María José; Chlamydas, Sarantis; Schwarz, Hans-Jürgen; Kremmer, Elisabeth; Heun, Patrick

    2013-04-25

    Centromere clustering during interphase is a phenomenon known to occur in many different organisms and cell types, yet neither the factors involved nor their physiological relevance is well understood. Using Drosophila tissue culture cells and flies, we identified a network of proteins, including the nucleoplasmin-like protein (NLP), the insulator protein CTCF, and the nucleolus protein Modulo, to be essential for the positioning of centromeres. Artificial targeting further demonstrated that NLP and CTCF are sufficient for clustering, while Modulo serves as the anchor to the nucleolus. Centromere clustering was found to depend on centric chromatin rather than specific DNA sequences. Moreover, unclustering of centromeres results in the spatial destabilization of pericentric heterochromatin organization, leading to partial defects in the silencing of repetitive elements, defects during chromosome segregation, and genome instability.

  8. Single-epitope recognition imaging of native chromatin

    Directory of Open Access Journals (Sweden)

    Wang Hongda

    2008-12-01

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

  9. Organization and evolution of Gorilla centromeric DNA from old strategies to new approaches.

    Science.gov (United States)

    Catacchio, C R; Ragone, R; Chiatante, G; Ventura, M

    2015-09-21

    The centromere/kinetochore interaction is responsible for the pairing and segregation of replicated chromosomes in eukaryotes. Centromere DNA is portrayed as scarcely conserved, repetitive in nature, quickly evolving and protein-binding competent. Among primates, the major class of centromeric DNA is the pancentromeric α-satellite, made of arrays of 171 bp monomers, repeated in a head-to-tail pattern. α-satellite sequences can either form tandem heterogeneous monomeric arrays or assemble in higher-order repeats (HORs). Gorilla centromere DNA has barely been characterized, and data are mainly based on hybridizations of human alphoid sequences. We isolated and finely characterized gorilla α-satellite sequences and revealed relevant structure and chromosomal distribution similarities with other great apes as well as gorilla-specific features, such as the uniquely octameric structure of the suprachromosomal family-2 (SF2). We demonstrated for the first time the orthologous localization of alphoid suprachromosomal families-1 and -2 (SF1 and SF2) between human and gorilla in contrast to chimpanzee centromeres. Finally, the discovery of a new 189 bp monomer type in gorilla centromeres unravels clues to the role of the centromere protein B, paving the way to solve the significance of the centromere DNA's essential repetitive nature in association with its function and the peculiar evolution of the α-satellite sequence.

  10. High quality maize centromere 10 sequence reveals evidence of frequent recombination events

    Directory of Open Access Journals (Sweden)

    Thomas Kai Wolfgruber

    2016-03-01

    Full Text Available The ancestral centromeres of maize contain long stretches of the tandemly arranged CentC repeat. The abundance of tandem DNA repeats and centromeric retrotransposons (CR have presented a significant challenge to completely assembling centromeres using traditional sequencing methods. Here we report a nearly complete assembly of the 1.85 Mb maize centromere 10 from inbred B73 using PacBio technology and BACs from the reference genome project. The error rates estimated from overlapping BAC sequences are 7 x 10-6 and 5 x 10-5 for mismatches and indels, respectively. The number of gaps in the region covered by the reassembly was reduced from 140 in the reference genome to three. Three expressed genes are located between 92 and 477 kb of the inferred ancestral CentC cluster, which lies within the region of highest centromeric repeat density. The improved assembly increased the count of full-length centromeric retrotransposons from 5 to 55 and revealed a 22.7 kb segmental duplication that occurred approximately 121,000 years ago. Our analysis provides evidence of frequent recombination events in the form of partial retrotransposons, deletions within retrotransposons, chimeric retrotransposons, segmental duplications including higher order CentC repeats, a deleted CentC monomer, centromere-proximal inversions, and insertion of mitochondrial sequences. Double-strand DNA break (DSB repair is the most plausible mechanism for these events and may be the major driver of centromere repeat evolution and diversity. This repair appears to be mediated by microhomology, suggesting that tandem repeats may have evolved to facilitate the repair of frequent DSBs in centromeres.

  11. The centromere geometry essential for keeping mitosis error free is controlled by spindle forces.

    Science.gov (United States)

    Loncarek, Jadranka; Kisurina-Evgenieva, Olga; Vinogradova, Tatiana; Hergert, Polla; La Terra, Sabrina; Kapoor, Tarun M; Khodjakov, Alexey

    2007-11-29

    Accurate segregation of chromosomes, essential for the stability of the genome, depends on 'bi-orientation'-simultaneous attachment of each individual chromosome to both poles of the mitotic spindle. On bi-oriented chromosomes, kinetochores (macromolecular complexes that attach the chromosome to the spindle) reside on the opposite sides of the chromosome's centromere. In contrast, sister kinetochores shift towards one side of the centromere on 'syntelic' chromosomes that erroneously attach to one spindle pole with both sister kinetochores. Syntelic attachments often arise during spindle assembly and must be corrected to prevent chromosome loss. It is assumed that restoration of proper centromere architecture occurs automatically owing to elastic properties of the centromere. Here we test this assumption by combining laser microsurgery and chemical biology assays in cultured mammalian cells. We find that kinetochores of syntelic chromosomes remain juxtaposed on detachment from spindle microtubules. These findings reveal that correction of syntelic attachments involves an extra step that has previously been overlooked: external forces must be applied to move sister kinetochores to the opposite sides of the centromere. Furthermore, we demonstrate that the shape of the centromere is important for spindle assembly, because bipolar spindles do not form in cells lacking centrosomes when multiple chromosomes with juxtaposed kinetochores are present. Thus, proper architecture of the centromere makes an important contribution to achieving high fidelity of chromosome segregation.

  12. Engineered human dicentric chromosomes show centromere plasticity.

    Science.gov (United States)

    Higgins, Anne W; Gustashaw, Karen M; Willard, Huntington F

    2005-01-01

    The centromere is essential for the faithful distribution of a cell's genetic material to subsequent generations. Despite intense scrutiny, the precise genetic and epigenetic basis for centromere function is still unknown. Here, we have used engineered dicentric human chromosomes to investigate mammalian centromere structure and function. We describe three classes of dicentric chromosomes isolated in different cell lines: functionally monocentric chromosomes, in which one of the two genetically identical centromeres is consistently inactivated; functionally dicentric chromosomes, in which both centromeres are consistently active; and dicentric chromosomes heterogeneous with respect to centromere activity. A study of serial single cell clones from heterogeneous cell lines revealed that while centromere activity is usually clonal, the centromere state (i.e. functionally monocentric or dicentric) in some lines can switch within a growing population of cells. Because pulsed field gel analysis indicated that the DNA at the centromeres of these chromosomes did not change detectably, this switching of the centromere state is most likely due to epigenetic changes. Inactivation of one of the two active centromeres in a functionally dicentric chromosome was observed in a percentage of cells after treatment with Trichostatin A, an inhibitor of histone deacetylation. This study provides evidence that the activity of human centromeres, while largely stable, can be subject to dynamic change, most likely due to epigenetic modification.

  13. DNMT3B interacts with constitutive centromere protein CENP-C to modulate DNA methylation and the histone code at centromeric regions.

    Science.gov (United States)

    Gopalakrishnan, Suhasni; Sullivan, Beth A; Trazzi, Stefania; Della Valle, Giuliano; Robertson, Keith D

    2009-09-01

    DNA methylation is an epigenetically imposed mark of transcriptional repression that is essential for maintenance of chromatin structure and genomic stability. Genome-wide methylation patterns are mediated by the combined action of three DNA methyltransferases: DNMT1, DNMT3A and DNMT3B. Compelling links exist between DNMT3B and chromosome stability as emphasized by the mitotic defects that are a hallmark of ICF syndrome, a disease arising from germline mutations in DNMT3B. Centromeric and pericentromeric regions are essential for chromosome condensation and the fidelity of segregation. Centromere regions contain distinct epigenetic marks, including dense DNA hypermethylation, yet the mechanisms by which DNA methylation is targeted to these regions remains largely unknown. In the present study, we used a yeast two-hybrid screen and identified a novel interaction between DNMT3B and constitutive centromere protein CENP-C. CENP-C is itself essential for mitosis. We confirm this interaction in mammalian cells and map the domains responsible. Using siRNA knock downs, bisulfite genomic sequencing and ChIP, we demonstrate for the first time that CENP-C recruits DNA methylation and DNMT3B to both centromeric and pericentromeric satellite repeats and that CENP-C and DNMT3B regulate the histone code in these regions, including marks characteristic of centromeric chromatin. Finally, we demonstrate that loss of CENP-C or DNMT3B leads to elevated chromosome misalignment and segregation defects during mitosis and increased transcription of centromeric repeats. Taken together, our data reveal a novel mechanism by which DNA methylation is targeted to discrete regions of the genome and contributes to chromosomal stability.

  14. Bacterial scaffold directs pole-specific centromere segregation.

    Science.gov (United States)

    Ptacin, Jerod L; Gahlmann, Andreas; Bowman, Grant R; Perez, Adam M; von Diezmann, Alexander R S; Eckart, Michael R; Moerner, W E; Shapiro, Lucy

    2014-05-13

    Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. As segregation proceeds, the ParB-bound centromere complex is moved by progressively disassembling ParA from a nucleoid-bound structure. Using superresolution microscopy, we show that released ParA is recruited directly to binding sites within a 3D ultrastructure composed of PopZ at the cell pole, whereas the ParB-centromere complex remains at the periphery of the PopZ structure. PopZ recruitment of ParA stimulates ParA to assemble on the nucleoid near the PopZ-proximal cell pole. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. PopZ therefore functions as a polar hub complex at the cell pole to directly regulate the directionality and destination of transfer of the mitotic segregation machine.

  15. Chromatin computation.

    Directory of Open Access Journals (Sweden)

    Barbara Bryant

    Full Text Available 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.

  16. Chromatin structure and ATRX function in mouse oocytes.

    Science.gov (United States)

    De La Fuente, Rabindranath; Baumann, Claudia; Viveiros, Maria M

    2012-01-01

    Differentiation of chromatin structure and function during oogenesis is essential to confer the mammalian oocyte with meiotic and developmental potential. Errors in chromosome segregation during female meiosis and subsequent transmission of an abnormal chromosome complement (aneuploidy) to the early conceptus are one of the leading causes of pregnancy loss in women. The chromatin remodeling protein ATRX (α-thalassemia mental retardation X-linked) has recently emerged as a critical factor involved in heterochromatin formation at mammalian centromeres during meiosis. In mammalian oocytes, ATRX binds to centromeric heterochromatin domains where it is required for accurate chromosome segregation. Loss of ATRX function induces abnormal meiotic chromosome morphology, reduces histone H3 phosphorylation, and promotes a high incidence of aneuploidy associated with severely reduced fertility. The presence of centromeric breaks during the transition to the first mitosis in the early embryo indicates that the role of ATRX in chromosome segregation is mediated through an epigenetic mechanism involving the maintenance of chromatin modifications associated with pericentric heterochromatin (PCH) formation and chromosome condensation. This is consistent with the existence of a potential molecular link between centromeric and PCH in the epigenetic control of centromere function and maintenance of chromosome stability in mammalian oocytes. Dissecting the molecular mechanisms of ATRX function during meiosis will have important clinical implications towards uncovering the epigenetic factors contributing to the onset of aneuploidy in the human oocyte.

  17. A regulatory effect of INMAP on centromere proteins: antisense INMAP induces CENP-B variation and centromeric halo.

    Directory of Open Access Journals (Sweden)

    Tan Tan

    Full Text Available CENP-B is a highly conserved protein that facilitates the assembly of specific centromere structures both in interphase nuclei and on mitotic chromosomes. INMAP is a conserved protein that localizes at nucleus in interphase cells and at mitotic apparatus in mitotic cells. Our previous results showed that INMAP over-expression leads to spindle defects, mitotic arrest and formation of polycentrosomal and multinuclear cells, indicating that INMAP may modulate the function of (a key protein(s in mitotic apparatus. In this study, we demonstrate that INMAP interacts with CENP-B and promotes cleavage of the N-terminal DNA binding domain from CENP-B. The cleaved CENP-B cannot associate with centromeres and thus lose its centromere-related functions. Consistent with these results, CENP-B in INMAP knockdown cells becomes more diffused around kinetochores. Although INMAP knockdown cells do not exhibit gross defects in mitotic spindle formation, these cells go through mitosis, especially prophase and metaphase, with different relative timing, indicating subtle abnormality. These results identify INMAP as a model regulator of CENP-B and support the notion that INMAP regulates mitosis through modulating CENP-B-mediated centromere organization.

  18. A regulatory effect of INMAP on centromere proteins: antisense INMAP induces CENP-B variation and centromeric halo.

    Science.gov (United States)

    Tan, Tan; Chen, Zhe; Lei, Yan; Zhu, Yan; Liang, Qianjin

    2014-01-01

    CENP-B is a highly conserved protein that facilitates the assembly of specific centromere structures both in interphase nuclei and on mitotic chromosomes. INMAP is a conserved protein that localizes at nucleus in interphase cells and at mitotic apparatus in mitotic cells. Our previous results showed that INMAP over-expression leads to spindle defects, mitotic arrest and formation of polycentrosomal and multinuclear cells, indicating that INMAP may modulate the function of (a) key protein(s) in mitotic apparatus. In this study, we demonstrate that INMAP interacts with CENP-B and promotes cleavage of the N-terminal DNA binding domain from CENP-B. The cleaved CENP-B cannot associate with centromeres and thus lose its centromere-related functions. Consistent with these results, CENP-B in INMAP knockdown cells becomes more diffused around kinetochores. Although INMAP knockdown cells do not exhibit gross defects in mitotic spindle formation, these cells go through mitosis, especially prophase and metaphase, with different relative timing, indicating subtle abnormality. These results identify INMAP as a model regulator of CENP-B and support the notion that INMAP regulates mitosis through modulating CENP-B-mediated centromere organization.

  19. A DNA polymerase alpha accessory protein, Mcl1, is required for propagation of centromere structures in fission yeast.

    Directory of Open Access Journals (Sweden)

    Toyoaki Natsume

    Full Text Available Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1 kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1 in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+, which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.

  20. Mislocalization of the Drosophila centromere-specific histone CIDpromotes formation of functional ectopic kinetochores

    Energy Technology Data Exchange (ETDEWEB)

    Heun, Patrick; Erhardt, Sylvia; Blower, Michael D.; Weiss,Samara; Skora, Andrew D.; Karpen, Gary H.

    2006-01-30

    The centromere-specific histone variant CENP-A (CID in Drosophila) is a structural and functional foundation for kinetochore formation and chromosome segregation. Here, we show that overexpressed CID is mislocalized into normally non-centromeric regions in Drosophila tissue culture cells and animals. Analysis of mitoses in living and fixed cells reveals that mitotic delays, anaphase bridges, chromosome fragmentation, and cell and organismal lethality are all direct consequences of CID mislocalization. In addition, proteins that are normally restricted to endogenous kinetochores assemble at a subset of ectopic CID incorporation regions. The presence of microtubule motors and binding proteins, spindle attachments, and aberrant chromosome morphologies demonstrate that these ectopic kinetochores are functional. We conclude that CID mislocalization promotes formation of ectopic centromeres and multicentric chromosomes, which causes chromosome missegregation, aneuploidy, and growth defects. Thus, CENP-A mislocalization is one possible mechanism for genome instability during cancer progression, as well as centromere plasticity during evolution.

  1. A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli.

    Science.gov (United States)

    Smith, Corey L; Matheson, Timothy D; Trombly, Daniel J; Sun, Xiaoming; Campeau, Eric; Han, Xuemei; Yates, John R; Kaufman, Paul D

    2014-09-15

    Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element-containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus.

  2. Centromere-independent accumulation of cohesin at ectopic heterochromatin sites induces chromosome stretching during anaphase.

    Directory of Open Access Journals (Sweden)

    Raquel A Oliveira

    2014-10-01

    Full Text Available Pericentric heterochromatin, while often considered as "junk" DNA, plays important functions in chromosome biology. It contributes to sister chromatid cohesion, a process mediated by the cohesin complex that ensures proper genome segregation during nuclear division. Long stretches of heterochromatin are almost exclusively placed at centromere-proximal regions but it remains unclear if there is functional (or mechanistic importance in linking the sites of sister chromatid cohesion to the chromosomal regions that mediate spindle attachment (the centromere. Using engineered chromosomes in Drosophila melanogaster, we demonstrate that cohesin enrichment is dictated by the presence of heterochromatin rather than centromere proximity. This preferential accumulation is caused by an enrichment of the cohesin-loading factor (Nipped-B/NIPBL/Scc2 at dense heterochromatic regions. As a result, chromosome translocations containing ectopic pericentric heterochromatin embedded in euchromatin display additional cohesin-dependent constrictions. These ectopic cohesion sites, placed away from the centromere, disjoin abnormally during anaphase and chromosomes exhibit a significant increase in length during anaphase (termed chromatin stretching. These results provide evidence that long stretches of heterochromatin distant from the centromere, as often found in many cancers, are sufficient to induce abnormal accumulation of cohesin at these sites and thereby compromise the fidelity of chromosome segregation.

  3. Chromatin challenges during DNA replication and repair

    DEFF Research Database (Denmark)

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

    2007-01-01

    Inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. To orchestrate DNA-replication and -repair processes in the context of chromatin is a challenge, both in terms of accessibility and maintenance of chromatin organization. To meet...... the challenge of maintenance, cells have evolved efficient nucleosome-assembly pathways and chromatin-maturation mechanisms that reproduce chromatin organization in the wake of DNA replication and repair. The aim of this Review is to describe how these pathways operate and to highlight how the epigenetic...

  4. Formation of functional CENP-B boxes at diverse locations in repeat units of centromeric DNA in New World monkeys.

    Science.gov (United States)

    Kugou, Kazuto; Hirai, Hirohisa; Masumoto, Hiroshi; Koga, Akihiko

    2016-06-13

    Centromere protein B, which is involved in centromere formation, binds to centromeric repetitive DNA by recognizing a nucleotide motif called the CENP-B box. Humans have large numbers of CENP-B boxes in the centromeric repetitive DNA of their autosomes and X chromosome. The current understanding is that these CENP-B boxes are located at identical positions in the repeat units of centromeric DNA. Great apes also have CENP-B boxes in locations that are identical to humans. The purpose of the present study was to examine the location of CENP-B box in New World monkeys. We recently identified CENP-B box in one species of New World monkeys (marmosets). In this study, we found functional CENP-B boxes in CENP-A-assembled repeat units of centromeric DNA in 2 additional New World monkeys (squirrel monkeys and tamarins) by immunostaining and ChIP-qPCR analyses. The locations of the 3 CENP-B boxes in the repeat units differed from one another. The repeat unit size of centromeric DNA of New World monkeys (340-350 bp) is approximately twice that of humans and great apes (171 bp). This might be, associated with higher-order repeat structures of centromeric DNA, a factor for the observed variation in the CENP-B box location in New World monkeys.

  5. A Positive Twist to the Centromeric Nucleosome

    Directory of Open Access Journals (Sweden)

    Josefina Ocampo

    2015-10-01

    Full Text Available Centromeric nucleosomes are critical for chromosome attachment to the mitotic spindle. In this issue of Cell Reports, Diaz-Ingelmo et al. (2015 propose that the yeast centromeric nucleosome is stabilized by a positively supercoiled loop formed by the sequence-specific CBF3 complex.

  6. Insights into centromeric transcription in mitosis.

    Science.gov (United States)

    Liu, Hong

    2016-01-01

    The major role of RNA polymerase II (RNAP II) is to generate mRNAs. I recently uncovered a novel function of RNAP II in chromosome segregation in mitosis, installing the cohesin protector, Shugoshin, at centromeres. Here I will discuss the current understanding of RNAP II-dependent centromeric transcription in mitosis.

  7. Correlation between centromere protein-F autoantibodies and cancer analyzed by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Welner, Simon; Trier, Nicole Hartwig; Morten Frisch, Morten

    2013-01-01

    Centromere protein-F (CENP-F) is a large nuclear protein of 367 kDa, which is involved in multiple mitosis-related events such as proper assembly of the kinetochores, stabilization of heterochromatin, chromosome alignment and mitotic checkpoint signaling. Several studies have shown a correlation...

  8. Dicentric Chromosome Formation and Epigenetics of Centromere Formation in Plants

    Institute of Scientific and Technical Information of China (English)

    Shulan Fu; Zhi Gao; James Birchler; Fangpu Han

    2012-01-01

    Plant centromeres are generally composed of tandem arrays of simple repeats that form a complex chromosome locus where the kinetochore forms and microtubules attach during mitosis and meiosis.Each chromosome has one centromere region,which is essential for accurate division of the genetic material.Recently,chromosomes containing two centromere regions (called dicentric chromosomes)have been found in maize and wheat.Interestingly,some dicentric chromosomes are stable because only one centromere is active and the other one is inactivated.Because such arrays maintain their typical structure for both active and inactive centromeres,the specification of centromere activity has an epigenetic component independent of the DNA sequence.Under some circumstances,the inactive centromeres may recover centromere function,which is called centromere reactivation.Recent studies have highlighted the important changes,such as DNA methylation and histone modification,that occur during centromere inactivation and reactivation.

  9. Dicentric chromosome formation and epigenetics of centromere formation in plants.

    Science.gov (United States)

    Fu, Shulan; Gao, Zhi; Birchler, James; Han, Fangpu

    2012-03-20

    Plant centromeres are generally composed of tandem arrays of simple repeats that form a complex chromosome locus where the kinetochore forms and microtubules attach during mitosis and meiosis. Each chromosome has one centromere region, which is essential for accurate division of the genetic material. Recently, chromosomes containing two centromere regions (called dicentric chromosomes) have been found in maize and wheat. Interestingly, some dicentric chromosomes are stable because only one centromere is active and the other one is inactivated. Because such arrays maintain their typical structure for both active and inactive centromeres, the specification of centromere activity has an epigenetic component independent of the DNA sequence. Under some circumstances, the inactive centromeres may recover centromere function, which is called centromere reactivation. Recent studies have highlighted the important changes, such as DNA methylation and histone modification, that occur during centromere inactivation and reactivation.

  10. Centromeres cluster de novo at the beginning of meiosis in Brachypodium distachyon.

    Directory of Open Access Journals (Sweden)

    Ruoyu Wen

    Full Text Available In most eukaryotes that have been studied, the telomeres cluster into a bouquet early in meiosis, and in wheat and its relatives and in Arabidopsis the centromeres pair at the same time. In Arabidopsis, the telomeres do not cluster as a typical telomere bouquet on the nuclear membrane but are associated with the nucleolus both somatically and at the onset of meiosis. We therefore assessed whether Brachypodium distachyon, a monocot species related to cereals and whose genome is approximately twice the size of Arabidopsis thaliana, also exhibited an atypical telomere bouquet and centromere pairing. In order to investigate the occurrence of a bouquet and centromere pairing in B distachyon, we first had to establish protocols for studying meiosis in this species. This enabled us to visualize chromosome behaviour in meiocytes derived from young B distachyon spikelets in three-dimensions by fluorescent in situ hybridization (FISH, and accurately to stage meiosis based on chromatin morphology in relation to spikelet size and the timing of sample collection. Surprisingly, this study revealed that the centromeres clustered as a single site at the same time as the telomeres also formed a bouquet or single cluster.

  11. Tension sensing by Aurora B kinase is independent of survivin-based centromere localization.

    Science.gov (United States)

    Campbell, Christopher S; Desai, Arshad

    2013-05-01

    Accurate segregation of the replicated genome requires chromosome biorientation on the spindle. Biorientation is ensured by Aurora B kinase (Ipl1), a member of the four-subunit chromosomal passenger complex (CPC). Localization of the CPC to the inner centromere is central to the current model for how tension ensures chromosome biorientation: kinetochore-spindle attachments that are not under tension remain close to the inner centromere and are destabilized by Aurora B phosphorylation, whereas kinetochores under tension are pulled away from the influence of Aurora B, stabilizing their microtubule attachments. Here we show that an engineered truncation of the Sli15 (known as INCENP in humans) subunit of budding yeast CPC that eliminates association with the inner centromere nevertheless supports proper chromosome segregation during both mitosis and meiosis. Truncated Sli15 suppresses the deletion phenotypes of the inner-centromere-targeting proteins survivin (Bir1), borealin (Nbl1), Bub1 and Sgo1 (ref. 6). Unlike wild-type Sli15, truncated Sli15 localizes to pre-anaphase spindle microtubules. Premature targeting of full-length Sli15 to microtubules by preventing Cdk1 (also known as Cdc28) phosphorylation also suppresses the inviability of Bir1 deletion. These results suggest that activation of Aurora B kinase by clustering either on chromatin or on microtubules is sufficient for chromosome biorientation.

  12. Recognition of the centromere-specific histone Cse4 by the chaperone Scm3

    OpenAIRE

    Cho, Uhn-Soo; Harrison, Stephen C.

    2011-01-01

    A specialized nucleosome is a component of all eukaryotic kinetochores. The core of this nucleosome contains a centromere-specific histone, CENP-A (the Cse4 gene product in budding yeast), instead of the usual H3. Assembly of a centromeric nucleosome depends on a specific chaperone, called Scm3 in yeast and HJURP in higher eukaryotes. We describe here the structure of a complex formed by an N-terminal fragment of Scm3 with the histone-fold domains of Cse4, and H4, all prepared as recombinant ...

  13. Chromatin associations in Arabidopsis interphase nuclei

    Directory of Open Access Journals (Sweden)

    Veit eSchubert

    2014-11-01

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

  14. High-affinity binding of Chp1 chromodomain to K9 methylated histone H3 is required to establish centromeric heterochromatin.

    Science.gov (United States)

    Schalch, Thomas; Job, Godwin; Noffsinger, Victoria J; Shanker, Sreenath; Kuscu, Canan; Joshua-Tor, Leemor; Partridge, Janet F

    2009-04-10

    In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced.

  15. 30 nm染色质的体外组装和电镜分析%In vitro Assembly and Electron Microscopic Analysis of 30 nm Chromatin Fibers

    Institute of Scientific and Technical Information of China (English)

    孙大鹏; 宋峰; 黄丽; 张阔; 季刚; 陈萍; 朱平

    2013-01-01

    Abstract Genomic DNA in the eukaryotic nucleus is hierarchically packaged by histones into chromatin.The plasticity and dynamics of higher-order chromatin fiber have been widely thought as the key regulators of transcription and other biological processes inherent to DNA.Elucidating how nucleosomal arrays can be folded into higher-order chromatin fibers is essential to understand the dynamics of chromatin structure.Although the structure of nucleosomes,the fundamental repeating unit of chromatin,which comprises 147 base pairs of DNA wrapped in 1.7 superhelical turns around an octamer ofhistones,has been solved at the atomic resolution,there is still much controversy over the chromatin structure at the higher-order level.Here,we built an in vitro chromatin reconstitution system which adopts histone octamers and arrays of 177 bp and 200 bp repeat of the Widom 601 DNA sequence.Taking advantage of this system,we have obtained highly regular spaced and soluble nucleosome arrays,and folded the arrays into 30 nm chromatin fibers with the existence of linker histone H1 or MgCh respectively.Several electron microscopic techniques,including metal shadowing,negative staining and Cryo-EM,have been used to investigate the morphology of the reconstituted 30 nm chromatin fibers.Our results suggest that both histone H1 and divalent Mg2+ can help the formation of 30 nm chromatin fibers,but the resulted chromatin fibers display different topologically architectures.To investigate how the length of linker histone may affect the architecture of chromatin,we measured the diameters of the reconstituted 30 nm chromatin fibers with different nucleosome repeat lengths (NRLs) of 177 and 200 bp and found that these two classes of chromatin fibers present different diameters (P < 0.05).%真核生物的基因组以染色质的形式存在,染色质在真核生物的基因表达调控及胚胎发育过程中起重要作用,为表观遗传提供一个重要的信息整合平台.染

  16. RNAi and heterochromatin repress centromeric meiotic recombination

    DEFF Research Database (Denmark)

    Ellermeier, Chad; Higuchi, Emily C; Phadnis, Naina

    2010-01-01

    During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essen......During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes....... Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between...... types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis....

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

    Directory of Open Access Journals (Sweden)

    Alexandra Lusser

    2011-10-01

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

  18. Role of transcription at centromeres in budding yeast.

    Science.gov (United States)

    Ohkuni, Kentaro; Kitagawa, Katsumi

    2012-01-01

    Centromeres are specialized chromosomal loci that are essential for proper chromosome segregation. Recent data show that a certain level of active transcription, regulated by transcription factors Cbf1 and Ste12, makes a direct contribution to centromere function in Saccharomyces cerevisiae. Here, we discuss the requirement and function of transcription at centromeres.

  19. ParB Partition Proteins: Complex Formation and Spreading at Bacterial and Plasmid Centromeres

    Directory of Open Access Journals (Sweden)

    Barbara Funnell

    2016-08-01

    Full Text Available In bacteria, active partition systems contribute to the faithful segregation of both chromosomes and low-copy-number plasmids. Each system depends on a site-specific DNA binding protein to recognize and assemble a partition complex at a centromere-like site, commonly called parS. Many plasmid and all chromosomal centromere-binding proteins are dimeric helix-turn-helix DNA binding proteins, which are commonly named ParB. Although the overall sequence conservation among ParBs is not high, the proteins share similar domain and functional organization, and they assemble into similar higher-order complexes. In vivo, ParBs spread; that is, DNA binding extends away from the parS site into the surrounding nonspecific DNA, a feature that reflects higher-order complex assembly. ParBs bridge and pair DNA at parS and nonspecific DNA sites. ParB dimers interact with each other via flexible conformations of an N-terminal region. This review will focus on the properties of the HTH centromere-binding protein, in light of recent experimental evidence and models that are adding to our understanding of how these proteins assemble into large and dynamic partition complexes at and around their specific DNA sites.

  20. Physical Characterization of human centromeric regions using transformation-associated recombination cloning technology

    Energy Technology Data Exchange (ETDEWEB)

    Vladimir Larionov, Ph D

    2007-06-05

    A special interest in the organization of human centromeric DNA was stimulated a few years ago when two independent groups succeeded in reconstituting a functional human centromere, using constructs carrying centromere-specific alphoid DNA arrays. This work demonstrated the importance of DNA components in mammalian centromeres and opened a way for studying the structural requirements for de novo kinetochore formation and for construction of human artificial chromosomes (HACs) with therapeutic potential. To elucidate the structural requirements for formation of HACs with a functional kinetochore, we developed a new method for cloning of large DNA fragments for human centromeric regions that can be used as a substrate for HAC formation. This method exploits in vivo recombination in yeast (TAR cloning). In addition, a new strategy for the construction of alphoid DNA arrays was developed in our lab. The strategy involves the construction of uniform or hybrid synthetic alphoid DNA arrays by the RCA-TAR technique. This technique comprises two steps: rolling circle amplification of an alphoid DNA dimer and subsequent assembling of the amplified fragments by in vivo homologous recombination in yeast (Figure 1). Using this system, we constructed a set of different synthetic alphoid DNA arrays with a predetermined sequence varying in size from 30 to 140 kb and demonstrated that some of the arrays are competent in HAC formation. Because any nucleotide can be changed in a dimer before its amplification, this new technique is optimal for identifying the structural requirements for de novo kinetochore formation in HACs. Moreover, the technique makes possible to introduce into alphoid DNA arrays recognition sites for DNA-binding proteins. We have made the following progress on the studying of human centromeric regions using transformation-associated recombination cloning technology: i) minimal size of alphoid DNA array required for de novo kinetochore formation was estimated; ii

  1. Genome-wide mapping of cytosine methylation revealed dynamic DNA methylation patterns associated with genes and centromeres in rice.

    Science.gov (United States)

    Yan, Huihuang; Kikuchi, Shinji; Neumann, Pavel; Zhang, Wenli; Wu, Yufeng; Chen, Feng; Jiang, Jiming

    2010-08-01

    We conducted genome-wide mapping of cytosine methylation using methylcytosine immunoprecipitation combined with Illumina sequencing. The chromosomal distribution pattern of methylated DNA is similar to the heterochromatin distribution pattern on rice chromosomes. The DNA methylation patterns of rice genes are similar to those in Arabidopsis thaliana, including distinct methylation patterns asssociated with gene bodies and promoters. The DNA sequences in the core domains of rice Cen4, Cen5 and Cen8 showed elevated methylation levels compared with sequences in the pericentromeric regions. In addition, elevated methylation levels were associated with the DNA sequences in the CENH3-binding subdomains, compared with the sequences in the flanking H3 subdomains. In contrast, the centromeric domain of Cen11, which is composed exclusively of centromeric satellite DNA, is hypomethylated compared with the pericentromeric domains. Thus, the DNA sequences associated with functional centromeres can be either hypomethylated or hypermethylated. The methylation patterns of centromeric DNA appear to be correlated with the composition of the associated DNA sequences. We propose that both hypomethylation and hypermethylation of CENH3-associated DNA sequences can serve as epigenetic marks to distinguish where CENH3 deposition will occur within the surrounding H3 chromatin.

  2. Chromatin is wonderful stuff.

    NARCIS (Netherlands)

    R. van Driel

    2007-01-01

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

  3. Stretching the rules: monocentric chromosomes with multiple centromere domains.

    Science.gov (United States)

    Neumann, Pavel; Navrátilová, Alice; Schroeder-Reiter, Elizabeth; Koblížková, Andrea; Steinbauerová, Veronika; Chocholová, Eva; Novák, Petr; Wanner, Gerhard; Macas, Jiří

    2012-01-01

    The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division and that can be reliably identified by the presence of the centromere-specific histone H3 variant CenH3. In monocentric chromosomes, the centromere is characterized by a single CenH3-containing region within a morphologically distinct primary constriction. This region usually spans up to a few Mbp composed mainly of centromere-specific satellite DNA common to all chromosomes of a given species. In holocentric chromosomes, there is no primary constriction; the centromere is composed of many CenH3 loci distributed along the entire length of a chromosome. Using correlative fluorescence light microscopy and high-resolution electron microscopy, we show that pea (Pisum sativum) chromosomes exhibit remarkably long primary constrictions that contain 3-5 explicit CenH3-containing regions, a novelty in centromere organization. In addition, we estimate that the size of the chromosome segment delimited by two outermost domains varies between 69 Mbp and 107 Mbp, several factors larger than any known centromere length. These domains are almost entirely composed of repetitive DNA sequences belonging to 13 distinct families of satellite DNA and one family of centromeric retrotransposons, all of which are unevenly distributed among pea chromosomes. We present the centromeres of Pisum as novel "meta-polycentric" functional domains. Our results demonstrate that the organization and DNA composition of functional centromere domains can be far more complex than previously thought, do not require single repetitive elements, and do not require single centromere domains in order to segregate properly. Based on these findings, we propose Pisum as a useful model for investigation of centromere architecture and the still poorly understood role of repetitive DNA in centromere evolution, determination, and function.

  4. Evolution of Centromeric Retrotransposons in Grasses

    Science.gov (United States)

    Sharma, Anupma; Presting, Gernot G.

    2014-01-01

    Centromeric retrotransposons (CRs) constitute a family of plant retroelements, some of which have the ability to target their insertion almost exclusively to the functional centromeres. Our exhaustive analysis of CR family members in four grass genomes revealed not only horizontal transfer (HT) of CR elements between the oryzoid and panicoid grass lineages but also their subsequent recombination with endogenous elements that in some cases created prolific recombinants in foxtail millet and sorghum. HT events are easily identifiable only in cases where host genome divergence significantly predates HT, thus documented HT events likely represent only a fraction of the total. If the more difficult to detect ancient HT events occurred at frequencies similar to those observable in present day grasses, the extant long terminal repeat retrotransposons represent the mosaic products of HT and recombination that are optimized for retrotransposition in their host genomes. This complicates not only phylogenetic analysis but also the establishment of a meaningful retrotransposon nomenclature, which we have nevertheless attempted to implement here. In contrast to the plant-centric naming convention used currently for CR elements, we classify elements primarily based on their phylogenetic relationships regardless of host plant, using the exhaustively studied maize elements assigned to six different subfamilies as a standard. The CR2 subfamily is the most widely distributed of the six CR subfamilies discovered in grass genomes to date and thus the most likely to play a functional role at grass centromeres. PMID:24814286

  5. Characterization of centromeric histone H3 (CENH3 variants in cultivated and wild carrots (Daucus sp..

    Directory of Open Access Journals (Sweden)

    Frank Dunemann

    Full Text Available In eukaryotes, centromeres are the assembly sites for the kinetochore, a multi-protein complex to which spindle microtubules are attached at mitosis and meiosis, thereby ensuring segregation of chromosomes during cell division. They are specified by incorporation of CENH3, a centromere specific histone H3 variant which replaces canonical histone H3 in the nucleosomes of functional centromeres. To lay a first foundation of a putative alternative haploidization strategy based on centromere-mediated genome elimination in cultivated carrots, in the presented research we aimed at the identification and cloning of functional CENH3 genes in Daucus carota and three distantly related wild species of genus Daucus varying in basic chromosome numbers. Based on mining the carrot transcriptome followed by a subsequent PCR-based cloning, homologous coding sequences for CENH3s of the four Daucus species were identified. The ORFs of the CENH3 variants were very similar, and an amino acid sequence length of 146 aa was found in three out of the four species. Comparison of Daucus CENH3 amino acid sequences with those of other plant CENH3s as well as their phylogenetic arrangement among other dicot CENH3s suggest that the identified genes are authentic CENH3 homologs. To verify the location of the CENH3 protein in the kinetochore regions of the Daucus chromosomes, a polyclonal antibody based on a peptide corresponding to the N-terminus of DcCENH3 was developed and used for anti-CENH3 immunostaining of mitotic root cells. The chromosomal location of CENH3 proteins in the centromere regions of the chromosomes could be confirmed. For genetic localization of the CENH3 gene in the carrot genome, a previously constructed linkage map for carrot was used for mapping a CENH3-specific simple sequence repeat (SSR marker, and the CENH3 locus was mapped on the carrot chromosome 9.

  6. A Structural Basis for BRD2/4-Mediated Host Chromatin Interaction and Oligomer Assembly of Kaposi Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus LANA Proteins

    Science.gov (United States)

    Krausze, Joern; Richter, Ulrike; Adler, Heiko; Fedorov, Roman; Pietrek, Marcel; Rückert, Jessica; Ritter, Christiane; Schulz, Thomas F.; Lührs, Thorsten

    2013-01-01

    Kaposi sarcoma-associated herpesvirus (KSHV) establishes a lifelong latent infection and causes several malignancies in humans. Murine herpesvirus 68 (MHV-68) is a related γ2-herpesvirus frequently used as a model to study the biology of γ-herpesviruses in vivo. The KSHV latency-associated nuclear antigen (kLANA) and the MHV68 mLANA (orf73) protein are required for latent viral replication and persistence. Latent episomal KSHV genomes and kLANA form nuclear microdomains, termed ‘LANA speckles’, which also contain cellular chromatin proteins, including BRD2 and BRD4, members of the BRD/BET family of chromatin modulators. We solved the X-ray crystal structure of the C-terminal DNA binding domains (CTD) of kLANA and MHV-68 mLANA. While these structures share the overall fold with the EBNA1 protein of Epstein-Barr virus, they differ substantially in their surface characteristics. Opposite to the DNA binding site, both kLANA and mLANA CTD contain a characteristic lysine-rich positively charged surface patch, which appears to be a unique feature of γ2-herpesviral LANA proteins. Importantly, kLANA and mLANA CTD dimers undergo higher order oligomerization. Using NMR spectroscopy we identified a specific binding site for the ET domains of BRD2/4 on kLANA. Functional studies employing multiple kLANA mutants indicate that the oligomerization of native kLANA CTD dimers, the characteristic basic patch and the ET binding site on the kLANA surface are required for the formation of kLANA ‘nuclear speckles’ and latent replication. Similarly, the basic patch on mLANA contributes to the establishment of MHV-68 latency in spleen cells in vivo. In summary, our data provide a structural basis for the formation of higher order LANA oligomers, which is required for nuclear speckle formation, latent replication and viral persistence. PMID:24146614

  7. Chromatin remodeling regulated by steroid and nuclear receptors

    Institute of Scientific and Technical Information of China (English)

    1997-01-01

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

  8. Regulation of centromere localization of the Drosophila Shugoshin MEI-S332 and sister-chromatid cohesion in meiosis.

    Science.gov (United States)

    Nogueira, Cristina; Kashevsky, Helena; Pinto, Belinda; Clarke, Astrid; Orr-Weaver, Terry L

    2014-07-31

    The Shugoshin (Sgo) protein family helps to ensure proper chromosome segregation by protecting cohesion at the centromere by preventing cleavage of the cohesin complex. Some Sgo proteins also influence other aspects of kinetochore-microtubule attachments. Although many Sgo members require Aurora B kinase to localize to the centromere, factors controlling delocalization are poorly understood and diverse. Moreover, it is not clear how Sgo function is inactivated and whether this is distinct from delocalization. We investigated these questions in Drosophila melanogaster, an organism with superb chromosome cytology to monitor Sgo localization and quantitative assays to test its function in sister-chromatid segregation in meiosis. Previous research showed that in mitosis in cell culture, phosphorylation of the Drosophila Sgo, MEI-S332, by Aurora B promotes centromere localization, whereas Polo phosphorylation promotes delocalization. These studies also suggested that MEI-S332 can be inactivated independently of delocalization, a conclusion supported here by localization and function studies in meiosis. Phosphoresistant and phosphomimetic mutants for the Aurora B and Polo phosphorylation sites were examined for effects on MEI-S332 localization and chromosome segregation in meiosis. Strikingly, MEI-S332 with a phosphomimetic mutation in the Aurora B phosphorylation site prematurely dissociates from the centromeres in meiosis I. Despite the absence of MEI-S332 on meiosis II centromeres in male meiosis, sister chromatids segregate normally, demonstrating that detectable levels of this Sgo are not essential for chromosome congression, kinetochore biorientation, or spindle assembly.

  9. Embryonic stem cell differentiation: a chromatin perspective.

    Science.gov (United States)

    Rasmussen, Theodore P

    2003-11-13

    Embryonic stem (ES) cells hold immense promise for the treatment of human degenerative disease. Because ES cells are pluripotent, they can be directed to differentiate into a number of alternative cell-types with potential therapeutic value. Such attempts at "rationally-directed ES cell differentiation" constitute attempts to recapitulate aspects of normal development in vitro. All differentiated cells retain identical DNA content, yet gene expression varies widely from cell-type to cell-type. Therefore, a potent epigenetic system has evolved to coordinate and maintain tissue-specific patterns of gene expression. Recent advances show that mechanisms that govern epigenetic regulation of gene expression are rooted in the details of chromatin dynamics. As embryonic cells differentiate, certain genes are activated while others are silenced. These activation and silencing events are exquisitely coordinated with the allocation of cell lineages. Remodeling of the chromatin of developmentally-regulated genes occurs in conjunction with lineage commitment. Oocytes, early embryos, and ES cells contain potent chromatin-remodeling activities, an observation that suggests that chromatin dynamics may be especially important for early lineage decisions. Chromatin dynamics are also involved in the differentiation of adult stem cells, where the assembly of specialized chromatin upon tissue-specific genes has been studied in fine detail. The next few years will likely yield striking advances in the understanding of stem cell differentiation and developmental biology from the perspective of chromatin dynamics.

  10. Embryonic stem cell differentiation: A chromatin perspective

    Directory of Open Access Journals (Sweden)

    Rasmussen Theodore P

    2003-11-01

    Full Text Available Abstract Embryonic stem (ES cells hold immense promise for the treatment of human degenerative disease. Because ES cells are pluripotent, they can be directed to differentiate into a number of alternative cell-types with potential therapeutic value. Such attempts at "rationally-directed ES cell differentiation" constitute attempts to recapitulate aspects of normal development in vitro. All differentiated cells retain identical DNA content, yet gene expression varies widely from cell-type to cell-type. Therefore, a potent epigenetic system has evolved to coordinate and maintain tissue-specific patterns of gene expression. Recent advances show that mechanisms that govern epigenetic regulation of gene expression are rooted in the details of chromatin dynamics. As embryonic cells differentiate, certain genes are activated while others are silenced. These activation and silencing events are exquisitely coordinated with the allocation of cell lineages. Remodeling of the chromatin of developmentally-regulated genes occurs in conjunction with lineage commitment. Oocytes, early embryos, and ES cells contain potent chromatin-remodeling activities, an observation that suggests that chromatin dynamics may be especially important for early lineage decisions. Chromatin dynamics are also involved in the differentiation of adult stem cells, where the assembly of specialized chromatin upon tissue-specific genes has been studied in fine detail. The next few years will likely yield striking advances in the understanding of stem cell differentiation and developmental biology from the perspective of chromatin dynamics.

  11. New mitotic regulators released from chromatin

    Directory of Open Access Journals (Sweden)

    Hideki eYokoyama

    2013-12-01

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

  12. Centromere activity in dicentric small supernumerary marker chromosomes.

    Science.gov (United States)

    Ewers, Elisabeth; Yoda, Kinya; Hamid, Ahmed B; Weise, Anja; Manvelyan, Marina; Liehr, Thomas

    2010-07-01

    Twenty-five dicentric small supernumerary marker chromosomes (sSMC) derived from #13/21, #14, #15, #18, and #22 were studied by immunohistochemistry for their centromeric activity. Centromere protein (CENP)-B was applied as marker for all centromeres and CENP-C to label the active ones. Three different 'predominant' activation patterns could be observed, i.e., centric fusion or either only one or all two centromeres were active. In one inherited case, the same activation pattern was found in mother and son. In acrocentric-derived sSMC, all three activation patterns could be present. In contrary, in chromosome 18-derived sSMC, only the fusion type was observed. In concordance with previous studies a certain centromeric plasticity was observed in up to 13% of the cells of an individual case. Surprisingly, the obtained data suggests a possible influence of the sSMC carrier's gender on the implementation of the predominant activation pattern; especially, only one active centromere was found more frequently in female than in male carriers. Also, it might be suggested that dicentric sSMC with one active centromere could be less stable than such with two active ones-centromeric plasticity might have an influence here, as well. Also, centromere activity in acrocentric-derived dicentrics could be influenced by heteromorphisms of the corresponding short arms. Finally, evidence is provided that the closer the centromeres of a dicentric are and if they are not fused, the more likely it was that both of them became active. In concordance and refinement with previous studies, a distance of 1.4 Mb up to about 13 Mb the two active centromere state was favored, while centromeric distance of over approximately 15 Mb lead to inactivation of one centromere. Overall, here, the first and largest ever undertaken study in dicentric sSMC is presented, providing evidence that the centromeric activation pattern is, and parental origin may be of interest for their biology. Influence of

  13. Centromere positions in chicken and Japanese quail chromosomes: de novo centromere formation versus pericentric inversions

    NARCIS (Netherlands)

    Zlotina, A.; Galkina, S.; Krasikova, A.; Crooijmans, R.P.M.A.; Groenen, M.; Gaginskaya, E.; Deryusheva, S.

    2012-01-01

    Chicken (Gallus gallus domesticus, GGA) and Japanese quail (Coturnix coturnix japonica, CCO) karyotypes are very similar. They have identical chromosome number (2n = 78) and show a high degree of synteny. Centromere positions on the majority of orthologous chromosomes are different in these two spec

  14. Neocentrics and holokinetics (holocentrics): chromosomes out of the centromeric rules.

    Science.gov (United States)

    Guerra, M; Cabral, G; Cuacos, M; González-García, M; González-Sánchez, M; Vega, J; Puertas, M J

    2010-07-01

    The centromere appears as a single constriction at mitotic metaphase in most eukaryotic chromosomes. Holokinetic chromosomes are the exception to this rule because they do not show any centromeric constrictions. Holokinetic chromosomes are usually forgotten in most reviews about centromeres, despite their presence in a number of animal and plant species. They are generally linked to very intriguing and unusual mechanisms of mitosis and meiosis. Holokinetic chromosomes differ from monocentric chromosomes not only in the extension of the kinetochore plate, but also in many other peculiar karyological features, which could be understood as the 'holokinetic syndrome' that is reviewed in detail. Together with holokinetic chromosomes we review neocentromeric activity, a similarly intriguing case of regions able to pull chromosomes towards the poles without showing the main components reported to be essential to centromeric function. A neocentromere is a chromosomal region different from the true centromere in structure, DNA sequence and location, but is able to lead chromosomes to the cell poles in special circumstances. Neocentromeres have been reported in plants and animals showing different features. Both in humans and Drosophila, neocentric activity appears in somatic cells with defective chromosomes lacking a functional centromere. In most cases in plants, neocentromeres appear in chromosomes which have normal centromeres, but are active only during meiosis. Because of examples such as spontaneous or induced neocentromeres and holokinetic chromosomes, it is becoming less surprising that different structures and DNA sequences of centromeres appear in evolution.

  15. Schizosaccharomyces pombe centromere protein Mis19 links Mis16 and Mis18 to recruit CENP-A through interacting with NMD factors and the SWI/SNF complex.

    Science.gov (United States)

    Hayashi, Takeshi; Ebe, Masahiro; Nagao, Koji; Kokubu, Aya; Sajiki, Kenichi; Yanagida, Mitsuhiro

    2014-07-01

    CENP-A is a centromere-specific variant of histone H3 that is required for accurate chromosome segregation. The fission yeast Schizosaccharomyces pombe and mammalian Mis16 and Mis18 form a complex essential for CENP-A recruitment to centromeres. It is unclear, however, how the Mis16-Mis18 complex achieves this function. Here, we identified, by mass spectrometry, novel fission yeast centromere proteins Mis19 and Mis20 that directly interact with Mis16 and Mis18. Like Mis18, Mis19 and Mis20 are localized at the centromeres during interphase, but not in mitosis. Inactivation of Mis19 in a newly isolated temperature-sensitive mutant resulted in CENP-A delocalization and massive chromosome missegregation, whereas Mis20 was dispensable for proper chromosome segregation. Mis19 might be a bridge component for Mis16 and Mis18. We isolated extragenic suppressor mutants for temperature-sensitive mis18 and mis19 mutants and used whole-genome sequencing to determine the mutated sites. We identified two groups of loss-of-function suppressor mutations in non-sense-mediated mRNA decay factors (upf2 and ebs1), and in SWI/SNF chromatin-remodeling components (snf5, snf22 and sol1). Our results suggest that the Mis16-Mis18-Mis19-Mis20 CENP-A-recruiting complex, which is functional in the G1-S phase, may be counteracted by the SWI/SNF chromatin-remodeling complex and non-sense-mediated mRNA decay, which may prevent CENP-A deposition at the centromere.

  16. Promiscuous stimulation of ParF protein polymerization by heterogeneous centromere binding factors.

    Science.gov (United States)

    Machón, Cristina; Fothergill, Timothy J G; Barillà, Daniela; Hayes, Finbarr

    2007-11-16

    The segrosome is the nucleoprotein complex that mediates accurate segregation of bacterial plasmids. The segrosome of plasmid TP228 comprises ParF and ParG proteins that assemble on the parH centromere. ParF, which exemplifies one clade of the ubiquitous ParA superfamily of segregation proteins, polymerizes extensively in response to ATP binding. Polymerization is modulated by the ParG centromere binding factor (CBF). The segrosomes of plasmids pTAR, pVT745 and pB171 include ParA homologues of the ParF subgroup, as well as diverse homodimeric CBFs with no primary sequence similarity to ParG, or each other. Centromere binding by these analogues is largely specific. Here, we establish that the ParF homologues of pTAR and pB171 filament modestly with ATP, and that nucleotide hydrolysis is not required for this polymerization, which is more prodigious when the cognate CBF is also present. By contrast, the ParF homologue of plasmid pVT745 did not respond appreciably to ATP alone, but polymerized extensively in the presence of both its cognate CBF and ATP. The co-factors also stimulated nucleotide-independent polymerization of cognate ParF proteins. Moreover, apart from the CBF of pTAR, the disparate ParG analogues promoted polymerization of non-cognate ParF proteins suggesting that filamentation of the ParF proteins is enhanced by a common mechanism. Like ParG, the co-factors may be modular, possessing a centromere-specific interaction domain linked to a flexible region containing determinants that promiscuously stimulate ParF polymerization. The CBFs appear to function as bacterial analogues of formins, microtubule-associated proteins or related ancillary factors that regulate eucaryotic cytoskeletal dynamics.

  17. In vitro kinetochore assembly

    Science.gov (United States)

    Miell, Matthew D D; Straight, Aaron F

    2016-01-01

    The kinetochore is the primary site of interaction between chromosomes and microtubules of the mitotic spindle during chromosome segregation. The kinetochore is a complex of more than 100 proteins that transiently assemble during mitosis at a single defined region on each chromosome, known as the centromere. Kinetochore assembly and activity must be tightly regulated to ensure proper microtubule interaction and faithful chromosome segregation because perturbation of kinetochores often results in aneuploidy and cell lethality. As such, cell free and reconstituted systems to analyze kinetochore formation and function are invaluable in probing the biochemical activities of kinetochores. In vitro approaches to studying kinetochores have enabled the manipulation of kinetochore protein structure, function, interactions and regulation that are not possible in cells. Here we outline a cell-free approach for the assembly of centromeres and recruitment of functional kinetochores that enables their manipulation and analysis. PMID:27193846

  18. Microsatellite-centromere mapping in Japanese scallop ( Patinopecten yessoensis) through half-tetrad analysis in gynogenetic diploid families

    Science.gov (United States)

    Li, Qi; Qi, Mingjun; Nie, Hongtao; Kong, Lingfeng; Yu, Hong

    2016-06-01

    Gene-centromere mapping is an essential prerequisite for understanding the composition and structure of genomes. Half-tetrad analysis is a powerful tool for mapping genes and understanding chromosomal behavior during meiosis. The Japanese scallop ( Patinopecten yessoensis), a cold-tolerant species inhabiting the northwestern Pacific coast, is a commercially important marine bivalve in Asian countries. In this study, inheritance of 32 informative microsatellite loci was examined in 70-h D-shaped larvae of three induced meiogynogenetic diploid families of P. yessoensis for centromere mapping using half-tetrad analysis. The ratio of gynogenetic diploids was proven to be 100%, 100% and 96% in the three families, respectively. Inheritance analysis in the control crosses showed that 51 of the 53 genotypic ratios observed were in accordance with Mendelian expectations at the 5% level after Bonferroni correction. Seven of the 32 microsatellite loci showed the existence of null alleles in control crosses. The second division segregation frequency ( y) of the microsatellite loci ranged from 0.07 to 0.85 with a mean of 0.38, suggesting the existence of positive interference after a single chiasma formation in some chromosomes in the scallop. Microsatellite-centromere distances ranged from 4 cM to 42 cM under the assumption of complete interference. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards the assembly of genetic maps in the commercially important scallop species.

  19. The centromeric/nucleolar chromatin protein ZFP-37 may function to specify neuronal nuclear domains

    NARCIS (Netherlands)

    E. Payen; T. Verkerk (Ton); D. Michalovich (David); S.D. Dreyer; A. Winterpacht; B. Lee (Brendan); C.I. de Zeeuw (Chris); N.J. Galjart (Niels); F.G. Grosveld (Frank)

    1998-01-01

    textabstractMurine ZFP-37 is a member of the large family of C2H2 type zinc finger proteins. It is characterized by a truncated NH2-terminal Kruppel-associated box and is thought to play a role in transcriptional regulation. During development Zfp-37 mRNA is most abunda

  20. Loss of maternal ATRX results in centromere instability and aneuploidy in the mammalian oocyte and pre-implantation embryo.

    Science.gov (United States)

    Baumann, Claudia; Viveiros, Maria M; De La Fuente, Rabindranath

    2010-09-23

    The α-thalassemia/mental retardation X-linked protein (ATRX) is a chromatin-remodeling factor known to regulate DNA methylation at repetitive sequences of the human genome. We have previously demonstrated that ATRX binds to pericentric heterochromatin domains in mouse oocytes at the metaphase II stage where it is involved in mediating chromosome alignment at the meiotic spindle. However, the role of ATRX in the functional differentiation of chromatin structure during meiosis is not known. To test ATRX function in the germ line, we developed an oocyte-specific transgenic RNAi knockdown mouse model. Our results demonstrate that ATRX is required for heterochromatin formation and maintenance of chromosome stability during meiosis. During prophase I arrest, ATRX is necessary to recruit the transcriptional regulator DAXX (death domain associated protein) to pericentric heterochromatin. At the metaphase II stage, transgenic ATRX-RNAi oocytes exhibit abnormal chromosome morphology associated with reduced phosphorylation of histone 3 at serine 10 as well as chromosome segregation defects leading to aneuploidy and severely reduced fertility. Notably, a large proportion of ATRX-depleted oocytes and 1-cell stage embryos exhibit chromosome fragments and centromeric DNA-containing micronuclei. Our results provide novel evidence indicating that ATRX is required for centromere stability and the epigenetic control of heterochromatin function during meiosis and the transition to the first mitosis.

  1. Loss of maternal ATRX results in centromere instability and aneuploidy in the mammalian oocyte and pre-implantation embryo.

    Directory of Open Access Journals (Sweden)

    Claudia Baumann

    2010-09-01

    Full Text Available The α-thalassemia/mental retardation X-linked protein (ATRX is a chromatin-remodeling factor known to regulate DNA methylation at repetitive sequences of the human genome. We have previously demonstrated that ATRX binds to pericentric heterochromatin domains in mouse oocytes at the metaphase II stage where it is involved in mediating chromosome alignment at the meiotic spindle. However, the role of ATRX in the functional differentiation of chromatin structure during meiosis is not known. To test ATRX function in the germ line, we developed an oocyte-specific transgenic RNAi knockdown mouse model. Our results demonstrate that ATRX is required for heterochromatin formation and maintenance of chromosome stability during meiosis. During prophase I arrest, ATRX is necessary to recruit the transcriptional regulator DAXX (death domain associated protein to pericentric heterochromatin. At the metaphase II stage, transgenic ATRX-RNAi oocytes exhibit abnormal chromosome morphology associated with reduced phosphorylation of histone 3 at serine 10 as well as chromosome segregation defects leading to aneuploidy and severely reduced fertility. Notably, a large proportion of ATRX-depleted oocytes and 1-cell stage embryos exhibit chromosome fragments and centromeric DNA-containing micronuclei. Our results provide novel evidence indicating that ATRX is required for centromere stability and the epigenetic control of heterochromatin function during meiosis and the transition to the first mitosis.

  2. Validation of the micronucleus-centromere assay for biological dosimetry

    Directory of Open Access Journals (Sweden)

    Wojcik A.

    2000-01-01

    Full Text Available The micronucleus assay is frequently used for purposes of biological dosimetry. Due to high interindividual variability in the spontaneous frequency of micronuclei, its sensitivity in the low dose region is poor. It has been suggested that this problem can be mitigated by selectively analyzing the frequency of those micronuclei which contain only acentric fragments. Using a pan-centromeric FISH probe we have studied the dose dependence of micronuclei with centromeres in peripheral lymphocytes of human donors. In contrast to previous publications, our approach is based on determining the relative frequency of micronuclei with and without centromeric signals. Our results confirm previous observations that in the low dose range of ionizing radiation, the micronucleus-centromere assay is more sensitive than the conventional micronucleus test.

  3. Dicentric chromosomes: unique models to study centromere function and inactivation

    OpenAIRE

    Kaitlin M Stimpson; Matheny, Justyne E.; Sullivan, Beth A.

    2012-01-01

    Dicentric chromosomes are products of genome rearrangement that place two centromeres on the same chromosome. Depending on the organism, dicentric stability varies after formation. In humans, dicentrics occur naturally in a substantial portion of the population and usually segregate successfully in mitosis and meiosis. Their stability has been attributed to inactivation of one of the two centromeres, creating a functionally monocentric chromosome that can segregate normally during cell divisi...

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

    Science.gov (United States)

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

    2003-10-24

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  6. Multilocus analysis for gene-centromere mapping using first polar bodies and secondary oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Da, Y.; Jarrell, V.L.; Wang, T.; Fernando, R.L.; Wheeler, M.B.; Lewin, H.A. [Univ. of Illinois, Urbana, IL (United States)

    1995-02-01

    Polar body and oocyte typing is a new technique for gene-centromere mapping and for generating female linkage maps. A maximum likelihood approach is presented for ordering multiple markers relative to the centromere and for estimating recombination frequencies between markers and between the centromere and marker loci. Three marker-centromere orders are possible for each pair of markers: two orders when the centromere flanks the two markers and one order when the centromere is flanked by the two markers. For each possible order, the likelihood was expressed as a function of recombination frequencies for two adjacent intervals. LOD score for recombination frequency between markers or between the centromere and a marker locus was derived based on the likelihood for each gene-centromere order. The methods developed herein provide a general solution to the problem of multilocus gene-centromere mapping that involves all theoretical crossover possibilities, including four-strand double crossovers. 24 refs., 2 figs., 2 tabs.

  7. Adaptive evolution of centromere proteins in plants and animals

    Directory of Open Access Journals (Sweden)

    Henikoff Steven

    2004-08-01

    Full Text Available Abstract Background Centromeres represent the last frontiers of plant and animal genomics. Although they perform a conserved function in chromosome segregation, centromeres are typically composed of repetitive satellite sequences that are rapidly evolving. The nucleosomes of centromeres are characterized by a special H3-like histone (CenH3, which evolves rapidly and adaptively in Drosophila and Arabidopsis. Most plant, animal and fungal centromeres also bind a large protein, centromere protein C (CENP-C, that is characterized by a single 24 amino-acid motif (CENPC motif. Results Whereas we find no evidence that mammalian CenH3 (CENP-A has been evolving adaptively, mammalian CENP-C proteins contain adaptively evolving regions that overlap with regions of DNA-binding activity. In plants we find that CENP-C proteins have complex duplicated regions, with conserved amino and carboxyl termini that are dissimilar in sequence to their counterparts in animals and fungi. Comparisons of Cenpc genes from Arabidopsis species and from grasses revealed multiple regions that are under positive selection, including duplicated exons in some grasses. In contrast to plants and animals, yeast CENP-C (Mif2p is under negative selection. Conclusions CENP-Cs in all plant and animal lineages examined have regions that are rapidly and adaptively evolving. To explain these remarkable evolutionary features for a single-copy gene that is needed at every mitosis, we propose that CENP-Cs, like some CenH3s, suppress meiotic drive of centromeres during female meiosis. This process can account for the rapid evolution and the complexity of centromeric DNA in plants and animals as compared to fungi.

  8. A Broad Set of Chromatin Factors Influences Splicing

    Science.gov (United States)

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

    2016-01-01

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

  9. Reprogramming the chromatin landscape

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  10. LINE retrotransposon RNA is an essential structural and functional epigenetic component of a core neocentromeric chromatin.

    Directory of Open Access Journals (Sweden)

    Anderly C Chueh

    2009-01-01

    Full Text Available We have previously identified and characterized the phenomenon of ectopic human centromeres, known as neocentromeres. Human neocentromeres form epigenetically at euchromatic chromosomal sites and are structurally and functionally similar to normal human centromeres. Recent studies have indicated that neocentromere formation provides a major mechanism for centromere repositioning, karyotype evolution, and speciation. Using a marker chromosome mardel(10 containing a neocentromere formed at the normal chromosomal 10q25 region, we have previously mapped a 330-kb CENP-A-binding domain and described an increased prevalence of L1 retrotransposons in the underlying DNA sequences of the CENP-A-binding clusters. Here, we investigated the potential role of the L1 retrotransposons in the regulation of neocentromere activity. Determination of the transcriptional activity of a panel of full-length L1s (FL-L1s across a 6-Mb region spanning the 10q25 neocentromere chromatin identified one of the FL-L1 retrotransposons, designated FL-L1b and residing centrally within the CENP-A-binding clusters, to be transcriptionally active. We demonstrated the direct incorporation of the FL-L1b RNA transcripts into the CENP-A-associated chromatin. RNAi-mediated knockdown of the FL-L1b RNA transcripts led to a reduction in CENP-A binding and an impaired mitotic function of the 10q25 neocentromere. These results indicate that LINE retrotransposon RNA is a previously undescribed essential structural and functional component of the neocentromeric chromatin and that retrotransposable elements may serve as a critical epigenetic determinant in the chromatin remodelling events leading to neocentromere formation.

  11. Lamin C and chromatin organization in Drosophila

    Indian Academy of Sciences (India)

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

    2010-04-01

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

  12. Making copies of chromatin: the challenge of nucleosomal organization and epigenetic information.

    Science.gov (United States)

    Corpet, Armelle; Almouzni, Geneviève

    2009-01-01

    Understanding the basic mechanisms underlying chromatin dynamics during DNA replication in eukaryotic cells is of fundamental importance. Beyond DNA compaction, chromatin organization represents a means to regulate genome function. Thus, the inheritance and maintenance of the DNA sequence, along with its organization into chromatin, is central for eukaryotic life. To orchestrate DNA replication in the context of chromatin is a challenge, both in terms of accessibility to the compact structures and maintenance of chromatin organization. To meet the challenge of maintenance, cells have evolved efficient nucleosome dynamics involving assembly pathways and chromatin maturation mechanisms that restore chromatin organization in the wake of DNA replication. In this review, we describe our current knowledge concerning how these pathways operate at the nucleosomal level and highlight the key players, such as histone chaperones, chromatin remodelers or modifiers, involved in the process of chromatin duplication. Major advances have been made recently concerning de novo nucleosome assembly and our understanding of its coordination with recycling of parental histones is progressing. Insights into the transmission of chromatin-based information during replication have important implications in the field of epigenetics to fully comprehend how the epigenetic landscape might, or at times might not, be stably maintained in the face of dramatic changes in chromatin structure.

  13. Chromatin dynamics in plants

    NARCIS (Netherlands)

    Fransz, P.F.; Jong, de J.H.

    2002-01-01

    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 netwo

  14. Proteomic interrogation of human chromatin.

    Directory of Open Access Journals (Sweden)

    Mariana P Torrente

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2002-07-01

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

  16. No longer a nuisance: long non-coding RNAs join CENP-A in epigenetic centromere regulation.

    Science.gov (United States)

    Rošić, Silvana; Erhardt, Sylvia

    2016-04-01

    Centromeres represent the basis for kinetochore formation, and are essential for proper chromosome segregation during mitosis. Despite these essential roles, centromeres are not defined by specific DNA sequences, but by epigenetic means. The histone variant CENP-A controls centromere identity epigenetically and is essential for recruiting kinetochore components that attach the chromosomes to the mitotic spindle during mitosis. Recently, a new player in centromere regulation has emerged: long non-coding RNAs transcribed from repetitive regions of centromeric DNA function in regulating centromeres epigenetically. This review summarizes recent findings on the essential roles that transcription, pericentromeric transcripts, and centromere-derived RNAs play in centromere biology.

  17. Organization and evolution of primate centromeric DNA from whole-genome shotgun sequence data.

    Directory of Open Access Journals (Sweden)

    Can Alkan

    2007-09-01

    Full Text Available The major DNA constituent of primate centromeres is alpha satellite DNA. As much as 2%-5% of sequence generated as part of primate genome sequencing projects consists of this material, which is fragmented or not assembled as part of published genome sequences due to its highly repetitive nature. Here, we develop computational methods to rapidly recover and categorize alpha-satellite sequences from previously uncharacterized whole-genome shotgun sequence data. We present an algorithm to computationally predict potential higher-order array structure based on paired-end sequence data and then experimentally validate its organization and distribution by experimental analyses. Using whole-genome shotgun data from the human, chimpanzee, and macaque genomes, we examine the phylogenetic relationship of these sequences and provide further support for a model for their evolution and mutation over the last 25 million years. Our results confirm fundamental differences in the dispersal and evolution of centromeric satellites in the Old World monkey and ape lineages of evolution.

  18. The cotton centromere contains a Ty3-gypsy-like LTR retroelement.

    Directory of Open Access Journals (Sweden)

    Song Luo

    Full Text Available The centromere is a repeat-rich structure essential for chromosome segregation; with the long-term aim of understanding centromere structure and function, we set out to identify cotton centromere sequences. To isolate centromere-associated sequences from cotton, (Gossypium hirsutum we surveyed tandem and dispersed repetitive DNA in the genus. Centromere-associated elements in other plants include tandem repeats and, in some cases, centromere-specific retroelements. Examination of cotton genomic survey sequences for tandem repeats yielded sequences that did not localize to the centromere. However, among the repetitive sequences we also identified a gypsy-like LTR retrotransposon (Centromere Retroelement Gossypium, CRG that localizes to the centromere region of all chromosomes in domestic upland cotton, Gossypium hirsutum, the major commercially grown cotton. The location of the functional centromere was confirmed by immunostaining with antiserum to the centromere-specific histone CENH3, which co-localizes with CRG hybridization on metaphase mitotic chromosomes. G. hirsutum is an allotetraploid composed of A and D genomes and CRG is also present in the centromere regions of other AD cotton species. Furthermore, FISH and genomic dot blot hybridization revealed that CRG is found in D-genome diploid cotton species, but not in A-genome diploid species, indicating that this retroelement may have invaded the A-genome centromeres during allopolyploid formation and amplified during evolutionary history. CRG is also found in other diploid Gossypium species, including B and E2 genome species, but not in the C, E1, F, and G genome species tested. Isolation of this centromere-specific retrotransposon from Gossypium provides a probe for further understanding of centromere structure, and a tool for future engineering of centromere mini-chromosomes in this important crop species.

  19. Hijacking the chromatin remodeling machinery: impact of SWI/SNF perturbations in cancer

    OpenAIRE

    Weissman, Bernard; Knudsen, Karen E

    2009-01-01

    There is increasing evidence that alterations in chromatin remodeling play a significant role in human disease. The SWI/SNF chromatin remodeling complex family mobilizes nucleosomes and functions as a master regulator of gene expression and chromatin dynamics whose functional specificity is driven by combinatorial assembly of a central ATPase and association with 10-12 unique subunits. While the biochemical consequence of SWI/SNF in model systems has been extensively reviewed, the present art...

  20. Toxoplasma gondii chromodomain protein 1 binds to heterochromatin and colocalises with centromeres and telomeres at the nuclear periphery.

    Directory of Open Access Journals (Sweden)

    Mathieu Gissot

    Full Text Available BACKGROUND: Apicomplexan parasites are responsible for some of the most deadly parasitic diseases afflicting humans, including malaria and toxoplasmosis. These obligate intracellular parasites exhibit a complex life cycle and a coordinated cell cycle-dependant expression program. Their cell division is a coordinated multistep process. How this complex mechanism is organised remains poorly understood. METHODS AND FINDINGS: In this study, we provide evidence for a link between heterochromatin, cell division and the compartmentalisation of the nucleus in Toxoplasma gondii. We characterised a T. gondii chromodomain containing protein (named TgChromo1 that specifically binds to heterochromatin. Using ChIP-on-chip on a genome-wide scale, we report TgChromo1 enrichment at the peri-centromeric chromatin. In addition, we demonstrate that TgChromo1 is cell-cycle regulated and co-localised with markers of the centrocone. Through the loci-specific FISH technique for T. gondii, we confirmed that TgChromo1 occupies the same nuclear localisation as the peri-centromeric sequences. CONCLUSION: We propose that TgChromo1 may play a role in the sequestration of chromosomes at the nuclear periphery and in the process of T. gondii cell division.

  1. SHUGOSHINs and PATRONUS protect meiotic centromere cohesion in Arabidopsis thaliana.

    Science.gov (United States)

    Zamariola, Linda; De Storme, Nico; Vannerum, Katrijn; Vandepoele, Klaas; Armstrong, Susan J; Franklin, F Christopher H; Geelen, Danny

    2014-03-01

    In meiosis, chromosome cohesion is maintained by the cohesin complex, which is released in a two-step manner. At meiosis I, the meiosis-specific cohesin subunit Rec8 is cleaved by the protease Separase along chromosome arms, allowing homologous chromosome segregation. Next, in meiosis II, cleavage of the remaining centromere cohesin results in separation of the sister chromatids. In eukaryotes, protection of centromeric cohesion in meiosis I is mediated by SHUGOSHINs (SGOs). The Arabidopsis genome contains two SGO homologs. Here we demonstrate that Atsgo1 mutants show a premature loss of cohesion of sister chromatid centromeres at anaphase I and that AtSGO2 partially rescues this loss of cohesion. In addition to SGOs, we characterize PATRONUS which is specifically required for the maintenance of cohesion of sister chromatid centromeres in meiosis II. In contrast to the Atsgo1 Atsgo2 double mutant, patronus T-DNA insertion mutants only display loss of sister chromatid cohesion after meiosis I, and additionally show disorganized spindles, resulting in defects in chromosome segregation in meiosis. This leads to reduced fertility and aneuploid offspring. Furthermore, we detect aneuploidy in sporophytic tissue, indicating a role for PATRONUS in chromosome segregation in somatic cells. Thus, ploidy stability is preserved in Arabidopsis by PATRONUS during both meiosis and mitosis.

  2. Recombination patterns reveal information about centromere location on linkage maps

    DEFF Research Database (Denmark)

    Limborg, Morten T.; McKinney, Garrett J.; Seeb, Lisa W.

    2016-01-01

    . mykiss) characterized by low and unevenly distributed recombination – a general feature of male meiosis in many species. Further, a high frequency of double crossovers along chromosome arms in barley reduced resolution for locating centromeric regions on most linkage groups. Despite these limitations...

  3. Dicentric chromosomes: unique models to study centromere function and inactivation.

    Science.gov (United States)

    Stimpson, Kaitlin M; Matheny, Justyne E; Sullivan, Beth A

    2012-07-01

    Dicentric chromosomes are products of genome rearrangement that place two centromeres on the same chromosome. Depending on the organism, dicentric stability varies after formation. In humans, dicentrics occur naturally in a substantial portion of the population and usually segregate successfully in mitosis and meiosis. Their stability has been attributed to inactivation of one of the two centromeres, creating a functionally monocentric chromosome that can segregate normally during cell division. The molecular basis for centromere inactivation is not well understood, although studies in model organisms and in humans suggest that genomic and epigenetic mechanisms can be involved. Furthermore, constitutional dicentric chromosomes ascertained in patients presumably represent the most stable chromosomes, so the spectrum of dicentric fates, if it exists, is not entirely clear. Studies of engineered or induced dicentrics in budding yeast and plants have provided significant insight into the fate of dicentric chromosomes. And, more recently, studies have shown that dicentrics in humans can also undergo multiple fates after formation. Here, we discuss current experimental evidence from various organisms that has deepened our understanding of dicentric behavior and the intriguingly complex process of centromere inactivation.

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

    Science.gov (United States)

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

    2015-02-18

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

  5. Total centromere size and genome size are strongly correlated in ten grass species.

    Science.gov (United States)

    Zhang, Han; Dawe, R Kelly

    2012-05-01

    It has been known for decades that centromere size varies across species, but the factors involved in setting centromere boundaries are unknown. As a means to address this question, we estimated centromere sizes in ten species of the grass family including rice, maize, and wheat, which diverged 60~80 million years ago and vary by 40-fold in genome size. Measurements were made using a broadly reactive antibody to rice centromeric histone H3 (CENH3). In species-wide comparisons, we found a clear linear relationship between total centromere size and genome size. Species with large genomes and few chromosomes tend to have the largest centromeres (e.g., rye) while species with small genomes and many chromosomes have the smallest centromeres (e.g., rice). However, within a species, centromere size is surprisingly uniform. We present evidence from three oat-maize addition lines that support this claim, indicating that each of three maize centromeres propagated in oat are not measurably different from each other. In the context of previously published data, our results suggest that the apparent correlation between chromosome and centromere size is incidental to a larger trend that reflects genome size. Centromere size may be determined by a limiting component mechanism similar to that described for Caenorhabditis elegans centrosomes.

  6. Cas9 Functionally Opens Chromatin.

    Directory of Open Access Journals (Sweden)

    Amira A Barkal

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

  7. Essential roles of Snf21, a Swi2/Snf2 family chromatin remodeler, in fission yeast mitosis.

    Science.gov (United States)

    Yamada, Kentaro; Hirota, Kouji; Mizuno, Ken-Ichi; Shibata, Takehiko; Ohta, Kunihiro

    2008-10-01

    ATP-dependent chromatin remodelers (ADCRs) convert local chromatin structure into both transcriptional active and repressive state. Recent studies have revealed that ADCRs play diverse regulatory roles in chromosomal events such as DNA repair and recombination. Here we have newly identified a fission yeast gene encoding a Swi2/Snf2 family ADCR. The amino acid sequence of this gene, snf21(+), implies that Snf21 is a fission yeast orthologue of the budding yeast Sth1, the catalytic core of the RSC chromatin remodeling complex. The snf21(+) gene product is a nuclear protein essential to cell viability: the null mutant cells stop growing after several rounds of cell divisions. A temperature sensitive allele of snf21(+), snf21-36 exhibits at non-permissive temperature (34 degrees C) a cell cycle arrest at G2-M phase and defects in chromosome segregation, thereby causing cell elongation, lack of cell growth, and death of some cell population. snf21-36 shows thiabendazole (TBZ) sensitivity even at permissive temperature (25 degrees C). The TBZ sensitivity becomes severer as snf21-36 is combined with the deletion of a centromere-localized Mad2 spindle checkpoint protein. The cell cycle arrest phenotype at 34 degrees C cannot be rescued by the mad2(+) deletion, although it is substantially alleviated at 30 degrees C in mad2Delta. These data suggest that Snf21 plays an essential role in mitosis possibly functioning in centromeric chromatin.

  8. Dicentric chromosome stretching during anaphase reveals roles of Sir2/Ku in chromatin compaction in budding yeast.

    Science.gov (United States)

    Thrower, D A; Bloom, K

    2001-09-01

    We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction. Escherichia coli lac operator DNA was placed between two centromeres on a conditional dicentric chromosome in budding yeast cells and made visible by expression of a lac repressor-green fluorescent fusion protein. Centromeres on the same chromatid of a dicentric chromosome attach to opposite poles approximately 50% of the time, resulting in chromosome bridges during anaphase. In cells deleted for yKU70, yKU80, or SIR2, a 10-kb region of the dicentric chromosome stretched along the spindle axis to a length of 6 microm during anaphase. On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation. Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the dicentric chromosome. These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a chromosome experiencing tension or DNA damage. The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA.

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

    Science.gov (United States)

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

    2013-11-21

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

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

    NARCIS (Netherlands)

    J.G. van Bemmel (Joke)

    2012-01-01

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

  11. Two siblings with immunodeficiency, facial abnormalities and chromosomal instability without mutation in DNMT3B gene but liability towards malignancy; a new chromatin disorder delineation?

    Directory of Open Access Journals (Sweden)

    Neitzel Heidemarie

    2010-03-01

    Full Text Available Abstract Background ICF syndrome (standing for Immunodeficiency, Centromere instability and Facial anomalies syndrome is a very rare autosomal recessive immune disorder caused by mutations of the gene de novo DNA-methyltransferase 3B (DNMT3B. However, in the literature similar clinical cases without such mutations are reported, as well. Results We report on a family in which the unrelated spouses had two female siblings sharing similar phenotypic features resembling ICF-syndrome, i.e. congenital abnormalities, immunodeficiency, developmental delay and high level of chromosomal instability, including high frequency of centromeric/pericentromeric rearrangements and breaks, chromosomal fragments despiralization or pulverization. However, mutations in DNMT3B could not be detected. Conclusion The discovery of a new so-called 'chromatin disorder' is suggested. Clinical, molecular genetic and cytogenetic characteristics are reported and compared to other 'chromatin disorders'.

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

    DEFF Research Database (Denmark)

    Jasencakova, Zusana; Groth, Anja

    2010-01-01

    replication and chromatin assembly processes in time and space. Dynamic recycling and de novo deposition of histones are fundamental for chromatin restoration. Histone post-translational modifications (PTMs) are thought to have a causal role in establishing distinct chromatin structures. Here we discuss PTMs......In dividing cells genome stability and function rely on faithful transmission of both DNA sequence and its organization into chromatin. In the course of DNA replication chromatin undergoes transient genome-wide disruption followed by restoration on new DNA. This involves tight coordination of DNA...... present on new and parental histones and how they influence genome stability and restoration of epigenetically defined domains. Newly deposited histones must change their signature in the process of chromatin restoration, this may occur in a step-wise fashion involving replication-coupled processes...

  13. Uncoupling of satellite DNA and centromeric function in the genus Equus.

    Directory of Open Access Journals (Sweden)

    Francesca M Piras

    2010-02-01

    Full Text Available In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1 several centromeres, including the previously described evolutionary new centromeres (ENCs, seem to be devoid of satellite DNA, and 2 satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs.

  14. Epigenetics & chromatin: Interactions and processes

    NARCIS (Netherlands)

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

    2013-01-01

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

  15. Epigenetically-inherited centromere and neocentromere DNA replicates earliest in S-phase.

    Directory of Open Access Journals (Sweden)

    Amnon Koren

    2010-08-01

    Full Text Available Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position.

  16. The chromosomal passenger complex activates Polo kinase at centromeres.

    Directory of Open Access Journals (Sweden)

    Mar Carmena

    2012-01-01

    Full Text Available The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. Our study concerns the coordination of the activities of these two kinases in vivo. We show that INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC, which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. We show here that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity and is critical for Polo function at kinetochores. Our results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation.

  17. The chromosomal passenger complex activates Polo kinase at centromeres.

    Science.gov (United States)

    Carmena, Mar; Pinson, Xavier; Platani, Melpi; Salloum, Zeina; Xu, Zhenjie; Clark, Anthony; Macisaac, Fiona; Ogawa, Hiromi; Eggert, Ulrike; Glover, David M; Archambault, Vincent; Earnshaw, William C

    2012-01-01

    The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. Our study concerns the coordination of the activities of these two kinases in vivo. We show that INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. We show here that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. Our results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation.

  18. Molecular characterization of flow-sorted mammalian centromeres

    Energy Technology Data Exchange (ETDEWEB)

    Hamkalo, B.A.; Henschen, A.; Parseghian, M.H. [Univ. of Calfornia, Irvine, CA (United States). Dept. of Molecular Biology and Biochemistry] [and others

    1998-12-31

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The project involved experiments directed towards developing a molecular characterization of the centromere region of mammalian chromosomes. Attempts to purify this essential chromosomal locus by conventional methods have thus far been unsuccessful. However, preliminary data obtained in collaboration with the National Flow Cytometry Resource (NFCR) showed that it is possible to purify a chromosome fragment that is present in certain cultured mouse cell lines and has all the properties expected of an intact centromere region. To begin sorting this minichromosome for the identification of proteins preferentially associated with centromere regions, standard buffers utilized in chromosome sorting were evaluated for potential effects on maintenance of chromosomal proteins during sorting. The data indicate that the presence of several buffer constituents results in the extraction of all but a few chromosomal proteins. The subsequent use of a magnesium sulfate buffer resulted in the sorting of mouse chromosomes that do not suffer a significant loss of proteins. Several DNA stains were also evaluated for causing protein dissociation, but no significant losses were observed. Although flow-sorted chromosomes have been used extensively for DNA analysis and cloning, this is a pioneering effort by the NFCR, and its collaborators, to exploit chromosome sorting capabilities for the analysis of chromosomal proteins.

  19. Chromatin Structure of Epstein-Barr Virus Latent Episomes.

    Science.gov (United States)

    Lieberman, Paul M

    2015-01-01

    EBV latent infection is characterized by a highly restricted pattern of viral gene expression. EBV can establish latent infections in multiple different tissue types with remarkable variation and plasticity in viral transcription and replication. During latency, the viral genome persists as a multi-copy episome, a non-integrated-closed circular DNA with nucleosome structure similar to cellular chromosomes. Chromatin assembly and histone modifications contribute to the regulation of viral gene expression, DNA replication, and episome persistence during latency. This review focuses on how EBV latency is regulated by chromatin and its associated processes.

  20. Structure of chromatin in spermatozoa.

    Science.gov (United States)

    Björndahl, Lars; Kvist, Ulrik

    2014-01-01

    The specialized structure of the sperm chromatin has a dual function - first to protect the DNA from damage during storage and transport to the oocyte, and then to enable a rapid and complete unpacking of the undamaged paternal genome in the ooplasm. It is evident that zinc has a pivotal role in maintaining the structural stability and in enabling a rapid decondensation at the appropriate time. It is important for the sperm chromatin structure that the spermatozoa are ejaculated together with the zinc-rich prostatic secretion. Early exposure to zinc-binding seminal vesicular fluid can deplete the sperm chromatin of zinc and most likely induce surplus formation of disulfide bridges, likely to cause incomplete and delayed decondensation of the sperm chromatin in the oocyte. A premature decrease in sperm chromatin structure stability is likely to increase the risk for damage to the DNA due to increased access to the genome for DNA damaging compounds. The status of the sperm chromatin structure can vary in vitro depending on the exposure to zinc-depleting conditions when spermatozoa are stored in semen after ejaculation. When sperm DNA damage tests are evaluated and validated, it is therefore essential to also take into account the dynamics of zinc-dependent and zinc-independent sperm chromatin stability.

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

    Science.gov (United States)

    Redi, Carlo Alberto; Zuccotti, Maurizio

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Martina Belli

    2014-01-01

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

  3. Vernalization-mediated chromatin changes.

    Science.gov (United States)

    Zografos, Brett R; Sung, Sibum

    2012-07-01

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

  4. Decoding chromatin goes high tech.

    Science.gov (United States)

    Levy, Dan; Gozani, Or

    2010-09-17

    Identifying proteins that recognize histone methylation is critical for understanding chromatin function. Vermeulen et al. (2010) now describe a cutting-edge strategy to identify and characterize several nuclear proteins and complexes that recognize five major histone trimethyl marks.

  5. Mis17 is a regulatory module of the Mis6-Mal2-Sim4 centromere complex that is required for the recruitment of CenH3/CENP-A in fission yeast.

    Directory of Open Access Journals (Sweden)

    Yoshiharu Shiroiwa

    Full Text Available BACKGROUND: The centromere is the chromosome domain on which the mitotic kinetochore forms for proper segregation. Deposition of the centromeric histone H3 (CenH3, CENP-A is vital for the formation of centromere-specific chromatin. The Mis6-Mal2-Sim4 complex of the fission yeast S. pombe is required for the recruitment of CenH3 (Cnp1, but its function remains obscure. METHODOLOGY/PRINCIPAL FINDINGS: Mass spectrometry was performed on the proteins precipitated with Mis6- and Mis17-FLAG. The results together with the previously identified Sim4- and Mal2-TAP precipitated proteins indicated that the complex contains 12 subunits, Mis6, Sim4, Mal2, Mis15, Mis17, Cnl2, Fta1-4, Fta6-7, nine of which have human centromeric protein (CENP counterparts. Domain dissection indicated that the carboxy-half of Mis17 is functional, while its amino-half is regulatory. Overproduction of the amino-half caused strong negative dominance, which led to massive chromosome missegregation and hypersensitivity to the histone deacetylase inhibitor TSA. Mis17 was hyperphosphorylated and overproduction-induced negative dominance was abolished in six kinase-deletion mutants, ssp2 (AMPK, ppk9 (AMPK, ppk15 (Yak1, ppk30 (Ark1, wis4 (Ssk2, and lsk1 (P-TEFb. CONCLUSIONS: Mis17 may be a regulatory module of the Mis6 complex. Negative dominance of the Mis17 fragment is exerted while the complex and CenH3 remain at the centromere, a result that differs from the mislocalization seen in the mis17-362 mutant. The known functions of the kinases suggest an unexpected link between Mis17 and control of the cortex actin, nutrition, and signal/transcription. Possible interpretations are discussed.

  6. Heterochromatic genes undergo epigenetic changes and escape silencing in immunodeficiency, centromeric instability, facial anomalies (ICF syndrome.

    Directory of Open Access Journals (Sweden)

    Marie-Elisabeth Brun

    Full Text Available Immunodeficiency, Centromeric Instability, Facial Anomalies (ICF syndrome is a rare autosomal recessive disorder that is characterized by a marked immunodeficiency, severe hypomethylation of the classical satellites 2 and 3 associated with disruption of constitutive heterochromatin, and facial anomalies. Sixty percent of ICF patients have mutations in the DNMT3B (DNA methyltransferase 3B gene, encoding a de novo DNA methyltransferase. In the present study, we have shown that, in ICF lymphoblasts and peripheral blood, juxtacentromeric heterochromatic genes undergo dramatic changes in DNA methylation, indicating that they are bona fide targets of the DNMT3B protein. DNA methylation in heterochromatic genes dropped from about 80% in normal cells to approximately 30% in ICF cells. Hypomethylation was observed in five ICF patients and was associated with activation of these silent genes. Although DNA hypomethylation occurred in all the analyzed heterochromatic genes and in all the ICF patients, gene expression was restricted to some genes, every patient having his own group of activated genes. Histone modifications were preserved in ICF patients. Heterochromatic genes were associated with histone modifications that are typical of inactive chromatin: they had low acetylation on H3 and H4 histones and were slightly enriched in H3K9Me(3, both in ICF and controls. This was also the case for those heterochromatic genes that escaped silencing. This finding suggests that gene activation was not generalized to all the cells, but rather was restricted to a clonal cell population that may contribute to the phenotypic variability observed in ICF syndrome. A slight increase in H3K27 monomethylation was observed both in heterochromatin and active euchromatin in ICF patients; however, no correlation between this modification and activation of heterochromatic genes was found.

  7. Hematopoietic stem cell transplantation corrects the immunologic abnormalities associated with immunodeficiency-centromeric instability-facial dysmorphism syndrome.

    NARCIS (Netherlands)

    Gennery, A.R.; Slatter, M.A.; Bredius, R.G.; Hagleitner, M.M.; Weemaes, C.M.R.; Cant, A.J.; Lankester, A.C.

    2007-01-01

    Immunodeficiency-centromeric instability-facial dysmorphism syndrome, characterized by variable immunodeficiency, centromeric instability, and facial anomalies caused by epigenetic dysregulation resulting in hypomethylation, is caused in many patients by mutations in DNMT3B, a DNA methyltransferase

  8. Chromosome segregation regulation in human zygotes : Altered mitotic histone phosphorylation dynamics underlying centromeric targeting of the chromosomal passenger complex

    NARCIS (Netherlands)

    Van De Werken, C.; Avo Santos, M.; Laven, J. S E; Eleveld, C.; Fauser, B. C J M; Lens, S. M A; Baart, E. B.

    2015-01-01

    STUDY QUESTION Are the kinase feedback loops that regulate activation and centromeric targeting of the chromosomal passenger complex (CPC), functional during mitosis in human embryos? SUMMARY ANSWER Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal

  9. Spreading chromatin into chemical biology.

    Science.gov (United States)

    Allis, C David; Muir, Tom W

    2011-01-24

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

  10. High frequency of centromere inactivation resulting in stable dicentric chromosomes of maize.

    Science.gov (United States)

    Han, Fangpu; Lamb, Jonathan C; Birchler, James A

    2006-02-28

    Somatic chromosome spreads from maize (Zea mays L.) plants containing B-A translocation chromosomes undergoing the chromosome type breakage-fusion-bridge cycle were examined by FISH. The size and type of extra chromosomes varied among cells of the same individual. A collection of minichromosomes derived from the chromosome type breakage-fusion-bridge cycle was examined for the presence of stable dicentric chromosomes. Six of 23 chromosomes in the collection contained two regions with DNA sequences typical of centromeres. Functional analysis and immunolabeling of CENH3, the centromere-specific histone H3 variant, revealed only one functional centromere per chromosome, despite the duplicate centromere sequences. One plant was found with an inactive B centromere that had been translocated to the short arm of chromosome 9. The translocated centromere region appeared identical to that of a normal B chromosome. The inactivation of the centromeres was stable for at least four generations. By using dicentrics from dispensable chromosomes, centromere inactivation was found to be quite common under these circumstances.

  11. MHF1–2/CENP-S-X performs distinct roles in centromere metabolism and genetic recombination

    Science.gov (United States)

    Bhattacharjee, Sonali; Osman, Fekret; Feeney, Laura; Lorenz, Alexander; Bryer, Claire; Whitby, Matthew C.

    2013-01-01

    The histone-fold proteins Mhf1/CENP-S and Mhf2/CENP-X perform two important functions in vertebrate cells. First, they are components of the constitutive centromere-associated network, aiding kinetochore assembly and function. Second, they work with the FANCM DNA translocase to promote DNA repair. However, it has been unclear whether there is crosstalk between these roles. We show that Mhf1 and Mhf2 in fission yeast, as in vertebrates, serve a dual function, aiding DNA repair/recombination and localizing to centromeres to promote chromosome segregation. Importantly, these functions are distinct, with the former being dependent on their interaction with the FANCM orthologue Fml1 and the latter not. Together with Fml1, they play a second role in aiding chromosome segregation by processing sister chromatid junctions. However, a failure of this activity does not manifest dramatically increased levels of chromosome missegregation due to the Mus81–Eme1 endonuclease, which acts as a failsafe to resolve DNA junctions before the end of mitosis. PMID:24026537

  12. Chromatin organization and remodeling of interstitial telomeric sites during meiosis in the Mongolian gerbil (Meriones unguiculatus).

    Science.gov (United States)

    de la Fuente, Roberto; Manterola, Marcia; Viera, Alberto; Parra, María Teresa; Alsheimer, Manfred; Rufas, Julio S; Page, Jesús

    2014-08-01

    Telomeric DNA repeats are key features of chromosomes that allow the maintenance of integrity and stability in the telomeres. However, interstitial telomere sites (ITSs) can also be found along the chromosomes, especially near the centromere, where they may appear following chromosomal rearrangements like Robertsonian translocations. There is no defined role for ITSs, but they are linked to DNA damage-prone sites. We were interested in studying the structural organization of ITSs during meiosis, a kind of cell division in which programmed DNA damage events and noticeable chromatin reorganizations occur. Here we describe the presence of highly amplified ITSs in the pericentromeric region of Mongolian gerbil (Meriones unguiculatus) chromosomes. During meiosis, ITSs show a different chromatin conformation than DNA repeats at telomeres, appearing more extended and accumulating heterochromatin markers. Interestingly, ITSs also recruit the telomeric proteins RAP1 and TRF1, but in a stage-dependent manner, appearing mainly at late prophase I stages. We did not find a specific accumulation of DNA repair factors to the ITSs, such as γH2AX or RAD51 at these stages, but we could detect the presence of MLH1, a marker for reciprocal recombination. However, contrary to previous reports, we did not find a specific accumulation of crossovers at ITSs. Intriguingly, some centromeric regions of metacentric chromosomes may bind the nuclear envelope through the association to SUN1 protein, a feature usually performed by telomeres. Therefore, ITSs present a particular and dynamic chromatin configuration in meiosis, which could be involved in maintaining their genetic stability, but they additionally retain some features of distal telomeres, provided by their capability to associate to telomere-binding proteins.

  13. 植物着丝粒区串联重复序列的研究进展%Research Progress of Tandem Repetitive Sequence in the Centromere of Plant

    Institute of Scientific and Technical Information of China (English)

    郝薇薇; 周岩

    2013-01-01

      着丝粒是细胞染色体的重要结构组成,控制姊妹染色单体的结合、动粒的组装和纺锤丝的附着,确保真核生物细胞在有丝分裂和减数分裂过程中染色体的正常分离及遗传信息的稳定传递。植物着丝粒DNA序列主要由反转录转座子和串联重复序列构成。串联重复序列在着丝粒功能实现和基因组进化过程中起重要作用。随着测序技术的成熟,近年来对串联重复序列的研究取得了很大的进展。综述了植物串联重复序列结构、分析方法及在进化中的作用,以期为相关研究提供参考。%Centromeres are the important domains of chromosomes that are responsible for sister chromatid cohesion, kinetochore assembly and spindle attachment, and are essential for proper chromosome segregation during mitosis and meiosis. Satellite DNA and retrotransposons are the most abundant DNA elements found in plant centromere regions. Centromeric tandem repeat play an important role in the centromere function and genome evolution. The study of centromeric tandem repeats got great progress for the development of sequencing technology. This paper introduces the development of centromeric tandem repeat of plants.

  14. Chromatin analysis of occluded genes

    Science.gov (United States)

    Lee, Jae Hyun; Gaetz, Jedidiah; Bugarija, Branimir; Fernandes, Croydon J.; Snyder, Gregory E.; Bush, Eliot C.; Lahn, Bruce T.

    2009-01-01

    We recently described two opposing states of transcriptional competency. One is termed ‘competent’ whereby a gene is capable of responding to trans-acting transcription factors of the cell, such that it is active if appropriate transcriptional activators are present, though it can also be silent if activators are absent or repressors are present. The other is termed ‘occluded’ whereby a gene is silenced by cis-acting, chromatin-based mechanisms in a manner that blocks it from responding to trans-acting factors, such that it is silent even when activators are present in the cellular milieu. We proposed that gene occlusion is a mechanism by which differentiated cells stably maintain their phenotypic identities. Here, we describe chromatin analysis of occluded genes. We found that DNA methylation plays a causal role in maintaining occlusion for a subset of occluded genes. We further examined a variety of other chromatin marks typically associated with transcriptional silencing, including histone variants, covalent histone modifications and chromatin-associated proteins. Surprisingly, we found that although many of these marks are robustly linked to silent genes (which include both occluded genes and genes that are competent but silent), none is linked specifically to occluded genes. Although the observation does not rule out a possible causal role of these chromatin marks in occlusion, it does suggest that these marks might be secondary effect rather than primary cause of the silent state in many genes. PMID:19380460

  15. Single Molecule Studies of Chromatin

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-02-06

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

  16. Hijacking the chromatin remodeling machinery: impact of SWI/SNF perturbations in cancer.

    Science.gov (United States)

    Weissman, Bernard; Knudsen, Karen E

    2009-11-01

    There is increasing evidence that alterations in chromatin remodeling play a significant role in human disease. The SWI/SNF chromatin remodeling complex family mobilizes nucleosomes and functions as a master regulator of gene expression and chromatin dynamics whose functional specificity is driven by combinatorial assembly of a central ATPase and association with 10 to 12 unique subunits. Although the biochemical consequence of SWI/SNF in model systems has been extensively reviewed, the present article focuses on the evidence linking SWI/SNF perturbations to cancer initiation and tumor progression in human disease.

  17. Chromatin Remodeling and Plant Immunity.

    Science.gov (United States)

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

    2017-01-01

    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?

  18. The coordination of centromere replication, spindle formation, and kinetochore-microtubule interaction in budding yeast.

    Directory of Open Access Journals (Sweden)

    Hong Liu

    2008-11-01

    Full Text Available The kinetochore is a protein complex that assembles on centromeric DNA to mediate chromosome-microtubule interaction. Most eukaryotic cells form the spindle and establish kinetochore-microtubule interaction during mitosis, but budding yeast cells finish these processes in S-phase. It has long been noticed that the S-phase spindle in budding yeast is shorter than that in metaphase, but the biological significance of this short S-phase spindle structure remains unclear. We addressed this issue by using ask1-3, a temperature-sensitive kinetochore mutant that exhibits partially elongated spindles at permissive temperature in the presence of hydroxyurea (HU, a DNA synthesis inhibitor. After exposure to and removal of HU, ask1-3 cells show a delayed anaphase entry. This delay depends on the spindle checkpoint, which monitors kinetochore-microtubule interaction defects. Overproduction of microtubule-associated protein Ase1 or Cin8 also induces spindle elongation in HU-arrested cells. The spindle checkpoint-dependent anaphase entry delay is also observed after ASE1 or CIN8 overexpression in HU-arrested cells. Therefore, the shorter spindle in S-phase cells is likely to facilitate proper chromosome-microtubule interaction.

  19. The activity regulation of the mitotic centromere-associated kinesin by Polo-like kinase 1.

    Science.gov (United States)

    Ritter, Andreas; Sanhaji, Mourad; Steinhäuser, Kerstin; Roth, Susanne; Louwen, Frank; Yuan, Juping

    2015-03-30

    The mitotic centromere-associated kinesin (MCAK), a potent microtubule depolymerase, is involved in regulating microtubule dynamics. The activity and subcellular localization of MCAK are tightly regulated by key mitotic kinases, such as Polo-like kinase 1 (Plk1) by phosphorylating multiple residues in MCAK. Since Plk1 phosphorylates very often different residues of substrates at different stages, we have dissected individual phosphorylation of MCAK by Plk1 and characterized its function in more depth. We have recently shown that S621 in MCAK is the major phosphorylation site of Plk1, which is responsible for regulating MCAK's degradation by promoting the association of MCAK with APC/CCdc20. In the present study, we have addressed another two residues phosphorylated by Plk1, namely S632/S633 in the C-terminus of MCAK. Our data suggest that Plk1 phosphorylates S632/S633 and regulates its catalytic activity in mitosis. This phosphorylation is required for proper spindle assembly during early phases of mitosis. The subsequent dephosphorylation of S632/S633 might be necessary to timely align the chromosomes onto the metaphase plate. Therefore, our studies suggest new mechanisms by which Plk1 regulates MCAK: the degradation of MCAK is controlled by Plk1 phosphorylation on S621, whereas its activity is modulated by Plk1 phosphorylation on S632/S633 in mitosis.

  20. Chromatin differentiation between Theobroma cacao L. and T. grandiflorum Schum

    Directory of Open Access Journals (Sweden)

    Liliane G. Dantas

    2010-01-01

    Full Text Available A comparative analysis of mitotic chromosomes of Theobroma cacao (cacao and T. grandiflorum (cupuaçu was performed aiming to identify cytological differences between the two most important species of this genus. Both species have symmetric karyotypes, with 2n = 20 metacentric chromosomes ranging in size from 2.00 to 1.19 µm (cacao and from 2.21 to 1.15 mm (cupuaçu. The interphase nuclei of both species were of the arreticulate type, displaying up to 20 chromocentres, which were more regularly shaped in cacao than in cupuaçu. Prophase chromosomes of both species were more condensed in the proximal region, sometimes including the whole short arm. Both species exhibited only one pair of terminal heterochromatic bands, positively stained with chromomycin A3, which co-localized with the single 45S rDNA site. Each karyotype displayed a single 5S rDNA site in the proximal region of another chromosome pair. Heterochromatic bands were also observed on the centromeric/pericentromeric regions of all 20 chromosomes of cacao after C-banding followed by Giemsa or DAPI staining, whereas in cupuaçu they were never detected. These data suggest that the chromosomes of both species have been largely conserved and their pericentromeric chromatin is the only citologically differentiated region.

  1. Widespread Positive Selection Drives Differentiation of Centromeric Proteins in the Drosophila melanogaster subgroup.

    Science.gov (United States)

    Beck, Emily A; Llopart, Ana

    2015-11-25

    Rapid evolution of centromeric satellite repeats is thought to cause compensatory amino acid evolution in interacting centromere-associated kinetochore proteins. Cid, a protein that mediates kinetochore/centromere interactions, displays particularly high amino acid turnover. Rapid evolution of both Cid and centromeric satellite repeats led us to hypothesize that the apparent compensatory evolution may extend to interacting partners in the Condensin I complex (i.e., SMC2, SMC4, Cap-H, Cap-D2, and Cap-G) and HP1s. Missense mutations in these proteins often result in improper centromere formation and aberrant chromosome segregation, thus selection for maintained function and coevolution among proteins of the complex is likely strong. Here, we report evidence of rapid evolution and recurrent positive selection in seven centromere-associated proteins in species of the Drosophila melanogaster subgroup, and further postulate that positive selection on these proteins could be a result of centromere drive and compensatory changes, with kinetochore proteins competing for optimal spindle attachment.

  2. Centromere inactivation on a neo-Y fusion chromosome in threespine stickleback fish.

    Science.gov (United States)

    Cech, Jennifer N; Peichel, Catherine L

    2016-12-01

    Having one and only one centromere per chromosome is essential for proper chromosome segregation during both mitosis and meiosis. Chromosomes containing two centromeres are known as dicentric and often mis-segregate during cell division, resulting in aneuploidy or chromosome breakage. Dicentric chromosome can be stabilized by centromere inactivation, a process which reestablishes monocentric chromosomes. However, little is known about this process in naturally occurring dicentric chromosomes. Using a combination of fluorescence in situ hybridization (FISH) and immunofluorescence combined with FISH (IF-FISH) on metaphase chromosome spreads, we demonstrate that centromere inactivation has evolved on a neo-Y chromosome fusion in the Japan Sea threespine stickleback fish (Gasterosteus nipponicus). We found that the centromere derived from the ancestral Y chromosome has been inactivated. Our data further suggest that there have been genetic changes to this centromere in the two million years since the formation of the neo-Y chromosome, but it remains unclear whether these genetic changes are a cause or consequence of centromere inactivation.

  3. Guarding against Collateral Damage during Chromatin Transactions

    DEFF Research Database (Denmark)

    Altmeyer, Matthias; Lukas, Jiri

    2013-01-01

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

  4. Biophysical studies of cholesterol effects on chromatin.

    Science.gov (United States)

    Silva, Isabel T G; Fernandes, Vinicius; Souza, Caio; Treptow, Werner; Santos, Guilherme Martins

    2017-03-22

    Changes in chromatin structure regulate gene expression and genome maintenance. Molecules that bind to the nucleosome, the complex of DNA and histone proteins, are key modulators of chromatin structure. Previous work indicated that cholesterol, a ubiquitous cellular lipid, may bind to chromatin in vivo, suggesting a potential function for lipids in modulating chromatin architecture. However, the molecular mechanisms of cholesterol action on chromatin structure have remained unclear. Here, we explored the biophysical impact of cholesterol on nucleosome and chromatin fibers reconstituted in vitro and characterized in silico the cholesterol binding to nucleosome. Our findings support that cholesterol assists 10nm and 30nm chromatin formation and induces folding of long chromatin fibers as a result of direct interaction of the cholesterol to six nucleosomal binding sites.

  5. Transcriptional networks and chromatin remodeling controlling adipogenesis

    DEFF Research Database (Denmark)

    Siersbæk, Rasmus; Nielsen, Ronni; Mandrup, Susanne

    2012-01-01

    remodeling have revealed 'snapshots' of this cascade and the chromatin landscape at specific time-points of differentiation. These studies demonstrate that multiple adipogenic transcription factors co-occupy hotspots characterized by an open chromatin structure and specific epigenetic modifications...

  6. Chromatin structure regulates gene conversion.

    Directory of Open Access Journals (Sweden)

    W Jason Cummings

    2007-10-01

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

  7. The Chd Family of Chromatin Remodelers

    OpenAIRE

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

    2007-01-01

    Chromatin remodeling enzymes contribute to the dynamic changes that occur in chromatin structure during cellular processes such as transcription, recombination, repair, and replication. Members of the chromodomain helicase DNA-binding (Chd) family of enzymes belong to the SNF2 superfamily of ATP-dependent chromatin remodelers. The Chd proteins are distinguished by the presence of two N-terminal chromodomains that function as interaction surfaces for a variety of chromatin components. Genetic,...

  8. Human-Chromatin-Related Protein Interactions Identify a Demethylase Complex Required for Chromosome Segregation

    Directory of Open Access Journals (Sweden)

    Edyta Marcon

    2014-07-01

    Full Text Available Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.

  9. Human-chromatin-related protein interactions identify a demethylase complex required for chromosome segregation.

    Science.gov (United States)

    Marcon, Edyta; Ni, Zuyao; Pu, Shuye; Turinsky, Andrei L; Trimble, Sandra Smiley; Olsen, Jonathan B; Silverman-Gavrila, Rosalind; Silverman-Gavrila, Lorelei; Phanse, Sadhna; Guo, Hongbo; Zhong, Guoqing; Guo, Xinghua; Young, Peter; Bailey, Swneke; Roudeva, Denitza; Zhao, Dorothy; Hewel, Johannes; Li, Joyce; Gräslund, Susanne; Paduch, Marcin; Kossiakoff, Anthony A; Lupien, Mathieu; Emili, Andrew; Wodak, Shoshana J; Greenblatt, Jack

    2014-07-10

    Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.

  10. Impact of chromatin structure on PR signaling

    DEFF Research Database (Denmark)

    Grøntved, Lars; Hager, Gordon L

    2012-01-01

    The progesterone receptor (PR) interacts with chromatin in a highly dynamic manner that requires ongoing chromatin remodeling, interaction with chaparones and activity of the proteasome. Here we discuss dynamic interaction of steroid receptor with chromatin, with special attention not only to PR...

  11. Adaptive changes in the kinetochore architecture facilitate proper spindle assembly.

    Science.gov (United States)

    Magidson, Valentin; Paul, Raja; Yang, Nachen; Ault, Jeffrey G; O'Connell, Christopher B; Tikhonenko, Irina; McEwen, Bruce F; Mogilner, Alex; Khodjakov, Alexey

    2015-09-01

    Mitotic spindle formation relies on the stochastic capture of microtubules at kinetochores. Kinetochore architecture affects the efficiency and fidelity of this process with large kinetochores expected to accelerate assembly at the expense of accuracy, and smaller kinetochores to suppress errors at the expense of efficiency. We demonstrate that on mitotic entry, kinetochores in cultured human cells form large crescents that subsequently compact into discrete structures on opposite sides of the centromere. This compaction occurs only after the formation of end-on microtubule attachments. Live-cell microscopy reveals that centromere rotation mediated by lateral kinetochore-microtubule interactions precedes the formation of end-on attachments and kinetochore compaction. Computational analyses of kinetochore expansion-compaction in the context of lateral interactions correctly predict experimentally observed spindle assembly times with reasonable error rates. The computational model suggests that larger kinetochores reduce both errors and assembly times, which can explain the robustness of spindle assembly and the functional significance of enlarged kinetochores.

  12. Clinical spectrum of immunodeficiency, centromeric instability and facial dysmorphism (ICF syndrome).

    NARCIS (Netherlands)

    Hagleitner, M.M.; Lankester, A.; Maraschio, P.; Hulten, M.; Fryns, J.P.; Schuetz, C.; Gimelli, G.; Davies, E.G.; Gennery, A.; Belohradsky, B.H.; Groot, R. de; Gerritsen, E.J.; Mattina, T.; Howard, P.J.; Fasth, A.; Reisli, I.; Furthner, D.; Slatter, M.A.; Cant, A.J.; Cazzola, G.; Dijken, P.J. van; Deuren, M. van; Greef, J.C. de; Maarel, S.M. van der; Weemaes, C.M.R.

    2008-01-01

    BACKGROUND: Immunodeficiency, centromeric instability and facial dysmorphism (ICF syndrome) is a rare autosomal recessive disease characterised by facial dysmorphism, immunoglobulin deficiency and branching of chromosomes 1, 9 and 16 after PHA stimulation of lymphocytes. Hypomethylation of DNA of a

  13. Organisation of subunits in chromatin.

    Science.gov (United States)

    Carpenter, B G; Baldwin, J P; Bradbury, E M; Ibel, K

    1976-07-01

    There is considerable current interest in the organisation of nucleosomes in chromatin. A strong X-ray and neutron semi-meridional diffraction peak at approximately 10 nm had previously been attributed to the interparticle specing of a linear array of nucleosomes. This diffraction peak could also result from a close packed helical array of nucleosomes. A direct test of these proposals is whether the 10 nm peak is truly meridional as would be expected for a linear array of nucleosomes or is slightly off the meridian as expected for a helical array. Neutron diffraction studies of H1-depleted chromatin support the latter alternative. The 10 nm peak has maxima which form a cross-pattern with semi-meridional angle of 8 to 9 degrees. This is consistent with a coil of nucleosomes of pitch 10 nm and outer diameter of approximately 30 nm. These dimensions correspond to about six nucleosomes per turn of the coli.

  14. The Role of Dicentric Chromosome Formation and Secondary Centromere Deletion in the Evolution of Myeloid Malignancy

    OpenAIRE

    MacKinnon, Ruth N.; Campbell, Lynda J.

    2011-01-01

    Dicentric chromosomes have been identified as instigators of the genome instability associated with cancer, but this instability is often resolved by one of a number of different secondary events. These include centromere inactivation, inversion, and intercentromeric deletion. Deletion or excision of one of the centromeres may be a significant occurrence in myeloid malignancy and other malignancies but has not previously been widely recognized, and our reports are the first describing centrom...

  15. How the cell cycle impacts chromatin architecture and influences cell fate

    Directory of Open Access Journals (Sweden)

    Yiqin eMa

    2015-02-01

    Full Text Available Since the earliest observations of cells undergoing mitosis, it has been clear that there is an intimate relationship between the cell cycle and nuclear chromatin architecture. The nuclear envelope and chromatin undergo robust assembly and disassembly during the cell cycle, and transcriptional and post-transcriptional regulation of histone biogenesis and chromatin modification is controlled in a cell cycle-dependent manner. Chromatin binding proteins and chromatin modifications in turn influence the expression of critical cell cycle regulators, the accessibility of origins for DNA replication, DNA repair, and cell fate. In this review we aim to provide an integrated discussion of how the cell cycle machinery impacts nuclear architecture and vice-versa. We highlight recent advances in understanding cell cycle-dependent histone biogenesis and histone modification deposition, how cell cycle regulators control histone modifier activities, the contribution of chromatin modifications to origin firing for DNA replication, and newly identified roles for nucleoporins in regulating cell cycle gene expression, gene expression memory and differentiation. We close with a discussion of how cell cycle status may impact chromatin to influence cell fate decisions, under normal contexts of differentiation as well as in instances of cell fate re-programming.

  16. CenH3 evolution reflects meiotic symmetry as predicted by the centromere drive model.

    Science.gov (United States)

    Zedek, František; Bureš, Petr

    2016-09-15

    The centromere drive model explaining rapid evolution of eukaryotic centromeres predicts higher frequency of positive selection acting on centromeric histone H3 (CenH3) in clades with asymmetric meiosis compared to the clades with only symmetric meiosis. However, despite the impression one might get from the literature, this key prediction of the centromere drive model has not only never been confirmed, but it has never been tested, because all the previous studies dealt only with the presence or absence instead of the frequency of positive selection. To provide evidence for or against different frequencies of positively selected CenH3 in asymmetrics and symmetrics, we have inferred the selective pressures acting on CenH3 in seventeen eukaryotic clades, including plants, animals, fungi, ciliates and apicomplexa, using codon-substitution models, and compared the inferred frequencies between asymmetrics and symmetrics in a quantitative manner. We have found that CenH3 has been evolving adaptively much more frequently in clades with asymmetric meiosis compared with clades displaying only symmetric meiosis which confirms the prediction of centromere drive model. Our findings indicate that the evolution of asymmetric meiosis required CenH3 to evolve adaptively more often to counterbalance the negative consequences of centromere drive.

  17. Historical perspectives on the discovery and elucidation of autoantibodies to centromere proteins (CENP) and the emerging importance of antibodies to CENP-F.

    Science.gov (United States)

    Fritzler, Marvin J; Rattner, Jerome B; Luft, LeeAnne M; Edworthy, Steven M; Casiano, Carlos A; Peebles, Carol; Mahler, Michael

    2011-02-01

    Autoantibodies to the centromere proteins (CENP), which are major constituents of the primary constriction of metaphase chromosomes, were first described in 1980. In those seminal publications and 30 years of research that have followed, a number of CENP have been identified as autoantibody targets in human diseases. Historically, autoantibodies directed to CENP-A, -B and -C have been considered relatively specific biomarkers for limited cutaneous systemic sclerosis (lcSSc) or the calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) syndrome. These autoantibodies, found in up to 40% of SSc sera, can be identified by indirect immunofluorescence (IIF) on a variety of tissue culture cell lines as a discrete speckled staining pattern of both interphase nuclei and metaphase chromatin. Early in the investigation of anti-CENP, it became apparent that some autoantibodies had a similar IIF pattern wherein as cells entered into the cell cycle, speckled staining of the metaphase chromatin could be observed but, unlike conventional CENP staining, interphase nuclei were not stained. Subsequent studies identified one of the targets of these autoantibodies to be CENP-F, a kinesin binding protein essential for completion of the cell cycle. Early clinical studies found that, unlike antibodies to the earlier described CENP, lcSSc rarely expressed anti-CENP-F and approximately 50% of these patients had a malignancy. This review provides a historical perspective of CENP autoantibodies and focuses on an update of the information on CENP-F and their clinical associations.

  18. Proteomics of a fuzzy organelle: interphase chromatin

    Science.gov (United States)

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

    2014-01-01

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

  19. Efficient cell migration requires global chromatin condensation.

    Science.gov (United States)

    Gerlitz, Gabi; Bustin, Michael

    2010-07-01

    Cell migration is a fundamental process that is necessary for the development and survival of multicellular organisms. Here, we show that cell migration is contingent on global condensation of the chromatin fiber. Induction of directed cell migration by the scratch-wound assay leads to decreased DNaseI sensitivity, alterations in the chromatin binding of architectural proteins and elevated levels of H4K20me1, H3K27me3 and methylated DNA. All these global changes are indicative of increased chromatin condensation in response to induction of directed cell migration. Conversely, chromatin decondensation inhibited the rate of cell migration, in a transcription-independent manner. We suggest that global chromatin condensation facilitates nuclear movement and reshaping, which are important for cell migration. Our results support a role for the chromatin fiber that is distinct from its known functions in genetic processes.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  1. Chromatin Modification and Remodeling in Heart Development

    Directory of Open Access Journals (Sweden)

    Paul Delgado-Olguín

    2006-01-01

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

  2. Chromatin remodeling in cardiovascular development and physiology

    OpenAIRE

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

    2011-01-01

    Chromatin regulation provides an important means of controlling cardiac gene expression under different physiological and pathological conditions. Processes that direct the development of normal embryonic hearts and pathology of stressed adult hearts may share general mechanisms that govern cardiac gene expression by chromatin-regulating factors. These common mechanisms may provide a framework for us to investigate the interactions among diverse chromatin remodelers/modifiers and various tran...

  3. Chromatin remodeling and human disease.

    Science.gov (United States)

    Huang, Cheng; Sloan, Emily A; Boerkoel, Cornelius F

    2003-06-01

    In the past few years, there has been a nascent convergence of scientific understanding of inherited human diseases with epigenetics. Identified epigenetic processes involved in human disease include covalent DNA modifications, covalent histone modifications, and histone relocation. Each of these processes influences chromatin structure and thereby regulates gene expression and DNA methylation, replication, recombination, and repair. The importance of these processes for nearly all aspects of normal growth and development is illustrated by the array of multi-system disorders and neoplasias caused by their dysregulation.

  4. Advances in chromatin remodeling and human disease.

    Science.gov (United States)

    Cho, Kyoung Sang; Elizondo, Leah I; Boerkoel, Cornelius F

    2004-06-01

    Epigenetic factors alter phenotype without changing genotype. A primary molecular mechanism underlying epigenetics is the alteration of chromatin structure by covalent DNA modifications, covalent histone modifications, and nucleosome reorganization. Remodeling of chromatin structure regulates DNA methylation, replication, recombination, and repair as well as gene expression. As these functions would predict, dysfunction of the proteins that remodel chromatin causes an array of multi-system disorders and neoplasias. Insights from these diseases suggest that during embryonic and fetal life, environmental distortions of chromatin remodeling encode a 'molecular memory' that predispose the individual to diseases in adulthood.

  5. Rsc4 Connects the Chromatin Remodeler RSC to RNA Polymerases‡

    Science.gov (United States)

    Soutourina, Julie; Bordas-Le Floch, Véronique; Gendrel, Gabrielle; Flores, Amando; Ducrot, Cécile; Dumay-Odelot, Hélène; Soularue, Pascal; Navarro, Francisco; Cairns, Bradley R.; Lefebvre, Olivier; Werner, Michel

    2006-01-01

    RSC is an essential, multisubunit chromatin remodeling complex. We show here that the Rsc4 subunit of RSC interacted via its C terminus with Rpb5, a conserved subunit shared by all three nuclear RNA polymerases (Pol). Furthermore, the RSC complex coimmunoprecipitated with all three RNA polymerases. Mutations in the C terminus of Rsc4 conferred a thermosensitive phenotype and the loss of interaction with Rpb5. Certain thermosensitive rpb5 mutations were lethal in combination with an rsc4 mutation, supporting the physiological significance of the interaction. Pol II transcription of ca. 12% of the yeast genome was increased or decreased twofold or more in a rsc4 C-terminal mutant. The transcription of the Pol III-transcribed genes SNR6 and RPR1 was also reduced, in agreement with the observed localization of RSC near many class III genes. Rsc4 C-terminal mutations did not alter the stability or assembly of the RSC complex, suggesting an impact on Rsc4 function. Strikingly, a C-terminal mutation of Rsc4 did not impair RSC recruitment to the RSC-responsive genes DUT1 and SMX3 but rather changed the chromatin accessibility of DNases to their promoter regions, suggesting that the altered transcription of DUT1 and SMX3 was the consequence of altered chromatin remodeling. PMID:16782880

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    To maintain genome function and stability, DNA sequence and its organization into chromatin must be duplicated during cell division. Understanding how entire chromosomes are copied remains a major challenge. Here, we use nascent chromatin capture (NCC) to profile chromatin proteome dynamics durin...

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

    NARCIS (Netherlands)

    Driessen, Rosalie Paula Catharina

    2014-01-01

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

  8. The Role of Dicentric Chromosome Formation and Secondary Centromere Deletion in the Evolution of Myeloid Malignancy

    Directory of Open Access Journals (Sweden)

    Ruth N. MacKinnon

    2011-01-01

    Full Text Available Dicentric chromosomes have been identified as instigators of the genome instability associated with cancer, but this instability is often resolved by one of a number of different secondary events. These include centromere inactivation, inversion, and intercentromeric deletion. Deletion or excision of one of the centromeres may be a significant occurrence in myeloid malignancy and other malignancies but has not previously been widely recognized, and our reports are the first describing centromere deletion in cancer cells. We review what is known about dicentric chromosomes and the mechanisms by which they can undergo stabilization in both constitutional and cancer genomes. The failure to identify centromere deletion in cancer cells until recently can be partly explained by the standard approaches to routine diagnostic cancer genome analysis, which do not identify centromeres in the context of chromosome organization. This hitherto hidden group of primary dicentric, secondary monocentric chromosomes, together with other unrecognized dicentric chromosomes, points to a greater role for dicentric chromosomes in cancer initiation and progression than is generally acknowledged. We present a model that predicts and explains a significant role for dicentric chromosomes in the formation of unbalanced translocations in malignancy.

  9. A cytogenetic study of nuclear power plant workers using the micronucleus-centromere assay.

    Science.gov (United States)

    Thierens, H; Vral, A; Barbé, M; Aousalah, B; De Ridder, L

    1999-09-15

    A cytogenetic study was performed in 215 nuclear power plant workers occupationally exposed to radiation using the micronucleus-centromere assay for peripheral blood lymphocytes. As control population served administrative staff with yearly doses below 1 mSv. The increase of the micronucleus frequency with age, observed in the non-smoking control population, is mainly due to an enhanced number of centromere-positive micronuclei, pointing to an increased chromosome loss. No differences in the number of micronuclei, centromere-positive and centromere-negative micronuclei between smokers and non-smokers are observed. An analysis of the micronucleus data vs. the dose accumulated over the 10 years preceding the venepuncture shows no significant clastogenic or aneuploidogenic effects of the exposure in the studied population which is representative for workers in the nuclear industry at present. According to the linear fits to our data an increase of the micronucleus frequency pro rata 0.5 per 1000 binucleated cells per year, related to the centromere-negative micronuclei, may be expected for workers with the maximal tolerable dose of 20 mSv/year.

  10. Sisters unbound is required for meiotic centromeric cohesion in Drosophila melanogaster.

    Science.gov (United States)

    Krishnan, Badri; Thomas, Sharon E; Yan, Rihui; Yamada, Hirotsugu; Zhulin, Igor B; McKee, Bruce D

    2014-11-01

    Regular meiotic chromosome segregation requires sister centromeres to mono-orient (orient to the same pole) during the first meiotic division (meiosis I) when homologous chromosomes segregate, and to bi-orient (orient to opposite poles) during the second meiotic division (meiosis II) when sister chromatids segregate. Both orientation patterns require cohesion between sister centromeres, which is established during meiotic DNA replication and persists until anaphase of meiosis II. Meiotic cohesion is mediated by a conserved four-protein complex called cohesin that includes two structural maintenance of chromosomes (SMC) subunits (SMC1 and SMC3) and two non-SMC subunits. In Drosophila melanogaster, however, the meiotic cohesion apparatus has not been fully characterized and the non-SMC subunits have not been identified. We have identified a novel Drosophila gene called sisters unbound (sunn), which is required for stable sister chromatid cohesion throughout meiosis. sunn mutations disrupt centromere cohesion during prophase I and cause high frequencies of non-disjunction (NDJ) at both meiotic divisions in both sexes. SUNN co-localizes at centromeres with the cohesion proteins SMC1 and SOLO in both sexes and is necessary for the recruitment of both proteins to centromeres. Although SUNN lacks sequence homology to cohesins, bioinformatic analysis indicates that SUNN may be a structural homolog of the non-SMC cohesin subunit stromalin (SA), suggesting that SUNN may serve as a meiosis-specific cohesin subunit. In conclusion, our data show that SUNN is an essential meiosis-specific Drosophila cohesion protein.

  11. The role of dicentric chromosome formation and secondary centromere deletion in the evolution of myeloid malignancy.

    Science.gov (United States)

    Mackinnon, Ruth N; Campbell, Lynda J

    2011-01-01

    Dicentric chromosomes have been identified as instigators of the genome instability associated with cancer, but this instability is often resolved by one of a number of different secondary events. These include centromere inactivation, inversion, and intercentromeric deletion. Deletion or excision of one of the centromeres may be a significant occurrence in myeloid malignancy and other malignancies but has not previously been widely recognized, and our reports are the first describing centromere deletion in cancer cells. We review what is known about dicentric chromosomes and the mechanisms by which they can undergo stabilization in both constitutional and cancer genomes. The failure to identify centromere deletion in cancer cells until recently can be partly explained by the standard approaches to routine diagnostic cancer genome analysis, which do not identify centromeres in the context of chromosome organization. This hitherto hidden group of primary dicentric, secondary monocentric chromosomes, together with other unrecognized dicentric chromosomes, points to a greater role for dicentric chromosomes in cancer initiation and progression than is generally acknowledged. We present a model that predicts and explains a significant role for dicentric chromosomes in the formation of unbalanced translocations in malignancy.

  12. Chromatin modification by PSC occurs at one PSC per nucleosome and does not require the acidic patch of histone H2A.

    Science.gov (United States)

    Lo, Stanley M; McElroy, Kyle A; Francis, Nicole J

    2012-01-01

    Chromatin architecture is regulated through both enzymatic and non-enzymatic activities. For example, the Polycomb Group (PcG) proteins maintain developmental gene silencing using an array of chromatin-based mechanisms. The essential Drosophila PcG protein, Posterior Sex Combs (PSC), compacts chromatin and inhibits chromatin remodeling and transcription through a non-enzymatic mechanism involving nucleosome bridging. Nucleosome bridging is achieved through a combination of nucleosome binding and self-interaction. Precisely how PSC interacts with chromatin to bridge nucleosomes is not known and is the subject of this work. We determine the stoichiometry of PSC-chromatin interactions in compact chromatin (in which nucleosomes are bridged) using Scanning Transmission Electron Microscopy (STEM). We find that full compaction occurs with one PSC per nucleosome. In addition to compacting chromatin, we show that PSC oligomerizes nucleosome arrays. PSC-mediated oligomerization of chromatin occurs at similar stoichiometry as compaction suggesting it may also involve nucleosome bridging. Interactions between the tail of histone H4 and the acidic patch of histone H2A are important for chromatin folding and oligomerization, and several chromatin proteins bind the histone H2A acidic patch. However, mutation of the acidic patch of histone H2A does not affect PSC's ability to inhibit chromatin remodeling or bridge nucleosomes. In fact, PSC does not require nucleosomes for bridging activity but can bridge naked DNA segments. PSC clusters nucleosomes on sparsely assembled templates, suggesting it interacts preferentially with nucleosomes over bare DNA. This may be due to the ability of PSC to bind free histones. Our data are consistent with a model in which each PSC binds a nucleosome and at least one other PSC to directly bridge nucleosomes and compact chromatin, but also suggest that naked DNA can be included in compacted structures. We discuss how our data highlight the diversity

  13. Chromatin modification by PSC occurs at one PSC per nucleosome and does not require the acidic patch of histone H2A.

    Directory of Open Access Journals (Sweden)

    Stanley M Lo

    Full Text Available Chromatin architecture is regulated through both enzymatic and non-enzymatic activities. For example, the Polycomb Group (PcG proteins maintain developmental gene silencing using an array of chromatin-based mechanisms. The essential Drosophila PcG protein, Posterior Sex Combs (PSC, compacts chromatin and inhibits chromatin remodeling and transcription through a non-enzymatic mechanism involving nucleosome bridging. Nucleosome bridging is achieved through a combination of nucleosome binding and self-interaction. Precisely how PSC interacts with chromatin to bridge nucleosomes is not known and is the subject of this work. We determine the stoichiometry of PSC-chromatin interactions in compact chromatin (in which nucleosomes are bridged using Scanning Transmission Electron Microscopy (STEM. We find that full compaction occurs with one PSC per nucleosome. In addition to compacting chromatin, we show that PSC oligomerizes nucleosome arrays. PSC-mediated oligomerization of chromatin occurs at similar stoichiometry as compaction suggesting it may also involve nucleosome bridging. Interactions between the tail of histone H4 and the acidic patch of histone H2A are important for chromatin folding and oligomerization, and several chromatin proteins bind the histone H2A acidic patch. However, mutation of the acidic patch of histone H2A does not affect PSC's ability to inhibit chromatin remodeling or bridge nucleosomes. In fact, PSC does not require nucleosomes for bridging activity but can bridge naked DNA segments. PSC clusters nucleosomes on sparsely assembled templates, suggesting it interacts preferentially with nucleosomes over bare DNA. This may be due to the ability of PSC to bind free histones. Our data are consistent with a model in which each PSC binds a nucleosome and at least one other PSC to directly bridge nucleosomes and compact chromatin, but also suggest that naked DNA can be included in compacted structures. We discuss how our data

  14. Common themes and cell type specific variations of higher order chromatin arrangements in the mouse

    Directory of Open Access Journals (Sweden)

    Cremer Thomas

    2005-12-01

    Full Text Available Abstract Background Similarities as well as differences in higher order chromatin arrangements of human cell types were previously reported. For an evolutionary comparison, we now studied the arrangements of chromosome territories and centromere regions in six mouse cell types (lymphocytes, embryonic stem cells, macrophages, fibroblasts, myoblasts and myotubes with fluorescence in situ hybridization and confocal laser scanning microscopy. Both species evolved pronounced differences in karyotypes after their last common ancestors lived about 87 million years ago and thus seem particularly suited to elucidate common and cell type specific themes of higher order chromatin arrangements in mammals. Results All mouse cell types showed non-random correlations of radial chromosome territory positions with gene density as well as with chromosome size. The distribution of chromosome territories and pericentromeric heterochromatin changed during differentiation, leading to distinct cell type specific distribution patterns. We exclude a strict dependence of these differences on nuclear shape. Positional differences in mouse cell nuclei were less pronounced compared to human cell nuclei in agreement with smaller differences in chromosome size and gene density. Notably, the position of chromosome territories relative to each other was very variable. Conclusion Chromosome territory arrangements according to chromosome size and gene density provide common, evolutionary conserved themes in both, human and mouse cell types. Our findings are incompatible with a previously reported model of parental genome separation.

  15. Role of ATRX in chromatin structure and function: implications for chromosome instability and human disease.

    Science.gov (United States)

    De La Fuente, Rabindranath; Baumann, Claudia; Viveiros, Maria M

    2011-08-01

    Functional differentiation of chromatin structure is essential for the control of gene expression, nuclear architecture, and chromosome stability. Compelling evidence indicates that alterations in chromatin remodeling proteins play an important role in the pathogenesis of human disease. Among these, α-thalassemia mental retardation X-linked protein (ATRX) has recently emerged as a critical factor involved in heterochromatin formation at mammalian centromeres and telomeres as well as facultative heterochromatin on the murine inactive X chromosome. Mutations in human ATRX result in an X-linked neurodevelopmental condition with various degrees of gonadal dysgenesis (ATRX syndrome). Patients with ATRX syndrome may exhibit skewed X chromosome inactivation (XCI) patterns, and ATRX-deficient mice exhibit abnormal imprinted XCI in the trophoblast cell line. Non-random or skewed XCI can potentially affect both the onset and severity of X-linked disease. Notably, failure to establish epigenetic modifications associated with the inactive X chromosome (Xi) results in several conditions that exhibit genomic and chromosome instability such as fragile X syndrome as well as cancer development. Insight into the molecular mechanisms of ATRX function and its interacting partners in different tissues will no doubt contribute to our understanding of the pathogenesis of ATRX syndrome as well as the epigenetic origins of aneuploidy. In turn, this knowledge will be essential for the identification of novel drug targets and diagnostic tools for cancer progression as well as the therapeutic management of global epigenetic changes commonly associated with malignant neoplastic transformation.

  16. Expression-dependent folding of interphase chromatin.

    Directory of Open Access Journals (Sweden)

    Hansjoerg Jerabek

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

  17. Chromatin dynamics resolved with force spectroscopy

    NARCIS (Netherlands)

    Chien, Fan-Tso

    2011-01-01

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

  18. Chromatin Remodelers: From Function to Dysfunction

    Directory of Open Access Journals (Sweden)

    Gernot Längst

    2015-06-01

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

  19. Chromatin-modifying proteins in cancer

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  20. A Long-Distance Chromatin Affair

    NARCIS (Netherlands)

    Denker, Annette; de Laat, Wouter

    2015-01-01

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

  1. Chromatin roadblocks to reprogramming 50 years on.

    Science.gov (United States)

    Skene, Peter J; Henikoff, Steven

    2012-10-29

    A half century after John Gurdon demonstrated nuclear reprogramming, for which he was awarded the 2012 Nobel Prize in Physiology or Medicine, his group provides insights into the molecular mechanisms whereby chromatin remodeling is required for nuclear reprogramming. Among the issues addressed in Gurdon's latest work are the chromatin impediments to artificially induced reprogramming, discovered by Shinya Yamanaka, who shared the award with Gurdon.

  2. Cultivation and differentiation change nuclear localization of chromosome centromeres in human mesenchymal stem cells.

    Science.gov (United States)

    Voldgorn, Yana I; Adilgereeva, Elmira P; Nekrasov, Evgeny D; Lavrov, Alexander V

    2015-01-01

    Chromosome arrangement in the interphase nucleus is not accidental. Strong evidences support that nuclear localization is an important mechanism of epigenetic regulation of gene expression. The purpose of this research was to identify differences in the localization of centromeres of chromosomes 6, 12, 18 and X in human mesenchymal stem cells depending on differentiation and cultivating time. We analyzed centromere positions in more than 4000 nuclei in 19 mesenchymal stem cell cultures before and after prolonged cultivation and after differentiation into osteogenic and adipogenic directions. We found a centromere reposition of HSAX at late passages and after differentiation in osteogenic direction as well as of HSA12 and HSA18 after adipogenic differentiation. The observed changes of the nuclear structure are new nuclear characteristics of the studied cells which may reflect regulatory changes of gene expression during the studied processes.

  3. Cultivation and differentiation change nuclear localization of chromosome centromeres in human mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Yana I Voldgorn

    Full Text Available Chromosome arrangement in the interphase nucleus is not accidental. Strong evidences support that nuclear localization is an important mechanism of epigenetic regulation of gene expression. The purpose of this research was to identify differences in the localization of centromeres of chromosomes 6, 12, 18 and X in human mesenchymal stem cells depending on differentiation and cultivating time. We analyzed centromere positions in more than 4000 nuclei in 19 mesenchymal stem cell cultures before and after prolonged cultivation and after differentiation into osteogenic and adipogenic directions. We found a centromere reposition of HSAX at late passages and after differentiation in osteogenic direction as well as of HSA12 and HSA18 after adipogenic differentiation. The observed changes of the nuclear structure are new nuclear characteristics of the studied cells which may reflect regulatory changes of gene expression during the studied processes.

  4. Interactions of transcription factors with chromatin.

    Science.gov (United States)

    van Bakel, Harm

    2011-01-01

    Sequence-specific transcription factors (TFs) play a central role in regulating transcription initiation by directing the recruitment and activity of the general transcription machinery and accessory factors. It is now well established that many of the effects exerted by TFs in eukaryotes are mediated through interactions with a host of coregulators that modify the chromatin state, resulting in a more open (in case of activation) or closed conformation (in case of repression). The relationship between TFs and chromatin is a two-way street, however, as chromatin can in turn influence the recognition and binding of target sequences by TFs. The aim of this chapter is to highlight how this dynamic interplay between TF-directed remodelling of chromatin and chromatin-adjusted targeting of TF binding determines where and how transcription is initiated, and to what degree it is productive.

  5. Chromatin domain boundaries: insulators and beyond

    Institute of Scientific and Technical Information of China (English)

    Gong Hong WEI; De Pei LIU; Chih Chuan LIANG

    2005-01-01

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

  6. Chromatin remodeling in cardiovascular development and physiology.

    Science.gov (United States)

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

    2011-02-04

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

  7. Sequence information encoded in DNA that may influence long-range chromatin structure correlates with human chromosome functions.

    Directory of Open Access Journals (Sweden)

    Taichi E Takasuka

    Full Text Available Little is known about the possible function of the bulk of the human genome. We have recently shown that long-range regular oscillation in the motif non-T, A/T, G (VWG existing at ten-nucleotide multiples influences large-scale nucleosome array formation. In this work, we have determined the locations of all 100 kb regions that are predicted to form distinctive chromatin structures throughout each human chromosome (except Y. Using these data, we found that a significantly greater fraction of 300 kb sequences lacked annotated transcripts in genomic DNA regions > or = 300 kb that contained nearly continuous chromatin organizing signals than in control regions. We also found a relationship between the meiotic recombination frequency and the presence of strong VWG chromatin organizing signals. Large (> or = 300 kb genomic DNA regions having low average recombination frequency are enriched in chromatin organizing signals. As additional controls, we show using chromosome 1 that the VWG motif signals are not enriched in randomly selected DNA regions having the mean size of the recombination coldspots, and that non-VWG motif sets do not generate signals that are enriched in recombination coldspots. We also show that tandemly repeated alpha satellite DNA contains strong VWG signals for the formation of distinctive nucleosome arrays, consistent with the low recombination activity of centromeres. Our correlations cannot be explained simply by variations in the GC content. Our findings suggest that a specific set of periodic DNA motifs encoded in genomic DNA, which provide signals for chromatin organization, influence human chromosome function.

  8. Computational strategies to address chromatin structure problems

    Science.gov (United States)

    Perišić, Ognjen; Schlick, Tamar

    2016-06-01

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

  9. Replication initiator DnaA binds at the Caulobacter centromere and enables chromosome segregation.

    Science.gov (United States)

    Mera, Paola E; Kalogeraki, Virginia S; Shapiro, Lucy

    2014-11-11

    During cell division, multiple processes are highly coordinated to faithfully generate genetically equivalent daughter cells. In bacteria, the mechanisms that underlie the coordination of chromosome replication and segregation are poorly understood. Here, we report that the conserved replication initiator, DnaA, can mediate chromosome segregation independent of replication initiation. It does so by binding directly to the parS centromere region of the chromosome, and mutations that alter this interaction result in cells that display aberrant centromere translocation and cell division. We propose that DnaA serves to coordinate bacterial DNA replication with the onset of chromosome segregation.

  10. DNA deformability changes of single base pair mutants within CDE binding sites in S. Cerevisiae centromere DNA correlate with measured chromosomal loss rates and CDE binding site symmetries

    Directory of Open Access Journals (Sweden)

    Marx Kenneth A

    2006-03-01

    Full Text Available Abstract Background The centromeres in yeast (S. cerevisiae are organized by short DNA sequences (125 bp on each chromosome consisting of 2 conserved elements: CDEI and CDEIII spaced by a CDEII region. CDEI and CDEIII are critical sequence specific protein binding sites necessary for correct centromere formation and following assembly with proteins, are positioned near each other on a specialized nucleosome. Hegemann et al. BioEssays 1993, 15: 451–460 reported single base DNA mutants within the critical CDEI and CDEIII binding sites on the centromere of chromosome 6 and quantitated centromere loss of function, which they measured as loss rates for the different chromosome 6 mutants during cell division. Olson et al. Proc Natl Acad Sci USA 1998, 95: 11163–11168 reported the use of protein-DNA crystallography data to produce a DNA dinucleotide protein deformability energetic scale (PD-scale that describes local DNA deformability by sequence specific binding proteins. We have used the PD-scale to investigate the DNA sequence dependence of the yeast chromosome 6 mutants' loss rate data. Each single base mutant changes 2 PD-scale values at that changed base position relative to the wild type. In this study, we have utilized these mutants to demonstrate a correlation between the change in DNA deformability of the CDEI and CDEIII core sites and the overall experimentally measured chromosome loss rates of the chromosome 6 mutants. Results In the CDE I and CDEIII core binding regions an increase in the magnitude of change in deformability of chromosome 6 single base mutants with respect to the wild type correlates to an increase in the measured chromosome loss rate. These correlations were found to be significant relative to 105 Monte Carlo randomizations of the dinucleotide PD-scale applied to the same calculation. A net loss of deformability also tends to increase the loss rate. Binding site position specific, 4 data-point correlations were also

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

    Science.gov (United States)

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

    2014-12-11

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

  12. Extensive Variation in Chromatin States Across Humans

    KAUST Repository

    Kasowski, M.

    2013-10-17

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

  13. Chromatin Fiber Dynamics under Tension and Torsion

    Directory of Open Access Journals (Sweden)

    Christophe Lavelle

    2010-04-01

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

  14. Chromatin targeting drugs in cancer and immunity.

    Science.gov (United States)

    Prinjha, Rab; Tarakhovsky, Alexander

    2013-08-15

    Recent advances in the enzymology of transcription and chromatin regulation have led to the discovery of proteins that play a prominent role in cell differentiation and the maintenance of specialized cell functions. Knowledge about post-synthetic DNA and histone modifications as well as information about the rules that guide the formation of multimolecular chromatin-bound complexes have helped to delineate gene-regulating pathways and describe how these pathways are altered in various pathological conditions. The present review focuses on the emerging area of therapeutic interference with chromatin function for the purpose of cancer treatment and immunomodulation.

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

    Science.gov (United States)

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

    2015-03-12

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

  16. Inter-domain Cooperation in INCENP Promotes Aurora B Relocation from Centromeres to Microtubules

    Directory of Open Access Journals (Sweden)

    Armando van der Horst

    2015-07-01

    Full Text Available The chromosomal passenger complex is essential for error-free chromosome segregation and proper execution of cytokinesis. To coordinate nuclear division with cytoplasmic division, its enzymatic subunit, Aurora B, relocalizes from centromeres in metaphase to the spindle midzone in anaphase. In budding yeast, this requires dephosphorylation of the microtubule-binding (MTB domain of the INCENP analog Sli15. The mechanistic basis for this relocalization in metazoans is incompletely understood. We demonstrate that the putative coiled-coil domain within INCENP drives midzone localization of Aurora B via a direct, electrostatic interaction with microtubules. Furthermore, we provide evidence that the CPC multimerizes via INCENP’s centromere-targeting domain (CEN box, which increases the MTB affinity of INCENP. In (prometaphase, the MTB affinity of INCENP is outcompeted by the affinity of its CEN box for centromeres, while at anaphase onset—when the histone mark H2AT120 is dephosphorylated—INCENP and Aurora B switch from centromere to microtubule localization.

  17. Inter-domain Cooperation in INCENP Promotes Aurora B Relocation from Centromeres to Microtubules

    NARCIS (Netherlands)

    van der Horst, Armando; Vromans, Martijn J M; Bouwman, Kim; van der Waal, Maike S; Hadders, Michael A; Lens, Susanne M A; Lens, SMA

    2015-01-01

    The chromosomal passenger complex is essential for error-free chromosome segregation and proper execution of cytokinesis. To coordinate nuclear division with cytoplasmic division, its enzymatic subunit, Aurora B, relocalizes from centromeres in metaphase to the spindle midzone in anaphase. In buddin

  18. Mutations in ZBTB24 are associated with immunodeficiency, centromeric instability, and facial anomalies syndrome type 2

    NARCIS (Netherlands)

    Greef, J.C. de; Wang, J.; Balog, J.; Dunnen, J.T. den; Frants, R.R.; Straasheijm, K.R.; Aytekin, C.; Burg, M. van der; Duprez, L.; Ferster, A.; Gennery, A.R.; Gimelli, G.; Reisli, I.; Schuetz, C.; Schulz, A.; Smeets, D.F.C.M.; Sznajer, Y.; Wijmenga, C.; Eggermond, M.C. van; Ostaijen-ten Dam, M.M. van; Lankester, A.C.; Tol, M.J. van; Elsen, P.J. van den; Weemaes, C.M.R.; Maarel, S.M. van der

    2011-01-01

    Autosomal-recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is mainly characterized by recurrent, often fatal, respiratory and gastrointestinal infections. About 50% of patients carry mutations in the DNA methyltransferase 3B gene (DNMT3B) (ICF1). The remaining

  19. [Chromosomal organization of centromeric Ty3/gypsy retrotransposons in Allium cepa L. and Allium fistulosum L].

    Science.gov (United States)

    Kiseleva, A V; Kirov, I V; Khrustaleva, L I

    2014-06-01

    This is the first report on the presence of Ty3/gypsy-like retrotransposons in the centromeric region of Allium cepa and Allium fistulosum. The paper identifies the putative Ty3/gypsy centromeric retrotransposons (CR) among the DNA sequences of A. cepa present in the NCBI database and evaluates their copy number in the genomes of Allium cepa and Allium fistulosum. The putative copy number of Ty3/gypsy CR constituted about 26000 for A. cepa and about 7000 for A. fistulosum. The chromosomal organization of Ty3/gypsy CR was analyzed with the help of fluorescent in situ hybridization (FISH). The 300-bp PCR products synthesized with genomic DNA of Allium cepa and Allium fistulosum and primers designed for the sequence ET645811 of A. cepa (Genome Survey Sequence database), displaying similarity to the reverse transcriptase of the CR Ty3/gypsy family, served as FISH hybridization probes. On the chromosomes of A. cepa, hybridization signals were mainly localized in the centromeric region. On the chromosomes of A. fistulosum the signals were less expressed in the centromeric regions, though they were abundant in other chromosomal regions. The pathways of evolution in these closely related species are discussed.

  20. Novel Centromeric Loci of the Wine and Beer Yeast Dekkera bruxellensis CEN1 and CEN2

    DEFF Research Database (Denmark)

    Ishchuk, Olena P.; Vojvoda Zeljko, Tanja; Schifferdecker, Anna J.

    2016-01-01

    The wine and beer yeast Dekkera bruxellensis thrives in environments that are harsh and limiting, especially in concentrations with low oxygen and high ethanol. Its different strains' chromosomes greatly vary in number (karyotype). This study isolates two novel centromeric loci (CEN1 and CEN2...

  1. Survivin mediates targeting of the chromosomal passenger complex to the centromere and midbody

    NARCIS (Netherlands)

    Vader, G; Kauw, JJW; Medema, RH; Lens, SMA

    2006-01-01

    The chromosomal passenger complex (CPC) coordinates chromosomal and cytoskeletal events of mitosis. The enzymatic core of this complex (Aurora-B) is guided through the mitotic cell by its companion chromosomal passenger proteins, inner centromere protein (INCENP), Survivin and Borealin/Dasra-B, ther

  2. Loss of pRB causes centromere dysfunction and chromosomal instability.

    Science.gov (United States)

    Manning, Amity L; Longworth, Michelle S; Dyson, Nicholas J

    2010-07-01

    Chromosome instability (CIN) is a common feature of tumor cells. By monitoring chromosome segregation, we show that depletion of the retinoblastoma protein (pRB) causes rates of missegregation comparable with those seen in CIN tumor cells. The retinoblastoma tumor suppressor is frequently inactivated in human cancers and is best known for its regulation of the G1/S-phase transition. Recent studies have shown that pRB inactivation also slows mitotic progression and promotes aneuploidy, but reasons for these phenotypes are not well understood. Here we describe the underlying mitotic defects of pRB-deficient cells that cause chromosome missegregation. Analysis of mitotic cells reveals that pRB depletion compromises centromeric localization of CAP-D3/condensin II and chromosome cohesion, leading to an increase in intercentromeric distance and deformation of centromeric structure. These defects promote merotelic attachment, resulting in failure of chromosome congression and an increased propensity for lagging chromosomes following mitotic delay. While complete loss of centromere function or chromosome cohesion would have catastrophic consequences, these more moderate defects allow pRB-deficient cells to proliferate but undermine the fidelity of mitosis, leading to whole-chromosome gains and losses. These observations explain an important consequence of RB1 inactivation, and suggest that subtle defects in centromere function are a frequent source of merotely and CIN in cancer.

  3. Aurora B kinase controls the separation of centromeric and telomeric heterochromatin

    OpenAIRE

    Gachet, Yannick; Reyes, Celine; Tournier, Sylvie

    2015-01-01

    The segregation of chromosomes is coordinated at multiple levels to prevent chromosome loss, a phenotype frequently observed in cancers. We recently described an essential role for telomeres in the physical separation of chromosomes and identified Aurora B kinase as a double agent involved in the separation of centromeric and telomeric heterochromatin.

  4. Aurora B kinase controls the separation of centromeric and telomeric heterochromatin.

    Science.gov (United States)

    Gachet, Yannick; Reyes, Celine; Tournier, Sylvie

    2016-03-01

    The segregation of chromosomes is coordinated at multiple levels to prevent chromosome loss, a phenotype frequently observed in cancers. We recently described an essential role for telomeres in the physical separation of chromosomes and identified Aurora B kinase as a double agent involved in the separation of centromeric and telomeric heterochromatin.

  5. Condensin HEAT subunits required for DNA repair, kinetochore/centromere function and ploidy maintenance in fission yeast.

    Directory of Open Access Journals (Sweden)

    Xingya Xu

    Full Text Available Condensin, a central player in eukaryotic chromosomal dynamics, contains five evolutionarily-conserved subunits. Two SMC (structural maintenance of chromosomes subunits contain ATPase, hinge, and coiled-coil domains. One non-SMC subunit is similar to bacterial kleisin, and two other non-SMC subunits contain HEAT (similar to armadillo repeats. Here we report isolation and characterization of 21 fission yeast (Schizosaccharomyces pombe mutants for three non-SMC subunits, created using error-prone mutagenesis that resulted in single-amino acid substitutions. Beside condensation, segregation, and DNA repair defects, similar to those observed in previously isolated SMC and cnd2 mutants, novel phenotypes were observed for mutants of HEAT-repeats containing Cnd1 and Cnd3 subunits. cnd3-L269P is hypersensitive to the microtubule poison, thiabendazole, revealing defects in kinetochore/centromere and spindle assembly checkpoints. Three cnd1 and three cnd3 mutants increased cell size and doubled DNA content, thereby eliminating the haploid state. Five of these mutations reside in helix B of HEAT repeats. Two non-SMC condensin subunits, Cnd1 and Cnd3, are thus implicated in ploidy maintenance.

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

    KAUST Repository

    Bigeard, Jean

    2014-07-10

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

  7. Chromatin proteins and modifications as drug targets

    DEFF Research Database (Denmark)

    Helin, Kristian; Dhanak, Dashyant

    2013-01-01

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

  8. Chromatin roadblocks to reprogramming 50 years on

    Directory of Open Access Journals (Sweden)

    Skene Peter J

    2012-10-01

    Full Text Available Abstract A half century after John Gurdon demonstrated nuclear reprogramming, for which he was awarded the 2012 Nobel Prize in Physiology or Medicine, his group provides insights into the molecular mechanisms whereby chromatin remodeling is required for nuclear reprogramming. Among the issues addressed in Gurdon's latest work are the chromatin impediments to artificially induced reprogramming, discovered by Shinya Yamanaka, who shared the award with Gurdon. See research article: http://www.epigeneticsandchromatin.com/content/5/1/17

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

    Science.gov (United States)

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

    2014-01-01

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

  10. Chromatin structure of repeating CTG/CAG and CGG/CCG sequences in human disease.

    Science.gov (United States)

    Wang, Yuh-Hwa

    2007-05-01

    In eukaryotic cells, chromatin structure organizes genomic DNA in a dynamic fashion, and results in regulation of many DNA metabolic processes. The CTG/CAG and CGG/CCG repeating sequences involved in several neuromuscular degenerative diseases display differential abilities for the binding of histone octamers. The effect of the repeating DNA on nucleosome assembly could be amplified as the number of repeats increases. Also, CpG methylation, and sequence interruptions within the triplet repeats exert an impact on the formation of nucleosomes along these repeating DNAs. The two most common triplet expansion human diseases, myotonic dystrophy 1 and fragile X syndrome, are caused by the expanded CTG/CAG and CGG/CCG repeats, respectively. In addition to the expanded repeats and CpG methylation, histone modifications, chromatin remodeling factors, and noncoding RNA have been shown to coordinate the chromatin structure at both myotonic dystrophy 1 and fragile X loci. Alterations in chromatin structure at these two loci can affect transcription of these disease-causing genes, leading to disease symptoms. These observations have brought a new appreciation that a full understanding of disease gene expression requires a knowledge of the structure of the chromatin domain within which the gene resides.

  11. Etiology and Evaluation of Sperm Chromatin Anomalies

    Directory of Open Access Journals (Sweden)

    Marziyeh Tavalaee

    2008-01-01

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

  12. Transgenerational propagation and quantitative maintenance of paternal centromeres depends on Cid/Cenp-A presence in Drosophila sperm.

    Directory of Open Access Journals (Sweden)

    Nitika Raychaudhuri

    Full Text Available In Drosophila melanogaster, as in many animal and plant species, centromere identity is specified epigenetically. In proliferating cells, a centromere-specific histone H3 variant (CenH3, named Cid in Drosophila and Cenp-A in humans, is a crucial component of the epigenetic centromere mark. Hence, maintenance of the amount and chromosomal location of CenH3 during mitotic proliferation is important. Interestingly, CenH3 may have different roles during meiosis and the onset of embryogenesis. In gametes of Caenorhabditis elegans, and possibly in plants, centromere marking is independent of CenH3. Moreover, male gamete differentiation in animals often includes global nucleosome for protamine exchange that potentially could remove CenH3 nucleosomes. Here we demonstrate that the control of Cid loading during male meiosis is distinct from the regulation observed during the mitotic cycles of early embryogenesis. But Cid is present in mature sperm. After strong Cid depletion in sperm, paternal centromeres fail to integrate into the gonomeric spindle of the first mitosis, resulting in gynogenetic haploid embryos. Furthermore, after moderate depletion, paternal centromeres are unable to re-acquire normal Cid levels in the next generation. We conclude that Cid in sperm is an essential component of the epigenetic centromere mark on paternal chromosomes and it exerts quantitative control over centromeric Cid levels throughout development. Hence, the amount of Cid that is loaded during each cell cycle appears to be determined primarily by the preexisting centromeric Cid, with little flexibility for compensation of accidental losses.

  13. Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

    Science.gov (United States)

    Earnshaw, W C; Allshire, R C; Black, B E; Bloom, K; Brinkley, B R; Brown, W; Cheeseman, I M; Choo, K H A; Copenhaver, G P; Deluca, J G; Desai, A; Diekmann, S; Erhardt, S; Fitzgerald-Hayes, M; Foltz, D; Fukagawa, T; Gassmann, R; Gerlich, D W; Glover, D M; Gorbsky, G J; Harrison, S C; Heun, P; Hirota, T; Jansen, L E T; Karpen, G; Kops, G J P L; Lampson, M A; Lens, S M; Losada, A; Luger, K; Maiato, H; Maddox, P S; Margolis, R L; Masumoto, H; McAinsh, A D; Mellone, B G; Meraldi, P; Musacchio, A; Oegema, K; O'Neill, R J; Salmon, E D; Scott, K C; Straight, A F; Stukenberg, P T; Sullivan, B A; Sullivan, K F; Sunkel, C E; Swedlow, J R; Walczak, C E; Warburton, P E; Westermann, S; Willard, H F; Wordeman, L; Yanagida, M; Yen, T J; Yoda, K; Cleveland, D W

    2013-04-01

    The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    the chromatin-associated scaffold attachment factor SAFB1 as a component of the DNA damage response and show that SAFB1 cooperates with histone acetylation to allow for efficient γH2AX spreading and genotoxic stress signaling. SAFB1 undergoes a highly dynamic exchange at damaged chromatin in a poly...

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

    Directory of Open Access Journals (Sweden)

    Daniël P. Melters

    2015-08-01

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

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

    Science.gov (United States)

    Polo, Sophie E

    2015-02-13

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

  17. Genetic and physical mapping of two centromere-proximal regions of chromosome IV in Aspergillus nidulans

    DEFF Research Database (Denmark)

    Aleksenko, Alexei Y.; Nielsen, Michael Lynge; Clutterbuck, A.J.

    2001-01-01

    The centromere-proximal portion of the chromosome was mapped physically using overlapping clones of a cosmid genomic library. Two contiguous segments of a physical map, based on restriction mapping of cosmid clones, were generated, together covering more than 0.4 Mb DNA. A reverse genetic mapping...... approach was used to establish a correlation between physical and genetic maps; i,e., marker genes were integrated into physically mapped segments and subsequently mapped by mitotic and meiotic recombination. The resulting data, together with additional classical genetic mapping, lead to a substantial...... revision of the genetic map of the chromosome, including the position of the centromere, Comparison of physical and genetic maps indicates that meiotic recombination is low in subcentromeric DNA, its frequency being reduced from 1 crossover per 0.8 Mb to approximately 1 crossover per 5 Mb per meiosis...

  18. Identification of a centromeric exchange of acrocentric chromosomes by fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Yu, C.W.; Immken, L.; Curry, C.J.R. [UCSF, Fresno, CA (United States)] [and others

    1994-09-01

    Exchanges of the peri-centromeric area of acrocentric chromosomes are difficult to identify using the conventional cytogenetic techniques. Fluorescence in situ hybridization (FISH) provides a new way for precisely identifying such rearrangements. Here we report a case of centromeric rearrangement in an amniotic fluid specimen with an extra marker chromosome. M.G., a 41-year-old G1, was referred for advanced maternal age. Chromosome studies revealed a 47,XX +mar karyotype. The marker appeared to be bi-satallited with a single C band. Chromosome studies from the parents were normal. The parents elected to terminate the pregnancy. Anatomical examination of the abortus revealed a very short neck, posteriorly rotated ears, high set cecum, absent hepatic lobation and low abdominal kidneys with short ureters. FISH studies with alpha-satellite probes of 13/21, 14/22, and 15, and the DiGeorge probe, indicated that there is a translocation of 21 alpha-satellite to the 22, and that the marker chromosome probably consists of 14/22 alpha-satellite material. FISH analysis of the parents chromosome revealed that father had the translocation of 21 alpha-satellite to the 22 as well. Exchanges of centromeric material among the acrocentric chromosomes may not be an uncommon event in humans. Although it probably has no clinical significance, it may result in non-disjunction or marker chromosome formation from an uncommon satellite association. With the use of FISH techniques, exchanges involving the centromeric regions of acrocentric chromosomes can be identified.

  19. Chromatin Regulators in Pancreas Development and Diabetes.

    Science.gov (United States)

    Campbell, Stephanie A; Hoffman, Brad G

    2016-03-01

    The chromatin landscape of a cell is dynamic and can be altered by chromatin regulators that control nucleosome placement and DNA or histone modifications. Together with transcription factors, these complexes help dictate the transcriptional output of a cell and, thus, balance cell proliferation and differentiation while restricting tissue-specific gene expression. In this review, we describe current research on chromatin regulators and their roles in pancreas development and the maintenance of mature β cell function, which, once elucidated, will help us better understand how β cell differentiation occurs and is maintained. These studies have so far implicated proteins from several complexes that regulate DNA methylation, nucleosome remodeling, and histone acetylation and methylation that could become promising targets for diabetes therapy and stem cell differentiation.

  20. Functions of the Proteasome on Chromatin

    Science.gov (United States)

    McCann, Tyler S.; Tansey, William P.

    2014-01-01

    The proteasome is a large self-compartmentalized protease complex that recognizes, unfolds, and destroys ubiquitylated substrates. Proteasome activities are required for a host of cellular functions, and it has become clear in recent years that one set of critical actions of the proteasome occur on chromatin. In this review, we discuss some of the ways in which proteasomes directly regulate the structure and function of chromatin and chromatin regulatory proteins, and how this influences gene transcription. We discuss lingering controversies in the field, the relative importance of proteolytic versus non-proteolytic proteasome activities in this process, and highlight areas that require further investigation. Our intention is to show that proteasomes are involved in major steps controlling the expression of the genetic information, that proteasomes use both proteolytic mechanisms and ATP-dependent protein remodeling to accomplish this task, and that much is yet to be learned about the full spectrum of ways that proteasomes influence the genome. PMID:25422899

  1. Functions of the Proteasome on Chromatin

    Directory of Open Access Journals (Sweden)

    Tyler S. McCann

    2014-11-01

    Full Text Available The proteasome is a large self-compartmentalized protease complex that recognizes, unfolds, and destroys ubiquitylated substrates. Proteasome activities are required for a host of cellular functions, and it has become clear in recent years that one set of critical actions of the proteasome occur on chromatin. In this review, we discuss some of the ways in which proteasomes directly regulate the structure and function of chromatin and chromatin regulatory proteins, and how this influences gene transcription. We discuss lingering controversies in the field, the relative importance of proteolytic versus non-proteolytic proteasome activities in this process, and highlight areas that require further investigation. Our intention is to show that proteasomes are involved in major steps controlling the expression of the genetic information, that proteasomes use both proteolytic mechanisms and ATP-dependent protein remodeling to accomplish this task, and that much is yet to be learned about the full spectrum of ways that proteasomes influence the genome.

  2. A stable acentric marker chromosome: Possible existence of an intercalary ancient centromere at distal 8p

    Energy Technology Data Exchange (ETDEWEB)

    Ohashi, Hirofumi; Fukushima, Yoshimitsu; Wakui, Keiko; Ogawa, Kioyshi [Saitama Children`s Medical Center, Iwatsuki (Japan); Okano, Tetsuroh [Kitazato Univ., Tokyo (Japan); Niikawa, Norio [Nagasaki Univ. School of Medicine (Japan)

    1994-12-01

    A centromere is considered to be an essential chromosomal component where microtubule-kinetochore interaction occurs to segregate sister chromatids faithfully and acentric chromosomes are unstable and lost through cell divisions. We report a novel marker chromosome that was acentric but stable through cell divisions. The patient was a 2-year-old girl with mental retardation, patent ductus arteriosus, and mild dysmorphic features. G-banded chromosome analysis revealed that an additional small marker chromosome was observed in all 100 cells examined. By the reverse-chromosome-painting method, the marker was found to originate from the distal region of 8p, and a subsequent two-color FISH analysis with cosmid probes around the region revealed that the marker was an inverted duplication interpreted as 8pter {yields} p23.1::p23.1 {yields} 8pter. No centromeric region was involved in the marker. By FISH, no {alpha}-satellite sequence was detected on the marker, while a telomere sequence was detected at each end. Anti-kinetochore immunostaining, using a serum from a patient with CREST (calcinosis, Raynaud syndrome, esophageal dismotility, sclerodactyly, and telangiectasia) syndrome, showed a pair of signals on the marker, which indicated that a functional kinetochore was present on the marker. The analysis of this patient might suggest the possibility that an ancient centromere sequence exists at distal 8p (8p23.1-pter) and was activated through the chromosome rearrangement in the patient.

  3. Centromere binding and a conserved role in chromosome stability for SUMO-dependent ubiquitin ligases.

    Directory of Open Access Journals (Sweden)

    Loes A L van de Pasch

    Full Text Available The Saccharomyces cerevisiae Slx5/8 complex is the founding member of a recently defined class of SUMO-targeted ubiquitin ligases (STUbLs. Slx5/8 has been implicated in genome stability and transcription, but the precise contribution is unclear. To characterise Slx5/8 function, we determined genome-wide changes in gene expression upon loss of either subunit. The majority of mRNA changes are part of a general stress response, also exhibited by mutants of other genome integrity pathways and therefore indicative of an indirect effect on transcription. Genome-wide binding analysis reveals a uniquely centromeric location for Slx5. Detailed phenotype analyses of slx5Δ and slx8Δ mutants show severe mitotic defects that include aneuploidy, spindle mispositioning, fish hooks and aberrant spindle kinetics. This is associated with accumulation of the PP2A regulatory subunit Rts1 at centromeres prior to entry into anaphase. Knockdown of the human STUbL orthologue RNF4 also results in chromosome segregation errors due to chromosome bridges. The study shows that STUbLs have a conserved role in maintenance of chromosome stability and links SUMO-dependent ubiquitination to a centromere-specific function during mitosis.

  4. Shearing of the CENP-A dimerization interface mediates plasticity in the octameric centromeric nucleosome.

    Science.gov (United States)

    Winogradoff, David; Zhao, Haiqing; Dalal, Yamini; Papoian, Garegin A

    2015-11-25

    The centromeric nucleosome is a key epigenetic determinant of centromere identity and function. Consequently, deciphering how CENP-A containing nucleosomes contribute structurally to centromere function is a fundamental question in chromosome biology. Here, we performed microsecond timescale all-atom molecular dynamics (MD) simulations of CENP-A and H3 nucleosomes, and report that the octameric CENP-A core particles and nucleosomes display different dynamics from their canonical H3-containing counterparts. The most significant motion observed is within key interactions at the heart of the CENP-A octameric core, wherein shearing of contacts within the CENP-A:CENP-A' dimerization interface results in a weaker four helix bundle, and an extrusion of 10-30 bp of DNA near the pseudo-dyad. Coupled to other local and global fluctuations, the CENP-A nucleosome occupies a more rugged free energy landscape than the canonical H3 nucleosome. Taken together, our data suggest that CENP-A encodes enhanced distortability to the octameric nucleosome, which may allow for enhanced flexing of the histone core in vivo.

  5. Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication

    Directory of Open Access Journals (Sweden)

    Yasushi Shiomi

    2017-01-01

    Full Text Available During cell division, genome integrity is maintained by faithful DNA replication during S phase, followed by accurate segregation in mitosis. Many DNA metabolic events linked with DNA replication are also regulated throughout the cell cycle. In eukaryotes, the DNA sliding clamp, proliferating cell nuclear antigen (PCNA, acts on chromatin as a processivity factor for DNA polymerases. Since its discovery, many other PCNA binding partners have been identified that function during DNA replication, repair, recombination, chromatin remodeling, cohesion, and proteolysis in cell-cycle progression. PCNA not only recruits the proteins involved in such events, but it also actively controls their function as chromatin assembles. Therefore, control of PCNA-loading onto chromatin is fundamental for various replication-coupled reactions. PCNA is loaded onto chromatin by PCNA-loading replication factor C (RFC complexes. Both RFC1-RFC and Ctf18-RFC fundamentally function as PCNA loaders. On the other hand, after DNA synthesis, PCNA must be removed from chromatin by Elg1-RFC. Functional defects in RFC complexes lead to chromosomal abnormalities. In this review, we summarize the structural and functional relationships among RFC complexes, and describe how the regulation of PCNA loading/unloading by RFC complexes contributes to maintaining genome integrity.

  6. Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication.

    Science.gov (United States)

    Shiomi, Yasushi; Nishitani, Hideo

    2017-01-26

    During cell division, genome integrity is maintained by faithful DNA replication during S phase, followed by accurate segregation in mitosis. Many DNA metabolic events linked with DNA replication are also regulated throughout the cell cycle. In eukaryotes, the DNA sliding clamp, proliferating cell nuclear antigen (PCNA), acts on chromatin as a processivity factor for DNA polymerases. Since its discovery, many other PCNA binding partners have been identified that function during DNA replication, repair, recombination, chromatin remodeling, cohesion, and proteolysis in cell-cycle progression. PCNA not only recruits the proteins involved in such events, but it also actively controls their function as chromatin assembles. Therefore, control of PCNA-loading onto chromatin is fundamental for various replication-coupled reactions. PCNA is loaded onto chromatin by PCNA-loading replication factor C (RFC) complexes. Both RFC1-RFC and Ctf18-RFC fundamentally function as PCNA loaders. On the other hand, after DNA synthesis, PCNA must be removed from chromatin by Elg1-RFC. Functional defects in RFC complexes lead to chromosomal abnormalities. In this review, we summarize the structural and functional relationships among RFC complexes, and describe how the regulation of PCNA loading/unloading by RFC complexes contributes to maintaining genome integrity.

  7. Replicating chromatin: a tale of histones

    DEFF Research Database (Denmark)

    Groth, Anja

    2009-01-01

    Chromatin serves structural and functional roles crucial for genome stability and correct gene expression. This organization must be reproduced on daughter strands during replication to maintain proper overlay of epigenetic fabric onto genetic sequence. Nucleosomes constitute the structural...... framework of chromatin and carry information to specify higher-order organization and gene expression. When replication forks traverse the chromosomes, nucleosomes are transiently disrupted, allowing the replication machinery to gain access to DNA. Histone recycling, together with new deposition, ensures...... reassembly on nascent DNA strands. The aim of this review is to discuss how histones - new and old - are handled at the replication fork, highlighting new mechanistic insights and revisiting old paradigms....

  8. Chromatin proteins and modifications as drug targets

    DEFF Research Database (Denmark)

    Helin, Kristian; Dhanak, Dashyant

    2013-01-01

    A plethora of groundbreaking studies have demonstrated the importance of chromatin-associated proteins and post-translational modifications of histones, proteins and DNA (so-called epigenetic modifications) for transcriptional control and normal development. Disruption of epigenetic control...... is a frequent event in disease, and the first epigenetic-based therapies for cancer treatment have been approved. A generation of new classes of potent and specific inhibitors for several chromatin-associated proteins have shown promise in preclinical trials. Although the biology of epigenetic regulation...

  9. CHD chromatin remodelers and the transcription cycle.

    Science.gov (United States)

    Murawska, Magdalena; Brehm, Alexander

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Saera Hihara

    2012-12-01

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

  11. Chromatin compaction protects genomic DNA from radiation damage.

    Directory of Open Access Journals (Sweden)

    Hideaki Takata

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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  13. Research Discovers Frequent Mutations of Chromatin

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

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

  14. Chromatin and epigenetics in all their states

    NARCIS (Netherlands)

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

    2016-01-01

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

  15. Epigenetic chromatin silencing: bistability and front propagation

    Science.gov (United States)

    Sedighi, Mohammad; Sengupta, Anirvan M.

    2007-12-01

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

  16. Histone variants: key players of chromatin.

    Science.gov (United States)

    Biterge, Burcu; Schneider, Robert

    2014-06-01

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

  17. Chromatin conformation capture strategies in molecular diagnostics

    NARCIS (Netherlands)

    Vree, P.J.P. de

    2015-01-01

    In this thesis I have explored the clinical potential of the 4C-technology and worked on development of a novel chromatin conformation capture based technology, called TLA. In chapter 2 I describe how the 4C-technology can be applied as a targeted strategy to identify putative fusion-genes or chromo

  18. The great repression: chromatin and cryptic transcription.

    Science.gov (United States)

    Hennig, Bianca P; Fischer, Tamás

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Nishat Aliya

    2012-01-01

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

  20. A study of the interaction between ethidium bromide and rye chromatin: comparison with calf thymus chromatin.

    Science.gov (United States)

    LaRue, H; Pallotta, D

    1976-09-01

    We studied the interaction of ethidium bromide with rye and calf thymus chromatin. Both types of chromatin have the same dye accessibility, which is about 50% of that of DNA. From this result we conclude that the molecular structure of these two chromatins is similar. For rye, the extraction of H1 produces no change in the binding of ethidium bromide. The subsequent extraction of H2A and H2B produces a 14% increase in the binding, and the removal of H3 and H4, another 54% increase. At this stage, the number of binding sites is still less than that of DNA. This is presumably due to the presence of some tightly bound non-histones. Thus, the arginine-rich histones and the tightly bound non-histones are most responsible for limiting the binding of ethidium bromide to rye chromatin.

  1. Separase Is Required for Homolog and Sister Disjunction during Drosophila melanogaster Male Meiosis, but Not for Biorientation of Sister Centromeres.

    Directory of Open Access Journals (Sweden)

    Ariane C Blattner

    2016-04-01

    Full Text Available Spatially controlled release of sister chromatid cohesion during progression through the meiotic divisions is of paramount importance for error-free chromosome segregation during meiosis. Cohesion is mediated by the cohesin protein complex and cleavage of one of its subunits by the endoprotease separase removes cohesin first from chromosome arms during exit from meiosis I and later from the pericentromeric region during exit from meiosis II. At the onset of the meiotic divisions, cohesin has also been proposed to be present within the centromeric region for the unification of sister centromeres into a single functional entity, allowing bipolar orientation of paired homologs within the meiosis I spindle. Separase-mediated removal of centromeric cohesin during exit from meiosis I might explain sister centromere individualization which is essential for subsequent biorientation of sister centromeres during meiosis II. To characterize a potential involvement of separase in sister centromere individualization before meiosis II, we have studied meiosis in Drosophila melanogaster males where homologs are not paired in the canonical manner. Meiosis does not include meiotic recombination and synaptonemal complex formation in these males. Instead, an alternative homolog conjunction system keeps homologous chromosomes in pairs. Using independent strategies for spermatocyte-specific depletion of separase complex subunits in combination with time-lapse imaging, we demonstrate that separase is required for the inactivation of this alternative conjunction at anaphase I onset. Mutations that abolish alternative homolog conjunction therefore result in random segregation of univalents during meiosis I also after separase depletion. Interestingly, these univalents become bioriented during meiosis II, suggesting that sister centromere individualization before meiosis II does not require separase.

  2. Identification and Preliminary Analysis of Several Centromere-associated Bacterial Artificial Chromosome Clones from a Diploid Wheat Library

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Although the centromeres of some plants have been investigated previously, our knowledge of the wheat centromere is still very limited. To understand the structure and function of the wheat centromere, we used two centromeric repeats (RCS1 and CCS1-5ab) to obtain some centromere-associated bacterial artificial chromosome (BAC) clones in 32 RCS1-related BAC clones that had been screened out from a diploid wheat (Triticum boeoticum Boiss.; 2n=2x=14) BAC library. Southern hybridization results indicated that, of the 32 candidates,there were 28 RCS1-positive clones. Based on gel blot patterns, the frequency of RCS1 was approximately one copy every 69.4 kb in these 28 RCS1-positive BAC clones. More bands were detected when the same filter was probed with CCS1-5ab. Furthermore, the CCS1 bands covered all the bands detected by RCS1, which suggests that some CCS1 repeats were distributed together with RCS1. The frequency of CCS1 families was once every 35.8 kb, nearly twice that of RCS1. Fluorescence in situ hybridization (FISH) analysis indicated that the five BAC clones containing RCS1 and CCS1 sequences all detected signals at the centromeric regions in hexaploid wheat, but the signal intensities on the A-genome chromosomes were stronger than those on the B- and/or D-genome chromosomes. The FISH analysis among nine Triticeae cereals indicated that there were A-genomespecific (or rich) sequences dispersing on chromosome arms in the BAC clone TbBAC5. In addition, at the interphase cells, the centromeres of diploid species usually clustered at one pole and formed a ring-like allocation in the period before metaphase.

  3. Overexpression of SETβ, a protein localizing to centromeres, causes precocious separation of chromatids during the first meiosis of mouse oocytes.

    Science.gov (United States)

    Qi, Shu-Tao; Wang, Zhen-Bo; Ouyang, Ying-Chun; Zhang, Qing-Hua; Hu, Meng-Wen; Huang, Xin; Ge, Zhaojia; Guo, Lei; Wang, Ya-Peng; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan

    2013-04-01

    Chromosome segregation in mammalian oocyte meiosis is an error-prone process, and any mistake in this process may result in aneuploidy, which is the main cause of infertility, abortion and many genetic diseases. It is now well known that shugoshin and protein phosphatase 2A (PP2A) play important roles in the protection of centromeric cohesion during the first meiosis. PP2A can antagonize the phosphorylation of rec8, a member of the cohesin complex, at the centromeres and thus prevent cleavage of rec8 and so maintain the cohesion of chromatids. SETβ is a protein that physically interacts with shugoshin and inhibits PP2A activity. We thus hypothesized that SETβ might regulate cohesion protection and chromosome segregation during oocyte meiotic maturation. Here we report for the first time the expression, subcellular localization and functions of SETβ during mouse oocyte meiosis. Immunoblotting analysis showed that the expression level of SETβ was stable from the germinal vesicle stage to the MII stage of oocyte meiosis. Immunofluorescence analysis showed SETβ accumulation in the nucleus at the germinal vesicle stage, whereas it was targeted mainly to the inner centromere area and faintly localized to the interchromatid axes from germinal vesicle breakdown to MI stages. At the MII stage, SETβ still localized to the inner centromere area, but could relocalize to kinetochores in a process perhaps dependent on the tension on the centromeres. SETβ partly colocalized with PP2A at the inner centromere area. Overexpression of SETβ in mouse oocytes caused precocious separation of sister chromatids, but depletion of SETβ by RNAi showed little effects on the meiotic maturation process. Taken together, our results suggest that SETβ, even though it localizes to centromeres, might not be essential for chromosome separation during mouse oocyte meiotic maturation, although its forced overexpression causes premature chromatid separation.

  4. Separase Is Required for Homolog and Sister Disjunction during Drosophila melanogaster Male Meiosis, but Not for Biorientation of Sister Centromeres.

    Science.gov (United States)

    Blattner, Ariane C; Chaurasia, Soumya; McKee, Bruce D; Lehner, Christian F

    2016-04-01

    Spatially controlled release of sister chromatid cohesion during progression through the meiotic divisions is of paramount importance for error-free chromosome segregation during meiosis. Cohesion is mediated by the cohesin protein complex and cleavage of one of its subunits by the endoprotease separase removes cohesin first from chromosome arms during exit from meiosis I and later from the pericentromeric region during exit from meiosis II. At the onset of the meiotic divisions, cohesin has also been proposed to be present within the centromeric region for the unification of sister centromeres into a single functional entity, allowing bipolar orientation of paired homologs within the meiosis I spindle. Separase-mediated removal of centromeric cohesin during exit from meiosis I might explain sister centromere individualization which is essential for subsequent biorientation of sister centromeres during meiosis II. To characterize a potential involvement of separase in sister centromere individualization before meiosis II, we have studied meiosis in Drosophila melanogaster males where homologs are not paired in the canonical manner. Meiosis does not include meiotic recombination and synaptonemal complex formation in these males. Instead, an alternative homolog conjunction system keeps homologous chromosomes in pairs. Using independent strategies for spermatocyte-specific depletion of separase complex subunits in combination with time-lapse imaging, we demonstrate that separase is required for the inactivation of this alternative conjunction at anaphase I onset. Mutations that abolish alternative homolog conjunction therefore result in random segregation of univalents during meiosis I also after separase depletion. Interestingly, these univalents become bioriented during meiosis II, suggesting that sister centromere individualization before meiosis II does not require separase.

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

    Directory of Open Access Journals (Sweden)

    Sergei Vatolin

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

  6. Chromatin Dynamics of the mouse β-globin locus

    NARCIS (Netherlands)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2014-04-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Jamy C. [Univ. of California, Berkeley, CA (United States)

    2007-01-01

    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) that 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 in

  10. [Sop proteins can cause transcriptional silencing of genes located close to the centromere sites of linear plasmid N15].

    Science.gov (United States)

    Mardanov, A V; Lane, D; Ravin, N V

    2010-01-01

    Stable inheritance of bacterial chromosomes and low copy number plasmids is ensured by accurate partitioning of replicated molecules between the daughter cells at division. Partitioning of the prophage of the temperate bacteriophage N15, which exists as a linear plasmid molecule with covalently closed ends, depends on the sop locus, comprising genes sopA and sopB, as well as four centromere sites located in different regions of the N15 genome essential for replication and the control of lysogeny. We found that binding of SopB to the centromere can silence centromere-proximal promoters, presumably due to subsequent polymerizing of SopB along the DNA. Close to the IR4 centromere site we identified a promoter, P59, able to drive expression of phage late genes encoding the structural proteins of virion. We found that following binding to IR4 the N15 Sop proteins can cause repression of this promoter. The repression depends on SopB and became stronger in the presence of SopA. Sop-dependent silencing of centromere-proximal promoters control gene expression in phage N15, particularly preventing undesired expression of late genes in the N15 prophage. Thus, the phage N15 sop system not only ensures plasmid partitioning but is also involved in the genetic network controlling prophage replication and the maintenance of lysogeny.

  11. Characterization of the genomic organization of the region bordering the centromere of chromosome V of Podospora anserina by direct sequencing.

    Science.gov (United States)

    Silar, Philippe; Barreau, Christian; Debuchy, Robert; Kicka, Sébastien; Turcq, Béatrice; Sainsard-Chanet, Annie; Sellem, Carole H; Billault, Alain; Cattolico, Laurence; Duprat, Simone; Weissenbach, Jean

    2003-08-01

    A Podospora anserina BAC library of 4800 clones has been constructed in the vector pBHYG allowing direct selection in fungi. Screening of the BAC collection for centromeric sequences of chromosome V allowed the recovery of clones localized on either sides of the centromere, but no BAC clone was found to contain the centromere. Seven BAC clones containing 322,195 and 156,244bp from either sides of the centromeric region were sequenced and annotated. One 5S rRNA gene, 5 tRNA genes, and 163 putative coding sequences (CDS) were identified. Among these, only six CDS seem specific to P. anserina. The gene density in the centromeric region is approximately one gene every 2.8kb. Extrapolation of this gene density to the whole genome of P. anserina suggests that the genome contains about 11,000 genes. Synteny analyses between P. anserina and Neurospora crassa show that co-linearity extends at the most to a few genes, suggesting rapid genome rearrangements between these two species.

  12. Differing requirements for RAD51 and DMC1 in meiotic pairing of centromeres and chromosome arms in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Olivier Da Ines

    Full Text Available During meiosis homologous chromosomes pair, recombine, and synapse, thus ensuring accurate chromosome segregation and the halving of ploidy necessary for gametogenesis. The processes permitting a chromosome to pair only with its homologue are not fully understood, but successful pairing of homologous chromosomes is tightly linked to recombination. In Arabidopsis thaliana, meiotic prophase of rad51, xrcc3, and rad51C mutants appears normal up to the zygotene/pachytene stage, after which the genome fragments, leading to sterility. To better understand the relationship between recombination and chromosome pairing, we have analysed meiotic chromosome pairing in these and in dmc1 mutant lines. Our data show a differing requirement for these proteins in pairing of centromeric regions and chromosome arms. No homologous pairing of mid-arm or distal regions was observed in rad51, xrcc3, and rad51C mutants. However, homologous centromeres do pair in these mutants and we show that this does depend upon recombination, principally on DMC1. This centromere pairing extends well beyond the heterochromatic centromere region and, surprisingly, does not require XRCC3 and RAD51C. In addition to clarifying and bringing the roles of centromeres in meiotic synapsis to the fore, this analysis thus separates the roles in meiotic synapsis of DMC1 and RAD51 and the meiotic RAD51 paralogs, XRCC3 and RAD51C, with respect to different chromosome domains.

  13. The Inner Centromere Protein (INCENP) Coil Is a Single α-Helix (SAH) Domain That Binds Directly to Microtubules and Is Important for Chromosome Passenger Complex (CPC) Localization and Function in Mitosis.

    Science.gov (United States)

    Samejima, Kumiko; Platani, Melpomeni; Wolny, Marcin; Ogawa, Hiromi; Vargiu, Giulia; Knight, Peter J; Peckham, Michelle; Earnshaw, William C

    2015-08-28

    The chromosome passenger complex (CPC) is a master regulator of mitosis. Inner centromere protein (INCENP) acts as a scaffold regulating CPC localization and activity. During early mitosis, the N-terminal region of INCENP forms a three-helix bundle with Survivin and Borealin, directing the CPC to the inner centromere where it plays essential roles in chromosome alignment and the spindle assembly checkpoint. The C-terminal IN box region of INCENP is responsible for binding and activating Aurora B kinase. The central region of INCENP has been proposed to comprise a coiled coil domain acting as a spacer between the N- and C-terminal domains that is involved in microtubule binding and regulation of the spindle checkpoint. Here we show that the central region (213 residues) of chicken INCENP is not a coiled coil but a ∼ 32-nm-long single α-helix (SAH) domain. The N-terminal half of this domain directly binds to microtubules in vitro. By analogy with previous studies of myosin 10, our data suggest that the INCENP SAH might stretch up to ∼ 80 nm under physiological forces. Thus, the INCENP SAH could act as a flexible "dog leash," allowing Aurora B to phosphorylate dynamic substrates localized in the outer kinetochore while at the same time being stably anchored to the heterochromatin of the inner centromere. Furthermore, by achieving this flexibility via an SAH domain, the CPC avoids a need for dimerization (required for coiled coil formation), which would greatly complicate regulation of the proximity-induced trans-phosphorylation that is critical for Aurora B activation.

  14. The Cyclophilin AtCYP71 Interacts with CAF-1 and LHP1 and Functions in Multiple Chromatin Remodeling Processes

    Institute of Scientific and Technical Information of China (English)

    Hong Li; Sheng Luan

    2011-01-01

    Chromatin is the primary carrier of epigenetic information in higher eukaryotes. AtCYP71 contains both cyclo-philin domain and WD40 repeats. Loss of AtCYP71 function causes drastic pleiotropic phenotypic defects. Here, we show that AtCYP71 physically interacts with FAS1 and LHP1, respectively, to modulate their distribution on chromatin. The Ihpl cyp71 double mutant showed more severe phenotypes than the single mutants, suggesting that AtCYP71 and LHP1 syn-ergistically control plant development. Such synergism was in part illustrated by the observation that LHP1 association with its specific target loci requires AtCYP71 function. We also demonstrate that AtCYP71 physically interacts with FAS1and is indispensable for FAS1 targeting to the KNAT1 locus. Together, our data suggest that AtCYP71 is involved in fun-damental processes of chromatin assembly and histone modification in plants.

  15. Histone density is maintained during transcription mediated by the chromatin remodeler RSC and histone chaperone NAP1 in vitro.

    Science.gov (United States)

    Kuryan, Benjamin G; Kim, Jessica; Tran, Nancy Nga H; Lombardo, Sarah R; Venkatesh, Swaminathan; Workman, Jerry L; Carey, Michael

    2012-02-01

    ATPases and histone chaperones facilitate RNA polymerase II (pol II) elongation on chromatin. In vivo, the coordinated action of these enzymes is necessary to permit pol II passage through a nucleosome while restoring histone density afterward. We have developed a biochemical system recapitulating this basic process. Transcription through a nucleosome in vitro requires the ATPase remodels structure of chromatin (RSC) and the histone chaperone nucleosome assembly protein 1 (NAP1). In the presence of NAP1, RSC generates a hexasome. Despite the propensity of RSC to evict histones, NAP1 reprograms the reaction such that the hexasome is retained on the template during multiple rounds of transcription. This work has implications toward understanding the mechanism of pol II elongation on chromatin.

  16. Stem-loop structures of the repetitive DNA sequences located at human centromeres

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, G.; Garcia, A.E.; Ratliff, R.; Moyzis, R.K. [Los Alamos National Lab., NM (United States); Catasti, P.; Hong, Lin; Yau, P. [California Univ., Davis, CA (United States). Dept. of Biological Chemistry; Bradbury, E.M. [Los Alamos National Lab., NM (United States)]|[California Univ., Davis, CA (United States). Dept. of Biological Chemistry

    1993-09-01

    The presence of the highly conserved repetitive DNA sequences in the human centromeres argues for a special role of these sequences in their biological functions - most likely achieved by the formation of unusual structures. This prompted us to carry out quantitative one- and two-dimensional nuclear magnetic resonance (lD/2D NMR) spectroscopy to determine the structural properties of the human centromeric repeats, d(AATGG){sub n.d}(CCATT){sub n}. The studies on centromeric DNAs reveal that the complementary sequence, d(AATGG){sub n.d}(CCATT){sub n}, adopts the usual Watson-Crick B-DNA duplex and the pyrimidine-rich d(CCATT){sub n} strand is essentially a random coil. However, the purine-rich d(AATGG){sub n} strand is shown to adopt unusual stem-loop structures for repeat lengths, n=2,3,4, and 6. In addition to normal Watson-Crick A{center_dot}T pairs, the stem-loop structures are stabilized by mismatch A{center_dot}G and G{center_dot}G pairs in the stem and G-G-A stacking in the loop. Stem-loop structures of d(AATGG)n are independently verified by gel electrophoresis and nuclease digestion studies. Thermal melting studies show that the DNA repeats, d(AATGG){sub n}, are as stable as the corresponding Watson-Crick duplex d(AATGG){sub n.d}(CCATT){sub n}. Therefore, the sequence d(AATGG){sub n} can, indeed, nucleate a stem-loop structure at little free-energy cost and if, during mitosis, they are located on the chromosome surface they can provide specific recognition sites for kinetochore function.

  17. Cytological identification of an isotetrasomic in rice and its application to centromere mapping

    Institute of Scientific and Technical Information of China (English)

    CHENGZHUKUAN; HENGXIUYU; 等

    1997-01-01

    The aneuploid with isochromosome or telochromosome is ideal material for exploring the position of centromere in lingkage map.For obtaining these aneuploids in rice,the primary trisomics from triplo-1 to triplo-12 and the aneuploids derived from a triploid of indica rice variety Zhongxiao 3037 were carefully investigated.From the offsprings of triplo-10,a primary trisomic of chromosome 10 of the variety,an isotetrasomic “triplo-10-1” was obtained.Cytological investigation revealed that a pair of extra isochromosomes of triplo-10-1 were come from the short arm of chromosome 10.In the offsprings of the isotetrasomic,a secondary trisomic “triplo-10-2”,in which the extra-chromosome was an isochromosome derived from the short arm of chromosome 10,was identified.With the isotetrasomic,secondary trisomic,primary trisomic and diploid of variety Zhongxiao 3037,different molecular markers were used for exploring the position of the centromere of chromosome 10.Based on the DNA dosage effect,it was verified that the molecular markers G1125,G333 and L169 were Located on the short arm,G1084 and other 16 available molecular markers were on the long arm of chromosome 10.So the centromere of chromosome 10 was located somewhere between G1125 and G1084 according to the RFLP linkage map given by Kurata et al[1].The distance from G1125 to G1084 was about 3.2cM.

  18. RNA Pol II promotes transcription of centromeric satellite DNA in beetles.

    Directory of Open Access Journals (Sweden)

    Zeljka Pezer

    Full Text Available Transcripts of centromeric satellite DNAs are known to play a role in heterochromatin formation as well as in establishment of the kinetochore. However, little is known about basic mechanisms of satellite DNA expression within constitutive heterochromatin and its regulation. Here we present comprehensive analysis of transcription of abundant centromeric satellite DNA, PRAT from beetle Palorus ratzeburgii (Coleoptera. This satellite is characterized by preservation and extreme sequence conservation among evolutionarily distant insect species. PRAT is expressed in all three developmental stages: larvae, pupae and adults at similar level. Transcripts are abundant comprising 0.033% of total RNA and are heterogeneous in size ranging from 0.5 kb up to more than 5 kb. Transcription proceeds from both strands but with 10 fold different expression intensity and transcripts are not processed into siRNAs. Most of the transcripts (80% are not polyadenylated and remain in the nucleus while a small portion is exported to the cytoplasm. Multiple, irregularly distributed transcription initiation sites as well as termination sites have been mapped within the PRAT sequence using primer extension and RLM-RACE. The presence of cap structure as well as poly(A tails in a portion of the transcripts indicate RNA polymerase II-dependent transcription and a putative polymerase II promoter site overlaps the most conserved part of the PRAT sequence. The treatment of larvae with alpha-amanitin decreases the level of PRAT transcripts at concentrations that selectively inhibit pol II activity. In conclusion, stable, RNA polymerase II dependant transcripts of abundant centromeric satellite DNA, not regulated by RNAi, have been identified and characterized. This study offers a basic understanding of expression of highly abundant heterochromatic DNA which in beetle species constitutes up to 50% of the genome.

  19. Novel Centromeric Loci of the Wine and Beer Yeast Dekkera bruxellensis CEN1 and CEN2

    Science.gov (United States)

    Ishchuk, Olena P.; Vojvoda Zeljko, Tanja; Schifferdecker, Anna J.; Mebrahtu Wisén, Sofia; Hagström, Åsa K.; Rozpędowska, Elżbieta; Rørdam Andersen, Mikael; Hellborg, Linda; Ling, Zhihao; Sibirny, Andrei A.

    2016-01-01

    The wine and beer yeast Dekkera bruxellensis thrives in environments that are harsh and limiting, especially in concentrations with low oxygen and high ethanol. Its different strains’ chromosomes greatly vary in number (karyotype). This study isolates two novel centromeric loci (CEN1 and CEN2), which support both the yeast’s autonomous replication and the stable maintenance of plasmids. In the sequenced genome of the D. bruxellensis strain CBS 2499, CEN1 and CEN2 are each present in one copy. They differ from the known “point” CEN elements, and their biological activity is retained within ~900–1300 bp DNA segments. CEN1 and CEN2 have features of both “point” and “regional” centromeres: They contain conserved DNA elements, ARSs, short repeats, one tRNA gene, and transposon-like elements within less than 1 kb. Our discovery of a miniature inverted-repeat transposable element (MITE) next to CEN2 is the first report of such transposons in yeast. The transformants carrying circular plasmids with cloned CEN1 and CEN2 undergo a phenotypic switch: They form fluffy colonies and produce three times more biofilm. The introduction of extra copies of CEN1 and CEN2 promotes both genome rearrangements and ploidy shifts, with these effects mediated by homologous recombination (between circular plasmid and genome centromere copy) or by chromosome breakage when integrated. Also, the proximity of the MITE-like transposon to CEN2 could translocate CEN2 within the genome or cause chromosomal breaks, so promoting genome dynamics. With extra copies of CEN1 and CEN2, the yeast’s enhanced capacities to rearrange its genome and to change its gene expression could increase its abilities for exploiting new and demanding niches. PMID:27560164

  20. Isolation of chromatin DNA tightly bound to the nuclear envelope of HeLa cells.

    Science.gov (United States)

    Kuvichkin, Vasily Vladimirovich

    2012-11-01

    Recent discovery of the role of nuclear pores in transcription, predicted by our early DNA-membrane complex (DMC) model, makes membrane-bound DNA (MBD) isolation from the cell nucleus and analysis of the MBD actual. The method of MBD isolation proposed by us retains DMC integrity during isolation. We used HeLa cells for DMC extraction. Changing the ionic composition of the isolation medium and replacing DNase I, used commonly for chromatin destruction, with a set of restriction enzymes allowed us to isolate the MBD. Treatment of a nuclear membrane with proteinase K and ultrasound has been used to increase the yield of MBD. Electron microscopic analysis of the purified fraction of isolated DMC supports our previous model of nuclear envelope lipid-chromatin interaction in the nuclear pore assembly.

  1. Quantification of chromatin condensation level by image processing.

    Science.gov (United States)

    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.

  2. Chromatin structure and DNA damage repair

    Directory of Open Access Journals (Sweden)

    Dinant Christoffel

    2008-11-01

    Full Text Available Abstract The integrity of the genome is continuously challenged by both endogenous and exogenous DNA damaging agents. These damaging agents can induce a wide variety of lesions in the DNA, such as double strand breaks, single strand breaks, oxidative lesions and pyrimidine dimers. The cell has evolved intricate DNA damage response mechanisms to counteract the genotoxic effects of these lesions. The two main features of the DNA damage response mechanisms are cell-cycle checkpoint activation and, at the heart of the response, DNA repair. For both damage signalling and repair, chromatin remodelling is most likely a prerequisite. Here, we discuss current knowledge on chromatin remodelling with respect to the cellular response to DNA damage, with emphasis on the response to lesions resolved by nucleotide excision repair. We will discuss the role of histone modifications as well as their displacement or exchange in nucleotide excision repair and make a comparison with their requirement in transcription and double strand break repair.

  3. Keystone Symposia on Epigenomics and Chromatin Dynamics

    DEFF Research Database (Denmark)

    Ravnskjær, Kim

    2012-01-01

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

  4. A new model of sperm nuclear architecture following assessment of the organization of centromeres and telomeres in three-dimensions

    Science.gov (United States)

    Ioannou, Dimitrios; Millan, Nicole M.; Jordan, Elizabeth; Tempest, Helen G.

    2017-01-01

    The organization of chromosomes in sperm nuclei has been proposed to possess a unique “hairpin-loop” arrangement, which is hypothesized to aid in the ordered exodus of the paternal genome following fertilization. This study simultaneously assessed the 3D and 2D radial and longitudinal organization of telomeres, centromeres, and investigated whether chromosomes formed the same centromere clusters in sperm cells. Reproducible radial and longitudinal non-random organization was observed for all investigated loci using both 3D and 2D approaches in multiple subjects. We report novel findings, with telomeres and centromeres being localized throughout the nucleus but demonstrating roughly a 1:1 distribution in the nuclear periphery and the intermediate regions with exodus of paternal chromosomes following fertilization. PMID:28139771

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

    KAUST Repository

    Jégu, Teddy

    2015-10-12

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

  6. De novo deciphering three-dimensional chromatin interaction and topological domains by wavelet transformation of epigenetic profiles.

    Science.gov (United States)

    Chen, Yong; Wang, Yunfei; Xuan, Zhenyu; Chen, Min; Zhang, Michael Q

    2016-06-20

    Defining chromatin interaction frequencies and topological domains is a great challenge for the annotations of genome structures. Although the chromosome conformation capture (3C) and its derivative methods have been developed for exploring the global interactome, they are limited by high experimental complexity and costs. Here we describe a novel computational method, called CITD, for de novo prediction of the chromatin interaction map by integrating histone modification data. We used the public epigenomic data from human fibroblast IMR90 cell and embryonic stem cell (H1) to develop and test CITD, which can not only successfully reconstruct the chromatin interaction frequencies discovered by the Hi-C technology, but also provide additional novel details of chromosomal organizations. We predicted the chromatin interaction frequencies, topological domains and their states (e.g. active or repressive) for 98 additional cell types from Roadmap Epigenomics and ENCODE projects. A total of 131 protein-coding genes located near 78 preserved boundaries among 100 cell types are found to be significantly enriched in functional categories of the nucleosome organization and chromatin assembly. CITD and its predicted results can be used for complementing the topological domains derived from limited Hi-C data and facilitating the understanding of spatial principles underlying the chromosomal organization.

  7. FACT prevents the accumulation of free histones evicted from transcribed chromatin and a subsequent cell cycle delay in G1.

    Directory of Open Access Journals (Sweden)

    Macarena Morillo-Huesca

    2010-05-01

    Full Text Available The FACT complex participates in chromatin assembly and disassembly during transcription elongation. The yeast mutants affected in the SPT16 gene, which encodes one of the FACT subunits, alter the expression of G1 cyclins and exhibit defects in the G1/S transition. Here we show that the dysfunction of chromatin reassembly factors, like FACT or Spt6, down-regulates the expression of the gene encoding the cyclin that modulates the G1 length (CLN3 in START by specifically triggering the repression of its promoter. The G1 delay undergone by spt16 mutants is not mediated by the DNA-damage checkpoint, although the mutation of RAD53, which is otherwise involved in histone degradation, enhances the cell-cycle defects of spt16-197. We reveal how FACT dysfunction triggers an accumulation of free histones evicted from transcribed chromatin. This accumulation is enhanced in a rad53 background and leads to a delay in G1. Consistently, we show that the overexpression of histones in wild-type cells down-regulates CLN3 in START and causes a delay in G1. Our work shows that chromatin reassembly factors are essential players in controlling the free histones potentially released from transcribed chromatin and describes a new cell cycle phenomenon that allows cells to respond to excess histones before starting DNA replication.

  8. Plant chromatin warms up in Madrid: meeting summary of the 3rd European Workshop on Plant Chromatin 2013, Madrid, Spain.

    Science.gov (United States)

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

    2014-04-01

    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.

  9. A Model of DNA Repeat-Assembled Mitotic Chromosomal Skeleton

    Directory of Open Access Journals (Sweden)

    Shao-Jun Tang

    2011-09-01

    Full Text Available Despite intensive investigation for decades, the principle of higher-order organization of mitotic chromosomes is unclear. Here, I describe a novel model that emphasizes a critical role of interactions of homologous DNA repeats (repetitive elements; repetitive sequences in mitotic chromosome architecture. According to the model, DNA repeats are assembled, via repeat interactions (pairing, into compact core structures that govern the arrangement of chromatins in mitotic chromosomes. Tandem repeat assemblies form a chromosomal axis to coordinate chromatins in the longitudinal dimension, while dispersed repeat assemblies form chromosomal nodes around the axis to organize chromatins in the halo. The chromosomal axis and nodes constitute a firm skeleton on which non-skeletal chromatins can be anchored, folded, and supercoiled.

  10. A model of DNA repeat-assembled mitotic chromosomal skeleton.

    Science.gov (United States)

    Tang, Shao-Jun

    2011-01-01

    Despite intensive investigation for decades, the principle of higher-order organization of mitotic chromosomes is unclear. Here, I describe a novel model that emphasizes a critical role of interactions of homologous DNA repeats (repetitive elements; repetitive sequences) in mitotic chromosome architecture. According to the model, DNA repeats are assembled, via repeat interactions (pairing), into compact core structures that govern the arrangement of chromatins in mitotic chromosomes. Tandem repeat assemblies form a chromosomal axis to coordinate chromatins in the longitudinal dimension, while dispersed repeat assemblies form chromosomal nodes around the axis to organize chromatins in the halo. The chromosomal axis and nodes constitute a firm skeleton on which non-skeletal chromatins can be anchored, folded, and supercoiled.

  11. Point Mutations in Centromeric Histone Induce Post-zygotic Incompatibility and Uniparental Inheritance.

    Directory of Open Access Journals (Sweden)

    Sundaram Kuppu

    2015-09-01

    Full Text Available The centromeric histone 3 variant (CENH3, aka CENP-A is essential for the segregation of sister chromatids during mitosis and meiosis. To better define CENH3 functional constraints, we complemented a null allele in Arabidopsis with a variety of mutant alleles, each inducing a single amino acid change in conserved residues of the histone fold domain. Many of these transgenic missense lines displayed wild-type growth and fertility on self-pollination, but exhibited frequent post-zygotic death and uniparental inheritance when crossed with wild-type plants. The failure of centromeres marked by these missense mutation in the histone fold domain of CENH3 reproduces the genome elimination syndromes described with chimeric CENH3 and CENH3 from diverged species. Additionally, evidence that a single point mutation is sufficient to generate a haploid inducer provide a simple one-step method for the identification of non-transgenic haploid inducers in existing mutagenized collections of crop species. As proof of the extreme simplicity of this approach to create haploid-inducing lines, we performed an in silico search for previously identified point mutations in CENH3 and identified an Arabidopsis line carrying the A86V substitution within the histone fold domain. This A87V non-transgenic line, while fully fertile on self-pollination, produced postzygotic death and uniparental haploids when crossed to wild type.

  12. Characterization of the oncogenic function of centromere protein F in hepatocellular carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Yongdong; Liu, Lulu; Zeng, Tingting; Zhu, Ying-Hui [State Key Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer Center, Guangzhou (China); Li, Jiangchao [Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou (China); Chen, Leilei [Department of Clinical Oncology, The University of Hong Kong, Pokfulam, Hong Kong (China); Li, Yan; Yuan, Yun-Fei [State Key Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer Center, Guangzhou (China); Ma, Stephanie, E-mail: stefma@hku.hk [Department of Clinical Oncology, The University of Hong Kong, Pokfulam, Hong Kong (China); State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong (China); Guan, Xin-Yuan, E-mail: xyguan@hkucc.hku.hk [State Key Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer Center, Guangzhou (China); Department of Clinical Oncology, The University of Hong Kong, Pokfulam, Hong Kong (China); State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong (China)

    2013-07-12

    Highlights: •Overexpression of CENPF is frequently detected in HCC. •Upregulation of CENPF serves as an independent prognosis factor in HCC patients. •CENPF functions as an oncogene in HCC by promoting cell G2/M transition. -- Abstract: Centromere protein F (CENPF) is an essential nuclear protein associated with the centromere-kinetochore complex and plays a critical role in chromosome segregation during mitosis. Up-regulation of CENPF expression has previously been detected in several solid tumors. In this study, we aim to study the expression and functional role of CENPF in hepatocellular carcinoma (HCC). We found CENPF was frequently overexpressed in HCC as compared with non-tumor tissue. Up-regulated CENPF expression in HCC was positively correlated with serum AFP, venous invasion, advanced differentiation stage and a shorter overall survival. Cox regression analysis found that overexpression of CENPF was an independent prognosis factor in HCC. Functional studies found that silencing CENPF could decrease the ability of the cells to proliferate, form colonies and induce tumor formation in nude mice. Silencing CENPF also resulted in the cell cycle arrest at G2/M checkpoint by down-regulating cell cycle proteins cdc2 and cyclin B1. Our data suggest that CENPF is frequently overexpressed in HCC and plays a critical role in driving HCC tumorigenesis.

  13. Identification of a high frequency of chromosomal rearrangements in the centromeric regions of prostate cancer patients

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The aim of the present investigation was to study the major chromosomal aberrations (CA) like deletion, translocation,inversion and mosaic in prostate cancer patients of Tamilnadu, Southern India. Totally 45 blood samples were collected from various hospitals in Tamilnadu, Southern India. Equal numbers of normal healthy subjects were chosen after signing a consent form. Volunteers provided blood samples (5 ml) to establish leukocyte cultures. Cytogenetic studies were performed by using Giemsa-banding technique and finally the results were ensured by spectral karyotyping (SKY) technique. In the present investigation, major CA like deletion, translocation, inversion and mosaic were identified in experimental subjects. Results showed frequent CA in chromosomes 1, 3, 5, 6, 7, 9, 13, 16, 18 and X. In comparison with experimental subjects, the control subjects exhibited very low levels of major CA (P<0.05). In the present study, the high frequency of centromeric rearrangements indicates a potential role for mitotic irregularities associated with the centromere in prostate cancer tumorigenesis. Identification of chromosome alterations may be helpful in understanding the molecular basis of the disease in better manner.

  14. Genome-Wide Association between Transcription Factor Expression and Chromatin Accessibility Reveals Regulators of Chromatin Accessibility

    Science.gov (United States)

    Rueedi, Rico

    2017-01-01

    To better understand genome regulation, it is important to uncover the role of transcription factors in the process of chromatin structure establishment and maintenance. Here we present a data-driven approach to systematically characterise transcription factors that are relevant for this process. Our method uses a linear mixed modelling approach to combine datasets of transcription factor binding motif enrichments in open chromatin and gene expression across the same set of cell lines. Applying this approach to the ENCODE dataset, we confirm already known and imply numerous novel transcription factors that play a role in the establishment or maintenance of open chromatin. In particular, our approach rediscovers many factors that have been annotated as pioneer factors. PMID:28118358

  15. Spectroscopic study of fast-neutron-irradiated chromatin

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-02-01

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

  16. PTEN Interacts with Histone H1 and Controls Chromatin Condensation

    Directory of Open Access Journals (Sweden)

    Zhu Hong Chen

    2014-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Justin W Walley

    2008-12-01

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

  18. Impact of chromatin structures on DNA processing for genomic analyses.

    Directory of Open Access Journals (Sweden)

    Leonid Teytelman

    Full Text Available Chromatin has an impact on recombination, repair, replication, and evolution of DNA. Here we report that chromatin structure also affects laboratory DNA manipulation in ways that distort the results of chromatin immunoprecipitation (ChIP experiments. We initially discovered this effect at the Saccharomyces cerevisiae HMR locus, where we found that silenced chromatin was refractory to shearing, relative to euchromatin. Using input samples from ChIP-Seq studies, we detected a similar bias throughout the heterochromatic portions of the yeast genome. We also observed significant chromatin-related effects at telomeres, protein binding sites, and genes, reflected in the variation of input-Seq coverage. Experimental tests of candidate regions showed that chromatin influenced shearing at some loci, and that chromatin could also lead to enriched or depleted DNA levels in prepared samples, independently of shearing effects. Our results suggested that assays relying on immunoprecipitation of chromatin will be biased by intrinsic differences between regions packaged into different chromatin structures - biases which have been largely ignored to date. These results established the pervasiveness of this bias genome-wide, and suggested that this bias can be used to detect differences in chromatin structures across the genome.

  19. Ndc10 is a platform for inner kinetochore assembly in budding yeast

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Uhn-Soo; Harrison, Stephen C. (Harvard-Med)

    2012-01-10

    Kinetochores link centromeric DNA to spindle microtubules and ensure faithful chromosome segregation during mitosis. In point-centromere yeasts, the CBF3 complex Skp1-Ctf13-(Cep3){sub 2}-(Ndc10){sub 2} recognizes a conserved centromeric DNA element through contacts made by Cep3 and Ndc10. We describe here the five-domain organization of Kluyveromyces lactis Ndc10 and the structure at 2.8 {angstrom} resolution of domains I-II (residues 1-402) bound to DNA. The structure resembles tyrosine DNA recombinases, although it lacks both endonuclease and ligase activities. Structural and biochemical data demonstrate that each subunit of the Ndc10 dimer binds a separate fragment of DNA, suggesting that Ndc10 stabilizes a DNA loop at the centromere. We describe in vitro association experiments showing that specific domains of Ndc10 interact with each of the known inner-kinetochore proteins or protein complexes in budding yeast. We propose that Ndc10 provides a central platform for inner-kinetochore assembly.

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

    Directory of Open Access Journals (Sweden)

    Mariliis Tark-Dame

    2014-10-01

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

  1. Inverstigation of chromatin folding patterns by atomic force microscopy

    Institute of Scientific and Technical Information of China (English)

    ZHANGYi; OUYANGZhenqian; 等

    1999-01-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

  3. Long Noncoding RNAs, Chromatin, and Development

    Directory of Open Access Journals (Sweden)

    Daniel P. Caley

    2010-01-01

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

  4. Sequence assembly

    DEFF Research Database (Denmark)

    Scheibye-Alsing, Karsten; Hoffmann, S.; Frankel, Annett Maria

    2009-01-01

    Despite the rapidly increasing number of sequenced and re-sequenced genomes, many issues regarding the computational assembly of large-scale sequencing data have remain unresolved. Computational assembly is crucial in large genome projects as well for the evolving high-throughput technologies and...... in genomic DNA, highly expressed genes and alternative transcripts in EST sequences. We summarize existing comparisons of different assemblers and provide a detailed descriptions and directions for download of assembly programs at: http://genome.ku.dk/resources/assembly/methods.html....

  5. NET23/STING promotes chromatin compaction from the nuclear envelope.

    Directory of Open Access Journals (Sweden)

    Poonam Malik

    Full Text Available 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.

  6. Assaying chromatin structure and remodeling by restriction enzyme accessibility

    OpenAIRE

    Trotter, Kevin W.; Archer, Trevor K.

    2012-01-01

    The packaging of eukaryotic DNA into nucleosomes, the fundamental unit of chromatin, creates a barrier to nuclear processes, such as transcription, DNA replication, recombination, and repair(1). This obstructive nature of chromatin can be overcome by the enzymatic activity of chromatin remodeling complexes which creates a more favorable environment for the association of essential factors and regulators to sequences within target genes. Here we describe a detailed approach for analyzing chrom...

  7. Mutations in Z8T824 Are Associated with Immunodeficiency, Centromeric Instability, and Facial Anomalies Syndrome Type 2

    NARCIS (Netherlands)

    de Greef, Jessica C.; Wang, Jun; Balog, Judit; den Dunnen, Johan T.; Frants, Rune R.; Straasheijm, Kirsten R.; Aytekin, Caner; van der Burg, Mirjam; Duprez, Laurence; Ferster, Alina; Gennery, Andrew R.; Gimelli, Giorgio; Reisli, Ismail; Schuetz, Catharina; Schulz, Ansgar; Smeets, Dominique F. C. M.; Sznajer, Yves; Wijmenga, Cisca; van Eggermond, Maria C.; van Ostaijen-ten Dam, Monique M.; Lankester, Arjan C.; van Tol, Maarten J. D.; van den Elsen, Peter J.; Weemaes, Corry M.; van der Maarel, Silvere M.

    2011-01-01

    Autosomal-recessive immunodeficiency, centromeric instability, and facial anomalies (ICE) syndrome is mainly characterized by recurrent, often fatal, respiratory and gastrointestinal infections. About 50% of patients carry mutations in the DNA methyltransferase 3B gene (DNMT3B) (ICF1). The remaining

  8. Is the time dimension of the cell cycle re-entry in AD regulated by centromere cohesion dynamics?

    Science.gov (United States)

    Bajić, Vladan P; Spremo-Potparević, Biljana; Zivković, Lada; Djelić, Ninoslav; Smith, Mark A

    2008-01-01

    Chromosomal involvement is a legitimate, yet not well understood, feature of Alzheimer disease (AD). Firstly, AD affects more women than men. Secondly, the amyloid-β protein precursor genetic mutations, responsible for a cohort of familial AD cases, reside on chromosome 21, the same chromosome responsible for the developmental disorder Down's syndrome. Thirdly, lymphocytes from AD patients display a novel chromosomal phenotype, namely premature centromere separation (PCS). Other documented morphological phenomena associated with AD include the occurrence of micronuclei, aneuploidy, binucleation, telomere instability, and cell cycle re-entry protein expression. Based on these events, here we present a novel hypothesis that the time dimension of cell cycle re-entry in AD is highly regulated by centromere cohesion dynamics. In view of the fact that neurons can re-enter the cell division cycle, our hypothesis predicts that alterations in the signaling pathway leading to premature cell death in neurons is a consequence of altered regulation of the separation of centromeres as a function of time. It is well known that centromeres in the metaphase-anaphase transition separate in a non-random, sequential order. This sequence has been shown to be deregulated in aging cells, various tumors, syndromes of chromosome instability, following certain chemical inductions, as well as in AD. Over time, premature chromosome separation is both a result of, and a driving force behind, further cohesion impairment, activation of cyclin dependent kinases, and mitotic catastrophe, a vicious circle resulting in cellular degeneration and death.

  9. Variation in Copy Number of Ty3/Gypsy Centromeric Retrotransposons in the Genomes of Thinopyrum intermedium and Its Diploid Progenitors.

    Directory of Open Access Journals (Sweden)

    Mikhail G Divashuk

    Full Text Available Speciation and allopolyploidization in cereals may be accompanied by dramatic changes in abundance of centromeric repeated transposable elements. Here we demonstrate that the reverse transcriptase part of Ty3/gypsy centromeric retrotransposon (RT-CR is highly conservative in the segmental hexaploid Thinopyrum intermedium (JrJvsSt and its possible diploid progenitors Th. bessarabicum (Jb, Pseudoroegneria spicata (St and Dasypyrum villosum (V but the abundance of the repeats varied to a large extent. Fluorescence in situ hybridization (FISH showed hybridization signals in centromeric region of all chromosomes in the studied species, although the intensity of the signals drastically differed. In Th. intermedium, the strongest signal of RT-CR probe was detected on the chromosomes of Jv, intermediate on Jr and faint on Js and St subgenome suggesting different abundance of RT-CR on the individual chromosomes rather than the sequence specificity of RT-CRs of the subgenomes. RT-CR quantification using real-time PCR revealed that its content per genome in Th. bessarabicum is ~ 2 times and P. spicata is ~ 1,5 times higher than in genome of D. villosum. The possible burst of Ty3/gypsy centromeric retrotransposon in Th. intermedium during allopolyploidization and its role in proper mitotic and meiotic chromosome behavior in a nascent allopolyploid is discussed.

  10. Effects of sense and antisense centromere/kinetochore complex protein-B (CENP-B) in cell cycle regulation

    Institute of Scientific and Technical Information of China (English)

    LUO Song; LIN Haiyan; QI Jianguo; WANG Yongchao

    2005-01-01

    This paper investigates the effects of sense and antisense centromere/kinetochore complex protein-B (CENP-B) in cell cycle regulation. Full-length cenpb cDNA was subcloned into pBI-EGFP eukaryotic expression vector in both sense and antisense orientation. HeLa-Tet-Off cells were transfected with sense or antisense cenpb vectors. Sense transfection of HeLa-Tet-Off cells resulted in the formation of a large centromere/kinetochore complex, and apoptosis of cells following several times of cell division. A stable antisense cenpb transfected cell line, named HACPB, was obtained. The centromere/kinetochore complex of HACPB cells became smaller than control HeLa-Tet-Off cells and scattered, and the expression of CENP-B was down-regulated. In addition, delayed cell cycle progression, inhibited malignant phenotype, restrained ability of tumor formation in nude mice, and delayed entry from G2/M phase into next G1 phase were observed in HACPB cells. Furthermore, the expression of cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors (CKIs) were modulated during different phases of the cell cycle. CENP-B is an essential protein for the maintenance of the structure and function of centromere/kinetochore complex, and plays important roles in cell cycle regulation.

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

    Directory of Open Access Journals (Sweden)

    Hyangyee Oh

    2013-02-01

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

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

    Science.gov (United States)

    Jafari, Mahvash; Nateghi, M; Rabbani, A

    2010-01-01

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

  13. Distribution of intercalative dye binding sites in chromatin.

    Science.gov (United States)

    Lurquin, P F; Seligy, V L

    1976-04-01

    Actinomycin D (AMD) and ethidium bromide (EB) were found to bind to chromatin isolated from a variety of gander tissues according to a strong and weak process analogous to that found for deproteinized DNA. Distribution of the dye intercalation sites in chromatin and DNA were evaluated at low r-values (dye bound per nucleotide) by following the appearance of free dye released from chromatin and DNA during thermal denaturation. The AMD dissociation profiles closely resembled the DNA or chromatin-DNA denaturation profiles; whereas the EB derivative dissociation profiles, indicated 3 major transitions for transcriptionally active chromatin with the main component corresponding to the single component which characterizes DNA. The DNA-like component was greatly reduced for mature erythrocyte chromatin but could be generated by removal of histone I and V. Removal of residual non acid-soluble proteins from dehistonized chromatin, urea treatment or dissociation and reconstitution of chromatin favoured conversion to the DNA-like component with loss of the other two. This study indicates that more than one type of binding exists generally in chromatin.

  14. Chromatin Landscape of the IRF Genes and Role of the Epigenetic Reader BRD4.

    Science.gov (United States)

    Bachu, Mahesh; Dey, Anup; Ozato, Keiko

    2016-07-01

    Histone post-translational modification patterns represent epigenetic states of genomic genes and denote the state of their transcription, past history, and future potential in gene expression. Genome-wide chromatin modification patterns reported from various laboratories are assembled in the ENCODE database, providing a fertile ground for understanding epigenetic regulation of any genes of interest across many cell types. The IRF family genes critically control innate immunity as they direct expression and activities of interferons. While these genes have similar structural and functional traits, their chromatin landscapes and epigenetic features have not been systematically evaluated. Here, by mining ENCODE database using an imputational approach, we summarize chromatin modification patterns for 6 of 9 IRF genes and show characteristic features that connote their epigenetic states. BRD4 is a BET bromodomain protein that "reads and translates" epigenetic marks into transcription. We review recent findings that BRD4 controls constitutive and signal-dependent transcription of many genes, including IRF genes. BRD4 dynamically binds to various genomic genes with a spatial and temporal specificity. Of particular importance, BRD4 is shown to critically regulate IRF-dependent anti-pathogen protection, inflammatory responses triggered by NF-κB, and the growth and spread of many cancers. The advent of small molecule inhibitors that disrupt binding of BET bromdomain to acetylated histone marks has opened new therapeutic possibilities for cancer and inflammatory diseases.

  15. Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster.

    Science.gov (United States)

    Sebald, Johanna; Willi, Michaela; Schoberleitner, Ines; Krogsdam, Anne; Orth-Höller, Dorothea; Trajanoski, Zlatko; Lusser, Alexandra

    2016-01-01

    The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster.

  16. Involvement of chromatin and histone acetylation in theregulation of HIV-LTR by thyroid hormone receptor

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The HIV-1 LTR controls the expression of HIV-1 viral genes and thus is critical for viral propagation and pathology.Numerous host factors have been shown to participate in the regulation of the LTR promoter.Among them is the thyroid hormone (T3) receptor (TR).TR has been shown to bind to the critical region of the promoter that contain the NFκB and Sp1 binding sites.Interestingly,earlier transient transfection studies in tissue culture cells have yielded contradicting conclusions on the role of TR in LTR regulation,likely due to the use of different cell types and/or lack of proper chromatin organization.Here,using the frog oocyte as a model system that allows replication-coupled chromatin assembly,mimicking that in somatic cells,we demonstrate that unliganded heterodimers of TR and RXR (9-cis retinoic acid receptor) repress LTR while the addition of T3 relieves the repression and further activates the promoter.More importantly,we show that chromatin and unliganded TR/RXR synergize to repress the promoter in a histone deacetylase-dependent manner.

  17. Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Johanna Sebald

    Full Text Available The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster.

  18. Structure and function insights into the NuRD chromatin remodeling complex.

    Science.gov (United States)

    Torchy, Morgan P; Hamiche, Ali; Klaholz, Bruno P

    2015-07-01

    Transcription regulation through chromatin compaction and decompaction is regulated through various chromatin-remodeling complexes such as nucleosome remodeling and histone deacetylation (NuRD) complex. NuRD is a 1 MDa multi-subunit protein complex which comprises many different subunits, among which histone deacetylases HDAC1/2, ATP-dependent remodeling enzymes CHD3/4, histone chaperones RbAp46/48, CpG-binding proteins MBD2/3, the GATAD2a (p66α) and/or GATAD2b (p66β) and specific DNA-binding proteins MTA1/2/3. Here, we review the currently known crystal and NMR structures of these subunits, the functional data and their relevance for biomedical research considering the implication of NuRD subunits in cancer and various other diseases. The complexity of this macromolecular assembly, and its poorly understood mode of interaction with the nucleosome, the repeating unit of chromatin, illustrate that this complex is a major challenge for structure-function relationship studies which will be tackled best by an integrated biology approach.

  19. The yeast histone chaperone hif1p functions with RNA in nucleosome assembly.

    Directory of Open Access Journals (Sweden)

    Amy R Knapp

    Full Text Available Hif1p is an H3/H4-specific histone chaperone that associates with the nuclear form of the Hat1p/Hat2p complex (NuB4 complex in the yeast Saccharomyces cerevisiae. While not capable of depositing histones onto DNA on its own, Hif1p can act in conjunction with a yeast cytosolic extract to assemble nucleosomes onto a relaxed circular plasmid.To identify the factor(s that function with Hif1p to carry out chromatin assembly, multiple steps of column chromatography were carried out to fractionate the yeast cytosolic extract. Analysis of partially purified fractions indicated that Hif1p-dependent chromatin assembly activity resided in RNA rather than protein. Fractionation of isolated RNA indicated that the chromatin assembly activity did not simply purify with bulk RNA. In addition, the RNA-mediated chromatin assembly activity was blocked by mutations in the human homolog of Hif1p, sNASP, that prevent the association of this histone chaperone with histone H3 and H4 without altering its electrostatic properties.These results suggest that specific RNA species may function in concert with histone chaperones to assemble chromatin.

  20. Cis-acting determinants affecting centromere function, sister-chromatid cohesion and reciprocal recombination during meiosis in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Sears, D.D.; Hieter, P. [Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States); Shero, J.H. [Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States)]|[Baylor College of Medicine, Houston, TX (United States); Hegemann, J.H. [Justus Liebig Universitaet, Giessen (Germany)

    1995-03-01

    We have employed a system that utilizes homologous pairs of human DNA-derived yeast artificial chromosomes (YACs) as marker chromosomes to assess the specific role(s) of conserved centromere DNA elements (CDEI, CDEII, and CDEIII) in meiotic chromosome disjunction fidelity. Thirteen different centromere (CEN) mutations were tested for their effects on meiotic centromere function. YACs containing a wild-type CEN DNA sequence segregate with high fidelity in meiosis I (99% normal segregation) and in meiosis II (96% normal segregation). YACs containing a 31-bp deletion mutation in centromere DNA element II (CDEII{Delta}31) in either a heterocentric (mutant/wild type), homocentric (mutant/mutant) or monosomic (mutant/-) YAC pair configuration exhibited high levels (16-28%) of precocious sister-chromatid segregation (PSS) and increased levels (1-6%) of nondisjunction meiosis I (NDI). YACs containing this mutation also exhibit high levels (21%) of meiosis II nondisjunction. Interestingly, significant alterations in homolog recombination frequency were observed in the exceptional PSS class of tetrads, suggesting unusual interactions between prematurely separated sister chromatids and their homologous nonsister chromatids. We also have assessed the meiotic segregation effects of rare gene conversion events occurring at sites located immediately adjacent to or distantly from the centromere region. Proximal gene conversion events were associated with extremely high levels (60%) of meiosis I segregation errors (including both PSS and NDI), whereas distal events had no apparent effect. Taken together, our results indicate a critical role for CDEII in meiosis and underscore the importance of maintaining sister-chromatid cohesion for proper recombination in meiotic prophase and for proper disjunction in meiosis I. 49 refs., 4 figs., 5 tabs.

  1. Centromere pairing by a plasmid-encoded type I ParB protein

    DEFF Research Database (Denmark)

    Ringgaard, Simon; Löwe, Jan; Gerdes, Kenn

    2007-01-01

    over the nucleoid. ParB ribbon-helix-helix dimers bind cooperatively to direct repeats in parC1 and parC2. Using four different assays we obtain solid evidence that ParB can pair parC1- and parC2-encoding DNA fragments in vitro. Convincingly, electron microscopy revealed that ParB mediates binary...... pairing of parC fragments. In addition to binary complexes, ParB mediated the formation of higher order complexes consisting of several DNA fragments joined by ParB at centromere site parC. N-terminal truncated versions of ParB still possessing specific DNA binding activity were incompetent in pairing...

  2. Isolation and molecular characterization of a highly polymorphic centromeric tandem repeat in the family Falconidae.

    Science.gov (United States)

    Longmire, J L; Lewis, A K; Brown, N C; Buckingham, J M; Clark, L M; Jones, M D; Meincke, L J; Meyne, J; Ratliff, R L; Ray, F A

    1988-01-01

    An abundant tandem repeat has been cloned from genomic DNA of the merlin (Falco columbarius). The cloned sequence is 174 bp in length, and maps by in situ hybridization to the centromeric regions of several of the large chromosomes within the merlin karyotype. Complementary sequences have been identified within a variety of falcon species; these sequences are either absent or in very low copy number in the family Accipitridae. The cloned merlin repeat reveals highly polymorphic restriction patterns in the peregrine falcon (Falco peregrinus). These polymorphisms, which have been shown to be stably inherited within a family of captive peregrines, can be used to differentiate the Greenland and Argentina populations of this endangered raptor species.

  3. A hemicentric inversion in the maize line knobless Tama flint created two sites of centromeric elements and moved the kinetochore-forming region.

    Science.gov (United States)

    Lamb, Jonathan C; Meyer, Julie M; Birchler, James A

    2007-06-01

    A maize line, knobless Tama flint (KTF), was found to contain a version of chromosome 8 with two spatially distinct regions of centromeric elements, one at the original genetic position and the other at a novel location on the long arm. The new site of centromeric elements functions as the kinetochore-forming region resulting in a change of arm length ratio. Examination of fluorescence in situ hybridization markers on chromosome 8 revealed an inversion between the two centromere sites relative to standard maize lines, indicating that this chromosome 8 resulted from a hemicentric inversion with one breakpoint approximately 20 centi-McClintocks (cMc) on the long arm (20% of the total arm length from the centromere) and the other in the original cluster of centromere repeats. This inversion moved the kinetochore-forming region but left the remainder of the centromere repeats. In a hybrid between a standard line (Mo17) and KTF, both chromosome 8 homologues were completely synapsed at pachytene despite the inversion. Although the homologous centromeres were not paired, they were always correctly oriented at anaphase and migrated to opposite poles. Additionally, recombination on 8L was severely repressed in the hybrid.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  5. Drosophila TAP/p32 is a core histone chaperone that cooperates with NAP-1, NLP, and nucleophosmin in sperm chromatin remodeling during fertilization.

    Science.gov (United States)

    Emelyanov, Alexander V; Rabbani, Joshua; Mehta, Monika; Vershilova, Elena; Keogh, Michael C; Fyodorov, Dmitry V

    2014-09-15

    Nuclear DNA in the male gamete of sexually reproducing animals is organized as sperm chromatin compacted primarily by sperm-specific protamines. Fertilization leads to sperm chromatin remodeling, during which protamines are expelled and replaced by histones. Despite our increased understanding of the factors that mediate nucleosome assembly in the nascent male pronucleus, the machinery for protamine removal remains largely unknown. Here we identify four Drosophila protamine chaperones that mediate the dissociation of protamine-DNA complexes: NAP-1, NLP, and nucleophosmin are previously characterized histone chaperones, and TAP/p32 has no known function in chromatin metabolism. We show that TAP/p32 is required for the removal of Drosophila protamine B in vitro, whereas NAP-1, NLP, and Nph share roles in the removal of protamine A. Embryos from P32-null females show defective formation of the male pronucleus in vivo. TAP/p32, similar to NAP-1, NLP, and Nph, facilitates nucleosome assembly in vitro and is therefore a histone chaperone. Furthermore, mutants of P32, Nlp, and Nph exhibit synthetic-lethal genetic interactions. In summary, we identified factors mediating protamine removal from DNA and reconstituted in a defined system the process of sperm chromatin remodeling that exchanges protamines for histones to form the nucleosome-based chromatin characteristic of somatic cells.

  6. Global chromatin fibre compaction in response to DNA damage

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-11-04

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

  7. Human sperm chromatin stabilization: a proposed model including zinc bridges.

    Science.gov (United States)

    Björndahl, Lars; Kvist, Ulrik

    2010-01-01

    The primary focus of this review is to challenge the current concepts on sperm chromatin stability. The observations (i) that zinc depletion at ejaculation allows a rapid and total sperm chromatin decondensation without the addition of exogenous disulfide cleaving agents and (ii) that the human sperm chromatin contains one zinc for every protamine for every turn of the DNA helix suggest an alternative model for sperm chromatin structure may be plausible. An alternative model is therefore proposed, that the human spermatozoon could at ejaculation have a rapidly reversible zinc dependent chromatin stability: Zn(2+) stabilizes the structure and prevents the formation of excess disulfide bridges by a single mechanism, the formation of zinc bridges with protamine thiols of cysteine and potentially imidazole groups of histidine. Extraction of zinc enables two biologically totally different outcomes: immediate decondensation if chromatin fibers are concomitantly induced to repel (e.g. by phosphorylation in the ooplasm); otherwise freed thiols become committed into disulfide bridges creating a superstabilized chromatin. Spermatozoa in the zinc rich prostatic fluid (normally the first expelled ejaculate fraction) represent the physiological situation. Extraction of chromatin zinc can be accomplished by the seminal vesicular fluid. Collection of the ejaculate in one single container causes abnormal contact between spermatozoa and seminal vesicular fluid affecting the sperm chromatin stability. There are men in infertile couples with low content of sperm chromatin zinc due to loss of zinc during ejaculation and liquefaction. Tests for sperm DNA integrity may give false negative results due to decreased access for the assay to the DNA in superstabilized chromatin.

  8. A Mutation in Plant-Specific SWI2/SNF2-Like Chromatin-Remodeling Proteins, DRD1 and DDM1, Delays Leaf Senescence in Arabidopsis thaliana.

    Science.gov (United States)

    Cho, Eun Ju; Choi, Seung Hee; Kim, Ji Hong; Kim, Ji Eun; Lee, Min Hee; Chung, Byung Yeoup; Woo, Hye Ryun; Kim, Jin-Hong

    2016-01-01

    Leaf senescence is a finely regulated complex process; however, evidence for the involvement of epigenetic processes in the regulation of leaf senescence is still fragmentary. Therefore, we chose to examine the functions of DRD1, a SWI2/SNF2 chromatin remodeling protein, in epigenetic regulation of leaf senescence, particularly because drd1-6 mutants exhibited a delayed leaf senescence phenotype. Photosynthetic parameters such as Fv/Fm and ETRmax were decreased in WT leaves compared to leaves of drd1-6 mutants after dark treatment. The WT leaves remarkably lost more chlorophyll and protein content during dark-induced senescence (DIS) than the drd1-6 leaves did. The induction of senescence-associated genes was noticeably inhibited in the drd1-6 mutant after 5-d of DIS. We compared changes in epigenetic regulation during DIS via quantitative expression analysis of 180-bp centromeric (CEN) and transcriptionally silent information (TSI) repeats. Their expression levels significantly increased in both the WT and the drd1-6 mutant, but did much less in the latter. Moreover, the delayed leaf senescence was observed in ddm1-2 mutants as well as the drd1-6, but not in drd1-p mutants. These data suggest that SWI2/SNF2 chromatin remodeling proteins such as DRD1 and DDM1 may influence leaf senescence possibly via epigenetic regulation.

  9. Chromatin architecture and gene expression in Escherichia coli

    DEFF Research Database (Denmark)

    Willenbrock, Hanni; Ussery, David

    2004-01-01

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

  10. Transcription upregulation via force-induced direct stretching of chromatin

    Science.gov (United States)

    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.

  11. The AID-induced DNA damage response in chromatin

    DEFF Research Database (Denmark)

    Daniel, Jeremy A; Nussenzweig, André

    2013-01-01

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

  12. Effect of DNA groove binder distamycin A upon chromatin structure.

    Directory of Open Access Journals (Sweden)

    Parijat Majumder

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

  13. Defining the multivalent functions of CTCF from chromatin state and three-dimensional chromatin interactions.

    Science.gov (United States)

    Lu, Yiming; Shan, Guangyu; Xue, Jiguo; Chen, Changsheng; Zhang, Chenggang

    2016-07-27

    CCCTC-binding factor (CTCF) is a multi-functional protein that is assigned various, even contradictory roles in the genome. High-throughput sequencing-based technologies such as ChIP-seq and Hi-C provided us the opportunity to assess the multivalent functions of CTCF in the human genome. The location of CTCF-binding sites with respect to genomic features provides insights into the possible roles of this protein. Here we present the first genome-wide survey and characterization of three important functions of CTCF: enhancer insulator, chromatin barrier and enhancer linker. We developed a novel computational framework to discover the multivalent functions of CTCF based on chromatin state and three-dimensional chromatin architecture. We applied our method to five human cell lines and identified ∼46 000 non-redundant CTCF sites related to the three functions. Disparate effects of these functions on gene expression were found and distinct genomic features of these CTCF sites were characterized in GM12878 cells. Finally, we investigated the cell-type specificities of CTCF sites related to these functions across five cell types. Our study provides new insights into the multivalent functions of CTCF in the human genome.

  14. PML bodies provide an important platform for the maintenance of telomeric chromatin integrity in embryonic stem cells.

    Science.gov (United States)

    Chang, Fiona T M; McGhie, James D; Chan, F Lyn; Tang, Michelle C; Anderson, Melissa A; Mann, Jeffrey R; Andy Choo, K H; Wong, Lee H

    2013-04-01

    We have previously shown that α-thalassemia mental retardation X-linked (ATRX) and histone H3.3 are key regulators of telomeric chromatin in mouse embryonic stem cells. The function of ATRX and H3.3 in the maintenance of telomere chromatin integrity is further demonstrated by recent studies that show the strong association of ATRX/H3.3 mutations with alternative lengthening of telomeres in telomerase-negative human cancer cells. Here, we demonstrate that ATRX and H3.3 co-localize with the telomeric DNA and associated proteins within the promyelocytic leukemia (PML) bodies in mouse ES cells. The assembly of these telomere-associated PML bodies is most prominent at S phase. RNA interference (RNAi)-mediated knockdown of PML expression induces the disassembly of these nuclear bodies and a telomere dysfunction phenotype in mouse ES cells. Loss of function of PML bodies in mouse ES cells also disrupts binding of ATRX/H3.3 and proper establishment of histone methylation pattern at the telomere. Our study demonstrates that PML bodies act as epigenetic regulators by serving as platforms for the assembly of the telomeric chromatin to ensure a faithful inheritance of epigenetic information at the telomere.

  15. Human artificial chromosome assembly by transposon-based retrofitting of genomic BACs with synthetic alpha-satellite arrays.

    Science.gov (United States)

    Basu, Joydeep; Willard, Huntington F; Stromberg, Gregory

    2007-01-01

    The development of methodologies for the rapid assembly of synthetic alpha-satellite arrays recapitulating the higher-order periodic organization of native human centromeres permits the systematic investigation of the significance of primary sequence and sequence organization in centromere function. Synthetic arrays with defined mutations affecting sequence and/or organization may be evaluated in a de novo human artificial chromosome assay. This unit describes strategies for the assembly of custom built alpha-satellite arrays containing any desired mutation as well as strategies for the construction and manipulation of alpha satellite-based transposons. Transposons permit the rapid and reliable retrofitting of any genomic bacterial artificial chromosome (BAC) with synthetic alpha-satellite arrays and other functional components, thereby facilitating conversion into BAC-based human artificial chromosome vectors. These techniques permit identification and optimization of the critical parameters underlying the unique ability of alpha-satellite DNA to facilitate de novo centromere assembly, and they will establish the foundation for the next generation of human artificial chromosome vectors.

  16. Fractal Characterization of Chromatin Decompaction in Live Cells.

    Science.gov (United States)

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

    2015-12-01

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

  17. Determinants of Sir2-Mediated, Silent Chromatin Cohesion.

    Science.gov (United States)

    Chen, Yu-Fan; Chou, Chia-Ching; Gartenberg, Marc R

    2016-08-01

    Cohesin associates with distinct sites on chromosomes to mediate sister chromatid cohesion. Single cohesin complexes are thought to bind by encircling both sister chromatids in a topological embrace. Transcriptionally repressed chromosomal domains in the yeast Saccharomyces cerevisiae represent specialized sites of cohesion where cohesin binds silent chromatin in a Sir2-dependent fashion. In this study, we investigated the molecular basis for Sir2-mediated cohesion. We identified a cluster of charged surface residues of Sir2, collectively termed the EKDK motif, that are required for cohesin function. In addition, we demonstrated that Esc8, a Sir2-interacting factor, is also required for silent chromatin cohesion. Esc8 was previously shown to associate with Isw1, the enzymatic core of ISW1 chromatin remodelers, to form a variant of the ISW1a chromatin remodeling complex. When ESC8 was deleted or the EKDK motif was mutated, cohesin binding at silenced chromatin domains persisted but cohesion of the domains was abolished. The data are not consistent with cohesin embracing both sister chromatids within silent chromatin domains. Transcriptional silencing remains largely intact in strains lacking ESC8 or bearing EKDK mutations, indicating that silencing and cohesion are separable functions of Sir2 and silent chromatin.

  18. PREDICTION OF CHROMATIN STATES USING DNA SEQUENCE PROPERTIES

    KAUST Repository

    Bahabri, Rihab R.

    2013-06-01

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

  19. Persistent Chromatin Modifications Induced by High Fat Diet.

    Science.gov (United States)

    Leung, Amy; Trac, Candi; Du, Juan; Natarajan, Rama; Schones, Dustin E

    2016-05-13

    Obesity is a highly heritable complex disease that results from the interaction of multiple genetic and environmental factors. Formerly obese individuals are susceptible to metabolic disorders later in life, even after lifestyle changes are made to mitigate the obese state. This is reminiscent of the metabolic memory phenomenon originally observed for persistent complications in diabetic patients, despite subsequent glycemic control. Epigenetic modifications represent a potential mediator of this observed memory. We previously demonstrated that a high fat diet leads to changes in chromatin accessibility in the mouse liver. The regions of greatest chromatin changes in accessibility are largely strain-dependent, indicating a genetic component in diet-induced chromatin alterations. We have now examined the persistence of diet-induced chromatin accessibility changes upon diet reversal in two strains of mice. We find that a substantial fraction of loci that undergo chromatin accessibility changes with a high fat diet remains in the remodeled state after diet reversal in C57BL/6J mice. In contrast, the vast majority of diet-induced chromatin accessibility changes in A/J mice are transient. Our data also indicate that the persistent chromatin accessibility changes observed in C57BL/6J mice are associated with specific transcription factors and histone post-translational modifications. The persistent loci identified here are likely to be contributing to the overall phenotype and are attractive targets for therapeutic intervention.

  20. Anti-chromatin antibodies in juvenile rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    V. Gerloni

    2011-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-01-14

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  3. Maximum-likelihood method identifies meiotic restitution mechanism from heterozygosity transmission of centromeric loci: application in citrus

    Science.gov (United States)

    Cuenca, José; Aleza, Pablo; Juárez, José; García-Lor, Andrés; Froelicher, Yann; Navarro, Luis; Ollitrault, Patrick

    2015-01-01

    Polyploidisation is a key source of diversification and speciation in plants. Most researchers consider sexual polyploidisation leading to unreduced gamete as its main origin. Unreduced gametes are useful in several crop breeding schemes. Their formation mechanism, i.e., First-Division Restitution (FDR) or Second-Division Restitution (SDR), greatly impacts the gametic and population structures and, therefore, the breeding efficiency. Previous methods to identify the underlying mechanism required the analysis of a large set of markers over large progeny. This work develops a new maximum-likelihood method to identify the unreduced gamete formation mechanism both at the population and individual levels using independent centromeric markers. Knowledge of marker-centromere distances greatly improves the statistical power of the comparison between the SDR and FDR hypotheses. Simulating data demonstrated the importance of selecting markers very close to the centromere to obtain significant conclusions at individual level. This new method was used to identify the meiotic restitution mechanism in nineteen mandarin genotypes used as female parents in triploid citrus breeding. SDR was identified for 85.3% of 543 triploid hybrids and FDR for 0.6%. No significant conclusions were obtained for 14.1% of the hybrids. At population level SDR was the predominant mechanisms for the 19 parental mandarins. PMID:25894579

  4. Maximum-likelihood method identifies meiotic restitution mechanism from heterozygosity transmission of centromeric loci: application in citrus.

    Science.gov (United States)

    Cuenca, José; Aleza, Pablo; Juárez, José; García-Lor, Andrés; Froelicher, Yann; Navarro, Luis; Ollitrault, Patrick

    2015-04-20

    Polyploidisation is a key source of diversification and speciation in plants. Most researchers consider sexual polyploidisation leading to unreduced gamete as its main origin. Unreduced gametes are useful in several crop breeding schemes. Their formation mechanism, i.e., First-Division Restitution (FDR) or Second-Division Restitution (SDR), greatly impacts the gametic and population structures and, therefore, the breeding efficiency. Previous methods to identify the underlying mechanism required the analysis of a large set of markers over large progeny. This work develops a new maximum-likelihood method to identify the unreduced gamete formation mechanism both at the population and individual levels using independent centromeric markers. Knowledge of marker-centromere distances greatly improves the statistical power of the comparison between the SDR and FDR hypotheses. Simulating data demonstrated the importance of selecting markers very close to the centromere to obtain significant conclusions at individual level. This new method was used to identify the meiotic restitution mechanism in nineteen mandarin genotypes used as female parents in triploid citrus breeding. SDR was identified for 85.3% of 543 triploid hybrids and FDR for 0.6%. No significant conclusions were obtained for 14.1% of the hybrids. At population level SDR was the predominant mechanisms for the 19 parental mandarins.

  5. Pregnancy-associated thrombotic thrombocytopenic purpura with anti-centromere antibody-positive Raynaud's syndrome.

    Science.gov (United States)

    Watanabe, Ryu; Shirai, Tsuyoshi; Tajima, Yumi; Ohguchi, Hiroto; Onishi, Yasushi; Fujii, Hiroshi; Takasawa, Naruhiko; Ishii, Tomonori; Harigae, Hideo

    2010-01-01

    Thrombotic thrombocytopenic purpura (TTP), scleroderma renal crisis (SRC), and hemolysis, elevated liver enzyme levels, and a low platelet count (HELLP) syndrome display common symptoms that include microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. Therefore, it is important to distinguish between them because their treatments vary: however, the differential diagnosis is sometimes difficult. We report a 32-year-old woman who was referred to our department for further examination of microangiopathic hemolytic anemia, thrombocytopenia, and a slightly elevated serum creatinine level with anti-centromere antibody-positive Raynaud's syndrome in the early puerperal period. TTP, SRC, and HELLP syndrome were considered in the differential diagnosis, but the measurement of a disintegrin-like metalloprotease with thrombospondin type 1 motifs 13 (ADAMTS 13) activity and its inhibitor level led to the diagnosis of TTP. She was successfully treated by plasma exchange and high-dose prednisolone and angiotensin-converting enzyme inhibitor. If microangiopathic hemolytic anemia and thrombocytopenia are observed in perinatal women or patients with signs of systemic sclerosis, the measurement of ADAMTS13 activity and its inhibitor level are essential for diagnosis and therapeutic choice.

  6. Expression and prognostic relevance of centromere protein A in primary osteosarcoma.

    Science.gov (United States)

    Gu, Xiao-Min; Fu, Jie; Feng, Xiao-Jun; Huang, Xue; Wang, Shou-Mei; Chen, Xin-Feng; Zhu, Ming-Hua; Zhang, Shu-Hui

    2014-04-01

    Centromere protein A (CENP-A) is one of the fundamental components of the human active kinetochore and plays important roles in cell-cycle regulation, cell survival, and genetic stability. The aim of the present study was to explore the expression and prognostic significance of CENP-A in osteosarcoma. The results of real-time quantitative PCR and Western blotting analysis revealed an enhanced expression of CENP-A in osteosarcomas relative to adjacent non-tumorous bone tissues at both mRNA and protein levels. Immunohistochemically, 72 of the 123 osteosarcoma specimens (58.5%) had high expression of CENP-A. CENP-A overexpression was significantly correlated with tumor size (P=0.002), poor response to neoadjuvant chemotherapy (P=0.016), local recurrence/lung metastasis (P=0.001), high Ki-67 index (P=0.004), and P53 positivity (P=0.005). Median overall and recurrence-free survival time was significantly shorter in patients with high-CENP-A osteosarcomas than in those with low-CENP-A osteosarcomas. Multivariate analysis identified CENP-A as an independent poor prognostic factor for osteosarcoma. In conclusion, our results demonstrate that elevated CENP-A expression is significantly associated with osteosarcoma progression and has an independent prognostic value in predicting overall and recurrence-free survival for patients with osteosarcoma.

  7. Overexpression of centromere protein H is significantly associated with breast cancer progression and overall patient survival

    Institute of Scientific and Technical Information of China (English)

    Wen-Ting Liao; Yan Feng; Men-Lin Li; Guang-Lin Liu; Man-Zhi Li; Mu-Sheng Zeng; Li-Bing Song

    2011-01-01

    Breast cancer is one of the leading causes of cancer death worldwide.This study aimed to analyze the expression of centromere protein H (CENP-H) in breast cancer and to correlate it with clinicopathologic data,including patient survival.Using reverse transcription-polymerase chain reaction and Westem blotting to detect the expression of CENP-H in normal mammary epithelial cells,immortalized mammary epithelial cell lines,and breast cancer cell lines,we observed that the mRNA and protein levels of CENP-H were higher in breast cancer cell lines and in immortalized mammary epithelial cells than in normal mammary epithelial cells.We next examined CENP-H expression in 307 paraffin-embedded archived samples of clinicopathologically characterized breast cancer using immunohistochemistry,and detected high CENP-H expression in 134 (43.6%) samples.Statistical analysis showed that CENP-H expression was related with clinical stage (P = 0.001),T classification (P = 0.032),N classification (P =0.018),and Ki-67 (P<0.001).Patients with high CENP-H expression had short overall survival.Multivariate analysis showed that CENP-H expression was an independent prognostic indicator for patient survival.Our results suggest that CENP-H protein is a valuable marker of breast cancer progression and prognosis.

  8. New tool for biological dosimetry: Reevaluation and automation of the gold standard method following telomere and centromere staining

    Energy Technology Data Exchange (ETDEWEB)

    M’kacher, Radhia [Laboratoire de Radiobiologie et Oncologie (LRO), Commissariat à l’Energie Atomique (CEA), Route du Panorama, 92265 Fontenay-aux-Roses (France); Maalouf, Elie E.L. [Laboratoire de Radiobiologie et Oncologie (LRO), Commissariat à l’Energie Atomique (CEA), Route du Panorama, 92265 Fontenay-aux-Roses (France); Laboratoire MIPS – Groupe TIIM3D, Université de Haute-Alsace, F-68093 Mulhouse (France); Ricoul, Michelle [Laboratoire de Radiobiologie et Oncologie (LRO), Commissariat à l’Energie Atomique (CEA), Route du Panorama, 92265 Fontenay-aux-Roses (France); Heidingsfelder, Leonhard [MetaSystems GmbH, Robert-Bosch-Str. 6, 68804 Altlussheim (Germany); Laplagne, Eric [Pole Concept, 61 Rue Erlanger, 75016 Paris (France); Cuceu, Corina; Hempel, William M. [Laboratoire de Radiobiologie et Oncologie (LRO), Commissariat à l’Energie Atomique (CEA), Route du Panorama, 92265 Fontenay-aux-Roses (France); Colicchio, Bruno; Dieterlen, Alain [Laboratoire MIPS – Groupe TIIM3D, Université de Haute-Alsace, F-68093 Mulhouse (France); Sabatier, Laure, E-mail: laure.sabatier@cea.fr [Laboratoire de Radiobiologie et Oncologie (LRO), Commissariat à l’Energie Atomique (CEA), Route du Panorama, 92265 Fontenay-aux-Roses (France)

    2014-12-15

    Graphical abstract: - Highlights: • We have applied telomere and centromere (TC) staining to the scoring of dicentrics. • TC staining renders the scoring of dicentrics more rapid and robust. • TC staining allows the scoring of not only dicentrics but all chromosomal anomalies. • TC staining has led to a reevaluation of the radiation dose–response curve. • TC staining allows automation of the scoring of chromosomal aberations. • Automated scoring of dicentrics after TC staining was as efficient as manual scoring. - Abstract: Purpose: The dicentric chromosome (dicentric) assay is the international gold-standard method for biological dosimetry and classification of genotoxic agents. The introduction of telomere and centromere (TC) staining offers the potential to render dicentric scoring more efficient and robust. In this study, we improved the detection of dicentrics and all unstable chromosomal aberrations (CA) leading to a significant reevaluation of the dose–effect curve and developed an automated approach following TC staining. Material and methods: Blood samples from 16 healthy donors were exposed to {sup 137}Cs at 8 doses from 0.1 to 6 Gy. CA were manually and automatically scored following uniform (Giemsa) or TC staining. The detection of centromeric regions and telomeric sequences using PNA probes allowed the detection of all unstable CA: dicentrics, centric and acentric rings, and all acentric fragments (with 2, 4 or no telomeres) leading to the precise quantification of estimated double strand breaks (DSB). Results: Manual scoring following TC staining revealed a significantly higher frequency of dicentrics (p < 10{sup −3}) (up to 30%) and estimated DSB (p < 10{sup −4}) compared to uniform staining due to improved detection of dicentrics with centromeres juxtaposed with other centromeres or telomeres. This improvement permitted the development of the software, TCScore, that detected 95% of manually scored dicentrics compared to 50% for

  9. SAP-like domain in nucleolar spindle associated protein mediates mitotic chromosome loading as well as interphase chromatin interaction

    Energy Technology Data Exchange (ETDEWEB)

    Verbakel, Werner, E-mail: werner.verbakel@chem.kuleuven.be [Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Celestijnenlaan 200G, Bus 2403, 3001 Heverlee (Belgium); Carmeliet, Geert, E-mail: geert.carmeliet@med.kuleuven.be [Laboratory of Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, Herestraat 49, Bus 902, 3000 Leuven (Belgium); Engelborghs, Yves, E-mail: yves.engelborghs@fys.kuleuven.be [Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Celestijnenlaan 200G, Bus 2403, 3001 Heverlee (Belgium)

    2011-08-12

    Highlights: {yields} The SAP-like domain in NuSAP is a functional DNA-binding domain with preference for dsDNA. {yields} This SAP-like domain is essential for chromosome loading during early mitosis. {yields} NuSAP is highly dynamic on mitotic chromatin, as evident from photobleaching experiments. {yields} The SAP-like domain also mediates NuSAP-chromatin interaction in interphase nucleoplasm. -- Abstract: Nucleolar spindle associated protein (NuSAP) is a microtubule-stabilizing protein that localizes to chromosome arms and chromosome-proximal microtubules during mitosis and to the nucleus, with enrichment in the nucleoli, during interphase. The critical function of NuSAP is underscored by the finding that its depletion in HeLa cells results in various mitotic defects. Moreover, NuSAP is found overexpressed in multiple cancers and its expression levels often correlate with the aggressiveness of cancer. Due to its localization on chromosome arms and combination of microtubule-stabilizing and DNA-binding properties, NuSAP takes a special place within the extensive group of spindle assembly factors. In this study, we identify a SAP-like domain that shows DNA binding in vitro with a preference for dsDNA. Deletion of the SAP-like domain abolishes chromosome arm binding of NuSAP during mitosis, but is not sufficient to abrogate its chromosome-proximal localization after anaphase onset. Fluorescence recovery after photobleaching experiments revealed the highly dynamic nature of this NuSAP-chromatin interaction during mitosis. In interphase cells, NuSAP also interacts with chromatin through its SAP-like domain, as evident from its enrichment on dense chromatin regions and intranuclear mobility, measured by fluorescence correlation spectroscopy. The obtained results are in agreement with a model where NuSAP dynamically stabilizes newly formed microtubules on mitotic chromosomes to enhance chromosome positioning without immobilizing these microtubules. Interphase NuSAP-chromatin

  10. Probing Chromatin-modifying Enzymes with Chemical Tools

    KAUST Repository

    Fischle, Wolfgang

    2016-02-04

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

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle. ...

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

    Institute of Scientific and Technical Information of China (English)

    Qais Al-Hadid; Yanzhong Yang

    2016-01-01

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

  13. Control of chromatin structure by long noncoding RNA

    Science.gov (United States)

    Böhmdorfer, Gudrun; Wierzbicki, Andrzej T.

    2015-01-01

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

  14. Neutron scattering studies on chromatin higher-order structure

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-12-31

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

  15. Chromatin remodeling and cancer, Part I: Covalent histone modifications.

    Science.gov (United States)

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

    2007-09-01

    Dynamic chromatin remodeling underlies many, if not all, DNA-templated biological processes, including gene transcription; DNA replication and repair; chromosome condensation; and segregation and apoptosis. Disruption of these processes has been linked to the development and progression of cancer. The mechanisms of dynamic chromatin remodeling include the use of covalent histone modifications, histone variants, ATP-dependent complexes and DNA methylation. Together, these mechanisms impart variation into the chromatin fiber, and this variation gives rise to an 'epigenetic landscape' that extends the biological output of DNA alone. Here, we review recent advances in chromatin remodeling, and pay particular attention to mechanisms that appear to be linked to human cancer. Where possible, we discuss the implications of these advances for disease-management strategies.

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

    Science.gov (United States)

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

    2013-01-01

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

  17. Ectopically tethered CP190 induces large-scale chromatin decondensation

    Science.gov (United States)

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

    2014-01-01

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

  18. Diversity of eukaryotic DNA replication origins revealed by genome-wide analysis of chromatin structure.

    Directory of Open Access Journals (Sweden)

    Nicolas M Berbenetz

    2010-09-01

    Full Text Available Eukaryotic DNA replication origins differ both in their efficiency and in the characteristic time during S phase when they become active. The biological basis for these differences remains unknown, but they could be a consequence of chromatin structure. The availability of genome-wide maps of nucleosome positions has led to an explosion of information about how nucleosomes are assembled at transcription start sites, but no similar maps exist for DNA replication origins. Here we combine high-resolution genome-wide nucleosome maps with comprehensive annotations of DNA replication origins to identify patterns of nucleosome occupancy at eukaryotic replication origins. On average, replication origins contain a nucleosome depleted region centered next to the ACS element, flanked on both sides by arrays of well-positioned nucleosomes. Our analysis identified DNA sequence properties that correlate with nucleosome occupancy at replication origins genome-wide and that are correlated with the nucleosome-depleted region. Clustering analysis of all annotated replication origins revealed a surprising diversity of nucleosome occupancy patterns. We provide evidence that the origin recognition complex, which binds to the origin, acts as a barrier element to position and phase nucleosomes on both sides of the origin. Finally, analysis of chromatin reconstituted in vitro reveals that origins are inherently nucleosome depleted. Together our data provide a comprehensive, genome-wide view of chromatin structure at replication origins and suggest a model of nucleosome positioning at replication origins in which the underlying sequence occludes nucleosomes to permit binding of the origin recognition complex, which then (likely in concert with nucleosome modifiers and remodelers positions nucleosomes adjacent to the origin to promote replication origin function.

  19. Different patterns of evolution in the centromeric and telomeric regions of group A and B haplotypes of the human killer cell Ig-like receptor locus.

    Directory of Open Access Journals (Sweden)

    Chul-Woo Pyo

    Full Text Available The fast evolving human KIR gene family encodes variable lymphocyte receptors specific for polymorphic HLA class I determinants. Nucleotide sequences for 24 representative human KIR haplotypes were determined. With three previously defined haplotypes, this gave a set of 12 group A and 15 group B haplotypes for assessment of KIR variation. The seven gene-content haplotypes are all combinations of four centromeric and two telomeric motifs. 2DL5, 2DS5 and 2DS3 can be present in centromeric and telomeric locations. With one exception, haplotypes having identical gene content differed in their combinations of KIR alleles. Sequence diversity varied between haplotype groups and between centromeric and telomeric halves of the KIR locus. The most variable A haplotype genes are in the telomeric half, whereas the most variable genes characterizing B haplotypes are in the centromeric half. Of the highly polymorphic genes, only the 3DL3 framework gene exhibits a similar diversity when carried by A and B haplotypes. Phylogenetic analysis and divergence time estimates, point to the centromeric gene-content motifs that distinguish A and B haplotypes having emerged ~6 million years ago, contemporaneously with the separation of human and chimpanzee ancestors. In contrast, the telomeric motifs that distinguish A and B haplotypes emerged more recently, ~1.7 million years ago, before the emergence of Homo sapiens. Thus the centromeric and telomeric motifs that typify A and B haplotypes have likely been present throughout human evolution. The results suggest the common ancestor of A and B haplotypes combined a B-like centromeric region with an A-like telomeric region.

  20. Chromatin domains and prediction of MAR sequences.

    Science.gov (United States)

    Boulikas, T

    1995-01-01

    Polynuceosomes are constrained into loops or domains and are insulated from the effects of chromatin structure and torsional strain from flanking domains by the cross-complexation of matrix-attached regions (MARs) and matrix proteins. MARs or SARs have an average size of 500 bp, are spaced about every 30 kb, and are control elements maintaining independent realms of gene activity. A fraction of MARs may cohabit with core origin replication (ORIs) and another fraction might cohabit with transcriptional enhancers. DNA replication, transcription, repair, splicing, and recombination seem to take place on the nuclear matrix. Classical AT-rich MARs have been proposed to anchor the core enhancers and core origins complexed with low abundancy transcription factors to the nuclear matrix via the cooperative binding to MARs of abundant classical matrix proteins (topoisomerase II, histone H1, lamins, SP120, ARBP, SATB1); this creates a unique nuclear microenvironment rich in regulatory proteins able to sustain transcription, replication, repair, and recombination. Theoretical searches and experimental data strongly support a model of activation of MARs and ORIs by transcription factors. A set of 21 characteristics are deduced or proposed for MAR/ORI sequences including their enrichment in inverted repeats, AT tracts, DNA unwinding elements, replication initiator protein sites, homooligonucleotide repeats (i.e., AAA, TTT, CCC), curved DNA, DNase I-hypersensitive sites, nucleosome-free stretches, polypurine stretches, and motifs with a potential for left-handed and triplex structures. We are establishing Banks of ORI and MAR sequences and have undertaken a large project of sequencing a large number of MARs in an effort to determine classes of DNA sequences in these regulatory elements and to understand their role at the origins of replication and transcriptional enhancers.

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

    Science.gov (United States)

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

    2015-01-01

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

  2. Sabot assembly

    Energy Technology Data Exchange (ETDEWEB)

    Bzorgi, Fariborz

    2016-11-08

    A sabot assembly includes a projectile and a housing dimensioned and configured for receiving the projectile. An air pressure cavity having a cavity diameter is disposed between a front end and a rear end of the housing. Air intake nozzles are in fluid communication with the air pressure cavity and each has a nozzle diameter less than the cavity diameter. In operation, air flows through the plurality of air intake nozzles and into the air pressure cavity upon firing of the projectile from a gun barrel to pressurize the air pressure cavity for assisting in separation of the housing from the projectile upon the sabot assembly exiting the gun barrel.

  3. Wolbachia-mediated male killing is associated with defective chromatin remodeling.

    Directory of Open Access Journals (Sweden)

    Maria Giovanna Riparbelli

    Full Text Available Male killing, induced by different bacterial taxa of maternally inherited microorganisms, resulting in highly distorted female-biased sex-ratios, is a common phenomenon among arthropods. Some strains of the endosymbiont bacteria Wolbachia have been shown to induce this phenotype in particular insect hosts. High altitude populations of Drosophila bifasciata infected with Wolbachia show selective male killing during embryonic development. However, since this was first reported, circa 60 years ago, the interaction between Wolbachia and its host has remained unclear. Herein we show that D. bifasciata male embryos display defective chromatin remodeling, improper chromatid segregation and chromosome bridging, as well as abnormal mitotic spindles and gradual loss of their centrosomes. These defects occur at different times in the early development of male embryos leading to death during early nuclear division cycles or large defective areas of the cellular blastoderm, culminating in abnormal embryos that die before eclosion. We propose that Wolbachia affects the development of male embryos by specifically targeting male chromatin remodeling and thus disturbing mitotic spindle assembly and chromosome behavior. These are the first observations that demonstrate fundamental aspects of the cytological mechanism of male killing and represent a solid base for further molecular studies of this phenomenon.

  4. Ephemeral Protein Binding to DNA Shapes Stable Nuclear Bodies and Chromatin Domains.

    Science.gov (United States)

    Brackley, Chris A; Liebchen, Benno; Michieletto, Davide; Mouvet, Francois; Cook, Peter R; Marenduzzo, Davide

    2017-03-28

    Fluorescence microscopy reveals that the contents of many (membrane-free) nuclear bodies exchange rapidly with the soluble pool while the underlying structure persists; such observations await a satisfactory biophysical explanation. To shed light on this, we perform large-scale Brownian dynamics simulations of a chromatin fiber interacting with an ensemble of (multivalent) DNA-binding proteins able to switch between an "on" (binding) and an "off" (nonbinding) state. This system provides a model for any DNA-binding protein that can be posttranslationally modified to change its affinity for DNA (e.g., through phosphorylation). Protein switching is a nonequilibrium process, and it leads to the formation of clusters of self-limiting size, where individual proteins in a cluster exchange with the soluble pool with kinetics similar to those seen in photobleaching experiments. This behavior contrasts sharply with that exhibited by nonswitching proteins, which are permanently in the on-state; when these bind to DNA nonspecifically, they form clusters that grow indefinitely in size. To explain these findings, we propose a mean-field theory from which we obtain a scaling relation between the typical cluster size and the protein switching rate. Protein switching also reshapes intrachromatin contacts to give networks resembling those seen in topologically associating domains, as switching markedly favors local (short-range) contacts over distant ones. Our results point to posttranslational modification of chromatin-bridging proteins as a generic mechanism driving the self-assembly of highly dynamic, nonequilibrium, protein clusters with the properties of nuclear bodies.

  5. Genome-wide profiling of nucleosome sensitivity and chromatin accessibility in Drosophila melanogaster.

    Science.gov (United States)

    Chereji, Răzvan V; Kan, Tsung-Wai; Grudniewska, Magda K; Romashchenko, Alexander V; Berezikov, Eugene; Zhimulev, Igor F; Guryev, Victor; Morozov, Alexandre V; Moshkin, Yuri M

    2016-02-18

    Nucleosomal DNA is thought to be generally inaccessible to DNA-binding factors, such as micrococcal nuclease (MNase). Here, we digest Drosophila chromatin with high and low concentrations of MNase to reveal two distinct nucleosome types: MNase-sensitive and MNase-resistant. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C-containing dinucleotides, whereas MNase-sensitive nucleosomes form on A/T-rich sequences found at transcription start and termination sites, enhancers and DNase I hypersensitive sites. Estimates of nucleosome formation energies indicate that MNase-sensitive nucleosomes tend to be less stable than MNase-resistant ones. Strikingly, a decrease in cell growth temperature of about 10°C makes MNase-sensitive nucleosomes less accessible, suggesting that observed variations in MNase sensitivity are related to either thermal fluctuations of chromatin fibers or the activity of enzymatic machinery. In the vicinity of active genes and DNase I hypersensitive sites nucleosomes are organized into periodic arrays, likely due to 'phasing' off potential barriers formed by DNA-bound factors or by nucleosomes anchored to their positions through external interactions. The latter idea is substantiated by our biophysical model of nucleosome positioning and energetics, which predicts that nucleosomes immediately downstream of transcription start sites are anchored and recapitulates nucleosome phasing at active genes significantly better than sequence-dependent models.

  6. Extremely Long-Range Chromatin Loops Link Topological Domains to Facilitate a Diverse Antibody Repertoire

    Directory of Open Access Journals (Sweden)

    Lindsey Montefiori

    2016-02-01

    Full Text Available Early B cell development is characterized by large-scale Igh locus contraction prior to V(DJ recombination to facilitate a highly diverse Ig repertoire. However, an understanding of the molecular architecture that mediates locus contraction remains unclear. We have combined high-resolution chromosome conformation capture (3C techniques with 3D DNA FISH to identify three conserved topological subdomains. Each of these topological folds encompasses a major VH gene family that become juxtaposed in pro-B cells via megabase-scale chromatin looping. The transcription factor Pax5 organizes the subdomain that spans the VHJ558 gene family. In its absence, the J558 VH genes fail to associate with the proximal VH genes, thereby providing a plausible explanation for reduced VHJ558 gene rearrangements in Pax5-deficient pro-B cells. We propose that Igh locus contraction is the cumulative effect of several independently controlled chromatin subdomains that provide the structural infrastructure to coordinate optimal antigen receptor assembly.

  7. Chromosome 17 centromere duplication and responsiveness to anthracycline-based neoadjuvant chemotherapy in breast cancer.

    Science.gov (United States)

    Tibau, Ariadna; López-Vilaró, Laura; Pérez-Olabarria, Maitane; Vázquez, Tania; Pons, Cristina; Gich, Ignasi; Alonso, Carmen; Ojeda, Belén; Ramón y Cajal, Teresa; Lerma, Enrique; Barnadas, Agustí; Escuin, Daniel

    2014-10-01

    Human epidermal growth factor receptor 2 (HER2) and topoisomerase II alpha (TOP2A) genes have been proposed as predictive biomarkers of sensitivity to anthracycline chemotherapy. Recently, chromosome 17 centromere enumeration probe (CEP17) duplication has also been associated with increased responsiveness to anthracyclines. However, reports are conflicting and none of these tumor markers can yet be considered a clinically reliable predictor of response to anthracyclines. We studied the association of TOP2A gene alterations, HER2 gene amplification, and CEP17 duplication with response to anthracycline-based neoadjuvant chemotherapy in 140 patients with operable or locally advanced breast cancer. HER2 was tested by fluorescence in situ hybridization and TOP2A and CEP17 by chromogenic in situ hybridization. Thirteen patients (9.3%) achieved pathologic complete response (pCR). HER2 amplification was present in 24 (17.5%) of the tumors. TOP2A amplification occurred in seven tumors (5.1%). CEP17 duplication was detected in 13 patients (9.5%). CEP17 duplication correlated with a higher rate of pCR [odds ratio (OR) 6.55, 95% confidence interval (95% CI) 1.25-34.29, P = .026], and analysis of TOP2A amplification showed a trend bordering on statistical significance (OR 6.97, 95% CI 0.96-50.12, P = .054). TOP2A amplification and CEP17 duplication combined were strongly associated with pCR (OR 6.71, 95% CI 1.66-27.01, P = .007). HER2 amplification did not correlate with pCR. Our results suggest that CEP17 duplication predicts pCR to primary anthracycline-based chemotherapy. CEP17 duplication, TOP2A amplifications, and HER2 amplifications were not associated with prognosis.

  8. Molecular and evolutionary characteristics of the fraction of human alpha satellite DNA associated with CENP-A at the centromeres of chromosomes 1, 5, 19, and 21

    Directory of Open Access Journals (Sweden)

    Roizès Gérard

    2010-03-01

    Full Text Available Abstract Background The mode of evolution of the highly homogeneous Higher-Order-Repeat-containing alpha satellite arrays is still subject to discussion. This is also true of the CENP-A associated repeats where the centromere is formed. Results In this paper, we show that the molecular mechanisms by which these arrays evolve are identical in multiple chromosomes: i accumulation of crossovers that homogenise and expand the arrays into different domains and subdomains that are mostly unshared between homologues and ii sporadic mutations and conversion events that simultaneously differentiate them from one another. Individual arrays are affected by these mechanisms to different extents that presumably increase with time. Repeats associated with CENP-A, where the centromere is formed, are subjected to the same evolutionary mechanisms, but constitute minor subsets that exhibit subtle sequence differences from those of the bulk repeats. While the DNA sequence per se is not essential for centromere localisation along an array, it appears that certain sequences can be selected against. On chromosomes 1 and 19, which are more affected by the above evolutionary mechanisms than are chromosomes 21 and 5, CENP-A associated repeats were also recovered from a second homogeneous array present on each chromosome. This could be a way for chromosomes to sustain mitosis and meiosis when the normal centromere locus is ineluctably undermined by the above mechanisms. Conclusion We discuss, in light of these observations, possible scenarios for the normal evolutionary fates of human centromeric regions.

  9. Ephemeral protein binding to DNA shapes stable nuclear bodies and chromatin domains

    CERN Document Server

    Brackley, C A; Michieletto, D; Mouvet, F; Cook, P R; Marenduzzo, D

    2016-01-01

    Fluorescence microscopy reveals that the contents of many (membrane-free) nuclear "bodies" exchange rapidly with the soluble pool whilst the underlying structure persists; such observations await a satisfactory biophysical explanation. To shed light on this, we perform large-scale Brownian dynamics simulations of a chromatin fiber interacting with an ensemble of (multivalent) DNA-binding proteins; these proteins switch between two states -- active (binding) and inactive (non-binding). This system provides a model for any DNA-binding protein that can be modified post-translationally to change its affinity for DNA (e.g., like the phosphorylation of a transcription factor). Due to this out-of-equilibrium process, proteins spontaneously assemble into clusters of self-limiting size, as individual proteins in a cluster exchange with the soluble pool with kinetics like those seen in photo-bleaching experiments. This behavior contrasts sharply with that exhibited by "equilibrium", or non-switching, proteins that exis...

  10. Using oocyte nuclei for studies on chromatin structure and gene expression.

    Science.gov (United States)

    Sommerville, John

    2010-05-01

    The giant nucleus of amphibian oocytes is generally referred to as the germinal vesicle (GV). Its size allows relatively easy manual isolation from the rest of the oocyte and also presents a large target in situ for microinjection of macromolecules including plasmid DNA, RNA species, antibodies and other proteins and even whole organelles, including somatic cell nuclei. Thus the use of GVs is excellent for two major types of study: the function of endogenous nuclear processes such as gene transcription, RNA processing and intra-nuclear dynamics; and the use of the nuclear components to effect processes such as chromatin assembly, expression of foreign genes and nucleocytoplasmic transport of injected biomolecules. This article outlines some basic techniques appropriate for GV studies, particularly the preparation of oocytes for microinjection and the isolation of germinal vesicles into an oil phase. As an aid to the targeting of the GV within the nucleus, descriptions are given of the use of oocytes from albino animals.

  11. The RSC Chromatin Remodeling Complex Bears an Essential Fungal-Specific Protein Module With Broad Functional Roles

    OpenAIRE

    Wilson, Boris; Erdjument-Bromage, Hediye; Tempst, Paul; Bradley R Cairns

    2006-01-01

    RSC is an essential and abundant ATP-dependent chromatin remodeling complex from Saccharomyces cerevisiae. Here we show that the RSC components Rsc7/Npl6 and Rsc14/Ldb7 interact physically and/or functionally with Rsc3, Rsc30, and Htl1 to form a module important for a broad range of RSC functions. A strain lacking Rsc7 fails to properly assemble RSC, which confers sensitivity to temperature and to agents that cause DNA damage, microtubule depolymerization, or cell wall stress (likely via tran...

  12. Differential repetitive DNA composition in the centromeric region of chromosomes of Amazonian lizard species in the family Teiidae.

    Science.gov (United States)

    Carvalho, Natalia D M; Carmo, Edson; Neves, Rogerio O; Schneider, Carlos Henrique; Gross, Maria Claudia

    2016-01-01

    Differences in heterochromatin distribution patterns and its composition were observed in Amazonian teiid species. Studies have shown repetitive DNA harbors heterochromatic blocks which are located in centromeric and telomeric regions in Ameiva ameiva (Linnaeus, 1758), Kentropyx calcarata (Spix, 1825), Kentropyx pelviceps (Cope, 1868), and Tupinambis teguixin (Linnaeus, 1758). In Cnemidophorus sp.1, repetitive DNA has multiple signals along all chromosomes. The aim of this study was to characterize moderately and highly repetitive DNA sequences by C ot1-DNA from Ameiva ameiva and Cnemidophorus sp.1 genomes through cloning and DNA sequencing, as well as mapping them chromosomally to better understand its organization and genome dynamics. The results of sequencing of DNA libraries obtained by C ot1-DNA showed that different microsatellites, transposons, retrotransposons, and some gene families also comprise the fraction of repetitive DNA in the teiid species. FISH using C ot1-DNA probes isolated from both Ameiva ameiva and Cnemidophorus sp.1 showed these sequences mainly located in heterochromatic centromeric, and telomeric regions in Ameiva ameiva, Kentropyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin chromosomes, indicating they play structural and functional roles in the genome of these species. In Cnemidophorus sp.1, C ot1-DNA probe isolated from Ameiva ameiva had multiple interstitial signals on chromosomes, whereas mapping of C ot1-DNA isolated from the Ameiva ameiva and Cnemidophorus sp.1 highlighted centromeric regions of some chromosomes. Thus, the data obtained showed that many repetitive DNA classes are part of the genome of Ameiva ameiva, Cnemidophorus sp.1, Kentroyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin, and these sequences are shared among the analyzed teiid species, but they were not always allocated at the same chromosome position.

  13. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.

    Science.gov (United States)

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J; Fox, Catherine A

    2016-04-07

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

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

    CERN Document Server

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

    2009-01-01

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

  15. Chromatin dynamics at DNA breaks: what, how and why?

    Directory of Open Access Journals (Sweden)

    Théo Lebeaupin

    2015-09-01

    Full Text Available Chromatin has a complex, dynamic architecture in the interphase nucleus, which regulates the accessibility of the underlying DNA and plays a key regulatory role in all the cellular functions using DNA as a template, such as replication, transcription or DNA damage repair. Here, we review the recent progresses in the understanding of the interplay between chromatin architecture and DNA repair mechanisms. Several reports based on live cell fluorescence imaging show that the activation of the DNA repair machinery is associated with major changes in the compaction state and the mobility of chromatin. We discuss the functional consequences of these changes in yeast and mammals in the light of the different repair pathways utilized by these organisms. In the final section of this review, we show how future developments in high-resolution light microscopy and chromatin modelling by polymer physics should contribute to a better understanding of the relationship between the structural changes in chromatin and the activity of the repair processes.

  16. Chromatin Modifications and the DNA Damage Response to Ionizing Radiation

    Directory of Open Access Journals (Sweden)

    Tej K Pandita

    2013-01-01

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

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

    Science.gov (United States)

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

    2016-04-21

    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.

  18. Dump assembly

    Science.gov (United States)

    Goldmann, Louis H.

    1986-01-01

    A dump assembly having a fixed conduit and a rotatable conduit provided with overlapping plates, respectively, at their adjacent ends. The plates are formed with openings, respectively, normally offset from each other to block flow. The other end of the rotatable conduit is provided with means for securing the open end of a filled container thereto. Rotation of the rotatable conduit raises and inverts the container to empty the contents while concurrently aligning the conduit openings to permit flow of material therethrough.

  19. General Assembly

    CERN Multimedia

    Staff Association

    2016-01-01

    5th April, 2016 – Ordinary General Assembly of the Staff Association! In the first semester of each year, the Staff Association (SA) invites its members to attend and participate in the Ordinary General Assembly (OGA). This year the OGA will be held on Tuesday, April 5th 2016 from 11:00 to 12:00 in BE Auditorium, Meyrin (6-2-024). During the Ordinary General Assembly, the activity and financial reports of the SA are presented and submitted for approval to the members. This is the occasion to get a global view on the activities of the SA, its financial management, and an opportunity to express one’s opinion, including taking part in the votes. Other points are listed on the agenda, as proposed by the Staff Council. Who can vote? Only “ordinary” members (MPE) of the SA can vote. Associated members (MPA) of the SA and/or affiliated pensioners have a right to vote on those topics that are of direct interest to them. Who can give his/her opinion? The Ordinary General Asse...

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

    Science.gov (United States)

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

    2016-08-05

    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.

  1. Interplay of Dynamic Transcription and Chromatin Remodeling: Lessons from Yeast

    Directory of Open Access Journals (Sweden)

    Eva Klopf

    2011-07-01

    Full Text Available Regulation of transcription involves dynamic rearrangements of chromatin structure. The budding yeast Saccharomyces cerevisiae has a variety of highly conserved factors necessary for these reconstructions. Chromatin remodelers, histone modifiers and histone chaperones directly associate to promoters and open reading frames of exposed genes and facilitate activation and repression of transcription. We compare two distinct patterns of induced transcription: Sustained transcribed genes switch to an activated state where they remain as long as the induction signal is present. In contrast, single pulsed transcribed genes show a quick and strong induction pulse resulting in high transcript levels followed by adaptation and repression to basal levels. We discuss intensively studied promoters and coding regions from both groups for their co-factor requirements during transcription. Interplay between chromatin restructuring factors and dynamic transcription is highly variable and locus dependent.

  2. H4K44 Acetylation Facilitates Chromatin Accessibility during Meiosis.

    Science.gov (United States)

    Hu, Jialei; Donahue, Greg; Dorsey, Jean; Govin, Jérôme; Yuan, Zuofei; Garcia, Benjamin A; Shah, Parisha P; Berger, Shelley L

    2015-12-01

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

  3. Oxidative stress signaling to chromatin in health and disease

    KAUST Repository

    Kreuz, Sarah

    2016-06-20

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

  4. Sliding and peeling of histone during chromatin remodelling

    CERN Document Server

    Garai, Ashok; Chowdhury, Debashish

    2011-01-01

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

  5. Human pescadillo induces large-scale chromatin unfolding

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    Science.gov (United States)

    Ruiz-Romero, Marina; Corominas, Montserrat; Guigó, Roderic

    2015-01-01

    The interplay of active and repressive histone modifications is assumed to play a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated to stable production of RNA, while unmarked chromatin would permit rapid gene activation and de-activation during development. In this case, regulation by transcription factors would play a comparatively more important regulatory role. PMID:26280901

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

    Science.gov (United States)

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

    2014-02-15

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

  8. The telomere binding protein TRF2 induces chromatin compaction.

    Directory of Open Access Journals (Sweden)

    Asmaa M Baker

    Full Text Available Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

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

    Directory of Open Access Journals (Sweden)

    Lasse Sinkkonen

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

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

    Science.gov (United States)

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Grøntved Lars

    2012-06-01

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

  12. Chromatin remodeling pathways in smooth muscle cell differentiation, and evidence for an integral role for p300.

    Directory of Open Access Journals (Sweden)

    Joshua M Spin

    Full Text Available BACKGROUND: Phenotypic alteration of vascular smooth muscle cells (SMC in response to injury or inflammation is an essential component of vascular disease. Evidence suggests that this process is dependent on epigenetic regulatory processes. P300, a histone acetyltransferase (HAT, activates crucial muscle-specific promoters in terminal (non-SMC myocyte differentiation, and may be essential to SMC modulation as well. RESULTS: We performed a subanalysis examining transcriptional time-course microarray data obtained using the A404 model of SMC differentiation. Numerous chromatin remodeling genes (up to 62% of such genes on our array platform showed significant regulation during differentiation. Members of several chromatin-remodeling families demonstrated involvement, including factors instrumental in histone modification, chromatin assembly-disassembly and DNA silencing, suggesting complex, multi-level systemic epigenetic regulation. Further, trichostatin A, a histone deacetylase inhibitor, accelerated expression of SMC differentiation markers in this model. Ontology analysis indicated a high degree of p300 involvement in SMC differentiation, with 60.7% of the known p300 interactome showing significant expression changes. Knockdown of p300 expression accelerated SMC differentiation in A404 cells and human SMCs, while inhibition of p300 HAT activity blunted SMC differentiation. The results suggest a central but complex role for p300 in SMC phenotypic modulation. CONCLUSIONS: Our results support the hypothesis that chromatin remodeling is important for SMC phenotypic switching, and detail wide-ranging involvement of several epigenetic modification families. Additionally, the transcriptional coactivator p300 may be partially degraded during SMC differentiation, leaving an activated subpopulation with increased HAT activity and SMC differentiation-gene specificity.

  13. Chromatin structure and evolution in the human genome

    Directory of Open Access Journals (Sweden)

    Dunlop Malcolm G

    2007-05-01

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

  14. SANS spectra of the fractal supernucleosomal chromatin structure models

    Science.gov (United States)

    Ilatovskiy, Andrey V.; Lebedev, Dmitry V.; Filatov, Michael V.; Petukhov, Michael G.; Isaev-Ivanov, Vladimir V.

    2012-03-01

    The eukaryotic genome consists of chromatin—a nucleoprotein complex with hierarchical architecture based on nucleosomes, the organization of higher-order chromatin structures still remains unknown. Available experimental data, including SANS spectra we had obtained for whole nuclei, suggested fractal nature of chromatin. Previously we had built random-walk supernucleosomal models (up to 106 nucleosomes) to interpret our SANS spectra. Here we report a new method to build fractal supernucleosomal structure of a given fractal dimension or two different dimensions. Agreement between calculated and experimental SANS spectra was significantly improved, especially for model with two fractal dimensions—3 and 2.

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

    Science.gov (United States)

    Abuelo, J G; Moore, D E

    1969-04-01

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

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

    Science.gov (United States)

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

    2012-05-01

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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Bo eDing

    2015-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Andreas H Ehrensberger

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

  20. Instability of trinucleotidic repeats during chromatin remodeling in spermatids.

    Science.gov (United States)

    Simard, Olivier; Grégoire, Marie-Chantal; Arguin, Mélina; Brazeau, Marc-André; Leduc, Frédéric; Marois, Isabelle; Richter, Martin V; Boissonneault, Guylain

    2014-11-01

    Transient DNA breaks and evidence of DNA damage response have recently been reported during the chromatin remodeling process in haploid spermatids, creating a potential window of enhanced genetic instability. We used flow cytometry to achieve separation of differentiating spermatids into four highly purified populations using transgenic mice harboring 160 CAG repeats within exon 1 of the human Huntington disease gene (HTT). Trinucleotic repeat expansion was found to occur immediately following the chromatin remodeling steps, confirming the genetic instability of the process and pointing to the origin of paternal anticipation observed in some trinucleotidic repeats diseases.

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

    DEFF Research Database (Denmark)

    Laugesen, Anne; Helin, Kristian

    2014-01-01

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

  2. The importance of topoisomerases for chromatin regulated genes

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  3. Human chromosome pellicle antibody recognizing centromere protein—C (CENP0C),the main component of the kinetochore

    Institute of Scientific and Technical Information of China (English)

    XIEYONG; ZUMEINI; 等

    1997-01-01

    Recently the antichromosome antisera from several sclerogerma patients have been found to recognize the pellicle of metaphase and anaphase chromosomes.In order to identify the pellicle components,we used these antichromosome antisera to screen a human embryonic cDNA library.The sequences of the positive clones are identical to the cDNA gene sequence of CENP-C (centromere protein C),a human centromere autoantigen.This result suggusts that CENP-C is a component of the pellicle of human metaphase and anaphase chromosomes.

  4. Misregulation of Scm3p/HJURP Causes Chromosome Instability in Saccharomyces cerevisiae and Human Cells

    OpenAIRE

    Prashant K. Mishra; Wei-Chun Au; John S. Choy; P Henning Kuich; Baker, Richard E.; Foltz, Daniel R.; Basrai, Munira A.

    2011-01-01

    The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevanc...

  5. Functional analysis of phosphorylation of the mitotic centromere-associated kinesin by Aurora B kinase in human tumor cells.

    Science.gov (United States)

    Ritter, Andreas; Sanhaji, Mourad; Friemel, Alexandra; Roth, Susanne; Rolle, Udo; Louwen, Frank; Yuan, Juping

    2015-01-01

    Mitotic centromere-associated kinesin (MCAK) is the best characterized member of the kinesin-13 family and plays important roles in microtubule dynamics during mitosis. Its activity and subcellular localization is tightly regulated by an orchestra of mitotic kinases, such as Aurora B. It is well known that serine 196 of MCAK is the major phosphorylation site of Aurora B in Xenopus leavis extracts and that this phosphorylation regulates its catalytic activity and subcellular localization. In the current study, we have addressed the conserved phosphorylation site serine 192 in human MCAK to characterize its function in more depth in human cancer cells. Our data confirm that S192 is the major phosphorylation site of Aurora B in human MCAK and that this phosphorylation has crucial roles in regulating its catalytic activity and localization at the kinetochore/centromere region in mitosis. Interfering with this phosphorylation leads to a delayed progression through prometa- and metaphase associated with mitotic defects in chromosome alignment and segregation. We show further that MCAK is involved in directional migration and invasion of tumor cells, and interestingly, interference with the S192 phosphorylation affects this capability of MCAK. These data provide the first molecular explanation for clinical observation, where an overexpression of MCAK was associated with lymphatic invasion and lymph node metastasis in gastric and colorectal cancer patients.

  6. Analysis of premature centromere division (PCD) of the X chromosome in Alzheimer patients through the cell cycle.

    Science.gov (United States)

    Spremo-Potparevic, Biljana; Zivkovic, Lada; Djelic, Ninoslav; Bajic, Vladan

    2004-05-01

    Cytogenetic analysis of the X chromosome in phytohaemagglutinin stimulated peripheral blood lymphocytes was evaluated in 12 sporadic Alzheimer disease (AD) patients and in 11 healthy subjects. For chromosome analysis two methods were used: (1) standard analysis of G-banded metaphase chromosomes and; (2) fluorescent in situ hybridization (FISH) for the detection of the X chromosome centromeric region in interphase nuclei. Cytogenetic analysis revealed that the X chromosome expresses premature centromere division (PCD) in AD females in 10.53% of metaphase cells and in 15.22% of interphase nuclei. In AD men the percentages were 3.98 and 6.06%, respectively. X chromosome PCD in the female control group showed a percentage of 7.46% in metaphase cells and 9.35% in interphase nuclei and in male controls the percentages were 2.84% in metaphases and 5.54% in interphase nuclei. The results of FISH analysis showed that PCD could occur much earlier than metaphase of mitosis, i.e. in interphase of the cell cycle, immediately after replication. The FISH method can be used for PCD verification in all phases of the cell cycle in various disorders including AD.

  7. Assembling consumption

    DEFF Research Database (Denmark)

    Assembling Consumption marks a definitive step in the institutionalisation of qualitative business research. By gathering leading scholars and educators who study markets, marketing and consumption through the lenses of philosophy, sociology and anthropology, this book clarifies and applies...... the investigative tools offered by assemblage theory, actor-network theory and non-representational theory. Clear theoretical explanation and methodological innovation, alongside empirical applications of these emerging frameworks will offer readers new and refreshing perspectives on consumer culture and market...... societies. This is an essential reading for both seasoned scholars and advanced students of markets, economies and social forms of consumption....

  8. Combinatorial depletion analysis to assemble the network architecture of the SAGA and ADA chromatin remodeling complexes.

    Science.gov (United States)

    Lee, Kenneth K; Sardiu, Mihaela E; Swanson, Selene K; Gilmore, Joshua M; Torok, Michael; Grant, Patrick A; Florens, Laurence; Workman, Jerry L; Washburn, Michael P

    2011-07-05

    Despite the availability of several large-scale proteomics studies aiming to identify protein interactions on a global scale, little is known about how proteins interact and are organized within macromolecular complexes. Here, we describe a technique that consists of a combination of biochemistry approaches, quantitative proteomics and computational methods using wild-type and deletion strains to investigate the organization of proteins within macromolecular protein complexes. We applied this technique to determine the organization of two well-studied complexes, Spt-Ada-Gcn5 histone acetyltransferase (SAGA) and ADA, for which no comprehensive high-resolution structures exist. This approach revealed that SAGA/ADA is composed of five distinct functional modules, which can persist separately. Furthermore, we identified a novel subunit of the ADA complex, termed Ahc2, and characterized Sgf29 as an ADA family protein present in all Gcn5 histone acetyltransferase complexes. Finally, we propose a model for the architecture of the SAGA and ADA complexes, which predicts novel functional associations within the SAGA complex and provides mechanistic insights into phenotypical observations in SAGA mutants.

  9. Mind the gap; seven reasons to close fragmented genome assemblies.

    Science.gov (United States)

    Thomma, Bart P H J; Seidl, Michael F; Shi-Kunne, Xiaoqian; Cook, David E; Bolton, Melvin D; van Kan, Jan A L; Faino, Luigi

    2016-05-01

    Like other domains of life, research into the biology of filamentous microbes has greatly benefited from the advent of whole-genome sequencing. Next-generation sequencing (NGS) technologies have revolutionized sequencing, making genomic sciences accessible to many academic laboratories including those that study non-model organisms. Thus, hundreds of fungal genomes have been sequenced and are publically available today, although these initiatives have typically yielded considerably fragmented genome assemblies that often lack large contiguous genomic regions. Many important genomic features are contained in intergenic DNA that is often missing in current genome assemblies, and recent studies underscore the significance of non-coding regions and repetitive elements for the life style, adaptability and evolution of many organisms. The study of particular types of genetic elements, such as telomeres, centromeres, repetitive elements, effectors, and clusters of co-regulated genes, but also of phenomena such as structural rearrangements, genome compartmentalization and epigenetics, greatly benefits from having a contiguous and high-quality, preferably even complete and gapless, genome assembly. Here we discuss a number of important reasons to produce gapless, finished, genome assemblies to help answer important biological questions.

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

    Science.gov (United States)

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

    2015-01-01

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

  11. General Assembly

    CERN Multimedia

    Staff Association

    2015-01-01

    Mardi 5 mai à 11 h 00 Salle 13-2-005 Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : 1- Adoption de l’ordre du jour. 2- Approbation du procès-verbal de l’Assemblée générale ordinaire du 22 mai 2014. 3- Présentation et approbation du rapport d’activités 2014. 4- Présentation et approbation du rapport financier 2014. 5- Présentation et approbation du rapport des vérificateurs aux comptes pour 2014. 6- Programme 2015. 7- Présentation et approbation du projet de budget 2015 et taux de cotisation pour 2015. 8- Pas de modifications aux Statuts de l'Association du personnel proposée. 9- Élections des membres de la Commission é...

  12. General Assembly

    CERN Multimedia

    Staff Association

    2016-01-01

    Mardi 5 avril à 11 h 00 BE Auditorium Meyrin (6-2-024) Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 5 mai 2015. Présentation et approbation du rapport d’activités 2015. Présentation et approbation du rapport financier 2015. Présentation et approbation du rapport des vérificateurs aux comptes pour 2015. Programme de travail 2016. Présentation et approbation du projet de budget 2016 Approbation du taux de cotisation pour 2017. Modifications aux Statuts de l'Association du personnel proposée. Élections des membres de la Commissio...

  13. General assembly

    CERN Multimedia

    Staff Association

    2015-01-01

    Mardi 5 mai à 11 h 00 Salle 13-2-005 Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 22 mai 2014. Présentation et approbation du rapport d’activités 2014. Présentation et approbation du rapport financier 2014. Présentation et approbation du rapport des vérificateurs aux comptes pour 2014. Programme 2015. Présentation et approbation du projet de budget 2015 et taux de cotisation pour 2015. Pas de modifications aux Statuts de l'Association du personnel proposée. Élections des membres de la Commission électorale. &am...

  14. Generation of bivalent chromatin domains during cell fate decisions

    Directory of Open Access Journals (Sweden)

    De Gobbi Marco

    2011-06-01

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

  15. Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization

    Directory of Open Access Journals (Sweden)

    Peterhansel Christoph

    2007-09-01

    Full Text Available Abstract Background Chromatin remodeling, histone modifications and other chromatin-related processes play a crucial role in gene regulation. A very useful technique to study these processes is chromatin immunoprecipitation (ChIP. ChIP is widely used for a few model systems, including Arabidopsis, but establishment of the technique for other organisms is still remarkably challenging. Furthermore, quantitative analysis of the precipitated material and normalization of the data is often underestimated, negatively affecting data quality. Results We developed a robust ChIP protocol, using maize (Zea mays as a model system, and present a general strategy to systematically optimize this protocol for any type of tissue. We propose endogenous controls for active and for repressed chromatin, and discuss various other controls that are essential for successful ChIP experiments. We experienced that the use of quantitative PCR (QPCR is crucial for obtaining high quality ChIP data and we explain why. The method of data normalization has a major impact on the quality of ChIP analyses. Therefore, we analyzed different normalization strategies, resulting in a thorough discussion of the advantages and drawbacks of the various approaches. Conclusion Here we provide a robust ChIP protocol and strategy to optimize the protocol for any type of tissue; we argue that quantitative real-time PCR (QPCR is the best method to analyze the precipitates, and present comprehensive insights into data normalization.

  16. Control of the Transition to Flowering by Chromatin Modifications

    Institute of Scientific and Technical Information of China (English)

    Yuehui He

    2009-01-01

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

  17. Chromatin Structure in Cell Differentiation, Aging and Cancer

    NARCIS (Netherlands)

    S. Kheradmand Kia (Sima)

    2009-01-01

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

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

    DEFF Research Database (Denmark)

    Lichota, J.; Grasser, Klaus D.

    2003-01-01

    maize HMGA and five different HMGB proteins with mononucleosomes (containing approx. 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin. The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA...

  19. Epigenetic regulation and chromatin remodeling in learning and memory

    Science.gov (United States)

    Kim, Somi; Kaang, Bong-Kiun

    2017-01-01

    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. PMID:28082740

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

    NARCIS (Netherlands)

    Kornet, N.G.

    2008-01-01

    Stem cells replenish the cells present in an organism throughout its lifetime and sustain growth. They have unique characteristics: the capability to self-renew and the potential to differentiate into several cell types. Recently, it has become clear that chromatin factors support these unique featu

  1. Chromatin remodelers in the DNA double strand break response

    NARCIS (Netherlands)

    Smeenk, Godelieve

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sascha eBeneke

    2012-09-01

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

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

    NARCIS (Netherlands)

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

    2004-01-01

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

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

    NARCIS (Netherlands)

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

    2015-01-01

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

  5. Analysis of chromatin structure at meiotic DSB sites in yeasts.

    Science.gov (United States)

    Hirota, Kouji; Fukuda, Tomoyuki; Yamada, Takatomi; Ohta, Kunihiro

    2009-01-01

    One of the major features of meiosis is a high frequency of homologous recombination that not only confers genetic diversity to a successive generation but also ensures proper segregation of chromosomes. Meiotic recombination is initiated by DNA double-strand breaks that require many proteins including the catalytic core, Spo11. In this regard, like transcription and repair, etc., recombination is hindered by a compacted chromatin structure because trans-acting factors cannot easily access the DNA. Such inhibitory effects must be alleviated prior to recombination initiation. Indeed, a number of groups showed that chromatin around recombination hotspots is less condensed, by using nucleases as a probe to assess local DNA accessibility. Here we describe a method to analyze chromatin structure of a recombination hotspot in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This method, combining micrococcal nuclease (MNase) digestion ofchromatin DNA and subsequent Southern blotting, is expected to provide information as to chromatin context around a hotspot. Moreover, by virtue of MNase preferentially targeting linker DNA, positions of several nucleosomes surrounding a hotspot can also be determined. Our protocol is a very powerful way to analyze several-kb regions of interest and can be applied to other purposes.

  6. Trichomonas vaginalis: chromatin and mitotic spindle during mitosis.

    Science.gov (United States)

    Gómez-Conde, E; Mena-López, R; Hernández-Jaúregui, P; González-Camacho, M; Arroyo, R

    2000-11-01

    The mitotic phases and the changes that the chromatin and mitotic microtubules undergo during mitosis in the sexually transmitted parasite Trichomonas vaginalis are described. Parasites arrested in the gap 2 phase of the cell cycle by nutrient starvation were induced to mitosis by addition of fresh whole medium. [(3)H] Thymidine labeling of trichomonad parasites for 24 h showed that parasites have at least four synchronic duplications after mitosis induction. Fixed or live and acridine orange (AO)-stained trichomonads analyzed at different times during mitosis by epifluorescence microscopy showed that mitosis took about 45 min and is divided into five stages: prophase, metaphase, early and late anaphase, early and late telophase, and cytokinesis. The AO-stained nucleus of live trichomonads showed green (DNA) and orange (RNA) fluorescence, and the nucleic acid nature was confirmed by DNase and RNase treatment, respectively. The chromatin appeared partially condensed during interphase. At metaphase, it appeared as six condensed chromosomes, as recently reported, which decondensed at anaphase and migrated to the nuclear poles at telophase. In addition, small bundles of microtubules (as hemispindles) were detected only in metaphase with the polyclonal antibody anti-Entamoeba histolytica alpha-tubulin. This antibody showed that the hemispindle and an atractophore-like structure seem to duplicate and polarize during metaphase. In conclusion, T. vaginalis mitosis involves five mitotic phases in which the chromatin undergoes different degrees of condensation, from chromosomes to decondensed chromatin, and two hemispindles that are observed only in the metaphase stage.

  7. Discovering enhancers by mapping chromatin features in primary tissue.

    Science.gov (United States)

    Bowman, Sarah K

    2015-09-01

    Enhancers work with promoters to refine the timing, location, and level of gene expression. As they perform these functions, active enhancers generate a chromatin environment that is distinct from other areas of the genome. Therefore, profiling enhancer-associated chromatin features can produce genome-wide maps of potential regulatory elements. This review focuses on current technologies used to produce maps of potential tissue-specific enhancers by profiling chromatin from primary tissue. First, cells are separated from whole organisms either by affinity purification, automated cell sorting, or microdissection. Isolating the tissue prior to analysis ensures that the molecular signature of active enhancers will not become lost in an averaged signal from unrelated cell types. After cell isolation, the molecular feature that is profiled will depend on the abundance and quality of the harvested material. The combination of tissue isolation plus genome-wide chromatin profiling has successfully identified enhancers in several pioneering studies. In the future, the regulatory apparatus of healthy and diseased tissues will be explored in this manner, as researchers use the combined techniques to gain insight into how active enhancers may influence disease progression.

  8. Sister chromatid tension and the spindle assembly checkpoint.

    Science.gov (United States)

    Nezi, Luigi; Musacchio, Andrea

    2009-12-01

    The spindle assembly checkpoint (SAC) is a feedback control system that monitors the state of kinetochore/microtubule attachment during mitosis and halts cell cycle progression until all chromosomes are properly aligned at the metaphase plate. The state of chromosome-microtubule attachment is implicated as a crucial factor in the checkpoint response. On the contrary, lack of tension in the centromere-kinetochore region of sister chromatids has been shown to regulate a pathway of correction of undesired chromosome-microtubule connections, while the presence of tension is believed to promote the stabilization of attachments. We discuss how tension-sensitive phenomena, such as attachment correction and stabilization, relate to the SAC and we speculate on the existence of a single pathway linking error correction and SAC activation.

  9. Complexity of chromatin folding is captured by the strings and binders switch model

    OpenAIRE

    Barbieri, Mariano; Chotalia, Mita; Fraser, James; Lavitas, Liron-Mark; Dostie, Josée; Pombo, Ana; Nicodemi, Mario

    2012-01-01

    Chromatin has a complex spatial organization in the cell nucleus that serves vital functional purposes. A variety of chromatin folding conformations has been detected by single-cell imaging and chromosome conformation capture-based approaches. However, a unified quantitative framework describing spatial chromatin organization is still lacking. Here, we explore the “strings and binders switch” model to explain the origin and variety of chromatin behaviors that coexist and dynamically change wi...

  10. Widespread Chromatin Accessibility at Repetitive Elements Links Stem Cells with Human Cancer

    OpenAIRE

    Nicholas C. Gomez; Austin J. Hepperla; Raluca Dumitru; Jeremy M. Simon; Fang Fang; Ian J. Davis

    2016-01-01

    Chromatin regulation is critical for differentiation and disease. However, features linking the chromatin environment of stem cells with disease remain largely unknown. We explored chromatin accessibility in embryonic and multipotent stem cells and unexpectedly identified widespread chromatin accessibility at repetitive elements. Integrating genomic and biochemical approaches, we demonstrate that these sites of increased accessibility are associated with well-positioned nucleosomes marked by ...

  11. A Combined Global and Local Approach to Elucidate Spatial Organization of the Mycobacterial ParB-parS Partition Assembly

    Energy Technology Data Exchange (ETDEWEB)

    B Chaudhuri; S Gupta; V Urban; M Chance; R DMello; L Smith; K Lyons; J Gee

    2011-12-31

    Combining diverse sets of data at global (size, shape) and local (residue) scales is an emerging trend for elucidating the organization and function of the cellular assemblies. We used such a strategy, combining data from X-ray and neutron scattering with H/D-contrast variation and X-ray footprinting with mass spectrometry, to elucidate the spatial organization of the ParB-parS assembly from Mycobacterium tuberculosis. The ParB-parS participates in plasmid and chromosome segregation and condensation in predivisional bacterial cells. ParB polymerizes around the parS centromere(s) to form a higher-order assembly that serves to recruit cyto-skeletal ParA ATPases and SMC proteins for chromosome segregation. A hybrid model of the ParB-parS was built by combining and correlating computational models with experiment-derived information about size, shape, position of the symmetry axis within the shape, internal topology, DNA-protein interface, exposed surface patches, and prior knowledge. This first view of the ParB-parS leads us to propose how ParB spread on the chromosome to form a larger assembly.

  12. A combined global and local approach to elucidate spatial organization of the Mycobacterial ParB-parS partition assembly.

    Science.gov (United States)

    Chaudhuri, Barnali N; Gupta, Sayan; Urban, Volker S; Chance, Mark R; D'Mello, Rhijuta; Smith, Lauren; Lyons, Kelly; Gee, Jessica

    2011-03-22

    Combining diverse sets of data at global (size, shape) and local (residue) scales is an emerging trend for elucidating the organization and function of the cellular assemblies. We used such a strategy, combining data from X-ray and neutron scattering with H/D-contrast variation and X-ray footprinting with mass spectrometry, to elucidate the spatial organization of the ParB-parS assembly from Mycobacterium tuberculosis. The ParB-parS participates in plasmid and chromosome segregation and condensation in predivisional bacterial cells. ParB polymerizes around the parS centromere(s) to form a higher-order assembly that serves to recruit cyto-skeletal ParA ATPases and SMC proteins for chromosome segregation. A hybrid model of the ParB-parS was built by combining and correlating computational models with experiment-derived information about size, shape, position of the symmetry axis within the shape, internal topology, DNA-protein interface, exposed surface patches, and prior knowledge. This first view of the ParB-parS leads us to propose how ParB spread on the chromosome to form a larger assembly.

  13. Citrullination regulates pluripotency and histone H1 binding to chromatin

    Science.gov (United States)

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

    2014-03-01

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

  14. The ING tumor suppressors in cellular senescence and chromatin.

    Science.gov (United States)

    Ludwig, Susann; Klitzsch, Alexandra; Baniahmad, Aria

    2011-07-18

    The Inhibitor of Growth (ING) proteins represent a type II tumor suppressor family comprising five conserved genes, ING1 to ING5. While ING1, ING2 and ING3 proteins are stable components of the mSIN3a-HDAC complexes, the association of ING1, ING4 and ING5 with HAT protein complexes was also reported. Among these the ING1 and ING2 have been analyzed more deeply. Similar to other tumor suppressor factors the ING proteins are also involved in many cellular pathways linked to cancer and cell proliferation such as cell cycle regulation, cellular senescence, DNA repair, apoptosis, inhibition of angiogenesis and modulation of chromatin.A common structural feature of ING factors is the conserved plant homeodomain (PHD), which can bind directly to the histone mark trimethylated lysine of histone H3 (H3K4me3). PHD mutants lose the ability to undergo cellular senescence linking chromatin mark recognition with cellular senescence. ING1 and ING2 are localized in the cell nucleus and associated with chromatin modifying enzymes, linking tumor suppression directly to chromatin regulation. In line with this, the expression of ING1 in tumors is aberrant or identified point mutations are mostly localized in the PHD finger and affect histone binding. Interestingly, ING1 protein levels increase in replicative senescent cells, latter representing an efficient pathway to inhibit cancer proliferation. In association with this, suppression of p33ING1 expression prolongs replicative life span and is also sufficient to bypass oncogene-induced senescence. Recent analyses of ING1- and ING2-deficient mice confirm a tumor suppressive role of ING1 and ING2 and also indicate an essential role of ING2 in meiosis.Here we summarize the activity of ING1 and ING2 as tumor suppressors, chromatin factors and in development.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    of the cell cycle. Sperm nuclei were incubated in Xenopus egg extracts, and chromatin-associated proteins were analyzed by mass spectrometry at different times. Approximately 75% of the proteins varied in abundance on chromatin by more than 15%, suggesting that the chromatin proteome is highly dynamic...

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

    Directory of Open Access Journals (Sweden)

    Wioletta Czaja

    2012-09-01

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

  17. Fluorescence In Situ Hybridization (FISH-Based Karyotyping Reveals Rapid Evolution of Centromeric and Subtelomeric Repeats in Common Bean (Phaseolus vulgaris and Relatives

    Directory of Open Access Journals (Sweden)

    Aiko Iwata-Otsubo

    2016-04-01

    Full Text Available Fluorescence in situ hybridization (FISH-based karyotyping is a powerful cytogenetics tool to study chromosome organization, behavior, and chromosome evolution. Here, we developed a FISH-based karyotyping system using a probe mixture comprised of centromeric and subtelomeric satellite repeats, 5S rDNA, and chromosome-specific BAC clones in common bean, which enables one to unambiguously distinguish all 11 chromosome pairs. Furthermore, we applied the karyotyping system to several wild relatives and landraces of common bean from two distinct gene pools, as well as other related Phaseolus species, to investigate repeat evolution in the genus Phaseolus. Comparison of karyotype maps within common bean indicates that chromosomal distribution of the centromeric and subtelomeric satellite repeats is stable, whereas the copy number of the repeats was variable, indicating rapid amplification/reduction of the repeats in specific genomic regions. In Phaseolus species that diverged approximately 2–4 million yr ago, copy numbers of centromeric repeats were largely reduced or diverged, and chromosomal distributions have changed, suggesting rapid evolution of centromeric repeats. We also detected variation in the distribution pattern of subtelomeric repeats in Phaseolus species. The FISH-based karyotyping system revealed that satellite repeats are actively and rapidly evolving, forming genomic features unique to individual common bean accessions and Phaseolus species.

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

    Directory of Open Access Journals (Sweden)

    Jillian M Pattison

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-06-01

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

  1. Yeast genetic analysis reveals the involvement of chromatin reassembly factors in repressing HIV-1 basal transcription.

    Directory of Open Access Journals (Sweden)

    Manuela Vanti

    2009-01-01

    Full Text Available Rebound of HIV viremia after interruption of anti-retroviral therapy is due to the small population of CD4+ T cells that remain latently infected. HIV-1 transcription is the main process controlling post-integration latency. Regulation of HIV-1 transcription takes place at both initiation and elongation levels. Pausing of RNA polymerase II at the 5' end of HIV-1 transcribed region (5'HIV-TR, which is immediately downstream of the transcription start site, plays an important role in the regulation of viral expression. The activation of HIV-1 transcription correlates with the rearrangement of a positioned nucleosome located at this region. These two facts suggest that the 5'HIV-TR contributes to inhibit basal transcription of those HIV-1 proviruses that remain latently inactive. However, little is known about the cell elements mediating the repressive role of the 5'HIV-TR. We performed a genetic analysis of this phenomenon in Saccharomyces cerevisiae after reconstructing a minimal HIV-1 transcriptional system in this yeast. Unexpectedly, we found that the critical role played by the 5'HIV-TR in maintaining low levels of basal transcription in yeast is mediated by FACT, Spt6, and Chd1, proteins so far associated with chromatin assembly and disassembly during ongoing transcription. We confirmed that this group of factors plays a role in HIV-1 postintegration latency in human cells by depleting the corresponding human orthologs with shRNAs, both in HIV latently infected cell populations and in particular single-integration clones, including a latent clone with a provirus integrated in a highly transcribed gene. Our results indicate that chromatin reassembly factors participate in the establishment of the equilibrium between activation and repression of HIV-1 when it integrates into the human genome, and they open the possibility of considering these factors as therapeutic targets of HIV-1 latency.

  2. Statistical analysis of 3D images detects regular spatial distributions of centromeres and chromocenters in animal and plant nuclei.

    Directory of Open Access Journals (Sweden)

    Philippe Andrey

    Full Text Available In eukaryotes, the interphase nucleus is organized in morphologically and/or functionally distinct nuclear "compartments". Numerous studies highlight functional relationships between the spatial organization of the nucleus and gene regulation. This raises the question of whether nuclear organization principles exist and, if so, whether they are identical in the animal and plant kingdoms. We addressed this issue through the investigation of the three-dimensional distribution of the centromeres and chromocenters. We investigated five very diverse populations of interphase nuclei at different differentiation stages in their physiological environment, belonging to rabbit embryos at the 8-cell and blastocyst stages, differentiated rabbit mammary epithelial cells during lactation, and differentiated cells of Arabidopsis thaliana plantlets. We developed new tools based on the processing of confocal images and a new statistical approach based on G- and F- distance functions used in spatial statistics. Our original computational scheme takes into account both size and shape variability by comparing, for each nucleus, the observed distribution against a reference distribution estimated by Monte-Carlo sampling over the same nucleus. This implicit normalization allowed similar data processing and extraction of rules in the five differentiated nuclei populations of the three studied biological systems, despite differences in chromosome number, genome organization and heterochromatin content. We showed that centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation, suggesting that repulsive constraints or spatial inhomogeneities underlay the spatial organization of heterochromatic compartments. The proposed technique should be useful for identifying further spatial features in a wide range of cell types.

  3. SGO1 maintains bovine meiotic and mitotic centromeric cohesions of sister chromatids and directly affects embryo development.

    Directory of Open Access Journals (Sweden)

    Feng-Xia Yin

    Full Text Available Shugoshin (SGO is a critical factor that enforces cohesion from segregation of paired sister chromatids during mitosis and meiosis. It has been studied mainly in invertebrates. Knowledge of SGO(s in a mammalian system has only been reported in the mouse and Hela cells. In this study, the functions of SGO1 in bovine oocytes during meiotic maturation, early embryonic development and somatic cell mitosis were investigated. The results showed that SGO1 was expressed from germinal vesicle (GV to the metaphase II stage. SGO1 accumulated on condensed and scattered chromosomes from pre-metaphase I to metaphase II. The over-expression of SGO1 did not interfere with the process of homologous chromosome separation, although once separated they were unable to move to the opposing spindle poles. This often resulted in the formation of oocytes with 60 replicated chromosomes. Depletion of SGO1 in GV oocytes affected chromosomal separation resulting in abnormal chromosome alignment at a significantly higher proportion than in control oocytes. Knockdown of SGO1 expression significantly decreased the embryonic developmental rate and quality. To further confirm the function(s of SGO1 during mitosis, bovine embryonic fibroblast cells were transfected with SGO1 siRNAs. SGO1 depletion induced the premature dissociation of chromosomal cohesion at the centromere and along the chromosome arm giving rise to abnormal appearing mitotic patterns. The results of this study infer that SGO1 is involved in the centromeric cohesion of sister chromatids and chromosomal movement towards the spindle poles. Depletion of SGO1 causes arrestment of cell division in meiosis and mitosis.

  4. Drosophila Yemanuclein and HIRA cooperate for de novo assembly of H3.3-containing nucleosomes in the male pronucleus.

    Directory of Open Access Journals (Sweden)

    Guillermo A Orsi

    Full Text Available The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM, the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male pronucleus. yem loss of function alleles affect male pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation.

  5. 4-D single particle tracking of synthetic and proteinaceous microspheres reveals preferential movement of nuclear particles along chromatin – poor tracks

    Directory of Open Access Journals (Sweden)

    Athale Chaitanya

    2004-11-01

    sorbitol. This effect correlated with the compaction of chromatin. We conclude that alteration in chromatin density directly influences the mobility of protein assemblies within the nucleus.

  6. Chromatin dynamics during cell cycle mediate conversion of DNA damage into chromatid breaks and affect formation of chromosomal aberrations: Biological and clinical significance

    Energy Technology Data Exchange (ETDEWEB)

    Terzoudi, Georgia I.; Hatzi, Vasiliki I. [Institute of Radioisotopes and Radiodiagnostic Products, National Centre for Scientific Research ' Demokritos' , 15310 Ag. Paraskevi Attikis, Athens (Greece); Donta-Bakoyianni, Catherine [Oral Diagnosis and Radiology, University of Athens Dental School, Athens (Greece); Pantelias, Gabriel E., E-mail: gabriel@ipta.demokritos.gr [Institute of Radioisotopes and Radiodiagnostic Products, National Centre for Scientific Research ' Demokritos' , 15310 Ag. Paraskevi Attikis, Athens (Greece)

    2011-06-03

    The formation of diverse chromosomal aberrations following irradiation and the variability in radiosensitivity at different cell-cycle stages remain a long standing controversy, probably because most of the studies have focused on elucidating the enzymatic mechanisms involved using simple DNA substrates. Yet, recognition, processing and repair of DNA damage occur within the nucleoprotein complex of chromatin which is dynamic in nature, capable of rapid unfolding, disassembling, assembling and refolding. The present work reviews experimental work designed to investigate the impact of chromatin dynamics and chromosome conformation changes during cell-cycle in the formation of chromosomal aberrations. Using conventional cytogenetics and premature chromosome condensation to visualize interphase chromatin, the data presented support the hypothesis that chromatin dynamic changes during cell-cycle are important determinants in the conversion of sub-microscopic DNA lesions into chromatid breaks. Consequently, the type and yield of radiation-induced chromosomal aberrations at a given cell-cycle-stage depends on the combined effect of DNA repair processes and chromatin dynamics, which is cell-cycle-regulated and subject to up- or down-regulation following radiation exposure or genetic alterations. This new hypothesis is used to explain the variability in radiosensitivity observed at various cell-cycle-stages, among mutant cells and cells of different origin, or among different individuals, and to revisit unresolved issues and unanswered questions. In addition, it is used to better understand hypersensitivity of AT cells and to provide an improved predictive G2-assay for evaluating radiosensitivity at individual level. Finally, experimental data at single cell level obtained using hybrid cells suggest that the proposed hypothesis applies only to the irradiated component of the hybrid.

  7. Widespread Chromatin Accessibility at Repetitive Elements Links Stem Cells with Human Cancer

    Directory of Open Access Journals (Sweden)

    Nicholas C. Gomez

    2016-11-01

    Full Text Available Chromatin regulation is critical for differentiation and disease. However, features linking the chromatin environment of stem cells with disease remain largely unknown. We explored chromatin accessibility in embryonic and multipotent stem cells and unexpectedly identified widespread chromatin accessibility at repetitive elements. Integrating genomic and biochemical approaches, we demonstrate that these sites of increased accessibility are associated with well-positioned nucleosomes marked by distinct histone modifications. Differentiation is accompanied by chromatin remodeling at repetitive elements associated with altered expression of genes in relevant developmental pathways. Remarkably, we found that the chromatin environment of Ewing sarcoma, a mesenchymally derived tumor, is shared with primary mesenchymal stem cells (MSCs. Accessibility at repetitive elements in MSCs offers a permissive environment that is exploited by the critical oncogene responsible for this cancer. Our data demonstrate that stem cells harbor a unique chromatin landscape characterized by accessibility at repetitive elements, a feature associated with differentiation and oncogenesis.

  8. Proteomics and the genetics of sperm chromatin condensation

    Institute of Scientific and Technical Information of China (English)

    Rafael Oliva; Judit Castillo

    2011-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

    Conrad, Thomas; Ørom, Ulf Andersson

    2017-01-01

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

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    with the DNA damaging agent methyl methanesulfonate (MMS). We have uncovered a dynamic set of 20 upregulated and 33 downregulated SUMO-2 conjugates, and 755 SUMO-2 sites, of which 362 were dynamic in response to MMS. In contrast to yeast, where a response is centered on homologous recombination, we identified......Small ubiquitin-like modifiers play critical roles in the DNA damage response (DDR). To increase our understanding of SUMOylation in the mammalian DDR, we employed a quantitative proteomics approach in order to identify dynamically regulated SUMO-2 conjugates and modification sites upon treatment...... dynamically SUMOylated interaction networks of chromatin modifiers, transcription factors, DNA repair factors, and nuclear body components. SUMOylated chromatin modifiers include JARID1B/KDM5B, JARID1C/KDM5C, p300, CBP, PARP1, SetDB1, and MBD1. Whereas SUMOylated JARID1B was ubiquitylated by the SUMO...

  12. A quantitative telomeric chromatin isolation protocol identifies different telomeric states

    Science.gov (United States)

    Grolimund, Larissa; Aeby, Eric; Hamelin, Romain; Armand, Florence; Chiappe, Diego; Moniatte, Marc; Lingner, Joachim

    2013-11-01

    Telomere composition changes during tumourigenesis, aging and in telomere syndromes in a poorly defined manner. Here we develop a quantitative telomeric chromatin isolation protocol (QTIP) for human cells, in which chromatin is cross-linked, immunopurified and analysed by mass spectrometry. QTIP involves stable isotope labelling by amino acids in cell culture (SILAC) to compare and identify quantitative differences in telomere protein composition of cells from various states. With QTIP, we specifically enrich telomeric DNA and all shelterin components. We validate the method characterizing changes at dysfunctional telomeres, and identify and validate known, as well as novel telomere-associated polypeptides including all THO subunits, SMCHD1 and LRIF1. We apply QTIP to long and short telomeres and detect increased density of SMCHD1 and LRIF1 and increased association of the shelterins TRF1, TIN2, TPP1 and POT1 with long telomeres. Our results validate QTIP to study telomeric states during normal development and in disease.

  13. ATRX: the case of a peculiar chromatin remodeler.

    Science.gov (United States)

    Ratnakumar, Kajan; Bernstein, Emily

    2013-01-01

    The SWI/SNF-like chromatin remodeler ATRX has recently garnered renewed attention. ATRX mutations were first identified in patients bearing the syndrome after which it is named, alpha thalassemia/mental retardation, X-linked. While ATRX has long been implicated in transcriptional regulation through multiple mechanisms, recent studies have identified a role for ATRX in the regulation of histone variant deposition. In addition, current reports describe ATRX to be mutated at high percentages in multiple tumor types, suggestive of a potential 'driver' role in cancer. Here we discuss the numerous and seemingly diverse roles for ATRX in transcriptional regulation and histone deposition and suggest that ATRX's effects are mediated by its regulation of histones within the chromatin template.

  14. The chromatin remodeller ATRX: a repeat offender in human disease.

    Science.gov (United States)

    Clynes, David; Higgs, Douglas R; Gibbons, Richard J

    2013-09-01

    The regulation of chromatin structure is of paramount importance for a variety of fundamental nuclear processes, including gene expression, DNA repair, replication, and recombination. The ATP-dependent chromatin-remodelling factor ATRX (α thalassaemia/mental retardation X-linked) has emerged as a key player in each of these processes. Exciting recent developments suggest that ATRX plays a variety of key roles at tandem repeat sequences within the genome, including the deposition of a histone variant, prevention of replication fork stalling, and the suppression of a homologous recombination-based pathway of telomere maintenance. Here, we provide a mechanistic overview of the role of ATRX in each of these processes, and propose how they may be connected to give rise to seemingly disparate human diseases.

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

    Science.gov (United States)

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

    1978-05-01

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

  16. Defining the budding yeast chromatin-associated interactome

    OpenAIRE

    Lambert, Jean-Philippe; Fillingham, Jeffrey; Siahbazi, Mojgan; Greenblatt, Jack; Baetz, Kristin; Figeys, Daniel

    2010-01-01

    The maintenance of cellular fitness requires living organisms to integrate multiple signals into coordinated outputs. Central to this process is the regulation of the expression of the genetic information encoded into DNA. As a result, there are numerous constraints imposed on gene expression. The access to DNA is restricted by the formation of nucleosomes, in which DNA is wrapped around histone octamers to form chromatin wherein the volume of DNA is considerably reduced. As such, nucleosome ...

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

    OpenAIRE

    Ding, Bo; Wang, Guo-Liang

    2015-01-01

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

  18. Chromatin Memory in the Development of Human Cancers

    OpenAIRE

    Yao, Yixin; Des Marais, Thomas L; Costa, Max

    2014-01-01

    Cancer is a complex disease with acquired genomic and epigenomic alterations that affect cell proliferation, viability and invasiveness. Almost all the epigenetic mechanisms including cytosine methylation and hydroxymethylation, chromatin remodeling and non-coding RNAs have been found associate with carcinogenesis and cancer specific expression profile. Altered histone modification as an epigenetic hallmark is frequently found in tumors. Understanding the epigenetic alterations induced by car...

  19. ATRX: The case of a peculiar chromatin remodeler

    OpenAIRE

    Ratnakumar, Kajan; Bernstein, Emily

    2013-01-01

    The SWI/SNF-like chromatin remodeler ATRX has recently garnered renewed attention. ATRX mutations were first identified in patients bearing the syndrome after which it is named, alpha thalassemia/mental retardation, X-linked. While ATRX has long been implicated in transcriptional regulation through multiple mechanisms, recent studies have identified a role for ATRX in the regulation of histone variant deposition. In addition, current reports describe ATRX to be mutated at high percentages in ...

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

    Science.gov (United States)

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

    2015-01-01

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

  1. ATM and KAT5 safeguard replicating chromatin against formaldehyde damage.

    Science.gov (United States)

    Ortega-Atienza, Sara; Wong, Victor C; DeLoughery, Zachary; Luczak, Michal W; Zhitkovich, Anatoly

    2016-01-08

    Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA.

  2. Predicting chromatin architecture from models of polymer physics.

    Science.gov (United States)

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

    2017-01-09

    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.

  3. Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses.

    Science.gov (United States)

    Teste, Bruno; Champ, Jerome; Londono-Vallejo, Arturo; Descroix, Stéphanie; Malaquin, Laurent; Viovy, Jean-Louis; Draskovic, Irena; Mottet, Guillaume

    2017-01-31

    Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.

  4. Signaling to the circadian clock: plasticity by chromatin remodeling.

    Science.gov (United States)

    Nakahata, Yasukazu; Grimaldi, Benedetto; Sahar, Saurabh; Hirayama, Jun; Sassone-Corsi, Paolo

    2007-04-01

    Circadian rhythms govern several fundamental physiological functions in almost all organisms, from prokaryotes to humans. The circadian clocks are intrinsic time-tracking systems with which organisms can anticipate environmental changes and adapt to the appropriate time of day. In mammals, circadian rhythms are generated in pacemaker neurons within the suprachiasmatic nuclei (SCN), a small area of the hypothalamus, and are entrained by environmental cues, principally light. Disruption of these rhythms can profoundly influence human health, being linked to depression, insomnia, jet lag, coronary heart disease and a variety of neurodegenerative disorders. It is now well established that circadian clocks operate via transcriptional feedback autoregulatory loops that involve the products of circadian clock genes. Furthermore, peripheral tissues also contain independent clocks, whose oscillatory function is orchestrated by the SCN. The complex program of gene expression that characterizes circadian physiology involves dynamic changes in chromatin transitions. These remodeling events are therefore of great importance to ensure the proper timing and extent of circadian regulation. How signaling influences chromatin remodeling through histone modifications is therefore highly relevant in the context of circadian oscillation. Recent advances in the field have revealed unexpected links between circadian regulators, chromatin remodeling and cellular metabolism.

  5. Balancing chromatin remodeling and histone modifications in transcription.

    Science.gov (United States)

    Petty, Emily; Pillus, Lorraine

    2013-11-01

    Chromatin remodelers use the energy of ATP hydrolysis to reposition or evict nucleosomes or to replace canonical histones with histone variants. By regulating nucleosome dynamics, remodelers gate access to the underlying DNA for replication, repair, and transcription. Nucleosomes are subject to extensive post-translational modifications that can recruit regulatory proteins or alter the local chromatin structure. Just as extensive crosstalk has been observed between different histone post-translational modifications, there is growing evidence for both coordinated and antagonistic functional relations between nucleosome remodeling and modifying machineries. Defining the combined functions of the complexes that alter nucleosome interactions, position, and stability is key to understanding processes that require access to DNA, particularly with growing appreciation of their contributions to human health and disease. Here, we highlight recent advances in the interactions between histone modifications and the imitation-switch (ISWI) and chromodomain helicase DNA-binding protein 1 (CHD1) chromatin remodelers from studies in budding yeast, fission yeast, flies, and mammalian cells, with a focus on yeast.

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

    Science.gov (United States)

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

    1996-04-15

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

  7. Biphasic Chromatin Structure and FISH Signals Reflect Intranuclear Order

    Directory of Open Access Journals (Sweden)

    Jyoti P. Chaudhuri

    2005-01-01

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

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

    Science.gov (United States)

    Ausió, Juan

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Antonio Rodríguez-Campos

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

  10. The linkage of chromatin remodeling to genome maintenance: contribution from a human disease gene BRIT1/MCPH1

    OpenAIRE

    Peng, Guang; Lin, Shiaw-Yih

    2009-01-01

    Genomic DNA is packed into a highly condensed chromatin structure, which acts as natural barrier preventing accessibility of DNA. In various processes to maintain genomic integrity such as DNA replication, DNA repair, telomere regulation, proteins need to overcome the barrier of condensed chromatin to gain access to DNA. ATP-dependent chromatin remodeling is one of the fundamental mechanisms used by cells to relax chromatin. However, the chromatin remodeling complex does not contain intrinsic...

  11. Why is chromosome segregation error in oocytes increased with maternal aging?

    Science.gov (United States)

    Wang, Zhen-Bo; Schatten, Heide; Sun, Qing-Yuan

    2011-10-01

    It is well documented that female fertility is decreased with advanced maternal age due to chromosome abnormality in oocytes. Increased chromosome missegregation is mainly caused by centromeric cohesion reduction. Other factors such as weakened homologous recombination, improper spindle organization, spindle assembly checkpoint (SAC) malfunction, chromatin epigenetic changes, and extra-oocyte factors may also cause chromosome errors.

  12. Probe tip heating assembly

    Science.gov (United States)

    Schmitz, Roger William; Oh, Yunje

    2016-10-25

    A heating assembly configured for use in mechanical testing at a scale of microns or less. The heating assembly includes a probe tip assembly configured for coupling with a transducer of the mechanical testing system. The probe tip assembly includes a probe tip heater system having a heating element, a probe tip coupled with the probe tip heater system, and a heater socket assembly. The heater socket assembly, in one example, includes a yoke and a heater interface that form a socket within the heater socket assembly. The probe tip heater system, coupled with the probe tip, is slidably received and clamped within the socket.

  13. Lack of independent prognostic and predictive value of centromere 17 copy number changes in breast cancer patients with known HER2 and TOP2A status

    DEFF Research Database (Denmark)

    Nielsen, Kirsten Vang; Ejlertsen, Bent; Møller, Susanne

    2011-01-01

    The clinical benefit of anthracyclines has been connected to HER2 status, TOP2A status and centromere 17 copy numbers (CEN-17). Data from a clinical trial randomizing patients to anthracyclines was used to assess whether the number of CEN-17 in breast cancers may predict incremental responsiveness...... to anthracyclines besides what is obtained when used relatively to TOP2A and HER2. As cut sections of paraffin-embedded tissue are prone to truncation of nuclei, strict definition of ploidy levels is lacking. We therefore used normal breast tissue to assist define ploidy levels in cut sections. Fluorescence in situ...... hybridization (FISH) with centromere 17 (CEN-17) and TOP2A was performed on 120 normal breast specimens. The diploid CEN-17 copy number was reduced from the expected two signals in whole nuclei to an average of 1.68 signals per nucleus in cut sections of normal breast. Ploidy levels determined in normal breast...

  14. Newnes electronics assembly handbook

    CERN Document Server

    Brindley, Keith

    2013-01-01

    Newnes Electronics Assembly Handbook: Techniques, Standards and Quality Assurance focuses on the aspects of electronic assembling. The handbook first looks at the printed circuit board (PCB). Base materials, basic mechanical properties, cleaning of assemblies, design, and PCB manufacturing processes are then explained. The text also discusses surface mounted assemblies and packaging of electromechanical assemblies, as well as the soldering process. Requirements for the soldering process; solderability and protective coatings; cleaning of PCBs; and mass solder/component reflow soldering are des

  15. Complexity of chromatin folding is captured by the strings and binders switch model.

    Science.gov (United States)

    Barbieri, Mariano; Chotalia, Mita; Fraser, James; Lavitas, Liron-Mark; Dostie, Josée; Pombo, Ana; Nicodemi, Mario

    2012-10-02

    Chromatin has a complex spatial organization in the cell nucleus that serves vital functional purposes. A variety of chromatin folding conformations has been detected by single-cell imaging and chromosome conformation capture-based approaches. However, a unified quantitative framework describing spatial chromatin organization is still lacking. Here, we explore the "strings and binders switch" model to explain the origin and variety of chromatin behaviors that coexist and dynamically change within living cells. This simple polymer model recapitulates the scaling properties of chromatin folding reported experimentally in different cellular systems, the fractal state of chromatin, the processes of domain formation, and looping out. Additionally, the strings and binders switch model reproduces the recently proposed "fractal-globule" model, but only as one of many possible transient conformations.

  16. Probing flagellar promoter occupancy in wild-type and mutant Caulobacter crescentus by chromatin immunoprecipitation.

    Science.gov (United States)

    Davis, Nicole J; Viollier, Patrick H

    2011-06-01

    In the asymmetric predivisional cell of Caulobacter crescentus, TipF and TipN mark the cellular pole for future flagellar development. TipF is essential for motility and contains a cyclic-di-GMP phosphodiesterase-like (EAL) domain that is necessary for proper function. TipN is localized to the flagellar pole before TipF and is essential for the proper placement of the flagellum in C. crescentus. Using β-galactosidase promoter-probe assays and quantitative chromatin immunoprecipitation, we investigated the influence of the C. crescentus flagellar assembly regulator TipF on flagellar gene transcription. We compared the transcriptional activity of class II-fliF-lacZ, class III-flgE-lacZ, and class IV-fljL-lacZ fusions in a ΔtipF mutant with that of other flagellar mutants and the wild-type strain. We subsequently verified the in vivo occupancy of the fliF, flgE, and fljL flagellar promoters by the flagellar regulators CtrA, FlbD, and FliX in addition to RNA polymerase. We deduce that TipF contributes to proper expression of flagellar genes in C. crescentus by acting both within and outside of the canonical flagellar gene expression hierarchy.

  17. Molecular architecture of the ATP-dependent chromatin-remodeling complex SWR1.

    Science.gov (United States)

    Nguyen, Vu Q; Ranjan, Anand; Stengel, Florian; Wei, Debbie; Aebersold, Ruedi; Wu, Carl; Leschziner, Andres E

    2013-09-12

    The ATP-dependent chromatin-remodeling complex SWR1 exchanges a variant histone H2A.Z/H2B dimer for a canonical H2A/H2B dimer at nucleosomes flanking histone-depleted regions, such as promoters. This localization of H2A.Z is conserved throughout eukaryotes. SWR1 is a 1 megadalton complex containing 14 different polypeptides, including the AAA+ ATPases Rvb1 and Rvb2. Using electron microscopy, we obtained the three-dimensional structure of SWR1 and mapped its major functional components. Our data show that SWR1 contains a single heterohexameric Rvb1/Rvb2 ring that, together with the catalytic subunit Swr1, brackets two independently assembled multisubunit modules. We also show that SWR1 undergoes a large conformational change upon engaging a limited region of the nucleosome core particle. Our work suggests an important structural role for the Rvbs and a distinct substrate-handling mode by SWR1, thereby providing a structural framework for understanding the complex dimer-exchange reaction.

  18. A stable acentric marker chromosome derived from distal 8p: Reactivation of a latent ancient centromere at 8p23.1?

    Energy Technology Data Exchange (ETDEWEB)

    Ohashi, H.; Wakui, K.; Ogawa, K. [Saitama Children`s Medical Ctr., Iwatsuki (Japan)] [and others

    1994-09-01

    Centromere is considered to be an essential chromosomal component for faithful segregation, and acentric chromosomes are unstable and lost through cell divisions. We report a novel marker chromosome that was acentric but stable through cell divisions. The patient was a 2-year-old girl with mental retardation, patent ductus arteriosus and mild dysmorphic features. G-banded chromosome analysis revealed an additional small marker chromosome in all 100 cells examined. Using the targeted chromosome-band painting method, the marker was found to originate from the distal region of 8p, and subsequent two color FISH analysis with cosmid probes around the region revealed the marker was a rearranged chromosome interpreted as 8pter{r_arrow}p23.1{r_arrow}8pter. No centromeric region was involved in the marker. By FISH, no {alpha}-satellite sequence was detected on the marker, while telomere sequence was detected at each end. Antikinetochore immunostaining using a serum from a patient with CREST syndrome showed a pair of signals on the marker, which indicated that a functional kinetochore was present on the marker, presumably at 8p23.1-corresponding region. The patient may provide evidence that an ancient centromere sequence exists at 8p23.1 and was reactivated through the chromosome rearrangement in the patient.

  19. Preferential recruitment of the maternal centromere-specific histone H3 (CENH3) in oat (Avena sativa L.) × pearl millet (Pennisetum glaucum L.) hybrid embryos.

    Science.gov (United States)

    Ishii, Takayoshi; Sunamura, Naohiro; Matsumoto, Ayaka; Eltayeb, Amin Elsadig; Tsujimoto, Hisashi

    2015-12-01

    Chromosome elimination occurs frequently in interspecific hybrids between distantly related species in Poaceae. However, chromosomes from both parents behave stably in a hybrid of female oat (Avena sativa L.) pollinated by pearl millet (Pennisetum glaucum L.). To analyze the chromosome behavior in this hybrid, we cloned the centromere-specific histone H3 (CENH3) genes of oat and pearl millet and produced a pearl millet-specific anti-CENH3 antibody. Application of this antibody together with a grass species common anti-CENH3 antibody revealed the dynamic CENH3 composition of the hybrid cells before and after fertilization. Despite co-expression of CENH3 genes encoded by oat and pearl millet, only an oat-type CENH3 was incorporated into the centromeres of both species in the hybrid embryo. Oat CENH3 enables a functional centromere in pearl millet chromosomes in an oat genetic background. Comparison of CENH3 genes among Poaceae species that show chromosome elimination in interspecific hybrids revealed that the loop 1 regions of oat and pearl millet CENH3 exhibit exceptionally high similarity.

  20. Phosphorylation of the chromatin binding domain of KSHV LANA.

    Directory of Open Access Journals (Sweden)

    Crystal Woodard

    Full Text Available The Kaposi sarcoma associated herpesvirus (KSHV latency associated nuclear antigen (LANA is expressed in all KSHV associated malignancies and is essential for maintenance of KSHV genomes in infected cells. To identify kinases that are potentially capable of modifying LANA, in vitro phosphorylation assays were performed using an Epstein Barr virus plus LANA protein microarray and 268 human kinases purified in active form from yeast. Interestingly, of the Epstein-Barr virus proteins on the array, the EBNA1 protein had the most similar kinase profile to LANA. We focused on nuclear kinases and on the N-terminus of LANA (amino acids 1-329 that contains the LANA chromatin binding domain. Sixty-three nuclear kinases phosphorylated the LANA N-terminus. Twenty-four nuclear kinases phosphorylated a peptide covering the LANA chromatin binding domain (amino acids 3-21. Alanine mutations of serine 10 and threonine 14 abolish or severely diminish chromatin and histone binding by LANA. However, conversion of these residues to the phosphomimetic glutamic acid restored histone binding suggesting that phosphorylation of serine 10 and threonine 14 may modulate LANA function. Serine 10 and threonine 14 were validated as substrates of casein kinase 1, PIM1, GSK-3 and RSK3 kinases. Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B. Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability. The data indicate that RSK phosphorylation affects both LANA accumulation and function.