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1

Osmotic challenge drives rapid and reversible chromatin condensation in chondrocytes.  

PubMed

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

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

2013-02-19

2

Premature chromatin condensation upon accumulation of NIMA.  

PubMed Central

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

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

1994-01-01

3

Chromatin condensation modulates access and binding of nuclear proteins  

PubMed Central

The condensation level of chromatin is controlled by epigenetic modifications and associated regulatory factors and changes throughout differentiation and cell cycle progression. To test whether changes of chromatin condensation levels per se affect access and binding of proteins, we used a hypertonic cell treatment. This shift to hyperosmolar medium increased nuclear calcium concentrations and induced a reversible chromatin condensation comparable to the levels in mitosis. However, this condensation was independent of mitotic histone H3 serine 10 phosphorylation. Photobleaching and photoactivation experiments with chromatin proteins—histone H2B-GFP and GFP-HP1?—before and after induced chromatin condensation demonstrated that hypercondensation reduced their dissociation rate and stabilized their chromatin binding. Finally, measuring the distribution of nucleoplasmic proteins in the size range from 30 to 230 kDa, we found that even relatively small proteins like GFP were excluded from highly condensed chromatin in living cells. These results suggest that structural changes in condensed chromatin by themselves affect chromatin access and binding of chromatin proteins independent of regulatory histone modifications.—Martin, R. M., Cardoso, M. C. Chromatin condensation modulates access and binding of nuclear proteins.

Martin, Robert M.; Cardoso, M. Cristina

2010-01-01

4

Sperm chromatin structure assay (SCSA®).  

PubMed

The SCSA(®) is the pioneering assay for the detection of damaged sperm DNA and altered proteins in sperm nuclei via flow cytometry of acridine orange (AO) stained sperm. The SCSA(®) is considered to be the most precise and repeatable test providing very unique, dual parameter data (red vs. green fluorescence) on a 1,024 × 1,024 channel scale, not only on DNA fragmentation but also on abnormal sperm characterized by lack of normal exchange of histones to protamines. Raw semen/sperm aliquots or purified sperm can be flash frozen, placed in a box with dry ice and shipped by overnight courier to an experienced SCSA(®) lab. The samples are individually thawed, prepared, and analyzed in ?10 min. Of significance, data on 5,000 individual sperm are recorded on a 1,024 × 1,024 dot plot of green (native DNA) and red (broken DNA) fluorescence. Repeat measurements have virtually identical dot plot patterns demonstrating that the low pH treatment that opens up the DNA strands at the sites of breaks and staining by acridine orange (AO) are highly precise and repeatable (CVs of 1-3%) and the same between fresh and frozen samples. SCSAsoft(®) software transforms the X-Y data to total DNA stainability versus red/red + green fluoresence (DFI) providing a more accurate determination of % DFI as well as the more sensitive value of standard deviation of DFI (SD DFI) as demonstrated by animal fertility and dose-response toxicology studies. The current established clinical threshold is 25% DFI for placing a man into a statistical probability of the following: (a) longer time to natural pregnancy, (b) low odds of IUI pregnancy, (c) more miscarriages, or (d) no pregnancy. Changes in lifestyle as well as medical intervention can lower the %DFI to increase the probability of natural pregnancy. Couples of men with >25% DFI are counseled to try ICSI and when in the >50% range may consider TESE/ICSI. The SCSA(®) simultaneously determines the % of sperm with high DNA stainability (%HDS) related to retained nuclear histones consistent with immature sperm; high HDS values are predictive of pregnancy failure.The SCSA(®) is considered to be the most technician friendly, time- and cost-efficient, precise and repeatable DNA fragmentation assay, with the most data and the only fragmentation assay with an accepted clinical threshold for placing a man at risk for infertility. SCSA(®) data are more predictive of male factor infertility than classical semen analyses. PMID:22992911

Evenson, Donald P

2013-01-01

5

Condensed chromatin domains in the mammalian nucleus are accessible to large macromolecules  

Microsoft Academic Search

Most chromatin in interphase nuclei is part of condensed chromatin domains. Previous work has indicated that transcription takes place predominantly at the surface of chromatin domains, that is, in the perichromatin region. It is possible that genes inside chromatin domains are silenced due to inaccessibility to macromolecular components of the transcription machinery. We have tested the accessibility of chromatin domains

Ineke van der Kraan; Erik M. M. Manders; Deborah Hoogstraten; Adriaan B. Houtsmuller; Roel van Driel; Pernette J. Verschure

2003-01-01

6

Chromatin immunoprecipitation assay of piwi in Drosophila.  

PubMed

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

Yin, Hang; Lin, Haifan

2014-01-01

7

Formation of mammalian erythrocytes: Chromatin condensation and enucleation  

PubMed Central

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

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

2011-01-01

8

Transcriptional Activation: Getting a Grip on Condensed Chromatin  

Microsoft Academic Search

Chromosomal transcription involves the concerted action of enormous, multisubunit chromatin remodeling complexes. A recent study, however, suggests a different and surprising viewpoint in which these protein assemblies are quite dynamic and individual subunits play key roles in chromatin remodeling.

Craig L. Peterson

2003-01-01

9

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

PubMed Central

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

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

2006-01-01

10

The role of histone H1 in chromatin condensation and transcriptional repression.  

PubMed

Linker histones are a major determinant of chromatin condensation. We discuss here the nature and position of the interaction of the globular domain of histone H5 with the core nucleosome and the relevance of this positioning to chromatin structure and the regulation of transcription of the Xenopus borealis 5S rRNA genes. PMID:10710717

Buttinelli, M; Panetta, G; Rhodes, D; Travers, A

1999-01-01

11

Possible mechanisms for early and intermediate stages of sperm chromatin condensation patterning involving phase separation dynamics  

Microsoft Academic Search

During spermiogenesis in some internally fertilizing molluscs and insects, the post-meiotic spermatid nucleus develops via a sequence of complex patterns of the nuclear contents (chromatin and nucleoplasm) on the way to final chromatin condensation. We have examined the TEM data on these sequences for three species: Philaenus spumarius (a homopteran insect), Murex brandaris (a gastropod mollusc), and Eledone cirrhosa (a

Lionel G. Harrison; Harold E. Kasinsky; Enric Ribes; Manel Chiva

2005-01-01

12

Identification of Mycobacterial ? Factor Binding Sites by Chromatin Immunoprecipitation Assays?  

PubMed Central

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for infections that cause a substantial amount of death, suffering, and loss around the world. Still, relatively little is known about the mechanisms of gene expression in these bacteria. Here, we used genome-wide location assays to identify direct target genes for mycobacterial ? factors. Chromatin immunoprecipitation assays were performed with M. bovis BCG for Myc-tagged proteins expressed using an anhydrotetracycline-inducible promoter, and enriched DNA fragments were hybridized to a microarray representing intergenic regions from the M. tuberculosis H37Rv genome. Several putative target genes were validated by quantitative PCR. The corresponding transcriptional start sites were identified for ?F, ?C, and ?K, and consensus promoter sequences are proposed. Our conclusions were supported by the results of in vitro transcription assays. We also examined the role of each holoenzyme in the expression of ? factor genes. Our results revealed that many ? factors are expressed from autoregulated promoters.

Rodrigue, Sebastien; Brodeur, Joelle; Jacques, Pierre-Etienne; Gervais, Alain L.; Brzezinski, Ryszard; Gaudreau, Luc

2007-01-01

13

Structure of the H1 C-terminal domain and function in chromatin condensation1  

PubMed Central

Linker histones are multifunctional proteins that are involved in a myriad of processes ranging from stabilizing the folding and condensation of chromatin to playing a direct role in regulating gene expression. However, how this class of enigmatic proteins binds in chromatin and accomplishes these functions remains unclear. Here we review data regarding the H1 structure and function in chromatin, with special emphasis on the C-terminal domain (CTD), which typically encompasses approximately half of the mass of the linker histone and includes a large excess of positively charged residues. Owing to its amino acid composition, the CTD was previously proposed to function in chromatin as an unstructured polycation. However, structural studies have shown that the CTD adopts detectable secondary structure when interacting with DNA and macromolecular crowding agents. We describe classic and recent experiments defining the function of this domain in chromatin folding and emerging data indicating that the function of this protein may be linked to intrinsic disorder.

Caterino, Tamara L.; Hayes, Jeffrey J.

2013-01-01

14

Condensed chromatin in diploid and allopolyploid microseris species with different genome size: a quantitative electron microscopic study  

Microsoft Academic Search

The species-specific proportion of chromatin in the condensed state was estimated by quantitative electron microscopic morphometry of nuclear sections in 9 diploid and 5 allopolyploid species of Microseris (Asteraceae). A positive correlation between the genome size (haploid DNA content, or C value) and the percentage of chromatin in the condensed state (as visible in ultrathin sections) was found in diploids

W. Nagl; K. Bachmann

1980-01-01

15

ATR inhibition selectively sensitizes G1 checkpoint-deficient cells to lethal premature chromatin condensation  

Microsoft Academic Search

Premature chromatin condensation (PCC) is a hallmark of mammalian cells that begin mitosis before completing DNA replication. This lethal event is prevented by a highly conserved checkpoint involving an unknown, caffeine-sensitive mediator. Here, we have examined the possible involvement of the caffeine-sensitive ATM and ATR protein kinases in this checkpoint. We show that caffeine's ability to inhibit ATR (but not

Paul Nghiem; Peter K. Park; Yong-Son Kim; Cyrus Vaziri; Stuart L. Schreiber

2001-01-01

16

Proposed mechanism for sperm chromatin condensation\\/decondensation in the male rat  

Microsoft Academic Search

Condensation of sperm chromatin occurs after spermatozoa have left the caput epididymis and are in transit to the cauda epididymis, during which time large numbers of disulfide bonds are formed. The formation of these disulfide bonds requires the repeated oxidation of the cofactor, NAD(P)H. To date, the means by which this oxidation is achieved has yet to be elucidated. Spermatozoa

John C Chapman; Sandra D Michael

2003-01-01

17

Feulgen-DNA response and chromatin condensation in Malpighian tubules of Melipona rufiventris and Melipona quadrifasciata (Hymenoptera, Apoidea).  

PubMed

Melipona quadrifasciata and Melipona rufiventris are stingless bee species which present low and high heterochromatin content, respectively, on their mitotic chromosomes as assessed visually after a C-banding assay. However, these species do not show differences in the C-banding responses of their Malpighian tubule interphase nuclei. In the present study, the Feulgen-DNA response, which could inform on differences in DNA depurination due to differences in chromatin condensation, was compared in the cell nuclei of the Malpighian tubules of these species. It was hypothesized that differences in acid hydrolysis kinetics patterns, as assessed by Feulgen reaction and studied microspectrophotometrically, could discriminate M. quadrifasciata and M. rufiventris interphase nuclei not distinguishable with the C-banding method. Feulgen-DNA values corresponding to more than one ploidy class were found in both species; these values at the hydrolysis time corresponding to the maximal DNA depurination for each ploidy degree were higher in M. quadrifasciata, reflecting a higher DNA content in the Malpighian tubule cell nuclei of this species compared to those of M. rufiventris at the same larval instar. The maximal Feulgen-DNA values of M. quadrifasciata after short (50 min) and long (90 min) hydrolysis times were found to be closer to each other, while those of M. rufiventris occurred sharply at the long hydrolysis time, indicating that DNA depurination in M. quadrifasciata occurred faster. This result is probably related to the involvement of differences in chromatin condensation; it agrees with the idea that M. rufiventris contains more heterochromatin than M. quadrifasciata, which is supported by the analysis of results obtained with the image analysis parameter average absorption ratio. The depurination kinetics studied here with the Feulgen reaction were revealed to be more pertinent than the C-banding technique in establishing differences in levels of chromatin condensation for these cell nuclei. PMID:18474437

Mampumbu, André Roberto; Mello, Maria Luiza S

2008-04-10

18

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

SciTech Connect

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

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

2008-08-21

19

Nuclear chromatin variations in human spermatozoa undergoing swim-up and cryopreservation evaluated by the flow cytometric sperm chromatin structure assay  

Microsoft Academic Search

The sperm chromatin structure assay (SCSA) is a flow cytometric (FCM) technique which exploits the metachromatic properties of Acridine Orange to monitor the susceptibility of sperm chromatin DNA to in- situ acid denaturation. SCSA was used to study the chromatin structure variations of human spermatozoa in semen, both before and after swim-up and after cryopreservation. Semen samples were provided by

E. Cordelli; G. Leter; F. Lombardo; A. Lenzi; L. Gandini

1999-01-01

20

Condensed chromatin surface and NORs surface enhancement in mitogen-stimulated lymphocytes of Down syndrome patients  

Microsoft Academic Search

Mitogen-stimulated lymphocytes of 20 Down syndrome (DS) patients with regular trisomy 21 contain more condensed chromatin surface (11.28 ± 2.64 % of the total nuclear surface: mean ± SD) and more nucleolus organiser regions surface (13.21 ± 3.45 %) than that of 12 healthy controls: (8.84 ± 2.23 and 9.12 ± 2.33 %, reciprocally). The source of this peculiarity has been investigated. A computer program was designed for the planimetric measurement of the

Halil Demirtas; Nalan Imamoglu; Hamiyet Dönmez; Nurhan Cücer; Alpaslan Yilmaz; Zühal Candemir

2001-01-01

21

Histone H1 Subtypes Differentially Modulate Chromatin Condensation without Preventing ATP-Dependent Remodeling by SWI/SNF or NURF  

PubMed Central

Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.

Clausell, Jaime; Happel, Nicole; Hale, Tracy K.; Doenecke, Detlef; Beato, Miguel

2009-01-01

22

Non-uniform chromatin condensation on chromosomes: Comparison of different loci by two-color FISH  

SciTech Connect

Models for higher-order folding of the chromatin fiber have been proposed. To date, however, many questions are still unanswered concerning the specificity of chromatin condensation along the entire genome. With the most advanced molecular cytogenetic techniques, we could gather data to help elucidate some aspects of the structural organization of chromosomes by analyzing the resolution limit of adjacent probes at different loci. To study this limit of resolution, we have used two-color FISH to determine the minimal distance at which two probes are individualized. We used probes of known molecular distances on the following loci: the dystrophin gene on Xp21, the HLA locus on 6p21, the RB1 on 13q14 and the HSTF1 and INT2 genes on 11q13. For the dystrophin gene, the limit of resolution was found to be 250 kb, for the HLA locus, 150 kb, for HSTF1 and INT2 on 11q13, 40 kb, and for RB1, 30 kb. There was a large difference in resolution between the loci studied. These results show that condensation does not affect chromatin equally in every loci. The non-uniform compaction of chromatin within loci on chromosomes could be related to the different activities of those loci. The smallest limits of resolution that we observed were 30 kb for the RB1 gene and 40 kb for the HSTF1 and INT2 genes. These distances are much smaller than expected, suggesting that these genes have a particularly decondensed structure. Since these genes are known to be very active, these results can also be interpreted to show the relationship between greater activity and smaller condensation.

Tihy, F.; Fetni, r.; Lemieux, N. [Universite de Montreal, Quebec (Canada)] [and others

1994-09-01

23

In Vivo HP1 Targeting Causes Large-Scale Chromatin Condensation and Enhanced Histone Lysine Methylation†  

PubMed Central

Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) ? (HP1?) and HP1? on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1? and HP1? in vivo targeting. HP1? targeting causes the recruitment of endogenous HP1? to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1? and HP1? targeting is sufficient to induce heterochromatin formation.

Verschure, Pernette J.; van der Kraan, Ineke; de Leeuw, Wim; van der Vlag, Johan; Carpenter, Anne E.; Belmont, Andrew S.; van Driel, Roel

2005-01-01

24

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

EPA Science Inventory

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

25

Proposed mechanism for sperm chromatin condensation/decondensation in the male rat  

PubMed Central

Condensation of sperm chromatin occurs after spermatozoa have left the caput epididymis and are in transit to the cauda epididymis, during which time large numbers of disulfide bonds are formed. The formation of these disulfide bonds requires the repeated oxidation of the cofactor, NAD(P)H. To date, the means by which this oxidation is achieved has yet to be elucidated. Spermatozoa lose the bulk of their cytoplasm prior to leaving the testis; and, as a result, any shuttle systems for removing and transferring reducing equivalents into the mitochondria are unlikely to be operational. In an apparent preparation for the loss of cytoplasm, however, the following events occur during spermatogenesis. First, androgen-binding protein (ABP) is produced by the Sertoli cells of the testis; second, high affinity binding sites for ABP are inserted into the membrane surrounding the nucleus; and third, a nuclear location is acquired for the enzyme, 3?-hydroxysteroid dehydrogenase (3?-HSD). We propose that after the loss of cytoplasm, the nuclear region of spermatozoa is directly accessible to constituents contained in the lumen of the caput epididymis. As a consequence, luminal ABP attaches itself to the nuclear membrane via its binding sites, and is internalized. After internalization, ABP exerts its principle function, which is to bind to luminal 5?-dihydrotestosterone (5?-DHT), thereby ensuring its availability to the enzyme, 3?-HSD. In the conversion of 5?-DHT to 3?-androstanediol (3?-Diol), NAD(P)H is oxidized. Spermatozoa that reach the cauda epididymis have fully condensed chromatin. In addition, the nuclear region retains appreciable amounts of 5?-DHT and 3?-Diol, both bound to ABP. During fertilization, the bound 3?-Diol is converted back to 5?-DHT, reducing equivalents are transferred to NAD(P)+, and disulfide bonds are broken. IVF clinics report that spermatozoa with incompletely condensed chromatin have a low percentage of fertilization. If our proposed mechanism for chromatin condensation/decondensation is borne out by further research, IVF clinics might consider preincubating spermatozoa with 5?-DHT in order to increase the efficiency of fertilization.

Chapman, John C; Michael, Sandra D

2003-01-01

26

Linker histones are not essential and affect chromatin condensation in vivo.  

PubMed

We have (separately) disrupted all of the expressed macronuclear copies of the HHO gene encoding macronuclear histone H1 and of the micronuclear linker histone (MLH) gene encoding the protein MicLH in Tetrahymena thermophila. These disruptions are shown to eliminate completely the expression of each protein. Strains without either linker histone grow at normal rates and reach near-normal cell densities, demonstrating that linker histones are not essential for cell survival. Histone H1 knockout (delta H1) cells have enlarged DAPI-stained macronuclei and normal-sized micronuclei, while MicLH knockout (delta MicLH) cells have enlarged micronuclei and normal-sized macronuclei. delta MicLH cells undergo mitosis normally. However, the micronuclear mitotic chromosome structure is less condensed. These studies provide evidence that linker histones are nonessential and are involved in chromatin packaging and condensation in vivo. PMID:7606784

Shen, X; Yu, L; Weir, J W; Gorovsky, M A

1995-07-14

27

Possible mechanisms for early and intermediate stages of sperm chromatin condensation patterning involving phase separation dynamics.  

PubMed

During spermiogenesis in some internally fertilizing molluscs and insects, the post-meiotic spermatid nucleus develops via a sequence of complex patterns of the nuclear contents (chromatin and nucleoplasm) on the way to final chromatin condensation. We have examined the TEM data on these sequences for three species: Philaenus spumarius(a homopteran insect), Murex brandaris (a gastropod mollusc), and Eledone cirrhosa(a cephalopod mollusc). For each of these, spatially quantitative study reveals a constant spacing between pattern repeats through changes from granular to fibrillar to lamellar pattern, followed finally by a shrinkage of the spacing. Therefore we distinguish a "patterning" stage followed by a "condensation" stage. The former appears to demand a dynamic explanation, because there is no sign of structural connections to establish the part of the spacing that crosses the nucleoplasm. We consider types of dynamic mechanism, and show that for "nanostructural" dimensions (tens of nanometers as pattern spacing) reaction-diffusion dynamics are quite inappropriate, but that separation of two fluid phases by a mechanism similar to what is known as "spinodal decomposition" is a very attractive possibility. PMID:15612004

Harrison, Lionel G; Kasinsky, Harold E; Ribes, Enric; Chiva, Manel

2005-01-01

28

Evaluation of Myc E-Box Phylogenetic Footprints in Glycolytic Genes by Chromatin Immunoprecipitation Assays  

Microsoft Academic Search

Prediction of gene regulatory sequences using phylogenetic footprinting has advanced considerably but lacks experimental validation. Here, we report whether transcription factor binding sites predicted by dot plotting or web-based Trafac analysis could be validated by chromatin immunoprecipitation assays. MYC overexpres- sion enhances glycolysis without hypoxia and hence may contribute to altered tumor metabolism. Because the full spectrum of glycolytic genes

Jung-whan Kim; Karen I. Zeller; Yunyue Wang; Anil G. Jegga; Bruce J. Aronow; Kathryn A. O'Donnell; Chi V. Dang

2004-01-01

29

Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA ®)  

Microsoft Academic Search

Studies over the past two decades have clearly shown that reproductive toxicants cause sperm DNA fragmentation. This DNA fragmentation can usually be detected prior to observing alterations of metaphase chromosomes in embryos. Thus, Sperm Chromatin Structure Assay (SCSA)-detected DNA damage is viewed as the molecular precursor to later gross chromosome damage observed under the light microscope.SCSA measurements of animal or

Donald P.. Evenson; Regina Wixon

2005-01-01

30

Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase  

Microsoft Academic Search

Chromatin condensation paralleled by DNA fragmentation is one of the most important criteria which are used to identify apoptotic cells. However, comparable changes are also observed in interphase nuclei which have been treated with cell extracts from mitotic cells. In this respect it is known that in mito- sis, the lamina structure is broken down as a result of lamin

Franziska A. Oberhammer; Karin Hochegger; Roman Tiefenbacher; Margit Pavelka

1994-01-01

31

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

SciTech Connect

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

Hittelman, W.N.; Pollard, M.

1984-10-01

32

Caspase-6 gene disruption reveals a requirement for lamin A cleavage in apoptotic chromatin condensation  

PubMed Central

To study the role of caspase-6 during nuclear disassembly, we generated a chicken DT40 cell line in which both alleles of the caspase-6 gene were disrupted. No obvious morphological differences were observed in the apoptotic process in caspase-6- deficient cells compared with the wild type. However, examination of apoptosis in a cell-free system revealed a block in chromatin condensation and apoptotic body formation when nuclei from HeLa cells expressing lamin A or lamin A-transfected Jurkat cells were incubated in caspase-6-deficient apoptotic extracts. Transfection of exogenous caspase-6 into the clone reversed this phenotype. Lamins A and C, which are caspase-6-only substrates, were cleaved by the wild-type and heterozygous apoptotic extracts but not by the extracts lacking caspase-6. Furthermore, the caspase-6 inhibitor z-VEID-fmk mimicked the effects of caspase-6 deficiency and prevented the cleavage of lamin A. Taken together, these observations indicate that caspase-6 activity is essential for lamin A cleavage and that when lamin A is present it must be cleaved in order for the chromosomal DNA to undergo complete condensation during apoptotic execution.

Ruchaud, Sandrine; Korfali, Nadia; Villa, Pascal; Kottke, Timothy J.; Dingwall, Colin; Kaufmann, Scott H.; Earnshaw, William C.

2002-01-01

33

Analysis of Chromatin Composition of Repetitive Sequences: The ChIP-Chop Assay.  

PubMed

Chromatin immunoprecipitation (ChIP) is a powerful method that allows to probe specific protein-DNA interactions in vivo and to estimate the occupancy of proteins at specific sites of the genome. However, the traditional ChIP assay is not able to distinguish whether repeats that share identical sequences display a different composition of associated factors and, consequently, different functions. The ChIP-chop method provides a useful application to analyze the interaction of proteins with repetitive sequences based on their CpG methylation content. The detailed ChIP-chop protocol that serves to determine the chromatin composition of active and silent ribosomal RNA (rRNA) genes, repeats that share identical sequences but display distinct functions and chromatin compositions, is reported here. PMID:24162999

Santoro, Raffaella

2014-01-01

34

Small human sperm vacuoles observed under high magnification are pocket-like nuclear concavities linked to chromatin condensation failure.  

PubMed

Since an embryo's ability to grow to the blastocyst stage and implant can be improved by selection of a normal spermatozoon with a vacuole-free head, this study set out to determine the nature of small sperm vacuoles observed under high magnification (>×6300). For 15 infertile men with various sperm profiles, high-magnification microscopy was used to select motile, morphometrically normal spermatozoa with no vacuoles (n=450) or more than two small vacuoles (each of which occupied less than 4% of the head's area; n=450). Spermatozoa acrosome reaction status and degree of chromatin condensation were analysed. Three-dimensional deconvolution microscopy was used to accurately image the nucleus and acrosome at all depths in all spermatozoa. In all 450 spermatozoa with small vacuoles, the latter were seen to be abnormal, DNA-free nuclear concavities. Spermatozoa with small vacuoles were significantly more likely than vacuole-free spermatozoa to have noncondensed chromatin (39.8% versus 9.3%, respectively; P<0.0001). There was no significant difference between the two groups of spermatozoa in terms of acrosome reaction status. No association between chromatin condensation and acrosome reaction status was observed. Small human sperm vacuoles observed under high magnification are pocket-like nuclear concavities related to failure of chromatin condensation. PMID:23797052

Boitrelle, F; Albert, M; Petit, J-M; Ferfouri, F; Wainer, R; Bergere, M; Bailly, M; Vialard, F; Selva, J

2013-05-16

35

Differential resistance of mammalian sperm chromatin to oxidative stress as assessed by a two-tailed comet assay.  

PubMed

Protamines of eutherian species are cysteine-rich molecules that become cross-linked by disulfide bonds during epididymal transit, whereas the protamines of most marsupial species lack cysteine residuals. The present study made use of the differences in protamine structure between eutherian and metatherian mammal spermatozoa to examine the comparative resistance of sperm DNA to oxidative damage in three eutherian species (Mus musculus, Homo sapiens, Sus domesticus) and three metatherian species (Vombatus ursinus, Phascolarctos cinereus, Macropus giganteus). Sperm DNA fragmentation of samples exposed to increasing concentrations of hydrogen peroxide was assessed by means of the two-tailed comet assay. The sperm DNA of the marsupial species studied were significantly more sensitive to oxidative stress than the spermatozoa of eutherian species. Such susceptibility is consistent with the lack of disulfide cross-linking in marsupial sperm chromatin and suggests that the oxidation of thiols to disulfides for chromatin condensation during epididymal transit in eutherian mammals is likely to be important in order to provide stability and protect these cells from the genotoxic effects of adverse environments. PMID:21635811

Enciso, María; Johnston, Stephen D; Gosálvez, Jaime

2011-01-01

36

Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay  

Microsoft Academic Search

ObjectiveTo investigate how moderate and\\/or high levels of DNA fragmentation (DFI), as measured by the sperm chromatin structure assay (SCSA), affect either IVF or IVF with intracytoplasmic sperm injection (ICSI) fertilization, cleavage, blastulation, implantation, and pregnancy.

Kjersten L Larson-Cook; John D Brannian; Keith A Hansen; Kay M Kasperson; Edward T Aamold; Donald P Evenson

2003-01-01

37

Nuclear growth and chromatin relaxation-condensation cycle in hepatocytes during the proliferative activation of rat liver  

Microsoft Academic Search

Summary  In order to quantify the changes in nucleolar and nuclear volumes and in chromatin condensation produced during proliferative\\u000a activation we have carried out morphometric studies on hepatocyte nuclei during rat liver regeneration using electron microscopy.\\u000a To minimize the artefactual effects produced by fixation on subcellular structures we have fixed the livers by perfusion with\\u000a gluteraldehyde. The mean values for the

Joan Serratosa; Jordi Domingo; Carles Enrich; Oriol Bachs

1988-01-01

38

Immunohistochemical distribution of hyperacetylated histone H4 in testis of paddlefish Polyodon spathula : ultrastructural correlation with chromatin condensation  

Microsoft Academic Search

The acetylation of core histones has been correlated with the deposition of free histones onto newly replicated DNA, transcriptional\\u000a activity and the displacement of histones by protamines during spermiogenesis. The aim of the present study was to investigate\\u000a the immunohistochemical distribution of hyperacetylated H4 during spermatogenesis in Polyodon spathula and to correlate these findings with the pattern of chromatin condensation

Otilia Zarnescu

2007-01-01

39

Inferring chromatin-bound protein complexes from genome-wide binding assays  

PubMed Central

Genome-wide binding assays can determine where individual transcription factors bind in the genome. However, these factors rarely bind chromatin alone, but instead frequently bind to cis-regulatory elements (CREs) together with other factors thus forming protein complexes. Currently there are no integrative analytical approaches that can predict which complexes are formed on chromatin. Here, we describe a computational methodology to systematically capture protein complexes and infer their impact on gene expression. We applied our method to three human cell types, identified thousands of CREs, inferred known and undescribed complexes recruited to these CREs, and determined the role of the complexes as activators or repressors. Importantly, we found that the predicted complexes have a higher number of physical interactions between their members than expected by chance. Our work provides a mechanism for developing hypotheses about gene regulation via binding partners, and deciphering the interplay between combinatorial binding and gene expression.

Giannopoulou, Eugenia G.; Elemento, Olivier

2013-01-01

40

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

PubMed Central

Diagnosis of male infertility has mainly been based on the World Health Organization (WHO) manual-based semen parameter's concentration, motility and morphology. It has, however, become apparent that none of these parameters are reliable markers for evaluation of the fertility potential of a couple. A search for better markers has led to an increased focus on sperm chromatin integrity testing in fertility work-up and assisted reproductive techniques. During the last couple of decades, numerous sperm DNA integrity tests have been developed. These are claimed to be characterized by a lower intraindividual variation, less intralaboratory and interlaboratory variation and thus less subjective than the conventional sperm analysis. However, not all the sperm chromatin integrity tests have yet been shown to be of clinical value. So far, the test that has been found to have the most stable clinical threshold values in relation to fertility is the sperm chromatin structure assay (SCSA), a flow cytometric test that measures the susceptibility of sperm DNA to acid-induced DNA denaturation in situ. Sperm DNA fragmentation as measured by SCSA has shown to be an independent predictor of successful pregnancy in first pregnancy planners as well as in couples undergoing intrauterine insemination, and can be used as a tool in investigation, counseling and treatment of involuntary childlessness. More conflicting data exist regarding the role of sperm DNA fragmentation in relation to fertilization, pre-embryo development and pregnancy outcome in in vitro fertilization and intracytoplasmic sperm injection (ICSI).

Bungum, Mona; Bungum, Leif; Giwercman, Aleksander

2011-01-01

41

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

NASA Astrophysics Data System (ADS)

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

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

2012-02-01

42

Building a nuclear envelope at the end of mitosis: coordinating membrane reorganization, nuclear pore complex assembly, and chromatin de-condensation.  

PubMed

The metazoan nucleus is disassembled and re-built at every mitotic cell division. The nuclear envelope, including nuclear pore complexes, breaks down at the beginning of mitosis to accommodate the capture of massively condensed chromosomes by the spindle apparatus. At the end of mitosis, a nuclear envelope is newly formed around each set of segregating and de-condensing chromatin. We review the current understanding of the membrane restructuring events involved in the formation of the nuclear membrane sheets of the envelope, the mechanisms governing nuclear pore complex assembly and integration in the nascent nuclear membranes, and the regulated coordination of these events with chromatin de-condensation. PMID:23104094

Schooley, Allana; Vollmer, Benjamin; Antonin, Wolfram

2012-10-27

43

In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation  

Microsoft Academic Search

Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) [alpha] (HP1[alpha]) and HP1[beta] on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge

Pernette J. Verschure; Ineke van der Kraan; Leeuw de W. C; Johan van der Vlag; Anne E. Carpenter; Andrew S. Belmont; Roel van Driel

2005-01-01

44

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

Microsoft Academic Search

The linker DNA accessibility of chicken erythrocyte chromatin was studied by diffusion-enhanced resonance energy transfer (DERET). The 4?-{9?-[((4-carboxy-3-hydroxyphenyl)-acetatamido)-3?,6?,9?-(triacetyl)-3?,6?,9?-triazanonamido]-2?,6?-diazanonyl}-4,5',8-trimethyl psoralen-terbium complex was photocovalently bound to linker DNA and transferred its energy to fluorescein free in solution or bound on proteins of different sizes. We observed a diminution of linker DNA accessibility in chromatin as the protein size increased. Free fluorescein and

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

1994-01-01

45

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

SciTech Connect

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

Evenson, D.; Jost, L.; Gandour, D. [South Dakota State Univ., Brookings, SD (United States); Gandour, D.; Rhodes, L. [Becton Dickinson Immunocytometry Systems, San Jose, CA (United States)] [and others

1995-04-01

46

Etoposide-induced apoptosis in lymphoblastoid leukaemic MOLT4 cells: Evidence that chromatin condensation, loss of phosphatidylserine asymmetry and apoptotic body formation can occur independently  

Microsoft Academic Search

Apoptosis is characterised by a series of typical morphological features, such as nuclear and cellular convolution, chromatin condensation and the final disintegration of the cell into membrane-bound apoptotic bodies, which are phagocytosed, by neighbouring cells. Relocation of phosphatidylserine residues from the inner leaflet of the cellular membrane to being exposed on the cell surface is a necessary event for the

C. D. Ramirez; D. R. Catchpoole

2002-01-01

47

Isolation of nuclei from skeletal muscle satellite cells and myofibers for use in chromatin immunoprecipitation assays.  

PubMed

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

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

2012-01-01

48

Evaluation of myc E-box phylogenetic footprints in glycolytic genes by chromatin immunoprecipitation assays.  

PubMed

Prediction of gene regulatory sequences using phylogenetic footprinting has advanced considerably but lacks experimental validation. Here, we report whether transcription factor binding sites predicted by dot plotting or web-based Trafac analysis could be validated by chromatin immunoprecipitation assays. MYC overexpression enhances glycolysis without hypoxia and hence may contribute to altered tumor metabolism. Because the full spectrum of glycolytic genes directly regulated by Myc is not known, we chose Myc as a model transcription factor to determine whether it binds target glycolytic genes that have conserved canonical Myc binding sites or E boxes (5'-CACGTG-3'). Conserved canonical E boxes in ENO1, HK2, and LDHA occur in 31- to 111-bp islands with high interspecies sequence identity (>65%). Trafac analysis revealed another region in ENO1 that corresponds to a murine region with a noncanonical E box. Myc bound all these conserved regions well in the human P493-6 B lymphocytes. We also determined whether Myc could bind nonconserved canonical E boxes found in the remaining human glycolytic genes. Myc bound PFKM, but it did not significantly bind GPI, PGK1, and PKM2. Binding to BPGM, PGAM2, and PKLR was not detected. Both GAPD and TPI1 do not have conserved E boxes but are induced and bound by Myc through regions with noncanonical E boxes. Our results indicate that Myc binds well to conserved canonical E boxes, but not nonconserved E boxes. However, the binding of Myc to unpredicted genomic regions with noncanonical E boxes reveals a limitation of phylogenetic footprinting. In aggregate, these observations indicate that Myc is an important regulator of glycolytic genes, suggesting that MYC plays a key role in a switch to glycolytic metabolism during cell proliferation or tumorigenesis. PMID:15199147

Kim, Jung-whan; Zeller, Karen I; Wang, Yunyue; Jegga, Anil G; Aronow, Bruce J; O'Donnell, Kathryn A; Dang, Chi V

2004-07-01

49

Evaluation of Myc E-Box Phylogenetic Footprints in Glycolytic Genes by Chromatin Immunoprecipitation Assays  

PubMed Central

Prediction of gene regulatory sequences using phylogenetic footprinting has advanced considerably but lacks experimental validation. Here, we report whether transcription factor binding sites predicted by dot plotting or web-based Trafac analysis could be validated by chromatin immunoprecipitation assays. MYC overexpression enhances glycolysis without hypoxia and hence may contribute to altered tumor metabolism. Because the full spectrum of glycolytic genes directly regulated by Myc is not known, we chose Myc as a model transcription factor to determine whether it binds target glycolytic genes that have conserved canonical Myc binding sites or E boxes (5?-CACGTG-3?). Conserved canonical E boxes in ENO1, HK2, and LDHA occur in 31- to 111-bp islands with high interspecies sequence identity (>65%). Trafac analysis revealed another region in ENO1 that corresponds to a murine region with a noncanonical E box. Myc bound all these conserved regions well in the human P493-6 B lymphocytes. We also determined whether Myc could bind nonconserved canonical E boxes found in the remaining human glycolytic genes. Myc bound PFKM, but it did not significantly bind GPI, PGK1, and PKM2. Binding to BPGM, PGAM2, and PKLR was not detected. Both GAPD and TPI1 do not have conserved E boxes but are induced and bound by Myc through regions with noncanonical E boxes. Our results indicate that Myc binds well to conserved canonical E boxes, but not nonconserved E boxes. However, the binding of Myc to unpredicted genomic regions with noncanonical E boxes reveals a limitation of phylogenetic footprinting. In aggregate, these observations indicate that Myc is an important regulator of glycolytic genes, suggesting that MYC plays a key role in a switch to glycolytic metabolism during cell proliferation or tumorigenesis.

Kim, Jung-whan; Zeller, Karen I.; Wang, Yunyue; Jegga, Anil G.; Aronow, Bruce J.; O'Donnell, Kathryn A.; Dang, Chi V.

2004-01-01

50

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

EPA Science Inventory

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

51

Alul in situ digestion of human alphoid and classical satellite DNA regions: High-resolution digital image analysis of FISH signals from condensed and extended chromatin  

Microsoft Academic Search

Human lymphocyte chromatin either extended or condensed in interphase nuclei and chromosomes was in situ digested by the restriction endonuclease AluI and then hybridized with alphoid probes specific for chromosome 1 (D1Z5 locus), for chromosome X (DXZ1 locus), and with a classical satellite DNA probe specific for chromosome 9 (D9Z1 locus). Fluorescent hybridization signals were quantified by digital analysis of

J. L. Fernández; D. Valverde; V. Goyanes; I. Buño; J. Gosálvez

1997-01-01

52

AluI in situ digestion of human alphoid and classical satellite DNA regions: high-resolution digital image analysis of FISH signals from condensed and extended chromatin.  

PubMed

Human lymphocyte chromatin either extended or condensed in interphase nuclei and chromosomes was in situ digested by the restriction endonuclease AluI and then hybridized with alphoid probes specific for chromosome 1 (D1Z5 locus), for chromosome X (DXZ1 locus), and with a classical satellite DNA probe specific for chromosome 9 (D9Z1 locus). Fluorescent hybridization signals were quantified by digital analysis of high-resolution images obtained by a Photo-CD system in an attempt to analyze the differential DNA removal produced by AluI in specific repetitive DNA sequences with known restriction site frequency and distribution. The analysis of area and average pixel grey count of hybridization signals suggests that the greater the degree of chromatin stretching, the higher the accessibility of the probe and/or reporter molecules to the target. Nevertheless, this greater hybridization efficiency does not result in a higher fluorescence intensity due to dispersion of individual signals. Specific repetitive DNA at D9Z1 locus (classical) remained impervious to digestion, while that at DXZ1 (alphoid) was extensively removed, according to the frequency and distribution of restriction sites. Nevertheless, though the restriction sites were at least as frequent as at the DXZ1 locus, DNA at the D1Z5 locus (alphoid) was only partially removed. This indicates that chromatin organization within the C-band partially prevents extraction of alphoid sequences, supporting the hypothesis that alphoid DNA sequences are differentially organized among chromosomes. Overall, the same results were obtained from condensed and extended chromatin, suggesting that higher-order chromatin organization does not influence the in situ DNA cleavage and removal by AluI. PMID:9154135

Fernández, J L; Valverde, D; Goyanes, V; Buño, I; Gosálvez, J

1997-01-01

53

Nuclear DNA methylation and chromatin condensation phenotypes are distinct between normally proliferating/aging, rapidly growing/immortal, and senescent cells.  

PubMed

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

Oh, Jin Ho; Gertych, Arkadiusz; Tajbakhsh, Jian

2013-03-01

54

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

PubMed Central

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

Gertych, Arkadiusz; Tajbakhsh, Jian

2013-01-01

55

Isolation of nuclei for use in genome-wide DNase hypersensitivity assays to probe chromatin structure.  

PubMed

DNase hypersensitivity (DHS) analysis coupled with high-throughput DNA sequencing (DNase-seq) has emerged as a powerful tool to analyze chromatin accessibility and identify regulatory sequences in genomic DNA on a global scale. In this method, intact nuclei are isolated from fresh tissue or cultured cells and then subjected to limited digestion using DNase I. The resulting short DNA fragments released by DNase digestion, which correspond to regions of open chromatin structure, are subsequently purified and identified by high throughput next generation DNA sequencing. This chapter describes methods used to isolate intact nuclei from mouse liver suitable for DNase-seq studies. The following chapter presents a detailed protocol for DNase I digestion of liver nuclei followed by the isolation of DNase-released fragments for sequencing and genome-wide mapping of DHS sites. PMID:23436350

Ling, Guoyu; Waxman, David J

2013-01-01

56

Condensation  

NSDL National Science Digital Library

In this activity, learners explore the process of condensation. After seeing water vapor condense, learners will help design a test to see if cooling water vapor has an effect on the rate of condensation.

Kessler, James H.; Galvan, Patricia M.

2007-01-01

57

Chromatin immunoprecipitation.  

PubMed

Chromatin plays important functions in regulating many biological processes, including DNA transcription, replication, and repair. The use of chromatin immunoprecipitation (ChIP) assays has contributed enormously to identify interactions between DNA and a wide range of nuclear proteins including histones and their different posttranslational modifications as well as a variety of transcription factors. ChIP assays have been successfully used to map histone modifications and histone variants, as well as binding of transcription factors and chromatin-modifying complexes in both, specific candidate loci and the entire genome. In this chapter, we provide a brief review of the variations in ChIP assays and a detailed explanation of the basic standard ChIP protocol. PMID:24162998

Rodríguez-Ubreva, Javier; Ballestar, Esteban

2014-01-01

58

Histone H1 Subtypes Differentially Modulate Chromatin Condensation without Preventing ATP-Dependent Remodeling by SWI\\/SNF or NURF  

Microsoft Academic Search

Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome

Jaime Clausell; Nicole Happel; Tracy K. Hale; Detlef Doenecke; Miguel Beato

2009-01-01

59

ABA-mediated inhibition of seed germination is associated with ribosomal DNA chromatin condensation, decreased transcription, and ribosomal RNA gene hypoacetylation.  

PubMed

Seed germination is a highly organized biological process accompanied by many cellular and metabolic changes. The ribosomal RNA (rRNA) gene, which forms the nucleolus at interphase and is transcribed for ribosome production and protein synthesis, has an important role during seed germination. In this study, we report that there is a decondensation of ribosomal DNA (rDNA) chromatin during seed germination accompanied with increased rRNA gene expression and overall genomic hyperacetylation. Analysis of the rRNA gene promoter region by using chromatin immunoprecipitation (ChIP) shows that there is an increase in acetylation levels at the rRNA gene promoter region. Application of seed germination inhibitor abscisic acid (ABA) suppresses rDNA chromatin decondensation, the expression of rRNA genes and global genomic acetylation. The further ChIP experiments show that ABA treatment hinders the elevation of acetylation levels in the promoter region of the rRNA gene. The data together indicate that ABA treatment inhibits seed germination, which is associated with rDNA chromatin condensation, decreased transcription and rRNA gene hypoacetylation. PMID:22527753

Zhang, Lu; Hu, Yong; Yan, Shihan; Li, Hui; He, Shibin; Huang, Min; Li, Lijia

2012-04-13

60

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

SciTech Connect

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

Evenson, D.P.; Baer, R.K.; Jost, L.K. (South Dakota State Univ., Brookings (USA))

1989-01-01

61

A Nonessential HP1-Like Protein Affects Starvation-Induced Assembly of Condensed Chromatin and Gene Expression in Macronuclei of Tetrahymena thermophila  

PubMed Central

Heterochromatin represents a specialized chromatin environment vital to both the repression and expression of certain eukaryotic genes. One of the best-studied heterochromatin-associated proteins is Drosophila HP1. In this report, we have disrupted all somatic copies of the Tetrahymena HHP1 gene, which encodes an HP1-like protein, Hhp1p, in macronuclei (H. Huang, E. A. Wiley, R. C. Lending, and C. D. Allis, Proc. Natl. Acad. Sci. USA 95:13624–13629, 1998). Unlike the Drosophila HP1 gene, HHP1 is not essential in Tetrahymena spp., and during vegetative growth no clear phenotype is observed in cells lacking Hhp1p (?HHP1). However, during a shift to nongrowth conditions, the survival rate of ?HHP1 cells is reduced compared to that of wild-type cells. Upon starvation, Hhp1p becomes hyperphosphorylated concomitant with a reduction in macronuclear volume and an increase in the size of electron-dense chromatin bodies; neither of these morphological changes occurs in the absence of Hhp1p. Activation of two starvation-induced genes (ngoA and CyP) is significantly reduced in ?HHP1 cells while, in contrast, the expression of several growth-related or constitutively expressed genes is comparable to that in wild-type cells. These results suggest that Hhp1p functions in the establishment and/or maintenance of a specialized condensed chromatin environment that facilitates the expression of certain genes linked to a starvation-induced response.

Huang, Hui; Smothers, James F.; Wiley, Emily A.; Allis, C. David

1999-01-01

62

Stably integrated and expressed retroviral sequences can influence nuclear location and chromatin condensation of the integration locus.  

PubMed

The large-scale chromatin organization of retrovirus and retroviral gene vector integration loci has attracted little attention so far. We compared the nuclear organization of transcribed integration loci with the corresponding loci on the homologous chromosomes. Loci containing gamma-retroviral gene transfer vectors in mouse hematopoietic precursor cells showed small but significant repositioning of the integration loci towards the nuclear interior. HIV integration loci in human cells showed a significant repositioning towards the nuclear interior in two out of five cases. Notably, repositioned HIV integration loci also showed chromatin decondensation. Transcriptional activation of HIV by sodium butyrate treatment did not lead to a further enhancement of the differences between integration and homologous loci. The positioning relative to splicing speckles was indistinguishable for integration and homologous control loci. Our data show that stable retroviral integration can lead to alterations of the nuclear chromatin organization, and has the potential to modulate chromatin structure of the host cell. We thus present an example where a few kb of exogenous DNA are sufficient to significantly alter the large-scale chromatin organization of an endogenous locus. PMID:22415776

Nagel, Jens; Gross, Birgit; Meggendorfer, Manja; Preiss, Carolin; Grez, Manuel; Brack-Werner, Ruth; Dietzel, Steffen

2012-03-14

63

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

Microsoft Academic Search

The aim of this study was to investigate whether the sperm chromatin structure assay (SCSA) results after swim-up are related to fertilization rates, embryo quality and pregnancy rates following in vitro fertilization (IVF). A total of 223 couples undergoing IVF in our hospital from October 2008 to September 2009 were included in this study. Data on the IVF process and

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

2011-01-01

64

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

PubMed

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

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

2011-09-15

65

Frozen-thawed rhinoceros sperm exhibit DNA damage shortly after thawing when assessed by the sperm chromatin dispersion assay.  

PubMed

This study reports on the successful validation (via in situ nick translation and neutral comet assay) of the equine Sperm-Halomax kit as an appropriate methodology for the assessment of sperm DNA fragmentation in three species of rhinoceros. Rhinoceros sperm nuclei with fragmented DNA (validated using in situ nick translation) were evident as large halos with dispersed DNA fragments, whereas those with nonfragmented DNA displayed small halos of nondispersed DNA within the microgel. There was a high correlation (r) of 0.974 (R(2) value=0.949; P<0.01; n=16) between the respective assessments of the Sperm Chromatin Dispersion test (SCDt) and the neutral comet assay. Application of the SCDt to determine the DNA fragmentation dynamics of rhinoceros (n=6) sperm frozen in liquid nitrogen vapor and incubated postthaw at 37 degrees C for up to 48 h to mimic in vitro conditions in the female reproductive tract, revealed an increase (P=0.001) in DNA damage, as soon as 4h after the start of incubation. Linear regression equations were calculated for all six rhinoceroses over the first 6h of incubation and revealed individual animal variation. Freshly collected and incubated (37 degrees C) rhinoceros (n=3) sperm had no increase in the basal level of DNA fragmentation for up to 48 h, indicating that the cryopreservation of rhinoceros sperm in liquid nitrogen vapor, as used in this study, appeared to result in freeze-thaw DNA damage. PMID:19560805

Portas, T; Johnston, S D; Hermes, R; Arroyo, F; López-Fernadez, C; Bryant, B; Hildebrandt, T B; Göritz, F; Gosalvez, J

2009-06-27

66

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

EPA Science Inventory

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

67

The influence of condensed tannin structure on rate of microbial mineralization and reactivity to chemical assays.  

PubMed

We examined how tannin structure influences reactivity in tannin assays and carbon and nitrogen mineralization. Condensed tannins from the foliage of ten tree and shrub species and from pecan shells (Carya illinoensis) had different proportions of: (a) epicatechin (cis) and catechin (trans) isomers, (b) procyanidin (PC) and prodelphinidin (PD) monomers, and (c) different chain lengths. The response of each tannin to several widely used tannin assays was determined. Although there was some variation in response to proanthocyanidin (butanol/HCl) and Folin Ciocalteu assays, we did not deduce any predictable relationship between tannin structure and response to either assay. There was little variation in protein precipitation among the different tannins. To assess biological activity, six of the tannins were incubated with forest humus for 22 days. We determined that, while PC-based tannins remained at least partly extractable for the duration of the incubation, tannins with a high proportion of PD subunits rapidly became unextractable from soil. There was a positive correlation between net nitrogen mineralization and cis chemical structure. Carbon mineralization was enhanced initially by the addition of tannins to humus, but after 22 days, a negative correlation between the proportion of cis subunits and respiration was determined. Overall, we were not able to demonstrate consistent effects of structure on either microbial mineralization or reactivity to chemical assays; such relationships remain elusive. PMID:21340461

Norris, Charlotte E; Preston, Caroline M; Hogg, Karen E; Titus, Brian D

2011-02-22

68

The Forkhead Transcription Factor FoxI1 Remains Bound to Condensed Mitotic Chromosomes and Stably Remodels Chromatin Structure  

Microsoft Academic Search

All forkhead (Fox) proteins contain a highly conserved DNA binding domain whose structure is remarkably similar to the winged-helix structures of histones H1 and H5. Little is known about Fox protein binding in the context of higher-order chromatin structure in living cells. We created a stable cell line expressing FoxI1-green fluorescent protein (GFP) or FoxI1-V5 fusion proteins under control of

Jizhou Yan; Lisha Xu; Gregory Crawford; Zenfeng Wang; Shawn M. Burgess

2006-01-01

69

Denaturation and condensation of DNA in situ induced by acridine orange in relation to chromatin changes during growth and differentiation of Friend erythroleukemia cells.  

PubMed

DNA in situ is progressively denatured when the cells or nuclei are treated with increasing concentration of acridine orange (AO). This transition can be monitored by flow cytometry as a decrease in green fluorescence. The complexes of denatured DNA and AO undergo immediate condensation and aggregation; this step is manifested by appearance of red luminescence and formation of precipitates that can be detected by electron microscopy. The precipitates form preferentially in heterochromatin as well as in ribosomes and polysomes. Their formation and further aggregation affects cellular light scatter properties in both the forward and right-angle direction. The AO-induced DNA denaturation and condensation was studied in nuclei of Friend erythroleukemia cells from exponentially growing, differentiated or quiescent cells. The DNA in nuclei of quiescent cells, from plateau-phase cultures, was the most sensitive to denaturation; it denatured (measured by changes in luminescence) at an AO concentration between 50 and 80 microM with the midpoint of the transition (Cd) at 70 microM. DNA in nuclei of differentiated cells (dimethyl-sulfoxide-induced erythroid differentiation) was more resistant (Cd = 77-83 microM), whereas DNA in exponentially growing cells was the most resistant (Cd = 86 microM). Extraction of proteins with 0.1 M HCl at 0 degree C abolished the differences between the cells and shifted the transition to a lower AO concentration (Cd = 46 microM). For comparison, the midpoint transitions representing condensation of free, nucleic acids measured as light scatter changes occurred at 13, 22, 31 and 53 microM of AO, for rRNA, tRNA, and denatured and native-calf thymus DNA, respectively. Denaturation and condensation of DNA, which can be induced by AO either in isolated nuclei or viable permeabilized or fixed cells provides a new approach to discriminate cell subpopulations with different chromatin structure by flow cytometry. The molecular mechanisms of this phenomenon are discussed. PMID:3858089

Darzynkiewicz, Z; Traganos, F; Kapuscinski, J; Melamed, M R

1985-05-01

70

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

PubMed Central

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

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

2007-01-01

71

Long-Term Effects of Triethylenemelamine Exposure on Mouse Testis Cells and Sperm Chromatin Structure Assayed by Flow Cytometry.  

National Technical Information Service (NTIS)

The toxic and potentially mutagenic actions of triethylenemelamine (TEM) on mouse body and testis weights, testicular cell kinetics, sperm production, sperm head morphology, and sperm chromatin structure were assessed in two experiments. The first experim...

D. P. Evenson R. K. Baer L. K. Jost

1989-01-01

72

LONG-TERM EFFECTS OF TRIETHYLENEMELAMINE EXPOSURE ON MOUSE TESTIS CELLS AND SPERM CHROMATIN STRUCTURE ASSAYED BY FLOW CYTOMETRY  

EPA Science Inventory

The toxic and potentially mutagenic actions of triethylenemelamine (TEM) on mouse body and testis weights, testicular cell kinetics, sperm production, sperm head morphology, and sperm chromatin structure were assessed in two experiments. he first experiment examined effects of fo...

73

TBP2 is essential for germ cell development by regulating transcription and chromatin condensation in the oocyte  

PubMed Central

Development of the germline requires consecutive differentiation events. Regulation of these has been associated with germ cell-specific and pluripotency-associated transcription factors, but the role of general transcription factors (GTFs) remains elusive. TATA-binding protein (TBP) is a GTF involved in transcription by all RNA polymerases. During ovarian folliculogenesis in mice the vertebrate-specific member of the TBP family, TBP2/TRF3, is expressed exclusively in oocytes. To determine TBP2 function in vivo, we generated TBP2-deficient mice. We found that Tbp2?/? mice are viable with no apparent phenotype. However, females lacking TBP2 are sterile due to defective folliculogenesis, altered chromatin organization, and transcriptional misregulation of key oocyte-specific genes. TBP2 binds to promoters of misregulated genes, suggesting that TBP2 directly regulates their expression. In contrast, TBP ablation in the female germline results in normal ovulation and fertilization, indicating that in these cells TBP is dispensable. We demonstrate that TBP2 is essential for the differentiation of female germ cells, and show the mutually exclusive functions of these key core promoter-binding factors, TBP and TBP2, in the mouse.

Gazdag, Emese; Santenard, Angele; Ziegler-Birling, Celine; Altobelli, Gioia; Poch, Olivier; Tora, Laszlo; Torres-Padilla, Maria-Elena

2009-01-01

74

Induction of stable protein-deoxyribonucleic acid adducts in Chinese hamster cell chromatin by ultraviolet light.  

PubMed

Ultraviolet (uv)-light-mediated formation of protein-DNA adducts in Chinese hamster cell chromatin was investigated in an attempt to compare chromatin alterations induced in vitro with those observed in vivo. Three independent methods of analysis indicated stable protein-DNA associations: (1) a membrane filter assay which retained DNA on the filter in the presence of high salt-detergent; (2) a Sepharose 4B column assay in which protein eluted cincident with DNA; and (3) a CsCl density gradient equilibrium assay which showed both protein and DNA banding at densities other than their respective native densities. Treatment of the irradiated chromatin with DNase provided further evidence that protein-DNA and not protein-protein adducts were being observed in the column assay. There is a fluence-dependent response of protein-DNA adduct formation when the chromatin is irradiated at low ionic strength and is linear for protein over the range studied. When the chromatin is exposed to differing conditions of pH, ionic strength, or divalent metal ion concentration, the quantity of adduct formed upon uv irradiation varies. Susceptibility to adduct formation can be partially explained in terms of the condensation state of the chromatin and other factors such as rearrangement, denaturation, and dissociation of the chromatin components. Besides providing information on the biological significance of these types of uv-induced lesions, this technique may be useful as a probe of chromatin structure. PMID:5110

Strniste, G F; Rall, S C

1976-04-20

75

Loss of protein phosphatase 1c{gamma} (PPP1CC) leads to impaired spermatogenesis associated with defects in chromatin condensation and acrosome development: an ultrastructural analysis.  

PubMed

Human male infertility affects approximately 5% of men, with one-third suffering from testicular failure, likely the result of an underlying genetic abnormality that disrupts spermatogenesis during development. Mouse models of male infertility such as the Ppp1cc knockout mouse display very similar phenotypes to humans with testicular failure. Male Ppp1cc mutant mice are sterile due to disruptions in spermatogenesis that begin during prepubertal testicular development, and continue into adulthood, often resulting in loss of germ cells to the point of Sertoli cell-only syndrome. The current study employs light and electron microscopy to identify new morphological abnormalities in Ppp1cc mutant seminiferous epithelium. This study reveals that germ cells become delayed in their development around stages VII and VIII of spermatogenesis. Loss of these cells likely results in the reduced numbers of elongating spermatids and spermatozoa previously observed in mutant animals. Interestingly, Ppp1cc mutants also display reduced numbers of spermatogonia compared with their wild-type counterparts. Using electron microscopy, we have shown that junction complexes in Ppp1cc mutants are ultrastructurally normal, and therefore do not contribute to the breakdown in tissue architecture seen in mutants. Electron microscopy revealed major acrosomal and chromatin condensation defects in Ppp1cc mutants. Our observations are discussed in the context of known molecular changes in Ppp1cc mutant testes. PMID:20385779

Forgione, Nicole; Vogl, A Wayne; Varmuza, Susannah

2010-04-12

76

Changes in cell nuclei during S phase: Progressive chromatin condensation and altered expression of the proliferation-associated nuclear proteins Ki-67, cyclin (PCNA), p105, and p34  

SciTech Connect

Using multiparameter flow cytometry the authors have measured the nuclear DNA content of exponentially growing HL-60 cells in conjunction with protein content, unclear forward light scatter, DNA in situ sensitivity to denaturation, DNA accessibility to 7-aminoactinomycin D (7-AMD), and content of the proliferation-associated proteins: cyclin (PCNA), p105, p34, and Ki-67. Multivariate analysis of the data made it possible to correlate changes in each parameter with the degree of cell advancement through S phase (amount of replicated DNA). A decrease of the protein/DNA ratio, lowered DNA accessibility to 7-AMD, increased sensitivity of DNA to denaturation, and increased ability of isolated nuclei to scatter light all paralleled cell progression through S phase. These changes indicate that during S phase chromatin progressively condenses and suggest that the condensation is associated with the efflux of nonhistone proteins from the nucleus. The data indicate that significant changes in structure and composition of chromatin take place during S phase and suggest that the composition of chromatin associated with the nonreplicated DNA is different compared to chromatin associated with the newly replicated DNA.

Bruno, S.; Darzynkiewicz, Z. (New York Medical College, Valhalla (United States)); Crissman, H.A. (Los Alamos National Lab., NM (United States)); Bauer, K.D. (Northwestern Univ., Chicago, IL (United States))

1991-09-01

77

Mutagenicity of wood smoke condensates in the Salmonella/microsome assay.  

PubMed

Smoke condensates of woods used for food preservation and aromatization in Nigeria were tested for mutagenic activity using Salmonella typhimurium TA98 and TA100. The woods were: white mangrove (Avicennia nitida), red mangrove (Rhizophora racemosa), mahogany Khaya sp.), abura (Mitragyna ciliata), alstonia (Alstonia boonei) and black afara (Terminalia ivorensis). Cigarette tar was tested for comparison. The condensates induced dose-dependent increases in the number of His+ revertants mainly with S9 mix. With the exception of mahogany and cigarette smoke condensate, the smoke condensates induced more revertants/microgram condensate in TA100 than in TA98. The number of revertants/microgram condensate ranged between 0.04 and 0.9 for the wood smoke condensates and was 0.12 for the cigarette smoke in TA100. The range was between 0.1 and 0.30 for the wood smoke condensates and 0.18 revertants/microgram condensate for cigarette smoke condensate in TA98. Concentrations of 7 polycyclic aromatic hydrocarbons (PAHs) in the condensates were determined namely, pyrene, benzo[a]pyrene, benz[a]anthracene, benzo[k]fluoranthene, benzo[b]chrysene, benzo[g,h,i]perylene and dibenzo[a,e]pyrene. The condensates contained varying concentrations of the individual PAHs and those with higher concentrations generally showed greater mutagenic activities. However, the order of mutagenic potency in the bacterial strains differed from the order of PAH concentrations, which were lower than the concentrations at which they are reported to induce mutations. When 6 of the PAHs were mixed in the concentrations in which they were found in the individual condensates, the mixtures did not induce mutation so that the contribution of the PAHs to the mutagenic activities of the condensates could not be determined. PMID:1881415

Asita, A O; Matsui, M; Nohmi, T; Matsuoka, A; Hayashi, M; Ishidate, M; Sofuni, T; Koyano, M; Matsushita, H

1991-09-01

78

Chromatin Remodeling  

NSDL National Science Digital Library

This Teaching Resource provides lecture notes and slides for a class covering chromatin remodeling mechanisms and is part of the course "Cell Signaling Systems: a Course for Graduate Students." The lecture begins with a discussion of chromatin organization and then proceeds to describe the process of chromatin remodeling through a review of chromatin remodeling complexes and methods used to study their function.

Neal F. Lue (Weill Medical College of Cornell University;Department of Microbiology and Immunology REV)

2005-07-26

79

Assessing cytogenotoxicity of cigarette smoke condensates using three in vitro assays  

Microsoft Academic Search

Cigarette smoke condensates (CSCs) are complex mixed compounds that contain both direct and indirect mutagens\\/carcinogens. To detect genotoxicity of CSCs in vitro, a combination of various enzymes (e.g. activation and detoxification enzymes) called S9 is usually added. However, as S9 may induce cytotoxicity in target cells, it is unclear whether the addition of S9 can impact CSC-induced toxicity. Here, differences

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

2009-01-01

80

Seasonal Changes in Sperm Chromatin Condensation in Ram (Ovis aries), Iberian Red Deer (Cervus elaphus hispanicus), and Brown Bear (Ursus arctos)  

Microsoft Academic Search

The effect of seasonality (temperate environment, Spain) on the chromatin status of ovine (Churra breed), Iberian red deer, and brown bear spermatozoa was studied. This work aims to improve genetic resource banks (GRBs) by enhancing existing knowledge of the effect of season on sperm quality. Samples were obtained by electroejaculation in Iberian red deer and brown bear and by artificial

VANESA GARCIA-MACIAS; FELIPE MARTINEZ-PASTOR; MERCEDES ALVAREZ; SANTIAGO BORRAGAN; CESAR A. CHAMORRO; ANA J. SOLER; LUIS ANEL; PAULINO DE PAZ

81

Chromatin Computation  

PubMed Central

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.

Bryant, Barbara

2012-01-01

82

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

Microsoft Academic Search

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

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

2010-01-01

83

Cloned calves from chromatin remodeled in vitro.  

PubMed

We have developed a novel system for remodeling mammalian somatic nuclei in vitro prior to cloning by nuclear transplantation. The system involves permeabilization of the donor cell and chromatin condensation in a mitotic cell extract to promote removal of nuclear factors solubilized during chromosome condensation. The condensed chromosomes are transferred into enucleated oocytes prior to activation. Unlike nuclei of nuclear transplant embryos, nuclei of chromatin transplant embryos exhibit a pattern of markers closely resembling that of normal embryos. Healthy calves were produced by chromatin transfer. Compared with nuclear transfer, chromatin transfer shows a trend toward greater survival of cloned calves up to at least 1 mo after birth. This is the first successful demonstration of a method for directly manipulating the somatic donor chromatin prior to transplantation. This procedure should be useful for investigating mechanisms of nuclear reprogramming and for making improvements in the efficiency of mammalian cloning. PMID:13679310

Sullivan, Eddie J; Kasinathan, Sriranjani; Kasinathan, Poothappillai; Robl, James M; Collas, Philippe

2003-09-17

84

Evaluation of eight in vitro assays for assessing the cytotoxicity of cigarette smoke condensate  

Microsoft Academic Search

The accurate assessment of cytotoxicity is important for evaluating the potential of a test agent to induce pathologies that result from cell killing and to determine appropriate doses for other endpoints, such as genetic toxicology studies. The objective of this work was to determine the most sensitive assays for assessing cytotoxicity in Chinese hamster ovary (CHO) cells following short-term (1

K. P. Putnam; D. W. Bombick; D. J. Doolittle

2002-01-01

85

Cytochemical evaluation of sperm chromatin and DNA integrity in couples with unexplained recurrent spontaneous abortions.  

PubMed

The aim of this study was to examine the possible relationship between sperm DNA integrity and chromatin packaging evaluated by cytochemical assays, traditional sperm parameters and recurrent spontaneous abortion (RSA) of unknown origin. In this cohort study, 40 couples with a history of RSA and 40 couples with proven fertility were considered as case and control groups respectively. The semen samples of all husbands were analysed for sperm parameters and also sperm chromatin and DNA integrity assessed using cytochemical tests including aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), acridine orange (AOT) and nuclear chromatin stability assay. Among different sperm parameters, only slow motility was significantly different between the two groups. In sperm chromatin evaluations, there were significant differences between the two groups in all of the tests. In addition, the majority of semen samples in RSA patients exhibited upper percentages of abnormal spermatozoa than the cut-off values regarding different cytochemical assays. Our study showed that in the cases of RSA, slow motility had a significant reduction in comparison with controls and also spermatozoa of men from RSA group had less chromatin condensation and poorer DNA integrity than spermatozoa that obtained from fertile men with no history of RSA. PMID:21806662

Talebi, A R; Vahidi, S; Aflatoonian, A; Ghasemi, N; Ghasemzadeh, J; Firoozabadi, R D; Moein, M R

2011-08-02

86

Chromatin Dynamics  

PubMed Central

The expression patterns of many protein-coding genes are orchestrated in response to exogenous stimuli, as well as cell-type-specific developmental programs. In recent years, researchers have shown that dynamic chromatin movements and interactions in the nucleus play a crucial role in gene regulation. In this review, we highlight our current understanding of the organization of chromatin in the interphase nucleus and the impact of chromatin dynamics on gene expression. We also discuss the current state of knowledge with regard to the localization of active and inactive genes within the three-dimensional nuclear space. Furthermore, we address recent findings that demonstrate the movements of chromosomal regions and genomic loci in association with changes in transcriptional activity. Finally, we discuss the role of intra-and interchromosomal interactions in the control of coregulated genes.

Hubner, Michael R.; Spector, David L.

2010-01-01

87

Mitotic Phosphorylation Prevents the Binding of HMGN Proteins to Chromatin  

PubMed Central

Condensation of the chromatin fiber and transcriptional inhibition during mitosis is associated with the redistribution of many DNA- and chromatin-binding proteins, including members of the high-mobility-group N (HMGN) family. Here we study the mechanism governing the organization of HMGN proteins in mitosis. Using site-specific antibodies and quantitative gel analysis with proteins extracted from synchronized HeLa cells, we demonstrate that, during mitosis, the conserved serine residues in the nucleosomal binding domain (NBD) of this protein family are highly and specifically phosphorylated. Nucleosome mobility shift assays with both in vitro-phosphorylated proteins and with point mutants bearing negative charges in the NBD demonstrate that the negative charge abolishes the ability of the proteins to bind to nucleosomes. Fluorescence loss of photobleaching demonstrates that, in living cells, the negative charge in the NBD increases the intranuclear mobility of the protein and significantly decreases the relative time that it is bound to chromatin. Expression of wild-type and mutant proteins in HmgN1?/? cells indicates that the negatively charged protein is not bound to chromosomes. We conclude that during mitosis the NBD of HMGN proteins is highly phosphorylated and that this modification regulates the interaction of the proteins with chromatin.

Prymakowska-Bosak, Marta; Misteli, Tom; Herrera, Julio E.; Shirakawa, Hitoshi; Birger, Yehudit; Garfield, Susan; Bustin, Michael

2001-01-01

88

A male-specific nuclease-resistant chromatin fraction in the mealybug Planococcus lilacinus  

Microsoft Academic Search

In mealybugs, chromatin condensation is related to both genomic imprinting and sex determination. The paternal chromosomal complement is condensed and genetically inactive in sons but not in daughters. During a study of chromatin organization in Planococcus lilacinus, digestion with micrococcal nuclease showed that 3% to 5% of the male genome is resistant to the enzyme. This Nuclease Resistant Chromatin (NRC)

Sanjeev Khosla; Prameelarani Kantheti; Vani Brahmachari; H. Sharat Chandra

1996-01-01

89

Experimental Visualization of Chromoneme as a Higher Level of Chromatin Compactization in the Mitotic Chromosome  

Microsoft Academic Search

We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1–0.2 µm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase

V. V. Burakov; A. V. Tvorogova; Yu. S. Chentsov

2005-01-01

90

Residual chromatin breaks as biodosimetry for cell killing by carbon ions  

NASA Astrophysics Data System (ADS)

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

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

1998-11-01

91

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

PubMed

Mobile and stationary combustion sources emit particle-bound organics that, after extraction, are mutagenic to Salmonella typhimurium. In this study, the organic emissions from diesel, cigarette smoke condensate (CSC), coke oven, and roofing tar were fractionated and compared for mutagenicity in the Ames assay. This study demonstrated major differences in the distribution of mutagenicity among the four emission sources. Within each source, the relative mutagenicity of each fraction was significantly different in the presence and absence of an exogenous metabolic activation. In the diesel sample, over 90% of the mutagenic activity is located in the aromatic and polar neutral (PN) fractions; nitrated polynuclear aromatic hydrocarbons (NO2-PNAs) can account for a significant portion of this activity. Most of the mutagenicity of the coke oven main sample was found in the organic base (BASE) and PN fractions, which contained aromatic amines and nitrogen heterocycles. The CSC sample also had a high percentage of the mutagenic activity in the BASE fraction. Chemical analysis, however, indicates that the components in the CSC differed from those of the coke oven main sample. The roofing tar sample, which was not mutagenic in the absence of metabolic activation, contained several components that were very mutagenic after fractionation. This may be due to the separation of toxic components from the mutagenic components. The roofing tar emissions contained aromatic and polar mutagenic constituents. Although the specific mutagens in these different sources are not identical, they all cause frameshift mutations and appear to be compounds that could be classified as polycyclic organic matter. PMID:2414094

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

1985-01-01

92

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

NASA Astrophysics Data System (ADS)

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

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

2013-04-01

93

Evaluation of Alternate Isotope-Coded Derivatization Assay (AIDA) in the LC–MS\\/MS analysis of aldehydes in exhaled breath condensate  

Microsoft Academic Search

We present the application of a novel isotope dilution method, named Alternate Isotope-Coded Derivatization Assay (AIDA), to the quantitative analysis of hydrazone derivatives of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in exhaled breath condensate (EBC) samples using liquid chromatography–tandem mass spectrometry. AIDA is based on the alternate derivatization of the analyte(s) with reagents that are available in two pure isotopic forms,

Paola Manini; Roberta Andreoli; Stefano Sforza; Chiara Dall’Asta; Gianni Galaverna; Antonio Mutti; Wilfried M. A. Niessen

2010-01-01

94

Nanostructure-induced DNA condensation  

NASA Astrophysics Data System (ADS)

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

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

2013-08-01

95

Nanostructure-induced DNA condensation.  

PubMed

The control of the DNA condensation process is essential for compaction of DNA in chromatin, as well as for biological applications such as nonviral gene therapy. This review endeavours to reflect the progress of investigations on DNA condensation effects of nanostructure-based condensing agents (such as nanoparticles, nanotubes, cationic polymer and peptide agents) observed by using atomic force microscopy (AFM) and other techniques. The environmental effects on structural characteristics of nanostructure-induced DNA condensates are also discussed. PMID:23838744

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

2013-09-21

96

Assessment of sperm quality through fluorometry and sperm chromatin structure assay in relation to field fertility of frozen-thawed semen from Swedish AI bulls  

Microsoft Academic Search

We investigated fluorometry to study sperm viability and flow cytometry to study sperm chromatin structure. We also assessed sperm quality after thawing relative to field fertility after AI as shown by 56-day non-return rates (56-d NRR). Frozen-thawed semen samples were obtained from 20 Swedish Red and White bulls (1 to 3 semen batches\\/bull) and the fertility data were based on

A. Januskauskas; A. Johannisson; H. Rodriguez-Martinez

2001-01-01

97

Chromatin fiber functional organization: Some plausible models  

NASA Astrophysics Data System (ADS)

We here present a modeling study of the chromatin fiber functional organization. Multi-scale modeling is required to unravel the complex interplay between the fiber and the DNA levels. It suggests plausible scenarios, including both physical and biological aspects, for fiber condensation, its targeted decompaction, and transcription regulation. We conclude that a major role of the chromatin fiber structure might be to endow DNA with allosteric potentialities and to control DNA transactions by an epigenetic tuning of its mechanical and topological constraints.

Lesne, A.; Victor, J.-M.

2006-03-01

98

What Determines the Folding of the Chromatin Fiber?  

Microsoft Academic Search

In this review, we attempt to summarize, in a critical manner, what is currently known about the processes of condensation and decondensation of chromatin fibers. We begin with a critical analysis of the possible mechanisms for condensation, considering both old and new evidence as to whether the linker DNA between nucleosomes bends or remains straight in the condensed structure. Concluding

Kensal van Holde; Jordanka Zlatanova

1996-01-01

99

Chromatin Compaction Protects Genomic DNA from Radiation Damage  

PubMed Central

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.

Takata, Hideaki; Hanafusa, Tomo; Mori, Toshiaki; Shimura, Mari; Iida, Yutaka; Ishikawa, Kenichi; Yoshikawa, Kenichi; Yoshikawa, Yuko; Maeshima, Kazuhiro

2013-01-01

100

Chromatin compaction protects genomic DNA from radiation damage.  

PubMed

Genomic DNA is organized three-dimensionally in the nucleus, and is thought to form compact chromatin domains. Although chromatin compaction is known to be essential for mitosis, whether it confers other advantages, particularly in interphase cells, remains unknown. Here, we report that chromatin compaction protects genomic DNA from radiation damage. Using a newly developed solid-phase system, we found that the frequency of double-strand breaks (DSBs) in compact chromatin after ionizing irradiation was 5-50-fold lower than in decondensed chromatin. Since radical scavengers inhibited DSB induction in decondensed chromatin, condensed chromatin had a lower level of reactive radical generation after ionizing irradiation. We also found that chromatin compaction protects DNA from attack by chemical agents. Our findings suggest that genomic DNA compaction plays an important role in maintaining genomic integrity. PMID:24130727

Takata, Hideaki; Hanafusa, Tomo; Mori, Toshiaki; Shimura, Mari; Iida, Yutaka; Ishikawa, Kenichi; Yoshikawa, Kenichi; Yoshikawa, Yuko; Maeshima, Kazuhiro

2013-10-09

101

Please do not disturb: Destruction of chromatin structure by supravital nucleic acid probes revealed by a novel assay of DNA-histone interaction  

PubMed Central

The biomarkers designed to be used supravitally are expected to have minimal effect on structure and function of the cell. Unfortunately nearly all fluorochromes developed to probe live cells interact in undesired way with cellular constituents and affect functional pathways. Herein we comment on potential applications of diverse DNA binding probes in view of the recent article by Wojcik & Dobrucki on DRAQ 5 and SYTO 17. The approach used by these authors to assess DNA-histone interactions using the cells having histones tagged with fluorescent proteins offers a valuable tool to study mechanism of action of antitumor drugs targeting DNA. While the effect of many intercalating drugs may be similar to that of DRAQ5, it may be of particular interest to observe the effects induced by intra-strand and inter-strand DNA crosslinking drugs, alkylating agents, histone deacetylase inhibitors or even anti-metabolites. The cells having histones tagged with fluorescent proteins thus may serve as biomarkers to probe mechanism of action of drugs targeting DNA or affecting chromatin structure. In fact, because such gross chromatin changes as revealed by dissociation and segregation of histones from DNA are most likely incompatible with long-term cell survival, the methodology may be applied for rapid screening of investigational antitumor agents.

Wlodkowic, Donald; Darzynkiewicz, Zbigniew

2008-01-01

102

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

103

Regulation of Chromatin Structure and Transcription Via Histone Modifications  

Microsoft Academic Search

\\u000a Chromatin, which was once considered merely a structural component required for DNA packaging, is now recognized as a dynamic\\u000a template governed by intricate regulation. Histone post-translational modifications (PTMs) contribute to chromatin dynamics\\u000a and regulate fundamental biological processes including transcription, mitotic chromatin condensation and DNA repair following\\u000a damage. To date, histone methylation, acetylation, phosphorylation, ubiquitination, sumoylation and ADP-ribosylation, among\\u000a others, have

Kajan Ratnakumar; Avnish Kapoor; Emily Bernstein

104

Chromatin remodeling and cancer, part I: covalent histone modifications  

Microsoft Academic Search

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

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

2007-01-01

105

Sperm Chromatin  

PubMed Central

Sperm are remarkably complex cells with a singularly important mission: to deliver paternal DNA and its associated factors to the oocyte to start a new life. The integrity of sperm DNA is a keystone of reproductive success, which includes fertilization and embryonic development. In addition, the significance in these processes of proteins that associate with sperm DNA is increasingly being appreciated. In this review, we highlight proteomic studies that have identified sperm chromatin proteins with fertility roles that have been validated by molecular studies in model organisms or correlations in the clinic. Up to 50% of male-factor infertility cases in the clinic have no known cause and therefore no direct treatment. In-depth study of the molecular basis of infertility has great potential to inform the development of sensitive diagnostic tools and effective therapies that will address this incongruity. Because sperm rely on testis-specific protein isoforms and post-translational modifications for their development and function, sperm-specific processes are ideal for proteomic explorations that can bridge the research lab and fertility clinic.

Wu, Tammy F.; Chu, Diana S.

2008-01-01

106

Cell-type-specific profiling of gene expression and chromatin binding without cell isolation: assaying RNA Pol II occupancy in neural stem cells.  

PubMed

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

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

2013-06-20

107

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

Microsoft Academic Search

Genome organization within the cell nucleus is a result of chromatin condensation achieved by histone tail-tail interactions and other nuclear proteins that counter the outward entropic pressure of the polymeric DNA. We probed the entropic swelling of chromatin driven by enzymatic disruption of these interactions in isolated mammalian cell nuclei. The large-scale decondensation of chromatin and the eventual rupture of

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

2008-01-01

108

DNA methylation-dependent chromatin fiber compaction in vivo and in vitro: requirement for linker histone  

Microsoft Academic Search

Dynamic alterations in chromatin struc- ture mediated by postsynthetic histone modifications and DNA methylation constitute a major regulatory mechanism in DNA functioning. DNA methylation has been implicated in transcriptional silencing, in part by inducing chromatin condensation. To understand the methylation-dependent chromatin structure, we per- formed atomic force microscope (AFM) studies of fibers isolated from cultured cells containing normal or elevated

MIKHAIL A. KARYMOV; MIROSLAV TOMSCHIK; SANFORD H. LEUBA; PAOLA CAIAFA; JORDANKA ZLATANOVA

2001-01-01

109

Effect of hyperthermia on replicating chromatin  

SciTech Connect

The extent of heat-induced structural alterations in chromatin containing nascent (pulse-labeled) DNA was assayed using the enzyme micrococcal nuclease. The basic nucleosome structure in nascent and mature chromatin of S-phase cells appeared unaltered for up to 16 hr after exposure to hyperthermic temperatures as high as 48/sup 0/C for 15 min. However, the rate of nuclease digestion of DNA in both nascent and mature chromatin is inhibited following exposure to hyperthermic temperatures. In unheated cells, pulse-labeled nascent DNA matured into mature chromatin structure with a half-time of 2.5 min. The half-time for the maturation of pulse-labeled DNA from nascent into mature chromatin increased in a linear manner as a function of increasing temperature of exposure with constant heating time at temperatures above 43/sup 0/C. Both the reduced nuclease digestibility of nascent DNA and the increased time for chromatin structural changes could be due to the increased protein mass of chromatin following hyperthermia.

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

1981-10-01

110

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

PubMed

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

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

2002-01-01

111

Chromatin remodelling during development  

PubMed Central

New methods for the genome-wide analysis of chromatin are providing insight into its roles in development and their underlying mechanisms. Current studies indicate that chromatin is dynamic, with its structure and its histone modifications undergoing global changes during transitions in development and in response to extracellular cues. In addition to DNA methylation and histone modification, ATP-dependent enzymes that remodel chromatin are important controllers of chromatin structure and assembly, and are major contributors to the dynamic nature of chromatin. Evidence is emerging that these chromatin-remodelling enzymes have instructive and programmatic roles during development. Particularly intriguing are the findings that specialized assemblies of ATP-dependent remodellers are essential for establishing and maintaining pluripotent and multipotent states in cells.

Ho, Lena; Crabtree, Gerald R.

2011-01-01

112

Bromodomain Protein Brd4 Associated with Acetylated Chromatin Is Important for Maintenance of Higher-order Chromatin Structure*  

PubMed Central

Chromatin structure organization is crucial for regulating many fundamental cellular processes. However, the molecular mechanism that regulates the assembly of higher-order chromatin structure remains poorly understood. In this study, we demonstrate that Brd4 (bromodomain-containing protein 4) protein participates in the maintenance of the higher-order chromatin structure. Brd4, a member of the BET family of proteins, has been shown to play important roles in cellular growth control, cell cycle progression, and cancer development. We apply in situ single cell chromatin imaging and micrococcal nuclease (MNase) assay to show that Brd4 depletion leads to a large scale chromatin unfolding. A dominant-negative inhibitor encoding the double bromodomains (BDI/II) of Brd4 can competitively dissociate endogenous Brd4 from chromatin to trigger severely fragmented chromatin morphology. Mechanistic studies using Brd4 truncation mutants reveal that the Brd4 C-terminal domain is crucial for maintaining normal chromatin structure. Using bimolecular fluorescence complementation technology, we demonstrate that Brd4 molecules interact intermolecularly on chromatin and that replacing Brd4 molecules by BDI/II causes abnormal nucleosome aggregation and chromatin fragmentation. These studies establish a novel structural role of Brd4 in supporting the higher chromatin architecture.

Wang, Ranran; Li, Qing; Helfer, Christine M.; Jiao, Jing; You, Jianxin

2012-01-01

113

Chromatin Configurations in the Ferret Germinal Vesicle that Reflect Developmental Competence for In Vitro Maturation  

PubMed Central

In several mammalian species, the configuration of germinal vesicle (GV) chromatin correlates with the developmental competence of oocytes. Yet, no study has been published on the configuration of GV chromatin in ferret, nor is it known whether a specific configuration predicts meiotic competence in this species, in spite of the potential importance of ferret cloning to the study of human disease and to species conservation efforts. Here, we report on an analysis of the chromatin configuration in ferret GV oocytes and on how they correlate with meiotic development. Three distinct configurations were identified based on the degree of chromatin condensation: (1) fibrillar chromatin (FC), featuring strands of intertwined chromatin occupying most of the visible GV region; (2) intermediate condensed chromatin (ICC), characterized by dense, irregular chromatin masses throughout the GV; and (3) condensed chromatin (CC), which is highly compact and centered around the nucleolus. We also found that chromatin configuration was related to the extent of association with cumulus cells in cumulus–oocyte complexes; CC-configured oocytes were most often surrounded by a compact cumulus layer and also a compact corona but FC-configured oocytes were associated with neither. In addition, increasing chromatin condensation corresponded to an increase in oocyte diameter. Finally, following in vitro culture, significantly more CC-configured oocytes underwent maturation to meiotic metaphase II than did FC- or ICC-configured oocytes. We conclude that, in ferret, chromatin condensation is related to the sequential achievement of meiotic competencies during oocyte growth and differentiation, and thus can be used as a predictor of competence.

Sun, X; Li, Z; Yi, Y; Ding, W; Chen, J; Engelhardt, JF; Leno, GH

2013-01-01

114

Genetic Landscape of Open Chromatin in Yeast  

PubMed Central

Chromatin regulation underlies a variety of DNA metabolism processes, including transcription, recombination, repair, and replication. To perform a quantitative genetic analysis of chromatin accessibility, we obtained open chromatin profiles across 96 genetically different yeast strains by FAIRE (formaldehyde-assisted isolation of regulatory elements) assay followed by sequencing. While 5?10% of open chromatin region (OCRs) were significantly affected by variations in their underlying DNA sequences, subtelomeric areas as well as gene-rich and gene-poor regions displayed high levels of sequence-independent variation. We performed quantitative trait loci (QTL) mapping using the FAIRE signal for each OCR as a quantitative trait. While individual OCRs were associated with a handful of specific genetic markers, gene expression levels were associated with many regulatory loci. We found multi-target trans-loci responsible for a very large number of OCRs, which seemed to reflect the widespread influence of certain chromatin regulators. Such regulatory hotspots were enriched for known regulatory functions, such as recombinational DNA repair, telomere replication, and general transcription control. The OCRs associated with these multi-target trans-loci coincided with recombination hotspots, telomeres, and gene-rich regions according to the function of the associated regulators. Our findings provide a global quantitative picture of the genetic architecture of chromatin regulation.

Kim, Kwoneel; Seo, Jungmin; Lee, Heun-Sik; Bogu, Gireesh K.; Kim, Dongsup; Lee, Sanghyuk; Lee, Byungwook; Choi, Jung Kyoon

2013-01-01

115

Chromatin Ionic Atmosphere Analyzed by a Mesoscale Electrostatic Approach  

PubMed Central

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

Gan, Hin Hark; Schlick, Tamar

2010-01-01

116

Differential chromatin packaging of genomic imprinted regions between expressed and non-expressed alleles  

Microsoft Academic Search

Chromosomal regions subject to genomic imprinting comprise a functional domain exhibiting parental- specific expression of genes and hence may take a unique chromatin structure. Here we have examined the chromatin packaging state of allelic sites in the Zfp127\\/Snrpn locus on mouse chromosome 7 and in the Igf2r locus on mouse chromosome 17 with an assay consisting of chromatin fractionation and

Takuya Watanabe; Akira Yoshimura; Yukio Mishima; Yoshiro Endo; Toshihiko Shiroishi; Tuyoshi Koide; Hiroyuki Sasaki; Hitoshi Asakura; Ryo Kominami

2000-01-01

117

Shaping chromatin for repair.  

PubMed

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

Gospodinov, Anastas; Herceg, Zdenko

2012-10-18

118

DNA condensation  

Microsoft Academic Search

Recent progress in our understanding of DNA condensation includes the observation of the collapse of single DNA molecules, greater insights into the intermolecular forces driving condensation, the recognition of helix-structure perturbation in condensed DNA, and the increasing recognition of the likely biological consequences of condensation. DNA condensed with cationic liposomes is an efficient agent for the transfection of eukaryotic cells,

Victor A Bloomfield

1996-01-01

119

Analysis of Chromatin Organisation  

ERIC Educational Resources Information Center

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

Szeberenyi, Jozsef

2011-01-01

120

Sperm Chromatin: An Overview  

Microsoft Academic Search

\\u000a The dramatic changes in the structure and function of sperm chromatin that occur during spermatogenesis have continued to\\u000a intrigue researchers for more than a century. In addition to wanting to understand how these changes in chromatin organization\\u000a affect genome function, many of the studies conducted in placental mammals have been driven by a desire to understand the\\u000a relationship between sperm

Rod Balhorn

121

Solenoidal model for superstructure in chromatin.  

PubMed Central

Chromatin prepared by brief digestion of nuclei with micrococcal nuclease, and extracted in 0.2 mM EDTA, appears in the electron microscope as filaments of about 100 A diameter which coil loosely. In 0.2 mM Mg++ these "nucleofilaments" condense into a supercoil or solenoidal structure of pitch about 110 A corresponding to the diameter of a nucleofilament. It is proposed that the x-ray reflections at orders of 110 A observed in chromatin originate in the spacing between turns of the solenoid rather than that between nucleosomes along the nucleofilament. The solenoidal structure appears to need histone H1 for its stabilization. Under certain conditions, isolated nucleosomes can also aggregate into a similar structure. The solenoidal structure can be correlated with the "thread" of diameter about 300 A observed by other workers in nuclei. Images

Finch, J T; Klug, A

1976-01-01

122

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

PubMed

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

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

2013-08-21

123

Epigenomics and chromatin dynamics  

PubMed Central

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

2012-01-01

124

Prediction of human cell radiosensitivity: Comparison of clonogenic assay with chromosome aberrations scored using premature chromosome condensation with fluorescence in situ hybridization  

Microsoft Academic Search

The purpose of the present investigation was to determine whether chromosome aberrations scored by premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) can predict the radiosensitivity of human cell lines, thereby providing a possible means of assessing the in situ radiosensitivity of normal tissues and the radiocurability of individual human cancers. We used four cells lines of different

K. Sasai; J. W. Evans; M. S. Kovacs

1994-01-01

125

Chromatin Structure Gene Expression  

Microsoft Academic Search

It is now well understood that chromatin structure is perturbed in the neighborhood of expressed genes. This is most obvious in the neighborhood of promoters and enhancers, where hypersensitivity to nucleases marks sites that no longer carry canonical nucleosomes, and to which transcription factors bind. To study the relationship between transcription factor binding and the generation of these hypersensitive regions,

Gary Felsenfeld; Joan Boyes; Jay Chung; David Clark; Vasily Studitsky

1996-01-01

126

Chromatin and DNA replication.  

PubMed

The size of a eukaryotic genome presents a unique challenge to the cell: package and organize the DNA to fit within the confines of the nucleus while at the same time ensuring sufficient dynamics to allow access to specific sequences and features such as genes and regulatory elements. This is achieved via the dynamic nucleoprotein organization of eukaryotic DNA into chromatin. The basic unit of chromatin, the nucleosome, comprises a core particle with 147 bp of DNA wrapped 1.7 times around an octamer of histones. The nucleosome is a highly versatile and modular structure, both in its composition, with the existence of various histone variants, and through the addition of a series of posttranslational modifications on the histones. This versatility allows for both short-term regulatory responses to external signaling, as well as the long-term and multigenerational definition of large functional chromosomal domains within the nucleus, such as the centromere. Chromatin organization and its dynamics participate in essentially all DNA-templated processes, including transcription, replication, recombination, and repair. Here we will focus mainly on nucleosomal organization and describe the pathways and mechanisms that contribute to assembly of this organization and the role of chromatin in regulating the DNA replication program. PMID:23751185

MacAlpine, David M; Almouzni, Geneviève

2013-08-01

127

The Telomere Binding Protein TRF2 Induces Chromatin Compaction  

PubMed Central

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.

Baker, Asmaa M.; Fu, Qiang; Hayward, William; Victoria, Samuel; Pedroso, Ilene M.; Lindsay, Stuart M.; Fletcher, Terace M.

2011-01-01

128

The telomere binding protein TRF2 induces chromatin compaction.  

PubMed

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. PMID:21526145

Baker, Asmaa M; Fu, Qiang; Hayward, William; Victoria, Samuel; Pedroso, Ilene M; Lindsay, Stuart M; Fletcher, Terace M

2011-04-19

129

Antimutagenic activity of some naturally occurring compounds towards cigarette-smoke condensate and benzo[a]pyrene in the Salmonella/microsome assay.  

PubMed

Several compounds, occurring in food, were tested for antimutagenic activity towards cigarette-smoke condensate (CSC) and benzo[a]pyrene (BaP). Antimutagenicity was determined in the Salmonella/microsome test, with tester strain TA98, in the presence of rat-liver homogenate. Dose-response curves did show reduction of CSC- and BaP-induced mutagenicity by ellagic acid, riboflavin and chlorophyllin. Chlorophyll a and chlorophyll b, although less distinct, also inhibited CSC- and BaP-induced mutagenicity. Ascorbic acid, beta-carotene, tocopherol acetate, chlorogenic acid and butyl hydroxyanisole did not have any influence on the mutagenicity of CSC and BaP. The similarity in results for cigarette-smoke condensate and for BaP indicates that a general mechanism may be involved in the inhibition of CSC- and BaP-induced mutagenicity. PMID:3900711

Terwel, L; van der Hoeven, J C

1985-10-01

130

Protamines: structural complexity, evolution and chromatin patterning.  

PubMed

Despite their relatively arginine-rich composition, protamines exhibit a high degree of structural variation. Indeed, the primary structure of these histone H1-related sperm nuclear basic proteins (SNBPs) is not random and is the depository of important phylogenetic information. This appears to be the result of their fast rate of evolution driven by positive selection. The way by which the protein variability participates in the transitions that lead to the final highly condensed chromatin organization of spermatozoa at the end of spermiogenesis is not clearly understood. In this paper we focus on the transient chromatin/nucleoplasm patterning that occurs in either a lamellar step or an inversion step during early and mid-spermiogenesis. This takes place in a small subset of protamines in internally fertilizing species of vertebrates, invertebrates and plants. It involves "complex" protamines that are processed, replaced, or undergo side chain modification (such as phosphorylation or disulfide bond formation) during the histone-to-protamine transition. Characteristic features of such patterning, as observed in TEM photomicrographs, include: constancy of the dominant pattern repeat distance ?(m) despite dynamic changes in developmental morphology, bicontinuity of chromatin and nucleoplasm, and chromatin orientation either perpendicular or parallel to the nuclear envelope. This supports the hypothesis that liquid - liquid phase separation by the mechanism of spinodal decomposition may be occurring during spermiogenesis in these species. Spinodal decomposition involves long wave fluctuations of the local concentration with a low energy barrier and thus differs from the mechanism of nucleation and growth that is known to occur during spermiogenesis in internally fertilizing mammals. PMID:21443489

Kasinsky, Harold E; Eirín-López, José María; Ausió, Juan

2011-08-01

131

CONDENSATION CAN  

DOEpatents

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

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

1962-03-01

132

p300 Forms a Stable, Template-Committed Complex with Chromatin: Role for the Bromodomain  

Microsoft Academic Search

The nature of the interaction of coactivator proteins with transcriptionally active promoters in chromatin is a fundamental question in transcriptional regulation by RNA polymerase II. In this study, we used a bio- chemical approach to examine the functional association of the coactivator p300 with chromatin templates. Using in vitro transcription template competition assays, we observed that p300 forms a stable,

E. TORY MANNING; TSUYOSHI IKEHARA; TAKASHI ITO; JAMES T. KADONAGA; W. L. Kraus

2001-01-01

133

Image analysis of chromatin remodelling.  

PubMed

Chromatin packaging plays a significant role in regulating gene transcription. Study of the higher-order packing states of chromatin by image analysis at the light microscope level, especially when validated by methods of molecular biology, immunochemistry, and/or immunocytochemistry, enabled the detection of changes involved in the processes associated with or preceding alterations in transcriptional activities. Here, we recommend and describe the use of relatively simple methods for staining and detecting chromatin remodelling by image analysis. PMID:24162983

de Campos Vidal, Benedicto; Felisbino, Marina B; Mello, Maria Luiza S

2014-01-01

134

Ultrastructural organization of yeast chromatin  

PubMed Central

The ultrastructural organization of yeast chromatin was examined in Miller spread preparations of samples prepared from spheroplasts or isolated nuclei of Saccharomyces cerevisiae. Micrographs from preparations dispersed in 1 mM Tris (pH 7.2) illustrate that the basic chromatin fiber in yeast exists in two ultrastructurally distinct conformations. The majority (up to 95%) of the chromatin displays a beaded nucleosomal organization, although adjacent nucleosomes are separated by internucleosomal linkers of variable lengths. Ribonucleoprotein (RNP) fibrils are only occasionally associated with chromatin displaying the conformation. The remaining 5-10% of the chromatin appears to be devoid of discrete nucleosomes and has a smooth contour with a fiber diameter of 30-40 A. Transcriptional units, including putative ribosomal precursor RNA genes, defined by the presence of nascent RNP fibrils are restricted to chromatin displaying this smooth morphology. Chromatin released from nuclei in the presence of 5 mM Mg++ displays higher-order chromatin fibers, 200-300 A in diameter, these fibers appear to be arranged in a manner than reflects the two forms of the basic chromatin fiber.

1982-01-01

135

Mapping chromatin modifications in nanochannels  

NASA Astrophysics Data System (ADS)

DNA and chromatin are elongated to a fixed fraction of their contour length when introduced into quasi-1d nanochannels. Because single molecules are analyzed, their hold great potential for the analysis for the genetic analysis of material from single cells. In this study, we have reconstituted chromatin with histones from a variety of sources, and mapped the modification profile of the chromatin. We monitored methylation and acetylation patterns of the histone tail protein residues using fluorescently labelled antibodies. Using those, we distinguished chromatin reconstituted from chicken erythrocytes, calf thymus, and HeLa cells. We discuss prospects for profiling histone modifications for whole chromosomes from single cells.

Lim, Shuang Fang; Karpusenko, Alena; Riehn, Robert

2013-03-01

136

Facteurs impliqués dans le remodelage de la chromatine au cours de la spermiogenèse  

Microsoft Academic Search

Resume  Les spermatides rondes sont les cellules issues de la méiose dont le noyau haploïde présente initialement une structure similaire\\u000a à celle d’une cellule somatique. Au cours de la maturation post-méiotique de la spermatide, ou spermiogenèse, la chromatine\\u000a subit un remodelage au cours duquel le noyau de la spermatide s’allonge puis se condense pour former le noyau spermatique,\\u000a dont la chromatine

Sophie Rousseaux; Cécile Caron; Christophe Pivot-Pajot; Anne Karen Faure; Mira Hazzouri; Bernard Sele; Saadi Khochbin

2003-01-01

137

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

Microsoft Academic Search

Isolated human buccal epithelium cell were irradiated by microwaves at frequency f=35 GHz and surface power density E=30 mcW\\/cm2. The state of chromatin in human cells was determined by methodsof light and electron microscopy. The state of cell membranes was evaluated by the method of vital indigo carmine staining. The microwave-induced condensation of chromatin in human cells was revealed. Left

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

2008-01-01

138

Circle ligation of in vitro assembled chromatin indicates a highly flexible structure  

Microsoft Academic Search

Evidence is provided that some condensed linker histone-containing chromatin structures are highly flexible in solutions containing 2 mM Mg2+. Chromatin assembled in vitro 6 histone H5 on a 6.3 kb linear DNA fragment in 90 mM NaCl using the polyglutamic acid method sedimented fairly homo- geneously. The H5-containing sample had s20, w values that were 58-69% greater than the sample

A. Stein; Y. Dalal; T. J. Fleury

2002-01-01

139

Sperm chromatin structure components are differentially repaired in cancer survivors.  

PubMed

Chemotherapy is often associated with male infertility. Our aim was to determine the effect of chemotherapy on sperm chromatin quality in cancer survivors. Sixteen men with advanced testicular cancer and 15 with Hodgkin lymphoma requiring chemotherapy were compared with 11 community volunteers. Eleven idiopathic infertile men with abnormal sperm chromatin were included as a positive control group. Semen analysis and sperm chromatin quality were determined prechemotherapy and at 6, 18, and 24 months posttreatment. DNA damage was determined by the sperm chromatin structure assay (SCSA). The level of DNA compaction was assayed by determining high DNA stainability (HDS, SCSA), the percentage of free thiols (monobromobimane-labeling assay), and the level of protamination (chromomycin A3-labeling assay). Sperm concentration and motility were dramatically decreased in cancer patients 6-18 months after chemotherapy compared with community volunteers but were not statistically different from community volunteers at 24 months posttreatment. High levels of DNA damage were observed prechemotherapy, with a tendency to remain high during the 24-month posttreatment period in testicular cancer patients; low DNA compaction (HDS, SCSA) persisted in testicular cancer patients 24 months postchemotherapy. Low levels of sperm DNA compaction were observed in cancer patients compared with community volunteers and infertile men. Sperm monobromobimane and chromomycin A3 labeling in cancer patients were similar to those from community volunteers by 18 months after treatment. Chemotherapy-induced damage to components of the sperm chromatin structure was repaired differentially over time. However, significant sperm DNA damage and low DNA compaction remained up to 24 months posttreatment. The assessment of complementary aspects of sperm chromatin quality is necessary to evaluate sperm samples in cancer survivors. PMID:22052778

O'Flaherty, Cristian M; Chan, Peter T; Hales, Barbara F; Robaire, Bernard

2011-11-03

140

Induction of stable protein-deoxyribonucleic acid adducts in Chinese hamster cell chromatin by ultraviolet light  

Microsoft Academic Search

Ultraviolet (uv)-light-mediated formation of protein-DNA adducts in Chinese hamster cell chromatin was investigated in an attempt to compare chromatin alterations induced in vitro with those observed in vivo. Three independent methods of analysis indicated stable protein-DNA associations: a membrane filter assay which retained DNA on the filter in the presence of high salt-detergent; a Sepharose 4B column assay in which

Gary F. Strniste; S. Carlton Rall

1976-01-01

141

Deciphering how the chromatin factor RCC1 recognizes the nucleosome: the importance of individuals in the scientific discovery process  

PubMed Central

The nucleosome repeating unit of chromatin is the target of chromatin enzymes and factors which regulate gene activity in a eukaryotic cell. How the nucleosome is recognized by chromatin enzymes and factors is poorly understood even though such interaction is fundamental to gene regulation and chromatin biology. My laboratory recently determined the structural basis for how the RCC1 (regulator of chromosome condensation) chromatin factor binds to the nucleosome, including the first atomic crystal structure of a chromatin protein complexed with the nucleosome core particle. I describe here how we developed and investigated structural models for RCC1 binding to the nucleosome using biochemical methods, and how we crystallized the 300 kDa complex of RCC1 with the nucleosome core particle. This article highlights the contributions made by key laboratory members and explains our thinking and rationale during the discovery process.

Tan, Song

2012-01-01

142

Histone modifications influence mediator interactions with chromatin  

PubMed Central

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

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

2011-01-01

143

Papillomavirus interaction with cellular chromatin  

PubMed Central

High-risk human papillomavirus (HPV) infection is the primary risk factor for cervical cancer. HPVs establish persistent infection by maintaining their genomes as extrachromosomal elements (episomes) that replicate along with host DNA in infected cells. The productive life cycle of HPV is intimately tied to the differentiation program of host squamous epithelium. This review examines the involvement of host chromatin in multiple aspects of the papillomavirus life cycle and the malignant progression of infected host cells. Papillomavirus utilizes host mitotic chromosomes as vehicles for transmitting its genetic materials across the cell cycle. By hitchhiking on host mitotic chromosomes, the virus ensures accurate segregation of the replicated viral episomes to the daughter cells during host cell division. This strategy allows persistent maintenance of the viral episome in the infected cells. In the meantime, the virus subverts the host chromatin-remodeling factors to promote viral transcription and efficient propagation of viral genomes. By associating with the host chromatin, papillomavirus redirects the normal cellular control of chromatin to create a cellular environment conducive to both its own survival and malignant progression of host cells. Comprehensive understanding of HPV-host chromatin interaction will offer new insights into the HPV life cycle as well as chromatin regulation. This virus-host interaction will also provide a paradigm for investigating other episomal DNA tumor viruses that share a similar mechanism for interacting with host chromatin.

You, Jianxin

2009-01-01

144

Single Molecule Studies of Chromatin  

SciTech Connect

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

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

2004-01-06

145

Conformational study of the binding of a high mobility group protein with chromatin  

SciTech Connect

The nature of the binding of a high mobility group protein (HMG 17) to native and H1-H5-depleted chicken erythrocyte chromatin was studied, as a function of ionic strength, using circular dichroism and thermal denaturation techniques. The circular dichroism properties of the HMG 17-reconstituted whole chromatin and H1-H5-depleted chromatin structure occurred upon HMG 17 binding at low ionic strength. Thermal denaturation profiles confirmed this change in the structure of chromatin induced by HMG 17. Thermal denaturation profiles were resolved into three-component transitions. These results indicate that the binding sites of HMG 17 are situated in the linker regions immediately adjacent to the core. The nature of the interaction of HMG 17 at higher ionic strength with whole chromatin and H1-H5-depleted chromatin was found to be different. These observations suggest that HMG 17 does not loosen chromatin structure but produces an overall stabilization and condensation of structure. The implications of these results to the currently accepted models of transcriptionally active chromatin are discussed.

Sasi, R.; Huvoes, P.E.; Fasman, G.D.

1982-10-10

146

Mesoscale Modeling of Chromatin Folding  

NASA Astrophysics Data System (ADS)

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

Schlick, Tamar

2009-03-01

147

Chromatin, Cancer and Drug Therapies  

PubMed Central

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

Cortez, Connie C.; Jones, Peter A.

2008-01-01

148

Chromatin and Transcription in Yeast  

PubMed Central

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

Rando, Oliver J.; Winston, Fred

2012-01-01

149

Measuring chromatin interaction dynamics on the second time scale at single-copy genes.  

PubMed

The chromatin immunoprecipitation (ChIP) assay is widely used to capture interactions between chromatin and regulatory proteins, but it is unknown how stable most native interactions are. Although live-cell imaging suggests short-lived interactions at tandem gene arrays, current methods cannot measure rapid binding dynamics at single-copy genes. We show, by using a modified ChIP assay with subsecond temporal resolution, that the time dependence of formaldehyde cross-linking can be used to extract in vivo on and off rates for site-specific chromatin interactions varying over a ~100-fold dynamic range. By using the method, we show that a regulatory process can shift weakly bound TATA-binding protein to stable promoter interactions, thereby facilitating transcription complex formation. This assay provides an approach for systematic, quantitative analyses of chromatin binding dynamics in vivo. PMID:24091704

Poorey, Kunal; Viswanathan, Ramya; Carver, Melissa N; Karpova, Tatiana S; Cirimotich, Shana M; McNally, James G; Bekiranov, Stefan; Auble, David T

2013-10-03

150

Chromatin Remodeling by RNA Polymerase II  

Microsoft Academic Search

Tight compaction of eukaryotic DNA in chromatin having several levels of organization results in formation of a structure that is barely accessible to regulatory protein complexes and enzymes such as DNA- and RNA-polymerases. To deal with this intricate chromatin organization, numerous chromatin remodeling mechanisms operating at various levels of chromatin structure have been developed. Recently, it has become apparent that

V. M. Studitsky

2005-01-01

151

Chromatin insulator elements: establishing barriers to set heterochromatin boundaries.  

PubMed

Epigenomic profiling has revealed that substantial portions of genomes in higher eukaryotes are organized into extensive domains of transcriptionally repressive chromatin. The boundaries of repressive chromatin domains can be fixed by DNA elements known as barrier insulators, to both shield neighboring gene expression and to maintain the integrity of chromosomal silencing. Here, we examine the current progress in identifying vertebrate barrier elements and their binding factors. We overview the design of the reporter assays used to define enhancer-blocking and barrier insulators. We look at the mechanisms vertebrate barrier proteins, such as USF1 and VEZF1, employ to counteract Polycomb- and heterochromatin-associated repression. We also undertake a critical analysis of whether CTCF could also act as a barrier protein. There is good evidence that barrier elements in vertebrates can form repressive chromatin domain boundaries. Future studies will determine whether barriers are frequently used to define repressive domain boundaries in vertebrates. PMID:22332659

Barkess, Gráinne; West, Adam G

2012-02-01

152

Making sense of transcribing chromatin  

PubMed Central

Eukaryotic cells package their genomes into a nucleoprotein form called chromatin. The basic unit of chromatin is the nucleosome, formed by the wrapping of ?147 bp of DNA around an octameric complex of core histones. Advances in genomic technologies have enabled the locations of nucleosomes to be mapped across genomes [1,2]. This has revealed a striking organisation with respect to transcribed genes in a diverse range of eukaryotes. This consists of a nucleosome depleted region upstream of promoters, with an array of well spaced nucleosomes extending into coding regions [2]. This observation reinforces the links between chromatin organisation and transcription. Central to this is the paradox that while chromatin is required by eukaryotes to restrict inappropriate access to DNA, this must be overcome in order for genetic information to be expressed. This conundrum is at its most flagrant when considering the need for nucleic acid polymerase's to transit 1000's of based pairs of DNA wrapped as arrays of nucleosomes.

Owen-Hughes, Tom; Gkikopoulos, Triantafyllos

2012-01-01

153

Epigenetic aspects of HP1 exchange kinetics in apoptotic chromatin.  

PubMed

Apoptotic bodies are the most condensed form of chromatin. In general, chromatin structure and function are mostly dictated by histone post-translational modifications. Thus, we have analyzed the histone signature in apoptotic cells, characterized by pronounced chromatin condensation. Here, H2B mono-acetylation, and H3K9 and H4 acetylation was significantly decreased in apoptotic cells, which maintained a high level of H3K9 methylation. This phenotype was independent of p53 function and distinct levels of anti-apoptotic Bcl2 protein. Interestingly, after etoposide treatment of leukemia and multiple myeloma cells, H3K9 and H4 hypoacetylation was accompanied by increased H3K9me2, but not H3K9me1 or H3K9me3. In adherent mouse fibroblasts, a high level of H3K9me3 and histone deacetylation in apoptotic bodies was likely responsible for the pronounced (?40%) recovery of GFP-HP1? and GFP-HP1? after photobleaching. HP1 mobility in apoptotic cells appeared to be unique because limited exchange after photobleaching was observed for other epigenetically important proteins, including GFP-JMJD2b histone demethylase (?10% fluorescence recovery) or Polycomb group-related GFP-BMI1 protein (?20% fluorescence recovery). These findings imply a novel fact that only certain subset of proteins in apoptotic bodies is dynamic. PMID:23023195

Legartová, So?a; Jugová, Alžb?ta; Stixová, Lenka; Kozubek, Stanislav; Fojtová, Miloslava; Zdráhal, Zbyn?k; Lochmanová, Gabriela; Bártová, Eva

2012-09-27

154

Differential Chromatin Structure Encompassing Replication Origins in Transformed and Normal Cells  

PubMed Central

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

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

2012-01-01

155

ATP-dependent Chromatin Remodelling  

Microsoft Academic Search

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

Parul Choudhary; Patrick Varga-Weisz

156

Chromatin immunoprecipitation of mouse embryos.  

PubMed

During prenatal development, a large number of different cell types are formed, the vast majority of which contain identical genetic material. The basis of the great variety in cell phenotype and function is the differential expression of the approximately 25,000 genes in the mammalian genome. Transcriptional activity is regulated at many levels by proteins, including members of the basal transcriptional apparatus, DNA-binding transcription factors, and chromatin-binding proteins. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency, with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method to assess if chromatin modifications or proteins are present at a specific locus. ChIP involves the cross linking of DNA and associated proteins and immunoprecipitation using specific antibodies to DNA-associated proteins followed by examination of the co-precipitated DNA sequences or proteins. In the last few years, ChIP has become an essential technique for scientists studying transcriptional regulation and chromatin structure. Using ChIP on mouse embryos, we can document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development. Here, we describe a ChIP technique adapted for mouse embryos. PMID:22113287

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

2012-01-01

157

Spontaneous access to DNA target sites in folded chromatin fibers  

PubMed Central

Summary DNA wrapped in nucleosomes is sterically occluded from many protein complexes that must act on it; how such complexes gain access to nucleosomal DNA is not known. In vitro studies on isolated nucleosomes show that they undergo spontaneous partial unwrapping conformational transitions, which make the wrapped nucleosomal DNA transiently accessible. Thus, site exposure might provide a general mechanism allowing access of protein complexes to nucleosomal DNA. However, existing quantitative analyses of site exposure focused on single nucleosomes, while the presence of neighbor-nucleosomes, and concomitant chromatin folding, might significantly influence site exposure. Here, we carry out quantitative studies on the accessibility of nucleosomal DNA in homogeneous nucleosome arrays. Two striking findings emerge. Organization into chromatin fibers changes the accessibility of nucleosomal DNA only modestly, from ~3-fold decreases in accessibility to ~8-fold increases. This means that nucleosome arrays are intrinsically dynamic and accessible even when they are visibly condensed. In contrast, chromatin folding decreases the accessibility of linker DNA by as much as ~50-fold. Thus, nucleosome positioning dramatically influences the accessibility of target sites located inside nucleosomes, while chromatin folding dramatically regulates access to target sites in linker DNA.

Poirier, Michael G.; Bussiek, Malte; Langowski, Jorg; Widom, Jonathan

2008-01-01

158

A model for the structure of chromatin in mammalian sperm  

PubMed Central

DNA in mammalian, and most vertebrate sperm, is packaged by protamines into a highly condensed, biochemically inert form of chromatin. A model is proposed for the structure of this DNA-protamine complex which describes the site and mode of protamine binding to DNA and postulates, for the first time, specific inter- and intraprotamine interactions essential for the organization of this highly specialized chromatin. In this model, the central polyarginine segment of protamine binds in the minor groove of DNA, crosslinking and neutralizing the phosphodiester backbone of DNA while the COOH- and NH2-terminal ends of protamine participate in the formation of inter- and intraprotamine hydrogen, hydrophobic, and disulfide bonds. Each protamine segment is of sufficient length to fill one turn of DNA, and adjacent protamines are locked in place around DNA by multiple disulfide bridges. Such an arrangement generates a neutral, insoluble chromatin complex, uses all protamine sulfhydryl groups for cross linking, conserves volume, and effectively renders the chromatin invulnerable to most external influences.

1982-01-01

159

Dropwise condensation  

PubMed Central

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

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

2008-01-01

160

Effect of the fungal mycotoxin patulin on the chromatin structure of fission yeast Schizosaccharomyces pombe.  

PubMed

The fungal mycotoxin patulin is produced by several molds, especially by Aspergillus and Penicillium. The aim of this study was to clarify whether patulin causes alterations in plasma membrane permeability of Schizosaccharomyces pombe lead to cellular shrinkage charateristic to apoptosis or increases cell size indicating necrosis in cells. Transmission and scanning electronmicroscopy revealed that lower concentrations of patulin induced cellular shrinkage and blebbing, higher concentration caused expansion without cellular disruption. Large-scale morphological changes of individual cells were followed by time lapse video microscopy. Patulin caused the elongation and stickiness of cells or rounded up their shapes. To visualize chromatin structures of S. pombe nuclei upon patulin treatment, protoplasts were isolated from S. pombe and subjected to fluorescent microscopy. Chromatin changes in the presence of 50 ?M patulin concentration were characterized by elongated nuclei containing sticky fibrillary chromatin and enlarged round shaped nuclei trapped at the fibrillary stage of chromatin condensation. Short (60 min) incubation of S. pombe cells in the presence of high (500 ?M) patulin concentration generated patches of condensed chromatin bodies inside the nucleus and caused nuclear expansion, with the rest of chromatin remaining in fibrillary form. Longer (90 min, 500 ?M) incubation resulted in fewer highly condensed chromatin patches and in nuclear fragmentation. Although, high patulin concentration increased the size of S. pombe size, it did not lead to necrotic explosion of cells, neither did the fragmented nuclei resemble apoptotic bodies that would have indicated programmed cell death. All these morphological changes and the high rate of cell survival point to rapid adaptation and mixed type of fungistatic effects. PMID:22359238

Horvath, Eszter; Nagy, Gabor; Turani, Melinda; Balogh, Eniko; Papp, Gabor; Pollak, Edit; Pocsi, Istvan; Pesti, Miklos; Banfalvi, Gaspar

2012-02-23

161

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

PubMed Central

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

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

2008-01-01

162

A Method to Study the Epigenetic Chromatin States of Rare Hematopoietic Stem and Progenitor Cells; MiniChIP–Chip  

Microsoft Academic Search

Dynamic chromatin structure is a fundamental property of gene transcriptional regulation, and has emerged as a critical modulator of physiological processes during cellular differentiation and development. Analysis of chromatin structure using molecular biology and biochemical assays in rare somatic stem and progenitor cells is key for understanding these processes but poses a great challenge because of their reliance on millions

Holger Weishaupt; Joanne L. Attema

2010-01-01

163

Dermatan sulfate synergizes with heparin in murine sperm chromatin decondensation.  

PubMed

The mammalian sperm nucleus contains an unusually condensed chromatin, due to replacement of the majority of histones by protamines. However, soon after the spermatozoon penetrates the ooplasm at fertilization, decondensation of this densely packed chromatin must occur to allow formation of the male pronucleus and syngamy. Decondensation is accomplished by protamine disulfide bond reduction by oocyte glutathione and replacement of protamines by oocyte histones with the aid of an acceptor molecule. Previous results from our laboratory have demonstrated that heparan sulfate (HS) present in the ooplasm functions as protamine acceptor during human sperm decondensation in vivo. In the present paper, we analyze the role of heparin, structural analogue of HS, and dermatan sulfate (DS) in murine sperm chromatin decondensation in vitro, including the possibility of a synergistic effect between both glycosaminoglycans. Decondensation was assessed under phase contrast microscopy following incubation of murine spermatozoa with glutathione and either heparin, DS, or a combination of both. Ultrastructural changes taking place during decondensation were analyzed by transmission electron microscopy. Both glycosaminoglycans were able to promote the decondensation of murine spermatozoa in vitro but the decondensing ability of heparin was significantly higher. Use of both glycosaminoglycans together revealed the existence of a synergistic effect. Transmission electron microscopy analysis of decondensing spermatozoa supported these findings. Synergism between heparin and DS was observed both in capacitated and non-capacitated spermatozoa but decondensation kinetics was faster in the former. The results obtained indicate a new potential role for dermatan sulfate in murine sperm decondensation at fertilization and provide evidence of differences in the degree of chromatin condensation throughout the murine sperm nucleus. PMID:23301776

Sanchez, Melisa Celeste; Sedo, Cristian Alvarez; Julianelli, Vanina Laura; Romanato, Marina; Calvo, Lucrecia; Calvo, Juan Carlos; Fontana, Vanina Andrea

2013-01-09

164

BRIT1/MCPH1 Links Chromatin Remodeling to DNA Damage Response  

PubMed Central

To detect and repair damaged DNA, DNA damage response proteins need to overcome the barrier of condensed chromatin to gain access to DNA lesions1. ATP-dependent chromatin remodeling is one of the fundamental mechanisms used by cells to relax chromatin in DNA repair2–3. However, the mechanism mediating their recruitment to DNA lesions remains largely unknown. BRIT1 (also known as MCPH1) is an early DNA damage response protein that is mutated in human primary microcephaly4–8. We report here a previously unknown function of BRIT1 as a regulator of ATP-dependent chromatin remodeling complex SWI/SNF in DNA repair. Upon DNA damage, BRIT1 increases its interaction with SWI/SNF through the ATM/ATR-dependent phosphorylation on the BAF170 subunit. This increase of binding affinity provides a means by which SWI/SNF can be specifically recruited to and maintained at DNA lesions. Loss of BRIT1 causes impaired chromatin relaxation owing to reduced association of SWI/SNF with chromatin. This explains the decreased recruitment of repair proteins to DNA lesions and reduced efficiency of repair in BRIT1-deficient cells, resulting in impaired survival from DNA damage. Our findings, therefore, identify BRIT1 as a key molecule that links chromatin remodeling with DNA damage response in the control of DNA repair, and its dysfunction contributes to human disease.

Peng, Guang; Yim, Eun-Kyoung; Dai, Hui; Jackson, Andrew P.; van der Burgt, Ineke; Pan, Mei-Ren; Hu, Ruozhen; Li, Kaiyi; Lin, Shiaw-Yih

2009-01-01

165

Single Molecule Studies of Chromatin  

SciTech Connect

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.

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

2006-02-06

166

Structural and functional genome analysis using extended chromatin  

SciTech Connect

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

Heaf, T.; Ward, D.C. [Yale Univ., New Haven, CT (United States)

1994-09-01

167

Imaging of DNA/Nanosphere Condensates  

NASA Astrophysics Data System (ADS)

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

Krishnan, R.

2005-03-01

168

Incorporation of 1-(14)C linoleic acid in rat liver nuclei and chromatin fractions.  

PubMed

The incorporation of 1-(14)C linoleic acid in several chromatin fractions of rat liver nuclei was investigated using two different procedures: (1) rat liver nuclei were incubated with ATP, CoASH, Mg(++) and 1-(14)C linoleic acid. After 40 min at 37 degrees C the chromatin obtained by sonication of nuclei suspended in 0.25 M sucrose was fractionated by differential sedimentation; (2) chromatin fractions obtained by differential sedimentation were incubated separately with ATP, CoASH, Mg(++) and 1-(14)C linoleic acid 40 min at 37 degrees C in order to characterize the fatty acid incorporation in isolated chromatin. A comparative study of the incorporation of 1-(14)C linoleic acid in microsomes and nuclei isolated from rat liver is also presented for the purpose of comparison. Linoleic acid was incorporated into nuclear lipids as well as in chromatin fractions. The fatty acid incorporation was stimulated considerably in the acylation system when compared to control, it appears to be highly dependent on the state of condensation of chromatin, being barely detectable in the lowest density fraction. The major proportion of 1-(14)C linoleic acid was found in phospholipids and in a lesser proportion it remained esterified to triglycerides and cholesteryl esters. The distribution of radioactivity in different classes of phospholipids present in microsomes and nuclei isolated from rat liver, showed a similar profile of distribution. The major proportion of radioactivity, approximately 50% was found in phosphatidylcholine and in a lesser proportion in sphingomyelin, phosphatidylserine and phosphatidylethanolamine. When chromatin fractions were incubated separately, it was observed that the major proportion of 1-(14)C linoleic acid in phospholipids was found in heavy chromatin fractions whereas low density chromatin fraction only incorporated in a lesser proportion. PMID:11311857

Marmunti, M; Catalá, A

2001-03-01

169

Chromatin-Diminution bei Copepoden  

Microsoft Academic Search

1.The somatic chromosomes of Cyclops strenuus and of some othes Copepoda become different from those of the germ line cells by a procers of chromatin diminution similar to the classical case of Ascaris.2.The germ line cells investigated include primordial germ cells of the 5th cleavage division and oocytes in diplotene. In the germ line all 22 chromosomes terminate in long

Sigrid Beermann

1959-01-01

170

Mechanically stretching single chromatin fibers  

Microsoft Academic Search

Summary We have used the recently developed MAC Mode Atomic Force Microscope (AFM) that operates in aqueous solution to mechanically stretch single chicken erythrocyte chromatin fibers. The fibers contained the full complement of histones, or, alternatively, were depleted of linker histones. The AFM was used to produce the so- called force curves, by monitoring the cantilever deflection (proportional to force)

Sanford H. Leuba; Mikhail A. Karymov; Yanzhang Liu; Stuart M. Lindsay; Jordanka Zlatanova

1999-01-01

171

Chromatin profiling using targeted DNA adenine methyltransferase  

Microsoft Academic Search

Chromatin is the highly complex structure consisting of DNA and hundreds of associated proteins. Most chromatin proteins exert their regulatory and structural functions by binding to specific chromosomal loci. Knowledge of the identity of these in vivo tar- get loci is essential for the understanding of the functions and mechanisms of action of chromatin proteins. We report here large-scale mapping

Jeffrey Delrow; Steven Henikoff

2001-01-01

172

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

PubMed Central

Background The putative regulatory role of the male reproductive hormones in the molecular mechanism underlying chromatin condensation remains poorly understood. In the past decade, we developed two adult male rat models wherein functional deficits of testosterone or FSH, produced after treatments with 20?mg/Kg/d of cyproterone acetate (CPA) per os, for a period of 15?days or 3?mg/Kg/d of fluphenazine decanoate (FD) subcutaneously, for a period of 60?days, respectively, affected the rate of sperm chromatin decondensation in vitro. These rat models have been used in the current study in order to delineate the putative roles of testosterone and FSH in the molecular mechanism underlying remodelling of sperm chromatin. Results We report that deficits of both testosterone and FSH affected the turnover of polyubiquitylated histones and led to their accumulation in the testis. Functional deficits of testosterone reduced expression of MIWI, the 5-methyl cap binding RNA-binding protein (PIWIlike murine homologue of the Drosophila protein PIWI/P-element induced wimpy testis) containing a PAZ/Piwi-Argonaut-Zwille domain and levels of histone deacetylase1 (HDAC1), ubiquitin ligating enzyme (URE-B1/E3), 20S proteasome ?1 concomitant with reduced expression of ubiquitin activating enzyme (ube1), conjugating enzyme (ube2d2), chromodomain Y like protein (cdyl), bromodomain testis specific protein (brdt), hdac6 (histone deacetylase6), androgen-dependent homeobox placentae embryonic protein (pem/RhoX5), histones h2b and th3 (testis-specific h3). Functional deficits of FSH reduced the expression of cdyl and brdt genes in the testis, affected turnover of ubiquitylated histones, stalled the physiological DNA repair mechanism and culminated in spermiation of DNA damaged sperm. Conclusions We aver that deficits of both testosterone and FSH differentially affected the process of sperm chromatin remodelling through subtle changes in the ‘chromatin condensation transcriptome and proteome’, thereby stalling the replacement of ‘dynamic’ histones with ‘inert’ protamines, and altering the epigenetic state of condensed sperm chromatin. The inappropriately condensed chromatin affected the sperm chromatin cytoarchitecture, evident from subtle ultrastructural changes in the nuclei of immature caput epididymal sperm of CPA- or FD-treated rats, incubated in vitro with dithiothreitol.

2012-01-01

173

Acceptor proteins in rat androgenic tissue chromatin.  

PubMed Central

Fractionation of chromatin into urea-soluble chromosomal nonhistone proteins (UP), histones (HP), and DNA-associated nonhistone proteins (NP) revealed that the NP fraction from testicular and prostatic chromatin contains organ-specific acceptors for complexes of 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) and its receptor. This acceptor capacity of androgenic tissue chromatin could be transferred to chromatins from non-target tissues with the NP fraction of DNA-associated proteins. Phosphorylation of chromatin enhanced its hormone-receptor binding capacity.

Klyzsejko-Stefanowicz, L; Chiu, J F; Tsai, Y H; Hnilica, L S

1976-01-01

174

Chromatin rings as products of chromatin diminution in Cyclops  

Microsoft Academic Search

Nuclei from the interphase preceding the 6th cleavage (=first diminution) division of Cyclops furcifer were subjected to a micro-spreading technique (Counce and Meyer, 1973) and examined by electron microscopy. In some preparations numerous chromatin rings formed by 250–300 Å fibers were discovered in sizes ranging from 0.25 µm to more than 6 µm. These structures are assumed to represent the

Sigrid Beermann; Gfinther F. Meyer

1980-01-01

175

Understanding Condensation  

NSDL National Science Digital Library

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

Hartman, Monica

2007-12-01

176

Modulation of Higher Order Chromatin Conformation in Mammalian Cell Nuclei Can Be Mediated by Polyamines and Divalent Cations  

PubMed Central

The organisation of the large volume of mammalian genomic DNA within cell nuclei requires mechanisms to regulate chromatin compaction involving the reversible formation of higher order structures. The compaction state of chromatin varies between interphase and mitosis and is also subject to rapid and reversible change upon ATP depletion/repletion. In this study we have investigated mechanisms that may be involved in promoting the hyper-condensation of chromatin when ATP levels are depleted by treating cells with sodium azide and 2-deoxyglucose. Chromatin conformation was analysed in both live and permeabilised HeLa cells using FLIM-FRET, high resolution fluorescence microscopy and by electron spectroscopic imaging microscopy. We show that chromatin compaction following ATP depletion is not caused by loss of transcription activity and that it can occur at a similar level in both interphase and mitotic cells. Analysis of both live and permeabilised HeLa cells shows that chromatin conformation within nuclei is strongly influenced by the levels of divalent cations, including calcium and magnesium. While ATP depletion results in an increase in the level of unbound calcium, chromatin condensation still occurs even in the presence of a calcium chelator. Chromatin compaction is shown to be strongly affected by small changes in the levels of polyamines, including spermine and spermidine. The data are consistent with a model in which the increased intracellular pool of polyamines and divalent cations, resulting from depletion of ATP, bind to DNA and contribute to the large scale hyper-compaction of chromatin by a charge neutralisation mechanism.

Visvanathan, Ashwat; Ahmed, Kashif; Even-Faitelson, Liron; Lleres, David; Bazett-Jones, David P.; Lamond, Angus I.

2013-01-01

177

Chromatin Structure in Telomere Dynamics  

PubMed Central

The establishment of a specific nucleoprotein structure, the telomere, is required to ensure the protection of chromosome ends from being recognized as DNA damage sites. Telomere shortening below a critical length triggers a DNA damage response that leads to replicative senescence. In normal human somatic cells, characterized by telomere shortening with each cell division, telomere uncapping is a regulated process associated with cell turnover. Nevertheless, telomere dysfunction has also been associated with genomic instability, cell transformation, and cancer. Despite the essential role telomeres play in chromosome protection and in tumorigenesis, our knowledge of the chromatin structure involved in telomere maintenance is still limited. Here we review the recent findings on chromatin modifications associated with the dynamic changes of telomeres from protected to deprotected state and their role in telomere functions.

Galati, Alessandra; Micheli, Emanuela; Cacchione, Stefano

2013-01-01

178

Sperm chromatin in beef bulls in tropical environments.  

PubMed

Sperm chromatin status was assessed in 565 Zebu and Zebu crossbred beef bulls in extensive tropical environments using the sperm chromatin structure assay (SCSA). The SCSA involved exposure of sperm to acid hydrolysis for 0.5 or 5.0 minutes, followed by flow cytometry to ascertain relative amounts of double-stranded (normal) and single-stranded (denatured) DNA, which was used to generate a DNA fragmentation index (%DFI). With conventional SCSA (0.5-minute SCSA), 513 bulls (91%) had <15 %DFI, 24 bulls (4%) had 15 to 27 %DFI, and 28 bulls (5%) had >27 %DFI. In 5.0-minute SCSA, 432 bulls (76%) had <15 %DFI, 68 bulls (12%) had 15 to 27 %DFI and 65 bulls (12%) had >27 %DFI. For most bulls, the SCSA was repeatable on two to four occasions; however, because most bulls had <15 %DFI, repeatability of the SCSA will need to be determined in a larger number of bulls in the 15 to 27 %DFI and >27 %DFI categories. The %DFI was negatively correlated with several bull semen parameters and the strongest negative correlation was with normal sperm. There was a strong positive correlation between %DFI and sperm head abnormalities. Based on these findings, most Zebu beef bulls in extensive tropical environments had relatively stable sperm chromatin. Based on the apparent negative correlations with conventional semen parameters, we inferred that the SCSA measured a unique feature of sperm quality, which has also been suggested for other species. Further studies on the relationships between sperm chromatin stability and fertility are required in beef bulls before chromatin status can be used as an additional predictor of the siring capacity of individual bulls in extensive multiple-sire herds. PMID:23434358

D'Occhio, Michael J; Hengstberger, Kirstin J; Tutt, Desmond; Holroyd, Richard G; Fordyce, Geoffry; Boe-Hansen, Gry B; Johnston, Steve D

2013-02-21

179

Transcription factor access to chromatin.  

PubMed Central

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

Beato, M; Eisfeld, K

1997-01-01

180

The utility of sperm DNA damage assay using toluidine blue and aniline blue staining in routine semen analysis  

PubMed Central

Objective The aim of the present study was to examine the relationship among male age, strict morphology, and sperm chromatin structure and condensation. Methods Sperm samples from a total of 100 men underwent semen analysis, and sperm chromatin structure and condensation were assessed with toluidine blue (TB) and aniline blue (AB) tests. Results Prevalence of strict morphology of less than 4%, and abnormal sperm chromatin structure and condensation did not show any statistically significant differences according to male age (p=0.605, p=0.235, and p=0.080). No significant correlation was demonstrated among age of male partners, strict morphology, and abnormal sperm chromatin structure using TB and AB tests. However, abnormal sperm chromatin condensation was positively associated with sperm chromatin structure (r=0.594, p=0.000) and showed negative correlation with strict morphology (r=-0.219, p=0.029). Conclusion The tests for sperm chromatin condensation showed a significant association with strict morphology. Further study is needed to elucidate the relationship between clinical outcome and sperm chromatin tests.

Kim, Hee-Sun; Kang, Moon Joo; Kim, Sung Ah; Oh, Sun Kyung; Kim, Hoon; Ku, Seung-Yup; Kim, Seok Hyun; Moon, Shin Yong

2013-01-01

181

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

PubMed

The chromatin structure dynamics of the Colpoda inflata macronucleus have been investigated in relation to its functional condition, concerning chromatin body extrusion regulating activity. Samples of 2- and 25-day-old resting cysts derived from a standard culture, and of 1-year-old resting cysts derived from a senescent culture, were examined by means of histogram analysis performed on acquired optical microscopy images. Three groups of histograms were detected in each sample. Histogram classification, clustering and matching were assessed in order to obtain the mean histogram of each group. Comparative analysis of the mean histogram showed a similarity in the grey level range of 25-day- and 1-year-old cysts, unlike the wider grey level range found in 2-day-old cysts. Moreover, the respective mean histograms of the three cyst samples appeared rather similar in shape. All this implies that macronuclear chromatin structural features of 1-year-old cysts are common to both cyst standard cultures. The evaluation of the acquired images and their respective histograms evidenced a dynamic state of the macronuclear chromatin, appearing differently condensed in relation to the chromatin body extrusion regulating activity of the macronucleus. The coexistence of a chromatin-decondensed macronucleus with a pycnotic extrusion body suggests that chromatin unable to decondense, thus inactive, is extruded. This finding, along with the presence of chromatin structural features common to standard and senescent cyst populations, supports the occurrence of 'rejuvenated' cell lines from 1-year-old encysted senescent cells, a phenomenon which could be a result of accomplished macronuclear renewal. PMID:10439214

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

1999-01-01

182

Neutron scatter studies of chromatin structures related to functions  

SciTech Connect

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

Bradbury, E.M.

1992-01-01

183

Neutron scatter studies of chromatin structures related to functions  

SciTech Connect

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

Bradbury, E.M.

1992-01-01

184

Analysis of chromatin boundary activity in Drosophila cells  

PubMed Central

Background Chromatin boundaries, also known as insulators, regulate gene activity by organizing active and repressive chromatin domains and modulate enhancer-promoter interactions. However, the mechanisms of boundary action are poorly understood, in part due to our limited knowledge about insulator proteins, and a shortage of standard assays by which diverse boundaries could be compared. Results We report here the development of an enhancer-blocking assay for studying insulator activity in Drosophila cultured cells. We show that the activities of diverse Drosophila insulators including suHw, SF1, SF1b, Fab7 and Fab8 are supported in these cells. We further show that double stranded RNA (dsRNA)-mediated knockdown of SuHw and dCTCF factors disrupts the enhancer-blocking function of suHw and Fab8, respectively, thereby establishing the effectiveness of using RNA interference in our cell-based assay for probing insulator function. Conclusion The novel boundary assay provides a quantitative and efficient method for analyzing insulator mechanism and can be further exploited in genome-wide RNAi screens for insulator components. It provides a useful tool that complements the transgenic and genetic approaches for studying this important class of regulatory elements.

Li, Mo; Belozerov, Vladimir E; Cai, Haini N

2008-01-01

185

Chromatin signatures of the Drosophila replication program.  

PubMed

DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events. PMID:21177973

Eaton, Matthew L; Prinz, Joseph A; MacAlpine, Heather K; Tretyakov, George; Kharchenko, Peter V; MacAlpine, David M

2010-12-22

186

Chromatin signatures of the Drosophila replication program  

PubMed Central

DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events.

Eaton, Matthew L.; Prinz, Joseph A.; MacAlpine, Heather K.; Tretyakov, George; Kharchenko, Peter V.; MacAlpine, David M.

2011-01-01

187

Histone H1 is located in the interior of the chromatin 30-nm filament.  

PubMed

The linker histone H1 binds to the nucleosome and is essential for the organization of nucleosomes into the 30-nm filament of chromatin. It has been implicated in the repression of transcription, and phosphorylation of H1 may be involved in cell-cycle-dependent chromatin condensation and decondensation. A long-standing issue concerns the location of H1 in the chromatin filament. The original solenoidal model proposes that H1 is inside the 30-nm filament, but other models, also helical, suggest a variable or more accessible location for H1. Investigations to determine the location of the linker histone based on its accessibility to antibodies or immobilized proteases under various ionic conditions have yielded conflicting results. Here we use neutron scattering in a direct structural determination to show that H1 is located in the interior of the filament. PMID:8127372

Graziano, V; Gerchman, S E; Schneider, D K; Ramakrishnan, V

1994-03-24

188

Dedifferentiation of Tobacco Cells Is Associated with Ribosomal RNA Gene Hypomethylation, Increased Transcription, and Chromatin Alterations1[w  

PubMed Central

Epigenetic changes accompanying plant cell dedifferentiation and differentiation are reported in 35S ribosomal DNA (rDNA) of tobacco (Nicotiana tabacum). There was a reduction of CG and CNG methylation in both intergenic and genic regions of the rDNA cistron in fully dedifferentiated callus and root compared to leaf. The rDNA hypomethylation was not random, but targeted to particular rDNA gene families at units that are clustered within the tandem array. The process of hypomethylation was initiated as early as 2 weeks after the callus induction and established epigenetic patterns were stably maintained throughout prolonged culture. However, regenerated plants and their progeny showed partial and complete remethylation of units, respectively. Nuclear run-on assays revealed a 2-fold increase of primary (unprocessed) ribosomal RNA transcripts in callus compared to leaf tissue. However, the abundance of mature transcripts in callus was elevated by only about 25%. Fluorescence in situ hybridization analysis of interphase nuclei showed high levels of rDNA chromatin condensation in both callus and leaf, with substantially less decondensed rDNA than is observed in meristematic root-tip cells. It is likely that the regions of the rDNA locus showing decondensation correspond to the clusters of hypomethylated units that occur in the tandem array at each locus. The data together indicate that the establishment of pluripotency and cell proliferation occurring with callus induction is associated with enhanced ribosomal RNA gene expression and overall rDNA hypomethylation, but is not associated with material-enhanced relaxation of chromatin structure (decondensation) at rDNA loci.

Koukalova, Blazena; Fojtova, Miloslava; Lim, Kar Yoong; Fulnecek, Jaroslav; Leitch, Andrew Rowland; Kovarik, Ales

2005-01-01

189

Characterization of the RNA content of chromatin  

PubMed Central

Noncoding RNA (ncRNA) constitutes a significant portion of the mammalian transcriptome. Emerging evidence suggests that it regulates gene expression in cis or trans by modulating the chromatin structure. To uncover the functional role of ncRNA in chromatin organization, we deep sequenced chromatin-associated RNAs (CARs) from human fibroblast (HF) cells. This resulted in the identification of 141 intronic regions and 74 intergenic regions harboring CARs. The intronic and intergenic CARs show significant conservation across 44 species of placental mammals. Functional characterization of one of the intergenic CARs, Intergenic10, revealed that it regulates gene expression of neighboring genes through modulating the chromatin structure in cis. Our data suggest that ncRNA is an integral component of chromatin and that it may regulate various biological functions through fine-tuning of the chromatin architecture.

Mondal, Tanmoy; Rasmussen, Markus; Pandey, Gaurav Kumar; Isaksson, Anders; Kanduri, Chandrasekhar

2010-01-01

190

Electron spectroscopic tomography of specific chromatin domains.  

PubMed

The eukaryotic genome is packaged within the nucleus as poly-nucleosome 10 nm chromatin fibres. The nucleosome core particle, the fundamental chromatin subunit, consists of a DNA molecule wrapped around a histone octamer. Biochemical modifications of both the DNA and histone proteins have been characterized that influence chromatin structure and function. These modifications include DNA methylation, histone variants and posttranslational modifications of the core histone protein tails. An outstanding area for investigation in the field of nuclear cell biology is the characterization of the functional relation between these biochemical modifications and the underlying chromatin structure and nuclear sub-compartmentalization. Electron spectroscopic tomography is a high-resolution microscopy technique that facilitates visualization of individual 10 nm chromatin fibres in three dimensions. The method, therefore, has a role to play in exploring the relationships of the epigenome and nuclear organization. Correlating immunofluorescence microscopy with electron spectroscopic tomography provides a powerful approach to relate epigenetic marks with high resolution chromatin organization. PMID:23980008

Even-Faitelson, Liron; Fussner, Eden; Li, Ren; Strauss, Mike; Bazett-Jones, David P

2013-01-01

191

Chromatin Structure and the Cell Cycle  

Microsoft Academic Search

Pancreatic DNase I is used to probe the structure of chromatin isolated from synchronized HeLa cells. The degree to which DNA in chromatin is protected from DNase attack varies during the G1, S, and G2 phases of the cell cycle. In addition, the DNase sensitivity of chromatin from contact-inhibited African green monkey kidney cells differs from that of actively dividing,

Thoru Pederson

1972-01-01

192

RNA chain elongation on a chromatin template.  

PubMed Central

The rate of RNA chain elongation has been measured with DNA and chromatin as template. RNA propagation on chromatin is about 50% of the rate found with DNA. Kinetic experiments demonstrate that the inhibition is not due to interference with the addition of the nucleoside triphosphates. Analysis of the dependence of propagation on the Tm of DNA shows that the chromatin proteins interfere with the translocation of the RNA polymerase along the DNA template.

Solage, A; Cedar, H

1976-01-01

193

Histone H3 phosphorylation - a versatile chromatin modification for different occasions.  

PubMed

Post-translation modifications of histones modulate the accessibility and transcriptional competence of specific chromatin regions within the eukaryotic genome. Phosphorylation of histone H3 is unique in the sense that it associates on one hand with open chromatin during gene activation and marks on the other hand highly condensed chromatin during mitosis. Phosphorylation of serine residues at histone H3 is a highly dynamic process that creates together with acetylation and methylation marks at neighboring lysine residues specific combinatorial patterns that are read by specific detector proteins. In this review we describe the importance of different histone H3 phosphorylation marks for chromatin condensation during mitosis. In addition, we review the signals that trigger histone H3 phosphorylation and the factors that control this reversible modification during interphase and mediate the biological readout of the signal. Finally, we discuss different models describing the role of histone H3 phosphorylation in the activation of transcription of poised genes or by transient derepression of epigenetically silenced genes. We propose that histone H3 phosphorylation in the context with lysine methylation might temporarily relieve the silencing of specific genes without affecting the epigenetic memory. PMID:22564826

Sawicka, Anna; Seiser, Christian

2012-04-28

194

Genome-wide views of chromatin structure.  

PubMed

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

Rando, Oliver J; Chang, Howard Y

2009-01-01

195

Chromatin staining of Drosophila testes.  

PubMed

This protocol describes chromatin staining of Drosophila testes. To visualize DNA, preparations fixed using methanol-acetone, paraformaldehyde, or formaldehyde can be stained with several DNA-binding dyes. If the slides are to be examined with a fluorescence microscope equipped with filters that permit ultraviolet (UV) excitation, suitable dyes for DNA staining are Hoechst 33258 or 4',6-diamidino-2-phenylindole (DAPI). If the slides are to be analyzed with a confocal microscope not equipped with a UV laser, DNA can be stained with either propidium iodide or TOTO-3 iodide. PMID:22854562

Bonaccorsi, Silvia; Giansanti, Maria G; Cenci, Giovanni; Gatti, Maurizio

2012-08-01

196

ATP-dependent chromatin assembly is functionally distinct from chromatin remodeling  

PubMed Central

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

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

2013-01-01

197

Steam condenser developments  

NASA Astrophysics Data System (ADS)

Factors determining condenser size and tube arrangement are reviewed, including steam side pressure drop; incondensible blanketing; effect of incondensibles on heat transfer; vent requirements; deaeration; condensate depression; cooling water velocity; tube material and diameter selection; fouling; and enhanced heat transfer tubes. Tube nest shapes and condenser concepts are described. Thermal design, and condenser acceptance testing are treated; field test results on "Church Window'' condensers are reported.

Lang, H. V.

198

Comparative study of smoke condensates from 1R4F cigarettes that burn tobacco versus ECLIPSE cigarettes that primarily heat tobacco in the SENCAR mouse dermal tumor promotion assay  

Microsoft Academic Search

Numerous chemical and toxicological studies indicate that smoke from ECLIPSE®, a cigarette that primarily heats rather than burns tobacco, is simplified and reduced in specific chemicals believed to be associated with smoking-related diseases, and demonstrates reduced smoke toxicity and biological activity in vitro when compared to conventional tobacco burning cigarettes. These data led to the hypothesis that cigarette smoke condensate

Daniel R. Meckley; Johnnie R. Hayes; K. R. Van Kampen; Paul H. Ayres; Arnold T. Mosberg; James E. Swauger

2004-01-01

199

The Prefoldin Complex Regulates Chromatin Dynamics during Transcription Elongation.  

PubMed

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

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

2013-09-19

200

The Prefoldin Complex Regulates Chromatin Dynamics during Transcription Elongation  

PubMed Central

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

Millan-Zambrano, Gonzalo; Rodriguez-Gil, Alfonso; Penate, Xenia; de Miguel-Jimenez, Lola; Morillo-Huesca, Macarena; Krogan, Nevan; Chavez, Sebastian

2013-01-01

201

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

PubMed Central

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

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

1990-01-01

202

Methods designed for the identification and characterization of in vitro and in vivo chromatin assembly mutants in Saccharomyces cerevisiae  

PubMed Central

Assembly of DNA into chromatin allows for the formation of a barrier that protects naked DNA from protein and chemical agents geared to degrade or metabolize DNA. Chromatin assembly occurs whenever a length of DNA becomes exposed to the cellular elements, whether during DNA synthesis or repair. This report describes tools to study chromatin assembly in the model system Saccharomyces cerevisiae. Modifications to an in vitro chromatin assembly assay are described that allowed a brute force screen of temperature sensitive (ts) yeast strains in order to identify chromatin assembly defective extracts. This screen yielded mutations in genes encoding two ubiquitin protein ligases (E3s): RSP5, and a subunit of the Anaphase Promoting Complex (APC), APC5. Additional modifications are described that allow for a rapid analysis and an in vivo characterization of yeast chromatin assembly mutants, as well as any other mutant of interest. Our analysis suggests that the in vitro and in vivo chromatin assembly assays are responsive to different cellular signals, including cell cycle cues that involve different molecular networks.

Arnason, Terra G.; Legrand, Charmaine; Lone, Ashley

2003-01-01

203

The Sperm Chromatin Dispersion Test: A Simple Method for the Determination of Sperm DNA Fragmentation  

Microsoft Academic Search

Sperm DNA fragmentation is being increasingly rec- ognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm

JOSE LUIS FERNANDEZ; LOURDES MURIEL; MARIA TERESA RIVERO; VICENTE GOYANES; ROSANA VAZQUEZ; JUAN G. ALVAREZ

2003-01-01

204

Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage  

Microsoft Academic Search

BACKGROUND: Sperm DNA integrity is essential for the accurate transmission of genetic information. The clinical significance of this assessment lies in its association with not only natural conception rates, but also the success of assisted reproduction technology (ART). It has been reported that sperm chromatin structure assay (SCSA) identified thresholds for negative pregnancy outcome after ART when the DNA fragmentation

L. Gandini; F. Lombardo; D. Paoli; F. Caruso; P. Eleuteri; G. Leter; R. Ciriminna; F. Culasso; F. Dondero; A. Lenzi; M. Spano; CR Casaccia

2004-01-01

205

Analysis of Chromatin Structure in the Control Regions of the Chlamydomonas HSP70A and RBCS2 Genes  

Microsoft Academic Search

We have used DNaseI and micrococcal nuclease sensitivity assays to determine the chromatin structures in the control regions of the Chlamydomonas reinhardtii HSP70A and RBCS2 genes. Both genes appear to be organized into nucleosome arrays, which exhibit shorter nucleosome repeat lengths than bulk chromatin. In HSP70A we have identified up to four confined DNaseI hypersensitive sites, three of them localize

Mukesh Lodha; Michael Schroda

2005-01-01

206

An Oestrogen Receptor ?-bound Human Chromatin Interactome  

PubMed Central

Genomes are organized into high-level 3-dimensional structures, and DNA elements separated by long genomic distances could functionally interact. Many transcription factors bind to regulatory DNA elements distant from gene promoters. While distal binding sites have been shown to regulate transcription by long-range chromatin interactions at a few loci, chromatin interactions and their impact on transcription regulation have not been investigated in a genome-wide manner. Therefore, we developed Chromatin Interaction Analysis by Paired-End Tag sequencing (ChIA-PET) for de novo detection of global chromatin interactions, and comprehensively mapped the chromatin interaction network bound by oestrogen receptor ? (ER?) in the human genome. We found that most high-confidence remote ER? binding sites are anchored at gene promoters through long-range chromatin interactions, suggesting that ER? functions by extensive chromatin looping to bring genes together for coordinated transcriptional regulation. We propose that chromatin interactions constitute a primary mechanism for regulating transcription in mammalian genomes.

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

2009-01-01

207

Expression-Dependent Folding of Interphase Chromatin  

PubMed Central

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.

Jerabek, Hansjoerg; Heermann, Dieter W.

2012-01-01

208

Regulation of chromatin by histone modifications  

Microsoft Academic Search

Chromatin is not an inert structure, but rather an instructive DNA scaffold that can respond to external cues to regulate the many uses of DNA. A principle component of chromatin that plays a key role in this regulation is the modification of histones. There is an ever-growing list of these modifications and the complexity of their action is only just

Andrew J Bannister; Tony Kouzarides

2011-01-01

209

Histone Phosphorylation Related to Chromatin Strucuture.  

National Technical Information Service (NTIS)

It has been proposed that the control of DNA activities (such as gene expression, gene replication, and gene segregation) may be mediated by changes in chromatin structure caused by modifications of the chromatin proteins. If this proposal is true, one mi...

L. R. Gurley R. A. Walters C. E. Hildebrand P. G. Hohmann S. S. Barham

1976-01-01

210

Chromatin patterns associated with lung adenocarcinoma progression  

PubMed Central

The development and progression of lung adenocarcinoma, one of the most common cancers, is driven by the interplay of genetic and epigenetic changes and the role of chromatin structure in malignant transformation remains poorly understood. We used systematic nucleosome distribution and chromatin accessibility microarray mapping platforms to analyze the genome-wide chromatin structure from normal tissues and from primary lung adenocarcinoma of different grades and stages. We identified chromatin-based patterns across different patients with lung adenocarcinoma of different cancer grade and stage. Low-grade cancers had nucleosome distributions very different compared with the corresponding normal tissue but had nearly identical chromatin accessibility. Conversely, nucleosome distributions of high-grade cancers showed few differences. Substantial disruptions in chromosomal accessibility were seen in a patient with a high-grade and high-stage tumor. These data imply that chromatin structure changes during the progression of lung adenocarcinoma. We have therefore developed a model in which low-grade lung adenocarcinomas are linked to changes in nucleosome distributions, whereas higher-grade tumors are linked to large-scale chromosomal changes. These results provide a foundation for the development of a comprehensive framework linking the general and locus-specific roles of chromatin structure to lung cancer progression. We propose that this strategy has the potential to identify a new class of chromatin-based diagnostic, prognostic and therapeutic markers in cancer progression.

Druliner, Brooke R.; Fincher, Justin A.; Sexton, Brittany S.; Vera, Daniel L.; Roche, Michael; Lyle, Stephen; Dennis, Jonathan H.

2013-01-01

211

Optimizing process vacuum condensers  

SciTech Connect

Vacuum condensers play a critical role in supporting vacuum processing operations. Although they may appear similar to atmospheric units, vacuum condensers have their own special designs, considerations and installation needs. By adding vacuum condensers, precondensers and intercondensers, system cost efficiency can be optimized. Vacuum-condensing systems permit reclamation of high-value product by use of a precondenser, or reduce operating costs with intercondensers. A precondenser placed between the vacuum vessel and ejector system will recover valuable process vapors and reduce vapor load to an ejector system--minimizing the system`s capital and operating costs. Similarly, an intercondenser positioned between ejector stages can condense motive steam and process vapors and reduce vapor load to downstream ejectors as well as lower capital and operating costs. The paper describes vacuum condenser systems, types of vacuum condensers, shellside condensing, tubeside condensing, noncondensable gases, precondenser pressure drop, system interdependency, equipment installation, and equipment layout.

Lines, J.R.; Tice, D.W. [Graham Mfg. Co., Batavia, NY (United States)

1997-09-01

212

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

PubMed Central

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

2011-01-01

213

TOPICAL REVIEW: The physics of chromatin  

NASA Astrophysics Data System (ADS)

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

Schiessel, Helmut

2003-05-01

214

Organic osmotic effectors and chromatin structure.  

PubMed

Organic amino compounds (taurine, glycine) and polyols (mannitol, sorbitol) are used as osmotic effectors by most animal cells, particularly by some marine invertebrates, but also to a limit extent by mammalian cells. Using physico-chemical techniques (circular dichroism, thermal denaturation, solubility, electrophoresis and electric linear dichroism), we demonstrated that some of these effectors prevent chromatin aggregation, without histone release. The influence of glycine on chromatin aggregation, dissociation and reconstitution was thoroughly investigated. Glycine at 2 M concentration does not in itself induce chromatin dissociation; it does hinder salt-induced histone dissociation from chromatin (especially at 1.2 M NaCl) but does not impede chromatin reconstitution. Several hypothesis may be put forward to explain the action of these effectors: (i) a modulation of histone conformation; (ii) a modification of fractional DNA charge, either directly by the zwitterions (glycine, taurine) or indirectly by alteration of cations counterions hydration. The physiological relevance of our experiments is also discussed. PMID:2100521

Buche, A; Colson, P; Houssier, C

1990-12-01

215

Chromatin modifications associated with diabetes.  

PubMed

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

Keating, Samuel T; El-Osta, Assam

2012-05-26

216

Condensate Mixtures and Tunneling  

SciTech Connect

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

Timmermans, E.

1998-09-14

217

Diazinon alters sperm chromatin structure in mice by phosphorylating nuclear protamines  

SciTech Connect

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

Pina-Guzman, B. [Seccion Externa de Toxicologia, CINVESTAV-IPN, Ave. IPN 2508, Col. Zacatenco, Mexico City 07360 (Mexico); Solis-Heredia, M.J. [Seccion Externa de Toxicologia, CINVESTAV-IPN, Ave. IPN 2508, Col. Zacatenco, Mexico City 07360 (Mexico); Quintanilla-Vega, B. [Seccion Externa de Toxicologia, CINVESTAV-IPN, Ave. IPN 2508, Col. Zacatenco, Mexico City 07360 (Mexico)]. E-mail: mquintan@mail.cinvestav.mx

2005-01-15

218

CTCF binding and higher order chromatin structure of the H19 locus are maintained in mitotic chromatin  

PubMed Central

Most of the transcription factors, RNA polymerases and enhancer binding factors are absent from condensed mitotic chromosomes. In contrast, epigenetic marks of active and inactive genes somehow survive mitosis, since the activity status from one cell generation to the next is maintained. For the zinc-finger protein CTCF, a role in interpreting and propagating epigenetic states and in separating expression domains has been documented. To test whether such a domain structure is preserved during mitosis, we examined whether CTCF is bound to mitotic chromatin. Here we show that in contrast to other zinc-finger proteins, CTCF indeed is bound to mitotic chromosomes. Mitotic binding is mediated by a portion of the zinc-finger DNA binding domain and involves sequence specific binding to target sites. Furthermore, the chromatin loop organized by the CTCF-bound, differentially methylated region at the Igf2/H19 locus can be detected in mitosis. In contrast, the enhancer/promoter loop of the same locus is lost in mitosis. This may provide a novel form of epigenetic memory during cell division.

Burke, Les J; Zhang, Ru; Bartkuhn, Marek; Tiwari, Vijay K; Tavoosidana, Gholamreza; Kurukuti, Sreenivasulu; Weth, Christine; Leers, Joerg; Galjart, Niels; Ohlsson, Rolf; Renkawitz, Rainer

2005-01-01

219

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

PubMed Central

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

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

2010-01-01

220

Condensed Matter Physics  

Microsoft Academic Search

A modern, unified treatment of condensed matter physics This new work presents for the first time in decades a sweeping review of the whole field of condensed matter physics. It consolidates new and classic topics from disparate sources, teaching \\

Michael P. Marder

2000-01-01

221

Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres  

PubMed Central

Background Mammalian centromere formation is dependent on chromatin that contains centromere protein (CENP)-A, which is the centromere-specific histone H3 variant. Human neocentromeres have acquired CENP-A chromatin epigenetically in ectopic chromosomal locations on low-copy complex DNA. Neocentromeres permit detailed investigation of centromeric chromatin organization that is not possible in the highly repetitive alpha satellite DNA present at endogenous centromeres. Results We have examined the distribution of CENP-A, as well as two additional centromeric chromatin-associated proteins (CENP-C and CENP-H), across neocentromeric DNA using chromatin immunoprecipitation (ChIP) on CHIP assays on custom genomic microarrays at three different resolutions. Analysis of two neocentromeres using a contiguous bacterial artificial chromosome (BAC) microarray spanning bands 13q31.3 to 13q33.1 shows that both CENP-C and CENP-H co-localize to the CENP-A chromatin domain. Using a higher resolution polymerase chain reaction (PCR)-amplicon microarray spanning the neocentromere, we find that the CENP-A chromatin is discontinuous, consisting of a major domain of about 87.8 kilobases (kb) and a minor domain of about 13.2 kb, separated by an approximately 158 kb region devoid of CENPs. Both CENP-A domains exhibit co-localization of CENP-C and CENP-H, defining a distinct inner kinetochore chromatin structure that is consistent with higher order chromatin looping models at centromeres. The PCR microarray data suggested varying density of CENP-A nucleosomes across the major domain, which was confirmed using a higher resolution oligo-based microarray. Conclusion Centromeric chromatin consists of several CENP-A subdomains with highly discontinuous CENP-A chromatin at both the level of individual nucleosomes and at higher order chromatin levels, raising questions regarding the overall structure of centromeric chromatin.

Alonso, Alicia; Fritz, Bjorn; Hasson, Dan; Abrusan, Gyorgy; Cheung, Fanny; Yoda, Kinya; Radlwimmer, Bernhard; Ladurner, Andreas G; Warburton, Peter E

2007-01-01

222

Biphasic chromatin binding of histone chaperone FACT during eukaryotic chromatin DNA replication.  

PubMed

The facilitates chromatin transcription (FACT) complex affects nuclear DNA transactions in a chromatin context. Though the involvement of FACT in eukaryotic DNA replication has been revealed, a clear understanding of its biochemical behavior during DNA replication still remains elusive. Here, we analyzed the chromatin-binding dynamics of FACT using Xenopus egg extract cell-free system. We found that FACT has at least two distinct chromatin-binding phases: (1) a rapid chromatin-binding phase at the onset of DNA replication that did not involve origin licensing and (2) a second phase of chromatin binding that initiated after origin licensing. Intriguingly, early-binding FACT dissociated from chromatin when DNA replication was blocked by the addition of Cdc6 in the licensed state before origin firing. Cdc6-induced removal of FACT was blocked by the inhibition of origin licensing with geminin, but not by suppressing the activity of DNA polymerases, CDK, or Cdc7. Furthermore, chromatin transfer experiments revealed that impairing the later binding of FACT severely compromises DNA replication activity. Taken together, we propose that even though FACT has rapid chromatin-binding activity, the binding pattern of FACT on chromatin changes after origin licensing, which may contribute to the establishment of its functional link to the DNA replication machinery. PMID:21232560

Kundu, Lena R; Seki, Masayuki; Watanabe, Nanae; Murofushi, Hiromu; Furukohri, Asako; Waga, Shou; Score, Alan J; Blow, J Julian; Horikoshi, Masami; Enomoto, Takemi; Tada, Shusuke

2011-01-11

223

Embryonic stem cell differentiation: A chromatin perspective  

PubMed Central

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.

Rasmussen, Theodore P

2003-01-01

224

Chromatin maintenance by a molecular motor protein  

PubMed Central

The kinesin motor protein KIF4 performs essential functions in mitosis. Like other mitotic kinesins, loss of KIF4 causes spindle defects, aneuploidy, genomic instability and ultimately tumor formation. However, KIF4 is unique among molecular motors in that it resides in the cell nucleus throughout interphase, suggesting a non-mitotic function as well. Here we identify a novel cellular function for a molecular motor protein by demonstrating that KIF4 acts as a modulator of large-scale chromatin architecture during interphase. KIF4 binds globally to chromatin and its absence leads to chromatin decondensation and loss of heterochromatin domains. KIF4-dependent chromatin decondensation has functional consequences by causing replication defects and global mis-regulation of gene expression programs. KIF4 exerts its function in chromatin architecture via regulation of ADP-ribosylation of core and linker histones and by physical interaction and recruitment of chromatin assembly proteins during S-phase. These observations document a novel function for a molecular motor protein in establishment and maintenance of higher order chromatin structure.

Sung, Myong-Hee; Misteli, Tom

2011-01-01

225

Chromatin remodelling initiation during human spermiogenesis.  

PubMed

During the last phase of spermatogenesis, spermiogenesis, haploid round spermatids metamorphose towards spermatozoa. Extensive cytoplasmic reduction and chromatin remodelling together allow a dramatic decrease of cellular, notably nuclear volume. DNA packing by a nucleosome based chromatin structure is largely replaced by a protamine based one. At the cytoplasmic level among others the acrosome and perinuclear theca (PNT) are formed. In this study we describe the onset of chromatin remodelling to occur concomitantly with acrosome and PNT development. In spread human round spermatid nuclei, we show development of a DAPI-intense doughnut-like structure co-localizing with the acrosomal sac and sub acrosomal PNT. At this structure we observe the first gradual decrease of nucleosomes and several histones. Histone post-translational modifications linked to chromatin remodelling such as H4K8ac and H4K16ac also delineate the doughnut, that is furthermore marked by H3K9me2. During the capping phase of acrosome development, the size of the doughnut-like chromatin domain increases, and this area often is marked by uniform nucleosome loss and the first appearance of transition protein 2 and protamine 1. In the acrosome phase at nuclear elongation, chromatin remodelling follows the downward movement of the marginal ring of the acrosome. Our results indicate that acrosome development and chromatin remodelling are interacting processes. In the discussion we relate chromatin remodelling to the available data on the nuclear envelope and the linker of nucleoskeleton and cytoskeleton (LINC) complex of spermatids, suggesting a signalling route for triggering chromatin remodelling. PMID:23213436

De Vries, Marieke; Ramos, Liliana; Housein, Zjwan; De Boer, Peter

2012-03-21

226

Condensation in Microchannels  

Microsoft Academic Search

Condensation in microchannels has applications in a wide variety of advanced microthermal devices. Presented here is a review of both experimental and theoretical analyses of condensation in these microchannels, with special attention given to the effects of channel diameter and surface conditions on the flow regimes of condensing flows occurring in these channels. This review suggests that surface tension, rather

Yongping Chen; Mingheng Shi; Ping Cheng; G. P. Peterson

2008-01-01

227

Efficient condensate removal  

Microsoft Academic Search

Achieving stable control of product temperatures on reboilers and other steam\\/product heat exchangers has long been a problem area where conventional steam control and condensate trapping methods were used. Application of a non-electric condensate pump and steam trap sidesteps the problem. Removal of condenser under all conditions, from full load to zero load, is readily achieved. Steam flow control then

Armer

1988-01-01

228

RNA traffic control of chromatin complexes  

PubMed Central

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

Koziol, Magdalena J; Rinn, John L.

2010-01-01

229

Chromatin fiber dynamics under tension and torsion.  

PubMed

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

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

2010-04-12

230

Chromatin Evolution and Molecular Drive in Speciation  

PubMed Central

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

Sawamura, Kyoichi

2012-01-01

231

Cell Cycle-dependent Binding of HMGN Proteins to Chromatin  

Microsoft Academic Search

Throughout the cell cycle, the histones remain associated with DNA, but the repertoire of proteins associated with the chromatin fiber continuously changes. The chromatin interaction of HMGNs, a family of nucleosome binding proteins that modulates the structure and activity of chromatin, during the cell cycle is controversial. Immunofluorescence studies demonstrated that HMGNs are not associated with chromatin, whereas live cell

Srujana Cherukuri; Robert Hock; Tetsuya Ueda; Frédéric Catez; Mark Rochman; Michael Bustin

2008-01-01

232

On the chromatin structure of eukaryotic telomeres.  

PubMed

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

Vaquero-Sedas, María I; Vega-Palas, Miguel A

2011-09-01

233

On the chromatin structure of eukaryotic telomeres  

PubMed Central

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

Vaquero-Sedas, Maria I

2011-01-01

234

Polarization microscopy of extended chromatin fibers.  

PubMed

Analysis of the formation of extended chromatin fibers (ECFs) in response to the action of gravity following lysis by hypertonic and detergent solutions is a useful technical procedure relevant for studies of the positioning of particular DNA signals on chromatin filaments. Additionally, if toluidine blue molecules are allowed to bind electrostatically to available DNA phosphates on ECFs, the birefringence brightness generated in these filaments, as observed by polarization microscopy, facilitates the description of the frequency of ECF formation and extension of the chromatin filaments generated. Thus, different patterns of DNA-nuclear matrix protein associations related to varying transcriptional activities and chromatin organization in isolated cells can be assessed. A technique for producing ECFs in different isolated cell types under variable physiological and/or pathological conditions is detailed in this chapter. PMID:24162980

Mello, Maria Luiza S; de Campos Vidal, Benedicto

2014-01-01

235

Chromatin remodeling: RSC Motors along the DNA.  

PubMed

Single-molecule experiments show that the chromatin-remodeling complex RSC, a member of the SNF2 ATPase family, induces formation of a negatively supercoiled DNA loop by active translocation. PMID:16631574

Strick, Terence; Quessada-Vial, Audrey

2006-04-18

236

Changes in Chromatin Compaction During the Cell Cycle Revealed by Micrometer-Scale Measurement of Molecular Flow in the Nucleus  

PubMed Central

We present a quantitative fluctuation-based assay to measure the degree of local chromatin compaction and investigate how chromatin density regulates the diffusive path adopted by an inert protein in dividing cells. The assay uses CHO-K1 cells coexpressing untagged enhanced green fluorescent protein (EGFP) and histone H2B tagged mCherry. We measure at the single-cell level the EGFP localization and molecular flow patterns characteristic of each stage of chromatin compaction from mitosis through interphase by means of pair-correlation analysis. We find that the naturally occurring changes in chromatin organization impart a regulation on the spatial distribution and temporal dynamics of EGFP within the nucleus. Combined with the analysis of Ca2+ intracellular homeostasis during cell division, EGFP flow regulation can be interpreted as the result of controlled changes in chromatin compaction. For the first time, to our knowledge, we were able to probe chromatin compaction on the micrometer scale, where the regulation of molecular diffusion may become relevant for many cellular processes.

Hinde, Elizabeth; Cardarelli, Francesco; Digman, Michelle A.; Gratton, Enrico

2012-01-01

237

Nuclear condensation and free radical scavenging: a dual mechanism of bisbenzimidazoles to modulate radiation damage to DNA  

Microsoft Academic Search

The complexing of histones with DNA and the resulting condensation of chromatin protects mammalian cell, from radiation-induced\\u000a strand breakage. In the present study, benzimidazoles DMA and TBZ showed marked radioprotection through drug-induced compaction\\u000a of chromatin and direct quenching of free radicals generated by radiation. The mammalian cells were incubated with 100 ?M\\u000a concentration of DMA and TBZ and irradiated at 5 Gy;

Urmila Tawar; Sandhya Bansal; Shiteshu Shrimal; Manish Singh; Vibha Tandon

2007-01-01

238

Functional interactions between nucleoporins and chromatin  

PubMed Central

As the gatekeepers of the eukaryotic cell nucleus, nuclear pore complexes (NPCs) mediate all molecular trafficking between the nucleoplasm and the cytoplasm. In recent years, transport-independent functions of NPC components, nucleoporins, have been identified including roles in chromatin organization and gene regulation. Here, we summarize our current view of the NPC as a dynamic hub for the integration of chromatin regulation and nuclear trafficking and discuss the functional interplay between nucleoporins and the nuclear genome.

Liang, Yun; Hetzer, Martin W

2013-01-01

239

Postnatal Effects of Sperm Chromatin Damage  

Microsoft Academic Search

\\u000a The use of spermatozoa with fragmented DNA has been linked to ­developmental and postnatal effects in animal models. Environmental\\u000a and toxic factors such as radiation, heat stress, air pollution, chemotherapeutic agents, etc. are known to have detrimental\\u000a effects on sperm chromatin. Sperm chromatin damage has also been observed following sperm manipulation techniques (freeze–thawing\\u000a without cryoprotectants, freeze-drying, preincubation under different conditions,

Miriam Pérez-Crespo; Raúl Fernández-González; Miguel Ángel Ramírez; Eva Pericuesta; Alexandra Calle; Alfonso Gutiérrez-Adán

240

Antiestrogenic effects of marijuana smoke condensate and cannabinoid compounds  

Microsoft Academic Search

The antiestrogenic effects of marijuana smoke condensate (MSC) and three major cannab-inoids, i.e., ?9-Metrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN), were evaluated usingin vitro bioassays,viz., the human breast cancer cell proliferation assay, the recombinant human estrogen receptor (ER) competitive binding assay,\\u000a and the reporter gene assay. The inhibitory effects on estrogen were also examined using the ethoxyresorufin-O-deethylase\\u000a (EROD) assay, the

Soo Yeun Lee; Seung Min Oh; Sang Ki Lee; Kyu Hyuck Chung

2005-01-01

241

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

PubMed

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

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

2013-07-18

242

Mitotic inactivation of a human SWI/SNF chromatin remodeling complex  

PubMed Central

During mitosis, chromatin is condensed into mitotic chromosomes and transcription is inhibited, processes that might be opposed by the chromatin remodeling activity of the SWI/SNF complexes. Brg1 and hBrm, which are components of human SWI/SNF (hSWI/SNF) complexes, were recently shown to be phosphorylated during mitosis. This suggested that phosphorylation might be used as a switch to modulate SWI/SNF activity. Using an epitope-tag strategy, we have purified hSWI/SNF complexes at different stages of the cell cycle, and found that hSWI/SNF was inactive in cells blocked in G2–M. Mitotic hSWI/SNF contained Brg1 but not hBrm, and was phosphorylated on at least two subunits, hSWI3 and Brg1. In vitro, active hSWI/SNF from asynchronous cells can be phosphorylated and inactivated by ERK1, and reactivated by dephosphorylation. hSWI/SNF isolated as cells traversed mitosis regained activity when its subunits were dephosphorylated either in vitro or in vivo. We propose that this transitional inactivation and reactivation of hSWI/SNF is required for formation of a repressed chromatin structure during mitosis and reformation of an active chromatin structure as cells leave mitosis.

Sif, Said; Stukenberg, P. Todd; Kirschner, Marc W.; Kingston, Robert E.

1998-01-01

243

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

PubMed

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

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

2013-10-07

244

Mass spectrometry-based proteomics for the analysis of chromatin structure and dynamics.  

PubMed

Chromatin is a highly structured nucleoprotein complex made of histone proteins and DNA that controls nearly all DNA-dependent processes. Chromatin plasticity is regulated by different associated proteins, post-translational modifications on histones (hPTMs) and DNA methylation, which act in a concerted manner to enforce a specific "chromatin landscape", with a regulatory effect on gene expression. Mass Spectrometry (MS) has emerged as a powerful analytical strategy to detect histone PTMs, revealing interplays between neighbouring PTMs and enabling screens for their readers in a comprehensive and quantitative fashion. Here we provide an overview of the recent achievements of state-of-the-art mass spectrometry-based proteomics for the detailed qualitative and quantitative characterization of histone post-translational modifications, histone variants, and global interactomes at specific chromatin regions. This synopsis emphasizes how the advances in high resolution MS, from "Bottom Up" to "Top Down" analysis, together with the uptake of quantitative proteomics methods by chromatin biologists, have made MS a well-established method in the epigenetics field, enabling the acquisition of original information, highly complementary to that offered by more conventional, antibody-based, assays. PMID:23466885

Soldi, Monica; Cuomo, Alessandro; Bremang, Michael; Bonaldi, Tiziana

2013-03-06

245

Nucleosome-depleted chromatin gaps recruit assembly factors for the H3.3 histone variant  

PubMed Central

Most nucleosomes that package eukaryotic DNA are assembled during DNA replication, but chromatin structure is routinely disrupted in active regions of the genome. Replication-independent nucleosome replacement using the H3.3 histone variant efficiently repackages these regions, but how histones are recruited to these sites is unknown. Here, we use an inducible system that produces nucleosome-depleted chromatin at the Hsp70 genes in Drosophila to define steps in the mechanism of nucleosome replacement. We find that the Xnp chromatin remodeler and the Hira histone chaperone independently bind nucleosome-depleted chromatin. Surprisingly, these two factors are only displaced when new nucleosomes are assembled. H3.3 deposition assays reveal that Xnp and Hira are required for efficient nucleosome replacement, and double-mutants are lethal. We propose that Xnp and Hira recognize exposed DNA and serve as a binding platform for the efficient recruitment of H3.3 predeposition complexes to chromatin gaps. These results uncover the mechanisms by which eukaryotic cells actively prevent the exposure of DNA in the nucleus.

Schneiderman, Jonathan I.; Orsi, Guillermo A.; Hughes, Kelly T.; Loppin, Benjamin; Ahmad, Kami

2012-01-01

246

Independent and complementary methods for large-scale structural analysis of mammalian chromatin  

PubMed Central

The fundamental building block of chromatin, the nucleosome, occupies 150 bp of DNA in a spaced arrangement that is a primary determinant in regulation of the genome. The nucleosomal organization of some regions of the human genome has been described, but mapping of these regions has been limited to a few kilobases. We have explored two independent and complementary methods for the high-throughput analysis of mammalian chromatin structure. Through adaptations to a protocol used to map yeast chromatin structure, we determined sites of nucleosomal protection over large regions of the mammalian genome using a tiling microarray. By modifying classical primer extension methods, we localized specific internucleosomally cleaved mammalian genomic sequences using a capillary electrophoresis sequencer in a manner that allows high-throughput nucleotide-resolution characterization of nucleosome protection patterns. We developed algorithms for the automated and unbiased analysis of the resulting data, a necessary step toward large-scale analysis. We validated these assays using the known positions of nucleosomes on the mouse mammary tumor virus LTR, and additionally, we characterized the previously unreported chromatin structure of the LCMT2 gene. These results demonstrate the effectiveness of the combined methods for reliable analysis of mammalian chromatin structure in a high-throughput manner.

Dennis, Jonathan H.; Fan, Hua-Ying; Reynolds, Sheila M.; Yuan, Guocheng; Meldrim, James C.; Richter, Daniel J.; Peterson, Daniel G.; Rando, Oliver J.; Noble, William S.; Kingston, Robert E.

2007-01-01

247

Proceedings: Condenser technology conference  

SciTech Connect

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

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

1991-08-01

248

Quantitative analysis of chromosome condensation in fission yeast.  

PubMed

Chromosomes undergo extensive conformational rearrangements in preparation for their segregation during cell divisions. Insights into the molecular mechanisms behind this still poorly understood condensation process require the development of new approaches to quantitatively assess chromosome formation in vivo. In this study, we present a live-cell microscopy-based chromosome condensation assay in the fission yeast Schizosaccharomyces pombe. By automatically tracking the three-dimensional distance changes between fluorescently marked chromosome loci at high temporal and spatial resolution, we analyze chromosome condensation during mitosis and meiosis and deduct defined parameters to describe condensation dynamics. We demonstrate that this method can determine the contributions of condensin, topoisomerase II, and Aurora kinase to mitotic chromosome condensation. We furthermore show that the assay can identify proteins required for mitotic chromosome formation de novo by isolating mutants in condensin, DNA polymerase ?, and F-box DNA helicase I that are specifically defective in pro-/metaphase condensation. Thus, the chromosome condensation assay provides a direct and sensitive system for the discovery and characterization of components of the chromosome condensation machinery in a genetically tractable eukaryote. PMID:23263988

Petrova, Boryana; Dehler, Sascha; Kruitwagen, Tom; Hériché, Jean-Karim; Miura, Kota; Haering, Christian H

2012-12-21

249

Phosphorylation of the Chromatin Binding Domain of KSHV LANA  

PubMed Central

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.

Liao, Gangling; Zhu, Jian; Ng, Ai Na; Li, Renfeng; Newman, Rob; Rho, Hee-Sool; Hu, Jianfei; Wan, Jun; Qian, Jiang; Zhu, Heng; Hayward, S. Diane

2012-01-01

250

Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes.  

PubMed

The accessibility of DNA in chromatin to psoralen was assayed to compare the chromatin structure of the rRNA coding and spacer regions of the two related frog species Xenopus laevis and Xenopus borealis. Isolated nuclei from tissue culture cells were photoreacted with psoralen, and the extent of cross-linking in the different rDNA regions was analyzed by using a gel retardation assay. In both species, restriction fragments from the coding regions showed two distinct extents of cross-linking, indicating the presence of two types of chromatin, one that contains nucleosomes and represents the inactive gene copies, and the other one which is more cross-linked and corresponds to the transcribed genes. A similar cross-linking pattern was obtained with restriction fragments from the enhancer region. Analysis of fragments including these sequences and the upstream portions of the genes suggests that active genes are preceded by nonnucleosomal enhancer regions. The spacer regions flanking the 3' end of the genes gave different results in the two frog species. In X. borealis, all these sequences are packaged in nucleosomes, whereas in X. laevis a distinct fraction, presumably those flanking the active genes, show a heterogeneous chromatin structure. This disturbed nucleosomal organization correlates with the presence of a weaker terminator at the 3' end of the X. laevis genes compared with those of X. borealis, which allows polymerases to transcribe into the downstream spacer. PMID:1406621

Lucchini, R; Sogo, J M

1992-10-01

251

The myc proteins are not associated with chromatin in mitotic cells.  

PubMed Central

The protein products of cellular and viral myc oncogenes are detected in nuclei by immunofluorescence. No myc fluorescence is found in nucleoli. In mitotic cells the myc antigens are not found associated with metaphase chromosomes, but are diffusely distributed throughout the cytoplasm. Cytoplasmic myc fluorescence is first observed when chromatin begins to condense in early prophase. Granular nuclear myc fluorescence is again discerned in telophase cells, when the nuclear envelope is formed and becomes more prominent upon cytokinesis; concomitantly the diffuse cytoplasmic myc staining is lost. These results suggest that myc proteins not only bind to DNA or chromatin, but are also associated with other structural systems in the nuclei. Images Fig. 1. Fig. 2. Fig. 3.

Winqvist, R; Saksela, K; Alitalo, K

1984-01-01

252

Two Distinct Mechanisms of Chromatin Interaction by the Isw2 Chromatin Remodeling Complex In Vivo  

PubMed Central

We have previously shown that Saccharomyces cerevisiae Isw2 complex slides nucleosomes to remodel chromatin in vivo. Our data suggested a model in which Isw2 complex binds the histone octamer and DNA separately to generate the force necessary for nucleosome movement. Here we find that the histone H4 “basic patch” is the only portion of any amino-terminal histone tail required for both target-specific association of Isw2 complex with chromatin and chromatin remodeling in vivo, whereas it is dispensable for basal levels of chromatin binding. Similarly, we find that nonremodeled chromatin structure and integrity of Isw2 complex are required only for target-specific association of Isw2 with chromatin. These data demonstrate fundamental differences between the target-specific and basal modes of chromatin binding by Isw2 complex in vivo and suggest that only the former involves contributions from DNA, histone H4, and sequence-specific DNA binding proteins. We propose a model for target recognition and chromatin remodeling by Isw2 complex in vivo.

Fazzio, Thomas G.; Gelbart, Marnie E.; Tsukiyama, Toshio

2005-01-01

253

Structure of active chromatin: isolation and characterization of transcriptionally active chromatin from rat liver.  

PubMed Central

Rat liver nuclei were isolated in low-ionic-strength buffer in the absence of bi- and multi-valent cations. Digestion of these nuclei by endogenous nuclease, micrococcal nuclease and DNase I revealed that a minor chromatin fraction was preferentially digested into poly- and oligo-nucleosomes. Southern blot hybridization with various active gene probes confirmed that these chromatin fragments represent coding and 5' upstream regions of transcriptionally active chromatin. Active chromatin fragments were released selectively into the medium, with inactive chromatin remaining inside the nuclei, under the above ionic conditions. The inclusion of bivalent cations during the digestion of nuclei reversed the solubility behaviour of active chromatin. Rearrangement and exchange of histone H1 between chromatin fragments was prevented by using low-salt conditions in all steps in the absence of bivalent cations. All histones, including H1, were present in stoichiometric amounts in this active chromatin fraction. Active nucleosomes showed a lower electrophoretic mobility than bulk nucleosomes in an acrylamide/agarose composite gel in the absence of Mg2+, but were selectively bound to the gel in the presence of this ion.

Tikoo, K; Gupta, S; Hamid, Q A; Shah, V; Chatterjee, B; Ali, Z

1997-01-01

254

Condensed Matter Physics  

NSDL National Science Digital Library

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

255

Chromatin States Accurately Classify Cell Differentiation Stages  

PubMed Central

Gene expression is controlled by the concerted interactions between transcription factors and chromatin regulators. While recent studies have identified global chromatin state changes across cell-types, it remains unclear to what extent these changes are co-regulated during cell-differentiation. Here we present a comprehensive computational analysis by assembling a large dataset containing genome-wide occupancy information of 5 histone modifications in 27 human cell lines (including 24 normal and 3 cancer cell lines) obtained from the public domain, followed by independent analysis at three different representations. We classified the differentiation stage of a cell-type based on its genome-wide pattern of chromatin states, and found that our method was able to identify normal cell lines with nearly 100% accuracy. We then applied our model to classify the cancer cell lines and found that each can be unequivocally classified as differentiated cells. The differences can be in part explained by the differential activities of three regulatory modules associated with embryonic stem cells. We also found that the “hotspot” genes, whose chromatin states change dynamically in accordance to the differentiation stage, are not randomly distributed across the genome but tend to be embedded in multi-gene chromatin domains, and that specialized gene clusters tend to be embedded in stably occupied domains.

Larson, Jessica L.; Yuan, Guo-Cheng

2012-01-01

256

Regulation of the Boundaries of Accessible Chromatin  

PubMed Central

Regulatory regions maintain nucleosome-depleted, open chromatin status but simultaneously require the presence of nucleosomes for specific histone modifications. It remains unclear how these can be achieved for proper regulatory function. Here we demonstrate that nucleosomes positioned within accessible chromatin regions near the boundaries provide platforms for histone modifications while preventing the occlusion of regulatory elements. These boundary nucleosomes were particularly enriched for active or poised regulatory marks in human, such as histone acetylations, H3K4 methylations, H3K9me3, H3K79me2, and H4K20me1. Additionally, we found that based on a genome-wide profiling of ?100 recombinant yeast strains, the location of open chromatin borders tends to vary mostly within 150 bp upon genetic perturbation whereas this positional variation increases in proportion to the sequence preferences of the underlying DNA for nucleosome formation. More than 40% of the local boundary shifts were associated with genetic variation in cis- or trans-acting factors. A sizeable fraction of the identified genetic factors was also associated with nearby gene expression, which was correlated with the distance between the transcription start site (tss) and the boundary that faces the tss. Taken together, the variation in the width of accessible chromatin regions may arise in conjunction with the modulation of the boundary nucleosomes by post-translational modifications or by chromatin regulators and in association with the activity of nearby gene transcription.

Kim, Kwoneel; Lee, Kibaick; Choi, Jung Kyoon

2013-01-01

257

Microscopic imaging of DNA condensation in the presence of charged nanospheres  

NASA Astrophysics Data System (ADS)

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

Krishnan, Rajagopal; Sandhu, Tejdev; Nordlund, Thomas

2004-11-01

258

Estrogenic effects of marijuana smoke condensate and cannabinoid compounds  

Microsoft Academic Search

Chronic exposure to marijuana produces adverse effects on the endocrine and reproductive systems in humans; however, the experimental evidence for this presented thus far has not been without controversy. In this study, the estrogenic effect of marijuana smoke condensate (MSC) was evaluated using in vitro bioassays, viz., the cell proliferation assay, the reporter gene assay, and the ER competitive binding

Soo Yeun Lee; Seung Min Oh; Kyu Hyuck. Chung

2006-01-01

259

Mammalian Sperm DNA Susceptibility to In Situ Denaturation Associated with the Presence of DNA Strand Breaks as Measured by the Terminal Deoxynucleotidyl Transferase Assay  

Microsoft Academic Search

Sperm from four mammalian species were analyzed by the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase assay (TdTA) using flow cytometry. The SCSA quantitates the susceptibility of sperm nuclear DNA to in situ acid denaturation, while the TdTA quantitates the presence of endogenous DNA strand breaks in sperm nuclear chromatin. Cor- relations were seen between the percentage

BRIAN L. SAILER; LORNA K. JOST; DONALD P. EVENSON

260

Linker Histones Incorporation Maintains Chromatin Fiber Plasticity  

PubMed Central

Genomic DNA in eukaryotic cells is organized in supercoiled chromatin fibers, which undergo dynamic changes during such DNA metabolic processes as transcription or replication. Indeed, DNA-translocating enzymes like polymerases produce physical constraints in vivo. We used single-molecule micromanipulation by magnetic tweezers to study the response of chromatin to mechanical constraints in the same range as those encountered in vivo. We had previously shown that under positive torsional constraints, nucleosomes can undergo a reversible chiral transition toward a state of positive topology. We demonstrate here that chromatin fibers comprising linker histones present a torsional plasticity similar to that of naked nucleosome arrays. Chromatosomes can undergo a reversible chiral transition toward a state of positive torsion (reverse chromatosome) without loss of linker histones.

Recouvreux, Pierre; Lavelle, Christophe; Barbi, Maria; Conde e Silva, Natalia; Le Cam, Eric; Victor, Jean-Marc; Viovy, Jean-Louis

2011-01-01

261

Cloud condensation nuclei  

Microsoft Academic Search

The state of knowledge of the particles upon which liquid droplets condense to form atmospheric water clouds is presented. The realization of cloud condensation nuclei (CCN) as a distinct aerosol subset originated with the cloud microphysical measurements and theoretical insights of Patrick Squires 40 years ago. He helped originate and continue the development of CCN counters and made significant CCN

James G. Hudson

1993-01-01

262

The Chromatin Fingerprint of Gene Enhancer Elements*  

PubMed Central

Different cell types within a single organism are generally distinguished by strikingly different patterns of gene expression, which are dynamic throughout development and adult life. Distal enhancer elements are key drivers of spatiotemporal specificity in gene regulation. Often located tens of kilobases from their target promoters and functioning in an orientation-independent manner, the identification of bona fide enhancers has proved a formidable challenge. With the development of ChIP-seq, global cataloging of putative enhancers has become feasible. Here, we review the current understanding of the chromatin landscape at enhancers and how these chromatin features enable robust identification of tissue-specific enhancers.

Zentner, Gabriel E.; Scacheri, Peter C.

2012-01-01

263

Chromatin proteins and modifications as drug targets.  

PubMed

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 is complex, new inhibitors such as these will hopefully be of clinical use in the coming years. PMID:24153301

Helin, Kristian; Dhanak, Dashyant

2013-10-24

264

Overcoming the chromatin barrier to end resection.  

PubMed

Repair of double-strand breaks by homologous recombination requires Repair of double-strand breaks by homologous recombination requires 5'-3' resection of the DNA ends to create 3' single-stranded DNA tails. While much progress has been made in identifying the proteins that directly participate in end resection, how this process occurs in the context of chromatin is not well understood. Two papers in Nature report that Fun30, a poorly characterized member of the Swi2/Snf2 family of chromatin remodelers, plays a role in end processing by facilitating the Exo1 and Sgs1-Dna2 resection pathways. PMID:23147792

Chen, Huan; Symington, Lorraine S

2012-11-13

265

Dynamics of Histone Tails within Chromatin  

NASA Astrophysics Data System (ADS)

Genetic information in humans is encoded within DNA molecules that is wrapped around histone octamer proteins and compacted into a highly conserved structural polymer, chromatin. The physical and material properties of chromatin appear to influence gene expression by altering the accessibility of proteins to the DNA. The tails of the histones are flexible domains that are thought to play a role in regulating DNA accessibility and compaction; however the molecular mechanisms for these phenomena are not understood. I will present CW-EPR studies on site directed spin labeled nucleosomes that probe the structure and dynamics of these histone tails within nucleosomes.

Bernier, Morgan; North, Justin; Page, Michael; Jaroniec, Christopher; Hammel, Christopher; Poirier, Michael

2012-02-01

266

CHD chromatin remodelers and the transcription cycle  

PubMed Central

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

Murawska, Magdalena

2011-01-01

267

Insights into Role of Bromodomain, Testis-specific (Brdt) in Acetylated Histone H4-dependent Chromatin Remodeling in Mammalian Spermiogenesis*  

PubMed Central

Mammalian spermiogenesis is of considerable biological interest especially due to the unique chromatin remodeling events that take place during spermatid maturation. Here, we have studied the expression of chromatin remodeling factors in different spermatogenic stages and narrowed it down to bromodomain, testis-specific (Brdt) as a key molecule participating in chromatin remodeling during rat spermiogenesis. Our immunocytochemistry experiments reveal that Brdt colocalizes with acetylated H4 in elongating spermatids. Remodeling assays showed an acetylation-dependent but ATP-independent chromatin reorganization property of Brdt in haploid round spermatids. Furthermore, Brdt interacts with Smarce1, a member of the SWI/SNF family. We have studied the genomic organization of smarce1 and identified that it has two splice variants expressed during spermatogenesis. The N terminus of Brdt is involved in the recognition of Smarce1 as well as in the reorganization of hyperacetylated round spermatid chromatin. Interestingly, the interaction between Smarce1 and Brdt increases dramatically upon histone hyperacetylation both in vitro and in vivo. Thus, our results indicate this interaction to be a vital step in the chromatin remodeling process during mammalian spermiogenesis.

Dhar, Surbhi; Thota, Anusha; Rao, Manchanahalli Rangaswamy Satyanarayana

2012-01-01

268

Delineation of the Protein Module That Anchors HMGN Proteins to Nucleosomes in the Chromatin of Living Cells?  

PubMed Central

Numerous nuclear proteins bind to chromatin by targeting unique DNA sequences or specific histone modifications. In contrast, HMGN proteins recognize the generic structure of the 147-bp nucleosome core particle. HMGNs alter the structure and activity of chromatin by binding to nucleosomes; however, the determinants of the specific interaction of HMGNs with chromatin are not known. Here we use systematic mutagenesis, quantitative fluorescence recovery after photobleaching, fluorescence imaging, and mobility shift assays to identify the determinants important for the specific binding of these proteins to both the chromatin of living cells and to purified nucleosomes. We find that several regions of the protein affect the affinity of HMGNs to chromatin; however, the conserved sequence RRSARLSA, is the sole determinant of the specific interaction of HMGNs with nucleosomes. Within this sequence, each of the 4 amino acids in the R-S-RL motif are the only residues absolutely essential for anchoring HMGN protein to nucleosomes, both in vivo and in vitro. Our studies identify a new chromatin-binding module that specifically recognizes nucleosome cores independently of DNA sequence or histone tail modifications.

Ueda, Tetsuya; Catez, Frederic; Gerlitz, Gabi; Bustin, Michael

2008-01-01

269

Transcription of in vitro assembled chromatin templates in a highly purified RNA polymerase II system  

PubMed Central

In mammalian cells RNA polymerase II efficiently transcribes nucleosome-packaged DNA. In this regard, a fundamental question concerns the nature and mechanism of action of the accessory factors that are necessary and sufficient for, or enhance, transcription through nucleosomal arrays by RNA polymerase II. Here we describe a highly purified system that allows for efficient activator-dependent transcription by RNA polymerase II from the promoter through several contiguous nucleosomes on defined chromatin templates. The system contains natural or recombinant histones, chromatin assembly factors, the histone-acetyltranferase p300, all components of the general transcription machinery, general coactivators and the elongation factor SII (TFIIS). As examples of the applicability of this system for mechanistic analyses of these and other factors, representative experiments show (i) that activated transcription from chromatin templates is concomitantly dependent on the activator, p300-mediated histone acetylation and elongation factor SII/TFIIS. (ii) that SII/TFIIS acts in a highly synergistic manner with p300 (and histone acetylation) at a step subsequent to preinitiation complex (PIC) formation and (iii) that SII/TFIIS works directly at the elongation step of chromatin transcription. Here we describe purification methods for the different factors employed and the specific transcriptional assays that led to the above-mentioned conclusions. This purified system will be very useful as an assay system for the discovery of new factors or the mechanistic analysis of known or candidate factors involved in transcription initiation or elongation on chromatin templates, including factors that effect specific histone modifications or nucleosomal remodeling.

Guermah, Mohamed; Kim, Jaehoon; Roeder, Robert G.

2009-01-01

270

Subtle membrane changes in cryopreserved bull semen in relation with sperm viability, chromatin structure, and field fertility  

Microsoft Academic Search

This study investigated the use of annexin-V\\/PI assay to assess sub lethal changes in bull spermatozoa post-thawing, and to further relate these changes to results obtained by fluorometric assessment of sperm viability and sperm chromatin structure assay (SCSA), as well as field fertility (as 56-day non-return rates, 56-day NRR) after AI. Frozen–thawed semen samples were obtained from 18 Swedish Red

A Januskauskas; A Johannisson; H Rodriguez-Martinez

2003-01-01

271

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

PubMed Central

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

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

2013-01-01

272

[Meiosis: role of a histone kinase in the condensation of ovarian oocyte chromosomes of Xenopus laevis and Ambystoma mexicanum].  

PubMed

By injecting heterologous histone-kinase preparations into ovarian Axolotl oocytes, it has been possible to speed up the progesterone-induced process of chromosome condensation. Moreover, in some instances, this condensation and even complete maturation have been obtained after injection of protein kinase alone, thus in the absence of hormone stimulation. Two different histone kinase preparations have been used: one was prepared from ascites cell chromatin and the other from in vitro ovulated Xenopus oocytes. PMID:177222

Wiblet, M; Baltus, E; Brachet, J

1975-12-15

273

Chromatin higher-order structures and gene regulation  

PubMed Central

Genomic DNA in the eukaryotic nucleus is hierarchically packaged by histones into chromatin to fit inside the nucleus. The dynamics of higher-order chromatin compaction play a critical role in transcription and other biological processes inherent to DNA. Many factors, including histone variants, histone modifications, DNA methylation and the binding of non-histone architectural proteins regulate the structure of chromatin. Although the structure of nucleosomes, the fundamental repeating unit of chromatin, is clear, there is still much discussion on the higher-order levels of chromatin structure. In this review, we focus on the recent progress in elucidating the structure of the 30-nm chromatin fiber. We also discuss the structural plasticity/dynamics and epigenetic inheritance of higher-order chromatin and the roles of chromatin higher-order organization in eukaryotic gene regulation.

Li, Guohong

2011-01-01

274

Local nucleosome dynamics facilitate chromatin accessibility in living mammalian cells.  

PubMed

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. PMID:23246002

Hihara, Saera; Pack, Chan-Gi; Kaizu, Kazunari; Tani, Tomomi; Hanafusa, Tomo; Nozaki, Tadasu; Takemoto, Satoko; Yoshimi, Tomohiko; Yokota, Hideo; Imamoto, Naoko; Sako, Yasushi; Kinjo, Masataka; Takahashi, Koichi; Nagai, Takeharu; Maeshima, Kazuhiro

2012-12-13

275

The influence of chromatin structure on initial DNA damage and radiosensitivity in CHO-K1 and xrs1 cells at low doses of irradiation (1-10 Gy)  

Microsoft Academic Search

Mitotic compaction of chromatin was generated by treatment of cells with nocodazole. Alternatively, chromatin structure was altered by incubating cells in 500 mM NaCl. The irradiation response in the dose range of 1-10 Gy was measured by colony assay and by a modified fluorometric analysis of DNA unwinding (FADU) assay which measures the amount of undamaged DNA by EtBr fluorescence.

W. P. Roos; A. Binder; L. Böhm

2002-01-01

276

Sex chromosomes, recombination, and chromatin conformation  

Microsoft Academic Search

We review what is known about the transcriptional inactivation and condensation of heteromorphic sex chromosomes in contrast to the activation of homomorphic sex chromosomes during meiotic prephase in animals. We relate these cytological and transcriptional features to the recombination status of the sex chromosomes. We propose that sex chromosome condensation is a meiotic adaptation to prevent the initiation of potentially

Bruce D. McKee; Mary Ann Handel

1993-01-01

277

Locus dependence in epigenetic chromatin silencing.  

PubMed

Current biological models of epigenetic switches built on chromatin modifications lead to strong constraints on the repertoire of dynamic behaviors for the system. We use the structure of the bifurcation diagram of the underlying dynamical system to explain the existing single cell data in silencing by the SIR system in yeast. PMID:20655355

Mukhopadhyay, Swagatam; Nagaraj, Vijayalakshmi H; Sengupta, Anirvan M

2010-07-22

278

Chromatin Organization of Gammaherpesvirus Latent Genomes  

PubMed Central

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

Tempera, Italo; Lieberman, Paul M.

2009-01-01

279

Locus dependence in epigenetic chromatin silencing  

PubMed Central

Current biological models of epigenetic switches built on chromatin modifications lead to strong constraints on the repertoire of dynamic behaviors for the system. We use the structure of the bifurcation diagram of the underlying dynamical system to explain the existing single cell data in silencing by the SIR system in yeast.

Mukhopadhyay, Swagatam; Nagaraj, Vijayalakshmi H.; Sengupta, Anirvan M.

2011-01-01

280

ATP-Dependent Chromatin-Remodeling Complexes  

Microsoft Academic Search

The importance of histones and chromatin structure in the regulation of eukaryotic gene transcription has become much more widely accepted over the past few years. It has been clear for a decade that histones contribute to the regulation of tran- scription both in vitro and in vivo (reviewed in references 14, 34, 50, 64, and 120). More recent studies have

MARISSA VIGNALI; AHMED H. HASSAN; KRISTEN E. NEELY; JERRY L. WORKMAN

2000-01-01

281

Chromosomes without a 30-nm chromatin fiber.  

PubMed

How is a long strand of genomic DNA packaged into a mitotic chromosome or nucleus? The nucleosome fiber (beads-on-a-string), in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fiber, and a further helically folded larger fiber. However, when frozen hydrated human mitotic cells were observed using cryoelectron microscopy, no higher-order structures that included 30-nm chromatin fibers were found. To investigate the bulk structure of mitotic chromosomes further, we performed small-angle X-ray scattering (SAXS), which can detect periodic structures in noncrystalline materials in solution. The results were striking: no structural feature larger than 11 nm was detected, even at a chromosome-diameter scale (~1 ?m). We also found a similar scattering pattern in interphase nuclei of HeLa cells in the range up to ~275 nm. Our findings suggest a common structural feature in interphase and mitotic chromatins: compact and irregular folding of nucleosome fibers occurs without a 30-nm chromatin structure. PMID:22825571

Joti, Yasumasa; Hikima, Takaaki; Nishino, Yoshinori; Kamada, Fukumi; Hihara, Saera; Takata, Hideaki; Ishikawa, Tetsuya; Maeshima, Kazuhiro

2012-07-31

282

Chromosomes without a 30-nm chromatin fiber  

PubMed Central

How is a long strand of genomic DNA packaged into a mitotic chromosome or nucleus? The nucleosome fiber (beads-on-a-string), in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fiber, and a further helically folded larger fiber. However, when frozen hydrated human mitotic cells were observed using cryoelectron microscopy, no higher-order structures that included 30-nm chromatin fibers were found. To investigate the bulk structure of mitotic chromosomes further, we performed small-angle X-ray scattering (SAXS), which can detect periodic structures in noncrystalline materials in solution. The results were striking: no structural feature larger than 11 nm was detected, even at a chromosome-diameter scale (~1 ?m). We also found a similar scattering pattern in interphase nuclei of HeLa cells in the range up to ~275 nm. Our findings suggest a common structural feature in interphase and mitotic chromatins: compact and irregular folding of nucleosome fibers occurs without a 30-nm chromatin structure.

Joti, Yasumasa; Hikima, Takaaki; Nishino, Yoshinori; Kamada, Fukumi; Hihara, Saera; Takata, Hideaki; Ishikawa, Tetsuya; Maeshima, Kazuhiro

2012-01-01

283

Electrolyte vapor condenser  

DOEpatents

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

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

1983-01-01

284

ATP-dependent chromatin remodeling factors and DNA damage repair  

Microsoft Academic Search

The organization of eukaryotic DNA into chromatin poses a barrier to all processes that require access of enzymes and regulatory factors to their sites of action. While the majority of studies in this area have concentrated on the role of chromatin in the regulation of transcription, there has been a recent emphasis on the relationship of chromatin to DNA damage

Mary Ann Osley; Toyoko Tsukuda; Jac A. Nickoloff

2007-01-01

285

The Telomere Binding Protein TRF2 Induces Chromatin Compaction  

Microsoft Academic Search

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

Asmaa M. Baker; Qiang Fu; William Hayward; Samuel Victoria; Ilene M. Pedroso; Stuart M. Lindsay; Terace M. Fletcher; Gil Ast

2011-01-01

286

Structure and function of bromodomains in chromatin-regulating complexes  

Microsoft Academic Search

Specific changes in chromatin structure are associated with transcriptional regulation. These chromatin alterations include both covalent modifications of the amino termini of histones as well as ATP-dependent non-covalent remodeling of nucleosomes. Certain protein domains, such as the bromodomains, are commonly associated with both of these classes of enzymes that alter chromatin. This review discusses recent advances in understanding the structure

Ronen Marmorstein; Shelley L. Berger

2001-01-01

287

Topoisomerase Assays  

PubMed Central

Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro.

Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

2012-01-01

288

Swi/Snf chromatin remodeling/tumor suppressor complex establishes nucleosome occupancy at target promoters.  

PubMed

Precise nucleosome-positioning patterns at promoters are thought to be crucial for faithful transcriptional regulation. However, the mechanisms by which these patterns are established, are dynamically maintained, and subsequently contribute to transcriptional control are poorly understood. The switch/sucrose non-fermentable chromatin remodeling complex, also known as the Brg1 associated factors complex, is a master developmental regulator and tumor suppressor capable of mobilizing nucleosomes in biochemical assays. However, its role in establishing the nucleosome landscape in vivo is unclear. Here we have inactivated Snf5 and Brg1, core subunits of the mammalian Swi/Snf complex, to evaluate their effects on chromatin structure and transcription levels genomewide. We find that inactivation of either subunit leads to disruptions of specific nucleosome patterning combined with a loss of overall nucleosome occupancy at a large number of promoters, regardless of their association with CpG islands. These rearrangements are accompanied by gene expression changes that promote cell proliferation. Collectively, these findings define a direct relationship between chromatin-remodeling complexes, chromatin structure, and transcriptional regulation. PMID:23723349

Tolstorukov, Michael Y; Sansam, Courtney G; Lu, Ping; Koellhoffer, Edward C; Helming, Katherine C; Alver, Burak H; Tillman, Erik J; Evans, Julia A; Wilson, Boris G; Park, Peter J; Roberts, Charles W M

2013-05-30

289

Models incorporating chromatin modification data identify functionally important p53 binding sites  

PubMed Central

Genome-wide prediction of transcription factor binding sites is notoriously difficult. We have developed and applied a logistic regression approach for prediction of binding sites for the p53 transcription factor that incorporates sequence information and chromatin modification data. We tested this by comparison of predicted sites with known binding sites defined by chromatin immunoprecipitation (ChIP), by the location of predictions relative to genes, by the function of nearby genes and by analysis of gene expression data after p53 activation. We compared the predictions made by our novel model with predictions based only on matches to a sequence position weight matrix (PWM). In whole genome assays, the fraction of known sites identified by the two models was similar, suggesting that there was little to be gained from including chromatin modification data. In contrast, there were highly significant and biologically relevant differences between the two models in the location of the predicted binding sites relative to genes, in the function of nearby genes and in the responsiveness of nearby genes to p53 activation. We propose that these contradictory results can be explained by PWM and ChIP data reflecting primarily biophysical properties of protein–DNA interactions, whereas chromatin modification data capture biologically important functional information.

Lim, Ji-Hyun; Iggo, Richard D.; Barker, Daniel

2013-01-01

290

Dynamic association of MLL1, H3K4 trimethylation with chromatin and Hox gene expression during the cell cycle.  

PubMed

Mixed lineage leukemias (MLLs) are histone H3 at lysine 4 (H3K4)-specific methylases that play a critical role in regulating gene expression in humans. As chromatin condensation, relaxation and differential gene expression are keys to correct cell cycle progression, we analyzed the dynamic association of MLL and H3K4 trimethylation at different stages of the cell cycle. Interestingly, MLL1, which is normally associated with transcriptionally active chromatins (G1 phase), dissociates from condensed mitotic chromatin and returns at the end of telophase when the nucleus starts to relax. In contrast, H3K4 trimethylation mark, which is also normally associated with euchromatins (in G1), remains associated, even with condensed chromatin, throughout the cell cycle. The global levels of MLL1 and H3K4 trimethylation are not affected during the cell cycle, and H3Ser28 phosphorylation is only observed during mitosis. Interestingly, MLL target homeobox-containing (Hox) genes (HoxA5, HoxA7 and HoxA10) are differentially expressed during the cell cycle, and the recruitment of MLL1 and H3K4 trimethylation levels are modulated in the promoter of these Hox genes as a function of their expression. In addition, down-regulation of MLL1 results in cell cycle arrest at the G2/M phase. The fluctuation of H3K4 trimethylation marks at specific promoters, but not at the global level, indicates that H3K4 trimethylation marks that are present in the G1 phase may not be the same as the marks in other phases of the cell cycle; rather, old marks are removed and new marks are introduced. In conclusion, our studies demonstrate that MLL1 and H3K4 methylation have distinct dynamics during the cell cycle and play critical roles in the differential expression of Hox genes associated with cell cycle regulation. PMID:19220463

Mishra, Bibhu P; Ansari, Khairul I; Mandal, Subhrangsu S

2009-02-07

291

Chromatin remodeling -- a novel strategy to control excessive alcohol drinking  

PubMed Central

Harmful excessive use of alcohol has a severe impact on society and it remains one of the major causes of morbidity and mortality in the population. However, mechanisms that underlie excessive alcohol consumption are still poorly understood, and thus available medications for alcohol use disorders are limited. Here, we report that changing the level of chromatin condensation by affecting DNA methylation or histone acetylation limits excessive alcohol drinking and seeking behaviors in rodents. Specifically, we show that decreasing DNA methylation by inhibiting the activity of DNA methyltransferase (DNMT) with systemic administration of the FDA-approved drug, 5-azacitidine (5-AzaC) prevents excessive alcohol use in mice. Similarly, we find that increasing histone acetylation via systemic treatment with several histone deacetylase (HDAC) inhibitors reduces mice binge-like alcohol drinking. We further report that systemic administration of the FDA-approved HDAC inhibitor, SAHA, inhibits the motivation of rats to seek alcohol. Importantly, the actions of both DNMT and HDAC inhibitors are specific for alcohol, as no changes in saccharin or sucrose intake were observed. In line with these behavioral findings, we demonstrate that excessive alcohol drinking increases DNMT1 levels and reduces histone H4 acetylation in the nucleus accumbens (NAc) of rodents. Together, our findings illustrate that DNA methylation and histone acetylation control the level of excessive alcohol drinking and seeking behaviors in preclinical rodent models. Our study therefore highlights the possibility that DNMT and HDAC inhibitors can be used to treat harmful alcohol abuse.

Warnault, V; Darcq, E; Levine, A; Barak, S; Ron, D

2013-01-01

292

Ghost condensate busting  

Microsoft Academic Search

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

Neven Bili?; Gary B. Tupper; Raoul D. Viollier

2008-01-01

293

Ghost condensate busting  

Microsoft Academic Search

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

Neven Bilic; Gary B. Tupper; Raoul D. Viollier

2008-01-01

294

Beware of condenser fouling  

SciTech Connect

Many chemical process plants generate steam for power production and process use. Recovering this steam as condensate, and returning it to the boiler, is an economical way to recycle heat. This is usually done in a watercooled, steam-surface condenser located at the exhaust of a turbine. Poor performance of such a condenser -- which is really a large heat exchanger -- can significantly decrease a plant`s heat-recycling efficiency. The most-common causes of condenser inefficiency are: microbiological growth on the water side, scale formation on the water side, tube pluggage by debris and air in-leakage on the steam side. These problems can cost a plant dearly. Well-planned treatment programs to combat these causes, however, pay for themselves many times over. The paper discusses these four types of fouling and solutions to their mitigation.

Buecker, B. [Burns and McDonnell Engineering, Kansas City, MO (United States)

1995-04-01

295

Forecasting Aircraft Condensation Trails.  

National Technical Information Service (NTIS)

Aircraft condensation trails (contrails) are caused by aircraft aerodynamics or engine exhaust in the proper atmospheric conditions. Engine-exhaust trails are the most common and are discussed in this report. Jet aircraft contrail-formation graphs facilit...

1981-01-01

296

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

PubMed

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

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

2013-01-22

297

Changes in chromatin structure correlate with transcriptional activity of nucleolar rDNA in polytene chromosomes.  

PubMed

Ribosomal DNA genes (rDNA) are found in tandem arrays of hundreds of repeated genes, but only a fraction of these genes are actively transcribed. The regulatory mechanism controlling the transition between active and inactive rDNA in higher eukaryotes is vital for cell survival. Here, we show that the nucleolus from Drosophila salivary gland cells contains two levels of chromatin organization reflecting differences in transcriptional activity: Decondensed chromatin is highly occupied with TATA-box-binding protein (TBP), phosphorylated H3S10, and acetylated H3K14, suggesting that rDNA in decondensed nucleolar areas is actively transcribed. Condensed chromatin lacks TBP, phosphorylated H3S10, or acetylated H3K14 and is enriched in the rDNA retrotransposons R1 and R2. The data show that R1 and R2 retrotransposons are not actively transcribed in salivary glands and may lead to the epigenetic silencing of flanking rDNA genes and that the silencing mechanisms of these sequences might be partially independent of heterochromatin formation by methylation of histone H3 at lysine 9 and binding of heterochromatin protein 1. PMID:19066928

Plata, Maria Piedad; Kang, Hyuck Joon; Zhang, Shaofei; Kuruganti, Srilalitha; Hsu, Shih-Jui; Labrador, Mariano

2008-12-09

298

Wapl is an essential regulator of chromatin structure and chromosome segregation.  

PubMed

Mammalian genomes contain several billion base pairs of DNA that are packaged in chromatin fibres. At selected gene loci, cohesin complexes have been proposed to arrange these fibres into higher-order structures, but how important this function is for determining overall chromosome architecture and how the process is regulated are not well understood. Using conditional mutagenesis in the mouse, here we show that depletion of the cohesin-associated protein Wapl stably locks cohesin on DNA, leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes. These findings reveal that the stability of cohesin-DNA interactions is an important determinant of chromatin structure, and indicate that cohesin has an architectural role in interphase chromosome territories. Furthermore, we show that regulation of cohesin-DNA interactions by Wapl is important for embryonic development, expression of genes such as c-myc (also known as Myc), and cell cycle progression. In mitosis, Wapl-mediated release of cohesin from DNA is essential for proper chromosome segregation and protects cohesin from cleavage by the protease separase, thus enabling mitotic exit in the presence of functional cohesin complexes. PMID:23975099

Tedeschi, Antonio; Wutz, Gordana; Huet, Sébastien; Jaritz, Markus; Wuensche, Annelie; Schirghuber, Erika; Davidson, Iain Finley; Tang, Wen; Cisneros, David A; Bhaskara, Venugopal; Nishiyama, Tomoko; Vaziri, Alipasha; Wutz, Anton; Ellenberg, Jan; Peters, Jan-Michael

2013-08-25

299

CTCF-Mediated Functional Chromatin Interactome in Pluripotent Cells  

PubMed Central

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

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

2011-01-01

300

ChIA-PET analysis of transcriptional chromatin interactions.  

PubMed

Long-range chromatin contacts between specific DNA regulatory elements play a pivotal role in gene expression regulation, and a global characterization of these interactions in the 3-dimensional (3D) chromatin structure is imperative in understanding signaling networks and cell states. Chromatin Interaction Analysis using Paired-End Tag sequencing (ChIA-PET) is a method which converts functional chromatin structure into millions of short tag sequences. Combining Chromatin Immunoprecipitation (ChIP), proximity ligation and high-throughput sequencing, ChIA-PET provides a global and unbiased interrogation of higher-order chromatin structures associated with specific protein factors. Here, we describe the detailed procedures of the ChIA-PET methodology, unraveling transcription-associated chromatin contacts in a model human cell line. PMID:22926262

Zhang, Jingyao; Poh, Huay Mei; Peh, Su Qin; Sia, Yee Yen; Li, Guoliang; Mulawadi, Fabianus Hendriyan; Goh, Yufen; Fullwood, Melissa J; Sung, Wing-Kin; Ruan, Xiaoan; Ruan, Yijun

2012-08-25

301

The bulk chromatin structure of a murine transgene does not vary with its transcriptional or DNA methylation status.  

PubMed Central

The DNA methylation status of HRD, a murine transgene, can be controlled by the genetic background upon which it is carried. We found the transgene to be transcribed in competent tissues only when undermethylated. Chromatin structure over the transgene was assayed by nuclear accessibility with DNase I, MspI, and PstI. While the transgene was up to fivefold more resistant to MspI when methylated than when not methylated, we observed no such difference with DNase I or PstI. We suggest that methyl-CpG-binding proteins are responsible for the difference observed with MspI, but that the chromatin structures are otherwise similarly compacted. Methylation could, therefore, play a regulatory role in gene expression beyond that which can be accomplished by bulk chromatin structure alone.

Weng, A; Engler, P; Storb, U

1995-01-01

302

Calorie restriction and the exercise of chromatin.  

PubMed

Since the earliest stages of evolution, organisms have faced the challenge of sensing and adapting to environmental changes for their survival under compromising conditions such as food depletion or stress. Implicit in these responses are mechanisms developed during evolution that include the targeting of chromatin to allow or prevent expression of fundamental genes and to protect genome integrity. Among the different approaches to study these mechanisms, the analysis of the response to a moderate reduction of energy intake, also known as calorie restriction (CR), has become one of the best sources of information regarding the factors and pathways involved in metabolic adaptation from lower to higher eukaryotes. Furthermore, responses to CR are involved in life span regulation-conserved from yeast to mammals-and therefore have garnered major research interest. Herein we review current knowledge of responses to CR at the molecular level and their functional link to chromatin. PMID:19608767

Vaquero, Alejandro; Reinberg, Danny

2009-07-16

303

[Folding of deoxyribonucleic acid in chromatin].  

PubMed

A structural model for the folding of deoxyribonucleic acid (DNA) in chromatin has been evolved on the basis of the X-ray diffraction patterns of deoxyribonucleoproteids (DNP). The DNA is oriented in the direction of DNP fibres and does not exhibit a superhelical structure. In the nu-bodies the DNA is folded 7 times to and fro on the envelope of a cylinder 10 nm in diameter. The height of the DNA-hairpins is 9 nm - 10 nm. The spacing between the refolded DNA segments is 3,6 nm. This supramolecular folding crystalization of the DNA is a general principle of organization and, through different types of morphological growth of the folding crystals, leads to the chromatin, to psi-DNA, to DNA monocrystals, and to DNA packing in some phage heads. PMID:1233835

Damaschun, G

1975-01-01

304

Long non-coding RNA modifies chromatin  

PubMed Central

Common themes are emerging in the molecular mechanisms of long non-coding RNA-mediated gene repression. Long non-coding RNAs (lncRNAs) participate in targeted gene silencing through chromatin remodelling, nuclear reorganisation, formation of a silencing domain and precise control over the entry of genes into silent compartments. The similarities suggest that these are fundamental processes of transcription regulation governed by lncRNAs. These findings have paved the way for analogous investigations on other lncRNAs and chromatin remodelling enzymes. Here we discuss these common mechanisms and provide our view on other molecules that warrant similar investigations. We also present our concepts on the possible mechanisms that may facilitate the exit of genes from the silencing domains and their potential therapeutic applications. Finally, we point to future areas of research and put forward our recommendations for improvements in resources and applications of existing technologies towards targeted outcomes in this active area of research.

Saxena, Alka; Carninci, Piero

2011-01-01

305

On the topology of chromatin fibres  

PubMed Central

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

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

2012-01-01

306

Calorie restriction and the exercise of chromatin  

PubMed Central

Since the earliest stages of evolution, organisms have faced the challenge of sensing and adapting to environmental changes for their survival under compromising conditions such as food depletion or stress. Implicit in these responses are mechanisms developed during evolution that include the targeting of chromatin to allow or prevent expression of fundamental genes and to protect genome integrity. Among the different approaches to study these mechanisms, the analysis of the response to a moderate reduction of energy intake, also known as calorie restriction (CR), has become one of the best sources of information regarding the factors and pathways involved in metabolic adaptation from lower to higher eukaryotes. Furthermore, responses to CR are involved in life span regulation—conserved from yeast to mammals—and therefore have garnered major research interest. Herein we review current knowledge of responses to CR at the molecular level and their functional link to chromatin.

Vaquero, Alejandro; Reinberg, Danny

2009-01-01

307

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

PubMed

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

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

1995-01-01

308

[The biological aspects of chromatin diminution].  

PubMed

The chromatine diminution (CD), first discovered by Boveri (1887) in ascarids, represents programmed elimination of a part of genetic material in the nuclei of the somatic cells in cyclops and ascarids, and in the protist macronuclei. The CD can be considered as a macromutation sharply changing chromosomal structure, though minimally effecting the phenotype. The analysis of CD is of significance for discussing mechanisms of origin of chromosomal organization, transformation of genome molecular structure in eucaryote evolution, role of the extra DNA. PMID:8484280

Akif'ev, A P; Grishanin, A K

309

Chromosomal proteins regulate steroid binding to chromatin  

Microsoft Academic Search

STEROID hormones accumulate in their respective target cells due to the presence of ``receptor'' proteins which bind the steroid with high affinity and specificity1-4. It seems that the steroid-receptor complexes then migrate and bind to nuclear chromatin2-5. Within about 15 min there is an alteration in transcription which is followed after 2 h by the appearance of specific messenger RNAs6-9.

Thomas C. Spelsberg; Robert A. Webster; George M. Pikler

1976-01-01

310

Chromatin remodeling during glucocoticoid receptor regulated transactivation  

PubMed Central

Steroid hormone receptor (SR) signaling leads to widespread changes in gene expression, and aberrant SR signaling can lead to malignancies including breast, prostate, and lung cancers. Chromatin remodeling is an essential component of SR signaling, and defining the process of chromatin and nucleosome remodeling during signaling is critical to the continued development of related therapies. The glucocorticoid receptor (GR) is a key SR that activates numerous promoters including the well defined MMTV promoter. The activation of MMTV by GR provides an excellent model for teasing apart the sequence of events between hormone treatment and changes in gene expression. Comparing hormone-induced transcription from stably integrated promoters with defined nucleosomal structure to that from transiently expressed, unstructured promoters permits key distinctions between interactions that require remodeling and those that do not. The importance of co-activators and histone modifications prior to remodeling and the formation of the preinitation complex that follows can also be clarified by defining key transition points in the propagation of hormonal signals. Combined with detailed mapping of proteins along the promoter, a temporal and spatial understanding of the signaling and remodeling processes begins to emerge. In this review, we examine SR signaling with a focus on GR activation of the MMTV promoter. We also discuss the ATP-dependent remodeling complex SWI/SNF, which provides the necessary remodeling activity during GR signaling and interacts with several SRs. BRG1, the central ATPase of SWI/SNF, also interacts with a set of BAF proteins that help determine the specialized function and fine-tuned regulation of BRG1 remodeling activity. BRG1 regulation comes from its own subdomains as well as its interactive partners. In particular, the HSA domain region of BRG1 and unique features of its ATPase homology appear to play key roles in regulating remodeling function. Details of the interworkings of this chromatin remodeling protein continue to be revealed and promise to improve our understanding of the role of chromatin remodeling during steroid hormone signaling.

King, Heather A.; Trotter, Kevin W.; Archer, Trevor K.

2012-01-01

311

Chromatin fine structure profiles for a developmentally regulated gene: reorganization of the lysozyme locus before trans-activator binding and gene expression  

PubMed Central

The chicken lysozyme locus is activated in a stepwise fashion during myeloid differentiation. We have used this locus as a model to study at high resolution changes in chromatin structure both in chicken cell lines representing various stages of macrophage differentiation and in primary cells from transgenic mice. In this study we have addressed the question of whether chromatin rearrangements can be detected in myeloid precursor cells at a stage well before overt transcription of the lysozyme gene begins. In addition to restriction enzyme accessibility assays and DMS footprinting, we have applied new, very sensitive techniques to assay for chromatin changes. Particularly informative was UV photofootprinting, using terminal transferase-dependent PCR and nonradioactive detection. We find that the basic chromatin structure in lysozyme nonexpressing hematopoietic precursor cells is highly similar to the pattern found in fully differentiated lysozyme-expressing cells. In addition, we find that only in nonexpressing cells are dimethylsulfate footprints and UV photofootprints affected by trichostatin, an inhibitor of histone deacetylation. These results are interpreted to mean that most chromatin pattern formation is complete before the binding of end-stage trans-activators, supporting the notion that heritable chromatin structure is central to the stable epigenetic programs that guide development.

Kontaraki, Joanna; Chen, Hsiu-Hua; Riggs, Arthur; Bonifer, Constanze

2000-01-01

312

Efficient condensate removal  

SciTech Connect

Achieving stable control of product temperatures on reboilers and other steam/product heat exchangers has long been a problem area where conventional steam control and condensate trapping methods were used. Application of a non-electric condensate pump and steam trap sidesteps the problem. Removal of condenser under all conditions, from full load to zero load, is readily achieved. Steam flow control then regulates heat flow to match load requirements, with stable product temperatures and fast response to control action. Avoidance of waterhammer and corrosion problems within the exchangers are added side benefits. Difficulties with conventional hookups begin when the surface area of the heater and the heat transfer coefficient means that the steam temperature within the heater must be lower than design levels. Part-load operation or even use of generous fouling factors at design stage both call for lowered steam temperatures and pressures. When steam pressures approach the back pressure on the traps, condensate flow slows down and can stop completely. Flooding of the heat exchange surface follows and heat transfer slows down. The temperature control then opens up the steam valve, as it responds to falling product temperature. With the increased steam pressure, the condensate clears through the trap. The exchanger is then filled with steam at a higher temperature and pressure than it needs, so the product temperature begins to climb again.

Armer, A.

1988-01-01

313

Condensed Genome Structure  

PubMed Central

Large, tailed dsDNA-containing bacteriophage genomes are packaged to a conserved and high density (~500 mg/ml), generally in ~2.5-nm, duplex-to-duplex, spaced, organized DNA shells within icosahedral capsids. Phages with these condensate properties, however, differ markedly in their inner capsid structures: (1) those with a naked condensed DNA, (2) those with many dispersed unstructured proteins embedded within the DNA, (3) those with a small number of localized proteins, and (4) those with a reduced or DNA-free internal protein structure of substantial volume. The DNA is translocated and condensed by a high-force ATPase motor into a procapsid already containing the proteins that are to be ejected together with the DNA into the infected host. The condensed genome structure of a single-phage type is unlikely to be precisely determined and can change without loss of function to fit an altered capsid size or internal structure. Although no such single-phage condensed genome structure is known exactly, it is known that a single general structure is unlikely to apply to all such phages.

Black, Lindsay W.

2013-01-01

314

Default assembly of early adenovirus chromatin  

SciTech Connect

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

Spector, David J. [Department of Microbiology and Immunology, Intercollege Graduate Degree Program in Genetics, and Cellular and Molecular Biology Program, Pennsylvania State University College of Medicine, Hershey, PA 17033 (United States)]. E-mail: dspector@psu.edu

2007-03-01

315

Higher order chromatin organization in cancer  

PubMed Central

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

Reddy, Karen L.; Feinberg, Andrew P.

2013-01-01

316

Titration and hysteresis in epigenetic chromatin silencing  

NASA Astrophysics Data System (ADS)

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

Dayarian, Adel; Sengupta, Anirvan M.

2013-06-01

317

Chromatin Immunoprecipitation (ChIP) on Unfixed Chromatin from Cells and Tissues to Analyze Histone Modifications.  

PubMed

INTRODUCTIONIn cells and tissues, the histone proteins that constitute the nucleosomes can present multiple post-translational modifications, such as lysine acetylation, lysine and arginine methylation, serine phosphorylation, and lysine ubiquitination. On their own, or in combination, these covalent modifications on the core histones are thought to play essential roles in chromatin organization and gene expression in eukaryotes. Importantly, patterns of histone modifications may be somatically conserved and can, thereby, maintain locus-specific repression/activity in defined lineages, or throughout development. Indirect immunofluorescence studies on cultured cells have been pivotal in unraveling the roles of histone modifications. However, to address in detail what happens at specific sites in vivo, chromatin immunoprecipitation (ChIP) is the method of choice. Here, we describe how ChIP can be performed on non-fixed chromatin from animal cells or tissues (fresh or frozen) to analyze histone modifications at specific chromosomal sites. These protocols are suitable only for analyzing histones and their modifications. For other applications, chromatin immunoprecipitation should be performed on cross-linked chromatin. PMID:21357105

Wagschal, Alexandre; Delaval, Katia; Pannetier, Maëlle; Arnaud, Philippe; Feil, Robert

2007-06-01

318

The PHD and Chromo Domains Regulate the ATPase Activity of the Human Chromatin Remodeler CHD4  

PubMed Central

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

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

2012-01-01

319

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

NASA Astrophysics Data System (ADS)

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

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

320

Cellulase Assays  

NASA Astrophysics Data System (ADS)

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

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

321

Effect of SWI/SNF chromatin remodeling complex on HIV-1 Tat activated transcription  

PubMed Central

Background Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of acquired immunodeficiency virus (AIDS). Following entry into the host cell, the viral RNA is reverse transcribed into DNA and subsequently integrated into the host genome as a chromatin template. The integrated proviral DNA, along with the specific chromatinized environment in which integration takes place allows for the coordinated regulation of viral transcription and replication. While the specific roles of and interplay between viral and host proteins have not been fully elucidated, numerous reports indicate that HIV-1 retains the ability for self-regulation via the pleiotropic effects of its viral proteins. Though viral transcription is fully dependent upon host cellular factors and the state of host activation, recent findings indicate a complex interplay between viral proteins and host transcription regulatory machineries including histone deacetylases (HDACs), histone acetyltransferases (HATs), cyclin dependent kinases (CDKs), and histone methyltransferases (HMTs). Results Here, we describe the effect of Tat activated transcription at the G1/S border of the cell cycle and analyze the interaction of modified Tat with the chromatin remodeling complex, SWI/SNF. HIV-1 LTR DNA reconstituted into nucleosomes can be activated in vitro using various Tat expressing extracts. Optimally activated transcription was observed at the G1/S border of the cell cycle both in vitro and in vivo, where chromatin remodeling complex, SWI/SNF, was present on the immobilized LTR DNA. Using a number of in vitro binding as well as in vivo chromatin immunoprecipitation (ChIP) assays, we detected the presence of both BRG1 and acetylated Tat in the same complex. Finally, we demonstrate that activated transcription resulted in partial or complete removal of the nucleosome from the start site of the LTR as evidenced by a restriction enzyme accessibility assay. Conclusion We propose a model where unmodified Tat is involved in binding to the CBP/p300 and cdk9/cyclin T1 complexes facilitating transcription initiation. Acetylated Tat dissociates from the TAR RNA structure and recruits bromodomain-binding chromatin modifying complexes such as p/CAF and SWI/SNF to possibly facilitate transcription elongation.

Agbottah, Emmanuel; Deng, Longwen; Dannenberg, Luke O; Pumfery, Anne; Kashanchi, Fatah

2006-01-01

322

Transcriptional repression mediated by polycomb group proteins and other chromatin-associated repressors is selectively blocked by insulators.  

PubMed

Polycomb group (PcG) proteins repress gene activity over a considerable distance, possibly by spreading along the chromatin fiber. Insulators or boundary elements, genetic elements within the chromatin, may serve to terminate the repressing action of PcG proteins. We studied the ability of insulators to block the action of chromatin-associated repressors such as PcG proteins, HP1, and MeCP2. We found that the Drosophila special chromatin structure insulator completely blocks transcriptional repression mediated by all of the repressors we tested. The Drosophila gypsy insulator was able to block the repression mediated by the PcG proteins Su(z)2 and RING1, as well as mHP1, but not the repression mediated by MeCP2 and the PcG protein HPC2. The 5'-located DNase I-hypersensitive site in the chicken beta-globin locus displayed a limited ability to block repression, and a matrix or scaffold attachment region element was entirely unable to block repression mediated by any repressor tested. Our results indicate that insulators can block repression mediated by PcG proteins and other chromatin-associated repressors, but with a high level of selectivity. This high degree of specificity may provide a useful assay to define and characterize distinct classes of insulators. PMID:10617669

van der Vlag, J; den Blaauwen, J L; Sewalt, R G; van Driel, R; Otte, A P

2000-01-01

323

Chromatin affinity-precipitation using a small metabolic molecule: its application to analysis of O-acetyl-ADP-ribose  

PubMed Central

In the cell, many small endogenous metabolic molecules are involved in distinct cellular functions such as modulation of chromatin structure and regulation of gene expression. O-acetyl-ADP-ribose (AAR) is a small metabolic molecule that is generated during NAD-dependent deacetylation by Sir2. Sir2 regulates gene expression, DNA repair, and genome stability. Here, we developed a novel chromatin affinity-precipitation (ChAP) method to detect the chromatin fragments at which small molecules interact with binding partners. We used this method to demonstrate that AAR associated with heterochromatin. Moreover, we applied the ChAP method to whole genome tiling array chips to compare the association of AAR and Sir2. We found that AAR and Sir2 displayed similar genomic binding patterns. Furthermore, we identified 312 potential association cluster regions of AAR. The ChAP assay may therefore be a generally useful strategy to study the small molecule association with chromosomal regions. Our results further suggest that the small metabolic molecule AAR associates with silent chromatin regions in a Sir2-dependent manner and provide additional support for the role of AAR in assembly of silent chromatin.

Tung, Shu-Yun; Hong, Jia-Yang; Walz, Thomas

2011-01-01

324

Chromatin remodeling during glucocorticoid receptor regulated transactivation.  

PubMed

Steroid hormone receptor (SR) signaling leads to widespread changes in gene expression, and aberrant SR signaling can lead to malignancies including breast, prostate, and lung cancers. Chromatin remodeling is an essential component of SR signaling, and defining the process of chromatin and nucleosome remodeling during signaling is critical to the continued development of related therapies. The glucocorticoid receptor (GR) is a key SR that activates numerous promoters including the well defined MMTV promoter. The activation of MMTV by GR provides an excellent model for teasing apart the sequence of events between hormone treatment and changes in gene expression. Comparing hormone-induced transcription from stably integrated promoters with defined nucleosomal structure to that from transiently expressed, unstructured promoters permits key distinctions between interactions that require remodeling and those that do not. The importance of co-activators and histone modifications prior to remodeling and the formation of the preinitiation complex that follows can also be clarified by defining key transition points in the propagation of hormonal signals. Combined with detailed mapping of proteins along the promoter, a temporal and spatial understanding of the signaling and remodeling processes begins to emerge. In this review, we examine SR signaling with a focus on GR activation of the MMTV promoter. We also discuss the ATP-dependent remodeling complex SWI/SNF, which provides the necessary remodeling activity during GR signaling and interacts with several SRs. BRG1, the central ATPase of SWI/SNF, also interacts with a set of BAF proteins that help determine the specialized function and fine-tuned regulation of BRG1 remodeling activity. BRG1 regulation comes from its own subdomains as well as its interactive partners. In particular, the HSA domain region of BRG1 and unique features of its ATPase homology appear to play key roles in regulating remodeling function. Details of the inter-workings of this chromatin remodeling protein continue to be revealed and promise to improve our understanding of the mechanism of chromatin remodeling during steroid hormone signaling. This article is part of a Special Issue entitled: Chromatin in time and space. PMID:22425674

King, Heather A; Trotter, Kevin W; Archer, Trevor K

2012-03-06

325

The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation.  

PubMed

Sperm DNA fragmentation is being increasingly recognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (4',6-diamidino-2-phenylindole) and/or the Diff-Quik reagent, and the percentages of sperm with nondispersed and dispersed chromatin loops were monitored by fluorescence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmentation, as measured by DBD-FISH. Conversely, all sperm with dispersed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had participated in a donor insemination program. The coefficient of variation obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly reproducible, and inexpensive method for the analysis of sperm DNA fragmentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test for the screening of sperm DNA fragmentation in the andrology laboratory. PMID:12514084

Fernández, Jose Luis; Muriel, Lourdes; Rivero, Maria Teresa; Goyanes, Vicente; Vazquez, Rosana; Alvarez, Juan G

326

Evaporation, Condensation, and Precipitation  

NSDL National Science Digital Library

After completion of this project students should have an understanding of evaporation, condensation, and precipitation in the water cycle. Use the websites provided to answer the questions. Record your answers on the spreadsheet provided. Do you understand how the water cycle works? Begin by watching this short video about the water cycle.water cycle video Use the website to define condensation, precipitation, and evaporation?water cycle List the different types of precipitation from the site.types of precipitation Follow the directions to the experiment on this website to get a better understanding of how evaporation takes ...

Brown, Miss

2009-10-21

327

Radial density distribution of chromatin: evidence that chromatin fibers have solid centers  

PubMed Central

Fiber diameter, radial distribution of density, and radius of gyration were determined from scanning transmission electron microscopy (STEM) of unstained, frozen-dried chromatin fibers. Chromatin fibers isolated under physiological conditions (ionic strength, 124 mM) from Thyone briareus sperm (DNA linker length, n = 87 bp) and Necturus maculosus erythrocytes (n = 48 bp) were analyzed by objective image-processing techniques. The mean outer diameters were determined to be 38.0 nm (SD = 3.7 nm; SEM = 0.36 nm) and 31.2 nm (SD = 3.6 nm; SEM = 0.32 nm) for Thyone and Necturus, respectively. These data are inconsistent with the twisted-ribbon and solenoid models, which predict constant diameters of approximately 30 nm, independent of DNA linker length. Calculated radial density distributions of chromatin exhibited relatively uniform density with no central hole, although the 4-nm hole in tobacco mosaic virus (TMV) from the same micrographs was visualized clearly. The existence of density at the center of chromatin fibers is in strong disagreement with the hollow-solenoid and hollow-twisted-ribbon models, which predict central holes of 16 and 9 nm for chromatin of 38 and 31 nm diameter, respectively. The cross-sectional radii of gyration were calculated from the radial density distributions and found to be 13.6 nm for Thyone and 11.1 nm for Necturus, in good agreement with x-ray and neutron scattering. The STEM data do not support the solenoid or twisted-ribbon models for chromatin fiber structure. They do, however, support the double-helical crossed-linker models, which exhibit a strong dependence of fiber diameter upon DNA linker length and have linker DNA at the center.

1990-01-01

328

Simple Simulations of DNA Condensation  

SciTech Connect

Molecular dynamics simulations of a simple, bead-spring model of semiflexible polyelectrolytes such as DNA are performed. All charges are explicitly treated. Starting from extended, noncondensed conformations, condensed structures form in the simulations with tetravalent or trivalent counterions. No condensates form or are stable for divalent counterions. The mechanism by which condensates form is described. Briefly, condensation occurs because electrostatic interactions dominate entropy, and the favored Coulombic structure is a charge ordered state. Condensation is a generic phenomena and occurs for a variety of polyelectrolyte parameters. Toroids and rods are the condensate structures. Toroids form preferentially when the molecular stiffness is sufficiently strong.

STEVENS,MARK J.

2000-07-12

329

Polariton Condensation in Photonic Molecules  

NASA Astrophysics Data System (ADS)

We report on polariton condensation in photonic molecules formed by two coupled micropillars. We show that the condensation process is strongly affected by the interaction with the cloud of uncondensed excitons and thus strongly depends on the exact localization of these excitons within the molecule. Under symmetric excitation conditions, condensation is triggered on both binding and antibinding polariton states of the molecule. On the opposite, when the excitonic cloud is injected in one of the two pillars, condensation on a metastable state is observed and a total transfer of the condensate into one of the micropillars can be achieved. Our results highlight the crucial role played by relaxation kinetics in the condensation process.

Galbiati, Marta; Ferrier, Lydie; Solnyshkov, Dmitry D.; Tanese, Dimitrii; Wertz, Esther; Amo, Alberto; Abbarchi, Marco; Senellart, Pascale; Sagnes, Isabelle; Lemaître, Aristide; Galopin, Elisabeth; Malpuech, Guillaume; Bloch, Jacqueline

2012-03-01

330

Detail of Bright Angel stone vault, containing condenser, Hoffman condensation ...  

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Detail of Bright Angel stone vault, containing condenser, Hoffman condensation pump, Jennings vacuum heating pump, and misc. pipes and valves. - Grand Canyon Village Utilities, Grand Canyon National Park, Grand Canyon Village, Coconino County, AZ

331

Meiotic Cohesion Requires Accumulation of ORD on Chromosomes before Condensation  

PubMed Central

Cohesion between sister chromatids is a prerequisite for accurate chromosome segregation during mitosis and meiosis. To allow chromosome condensation during prophase, the connections that hold sister chromatids together must be maintained but still permit extensive chromatin compaction. In Drosophila, null mutations in the orientation disruptor (ord) gene lead to meiotic nondisjunction in males and females because cohesion is absent by the time that sister kinetochores make stable microtubule attachments. We provide evidence that ORD is concentrated within the extrachromosomal domains of the nuclei of Drosophila primary spermatocytes during early G2, but accumulates on the meiotic chromosomes by mid to late G2. Moreover, using fluorescence in situ hybridization to monitor cohesion directly, we show that cohesion defects first become detectable in ordnull spermatocytes shortly after the time when wild-type ORD associates with the chromosomes. After condensation, ORD remains bound at the centromeres of wild-type spermatocytes and persists there until centromeric cohesion is released during anaphase II. Our results suggest that association of ORD with meiotic chromosomes during mid to late G2 is required to maintain sister-chromatid cohesion during prophase condensation and that retention of ORD at the centromeres after condensation ensures the maintenance of centromeric cohesion until anaphase II.

Balicky, Eric M.; Endres, Matthew W.; Lai, Cary; Bickel, Sharon E.

2002-01-01

332

Luciferase assay.  

PubMed

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

Smale, Stephen T

2010-05-01

333

[The structure of chromatin at meiotic prophase I].  

PubMed

The qualitative and quantitative changes in molecular chromatin structures during the meiotic prophase I were studied. The following patterns were discovered: (1) unlike somatic cells, the syntheses of total histone and DNA and its integration into the chromatin occur independently and asynchronously: DNA replication is completed by the interphase, whereas the synthesis of histone and its integration into the chromatin continue to late meiotic prophase I, and (2) individual histone fractions are synthesized and integrated into the chromatin during meiotic prophase independently and asynchronously. Chromatin hydrolysis with nucleases DNI, STN, and SI demonstrated considerable differences in the hydrolysis products obtained at different stages of the meiotic prophase I; presumably, this reflects the differences between the structures of initial chromatin at different stages of the meiotic prophase I. PMID:18186185

Strokov, A A

2007-11-01

334

RNAs templating chromatin structure for dosage compensation in animals.  

PubMed

The role of RNA as a messenger in the expression of the genome has been long appreciated, but its functions in regulating chromatin and chromosome structure are no less interesting. Recent results have shown that small RNAs guide chromatin-modifying complexes to chromosomal regions in a sequence-specific manner to elicit transcriptional repression. However, sequence-specific targeting by means of base pairing seems to be only one mechanism by which RNA is employed for epigenetic regulation. The focus of this review is on large RNAs that act in the dosage-compensation pathways of flies and mammals. These RNAs associate with chromatin over the length of whole chromosomes and are crucial for spreading epigenetic changes in chromatin structure. They do not appear to act in a sequence-specific manner but might provide scaffolds for co-operative binding of chromatin-associated complexes that enable spreading of chromatin modifications. PMID:12717814

Wutz, Anton

2003-05-01

335

Bayesian network analysis of targeting interactions in chromatin  

PubMed Central

In eukaryotes, many chromatin proteins together regulate gene expression. Chromatin proteins often direct the genomic binding pattern of other chromatin proteins, for example, by recruitment or competition mechanisms. The network of such targeting interactions in chromatin is complex and still poorly understood. Based on genome-wide binding maps, we constructed a Bayesian network model of the targeting interactions among a broad set of 43 chromatin components in Drosophila cells. This model predicts many novel functional relationships. For example, we found that the homologous proteins HP1 and HP1C each target the heterochromatin protein HP3 to distinct sets of genes in a competitive manner. We also discovered a central role for the remodeling factor Brahma in the targeting of several DNA-binding factors, including GAGA factor, JRA, and SU(VAR)3-7. Our network model provides a global view of the targeting interplay among dozens of chromatin components.

van Steensel, Bas; Braunschweig, Ulrich; Filion, Guillaume J.; Chen, Menzies; van Bemmel, Joke G.; Ideker, Trey

2010-01-01

336

G2 phase chromatin lacks determinants of replication timing  

PubMed Central

DNA replication in all eukaryotes follows a defined replication timing program, the molecular mechanism of which remains elusive. Using a Xenopus laevis egg extract replication system, we previously demonstrated that replication timing is established during early G1 phase of the cell cycle (timing decision point [TDP]), which is coincident with the repositioning and anchorage of chromatin in the newly formed nucleus. In this study, we use this same system to show that G2 phase chromatin lacks determinants of replication timing but maintains the overall spatial organization of chromatin domains, and we confirm this finding by genome-wide analysis of rereplication in vivo. In contrast, chromatin from quiescent cells retains replication timing but exhibits disrupted spatial organization. These data support a model in which events at the TDP, facilitated by chromatin spatial organization, establish determinants of replication timing that persist independent of spatial organization until the process of chromatin replication during S phase erases those determinants.

Lu, Junjie; Li, Feng; Murphy, Christopher S.; Davidson, Michael W.

2010-01-01

337

Chromatin interaction analysis using paired-end tag sequencing.  

PubMed

Chromatin Interaction Analysis using Paired-End Tag sequencing (ChIA-PET) is a technique developed for large-scale, de novo analysis of higher-order chromatin structures. Cells are treated with formaldehyde to cross-link chromatin interactions, DNA segments bound by protein factors are enriched by chromatin immunoprecipitation, and interacting DNA fragments are then captured by proximity ligation. The Paired-End Tag (PET) strategy is applied to the construction of ChIA-PET libraries, which are sequenced by high-throughput next-generation sequencing technologies. Finally, raw PET sequences are subjected to bioinformatics analysis, resulting in a genome-wide map of binding sites and chromatin interactions mediated by the protein factor under study. This unit describes ChIA-PET for genome-wide analysis of chromatin interactions in mammalian cells, with the application of Roche/454 and Illumina sequencing technologies. PMID:20069536

Fullwood, Melissa J; Han, Yuyuan; Wei, Chia-Lin; Ruan, Xiaoan; Ruan, Yijun

2010-01-01

338

Diversity of Operation in ATP-dependent Chromatin Remodelers  

PubMed Central

Chromatin is actively restructured by a group of proteins that belong to the family of ATP-dependent DNA translocases. These chromatin remodelers can assemble, relocate or remove nucleosomes, the fundamental building blocks of chromatin. The family of ATP-dependent chromatin remodelers has many properties in common, but there are also important differences that may account for their varying roles in the cell. Some of the important characteristics of these complexes have begun to be revealed such as their interactions with chromatin and their mechanism of operation. The different domains of chromatin remodelers are discussed in terms of their targets and functional roles in mobilizing nucleosomes. The techniques that have driven these findings are discussed and how these have helped develop the current models for how nucleosomes are remodeled.

Hota, Swetansu K.; Bartholomew, Blaine

2011-01-01

339

Chromatin dynamics associated with HIV-1 Tat activated transcription  

PubMed Central

Summary Chromatin remodeling is an essential event for HIV-1 transcription. Over the last two decades this field of research has come to the forefront, as silencing of the HIV-1 provirus through chromatin modifications has been linked to latency. Here, we focus on chromatin remodeling, especially in relation to the transactivator Tat, and review the most important and newly emerging studies that investigate remodeling mechanisms. We begin by discussing covalent modifications that can alter chromatin structure including acetylation, deacetylation, and methylation, as well as topics addressing the interplay between chromatin remodeling and splicing. Next, we focus on complexes that use the energy of ATP to remove or secure nucleosomes and can additionally act to control HIV-1 transcription. Finally, we cover recent literature on viral microRNAs which have been shown to alter chromatin structure by inducing methylation or even by remodeling nucleosomes.

Easley, Rebecca; Van Duyne, Rachel; Coley, Will; Guendel, Irene; Dadgar, Sherry; Kehn-Hall, Kylene; Kashanchi, Fatah

2009-01-01

340

Condensed matter physics journals  

Microsoft Academic Search

On the basis of a citation\\/reference criterion, 20 core journals are selected in the field of condensed matter physics. Citation data and indicators from 1980Journal Citation Reports reveal their different characteristic features such as applied orientation, communication function and longevity. The manually obtained data for the core journals are written into a matrix in order to determine an appropriate ranking

R. Todorov

1983-01-01

341

Soft condensed matter physics  

Microsoft Academic Search

Soft condensed matter physics is the study of materials, such as fluids, liquid crystals, polymers, colloids and emulsions, that are “soft” to the touch. This article will review some properties, such as the dominance of entropy, that are unique to soft materials and some properties such as the interplay between broken-symmetry, dynamic mode structure and topological defects that are common

T. C. Lubensky

1997-01-01

342

Condensed Matter Physics  

Microsoft Academic Search

Condensed matter physics constitutes nowadays an enormous field of knowledge. To write a good textbook covering all main topics in that field in a suitable way and in a reasonable volume is very hard indeed. I believe Michael Marder has achieved this goal with great success. The text is arranged in six parts. Part I describes the atomic structure of

I Strzalkowski

2000-01-01

343

Condensate removal device  

SciTech Connect

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

Maddox, J.W.; Berger, D.D.

1984-10-23

344

MUNICIPAL LANDFILL GAS CONDENSATE  

EPA Science Inventory

New regulations relative to air emissions from municipal landfills may require the installation of gas collection systems at landfills. As landfill gas (LFG) is collected, water and other vapors in the gas condense in the system or are purposely removed in the normal treatment of...

345

Soft Condensed Matter  

Microsoft Academic Search

The author states in the preface of the book that the aim is '...to give a unified overview of the various aspects of the physics of soft condensed matter'. The book succeeds in fulfilling this aim in many respects.i) The style is fluent and concise and gives the necessary explanations to make its content understandable to people with some knowledge

Richard A L Jones

2002-01-01

346

Modular invariant gaugino condensation  

SciTech Connect

The construction of effective supergravity lagrangians for gaugino condensation is reviewed and recent results are presented that are consistent with modular invariance and yield a positive definite potential of the noscale type. Possible implications for phenomenology are briefly discussed. 29 refs.

Gaillard, M.K.

1991-05-09

347

Macrocephaly in bull spermatozoa is associated with nuclear vacuoles, diploidy and alteration of chromatin condensation.  

PubMed

Spermatozoa from 2 dairy AI (artificial insemination) bulls (A and B), identified by their abnormal spermiogram with cells depicting frequent macrocephaly, double tails and nuclear vacuoles, were case-investigated and compared to normal spermatozoa from a control AI sire (C). Head sizes were measured and morphological abnormalities scored using brightfield and differential interference contrast microscopy. The degree of sperm maturation and of resistance to acid-induced DNA denaturation in situ were determined after uploading of acridine orange using flow cytometry of 5,000 cells/sample. Nuclear fragmentation, i.e. the ratio of red to total (red + green) fluorescence, reached 7.1% and 31% in bulls A and B, compared to 2% in bull C. The proportion of immature spermatozoa, i.e. those with incomplete histone-protamine exchange and depicting higher green fluorescence compared to the main population of the control bull, reached 9.54% in A and 7.75% in B, compared to only 0.47% in the control. In the second part of this study the previously unknown chromosomal constitution of large-headed spermatozoa of bull A was investigated by fluorescence in situ hybridization using an X-Y painting probe set. The 7.5% XY-bearing cells and the presence of diploid spermatozoa detected by flow cytometry indicate a meiotic arrest in the first division in bull A, becoming the first proven case of association of macrocephaly and M1 diploidy. The diverse approaches used for the investigation of spermatozoal DNA provide insights into the etiology of macrocephaly. PMID:20016171

Revay, T; Nagy, S; Kopp, C; Flyckt, A; Rens, W; Rath, D; Hidas, A; Kovacs, A; Johannisson, A; Rodriguez-Martinez, H; Andersson, M

2009-12-09

348

Linker histories are not essential and affect chromatin condensation in vivo  

Microsoft Academic Search

We have (separately) disrupted all of the expressed macronuclear copies of the HHO gene encoding macronuclear histone H1 and of the micronuclear linker histone (MLH) gene encoding the protein MicLH in Tetrahymena thermophila. These disruptions are shown to eliminate completely the expression of each protein. Strains without either linker histone grow at normal rates and reach near-normal cell densities, demonstrating

Xuetong Shen; Lanlan Yu; Joyce W. Weir; Martin A. Gorovsky

1995-01-01

349

Biomarker reproducibility in exhaled breath condensate collected with different condensers  

Microsoft Academic Search

Optimal collection and analysis of exhaled breath condensate (EBC) are prerequisites for standardisation and reproducibility of assessments. The present study aimed to assess reproducibility of EBC volume, hydrogen peroxide (H2O2), 8-isoprostane and cytokine measurements using different condensers, including a newly developed glass condenser. At four points in time, 30 healthy subjects performed sequential EBC collections randomly using the following four

P. P. Rosias; C. M. Robroeks; A. Kester; G. J. den Hartog; W. K. Wodzig; G. T. Rijkerse; L. J. Zimmermann; C. P. van Schayck; Q. Jobsis; E. Dompeling

2008-01-01

350

Resolution of a Spectrum of Nucleoprotein Species in Sonicated Chromatin  

Microsoft Academic Search

Sonicated rabbit-liver chromatin is fractionated by ion-exchange chromatography on ECTHAM-cellulose, a weakly cationic adsorbent. The chromatographic procedure provides a series of fractions having a spectrum of thermal denaturation profiles. The earliest-eluting fractions melt cooperatively at a significantly higher temperature than bulk chromatin, and totally lack any of the components of chromatin that melt at low temperatures. In contrast, the latest-eluting

Gerald R. Reeck; Robert T. Simpson; Herbert A. Sober

1972-01-01

351

Homology-driven chromatin remodeling by human RAD54  

Microsoft Academic Search

Human RAD51 and RAD54 are key players in homologous recombination, a process that requires homology recognition and strand invasion by a RAD51–single-stranded DNA (ssDNA) nucleoprotein filament and chromatin remodeling by RAD54. Here we use in vitro chromatin reconstitution systems to show that RAD51-ssDNA stimulates RAD54-dependent chromatin remodeling in a homology-dependent, polarity-independent manner. This stimulation was not seen with RAD54B or

Zhaoqing Zhang; Hua-Ying Fan; Joseph A Goldman; Robert E Kingston

2007-01-01

352

Aging by epigenetics--a consequence of chromatin damage?  

PubMed

Chromatin structure is not fixed. Instead, chromatin is dynamic and is subject to extensive developmental and age-associated remodeling. In some cases, this remodeling appears to counter the aging and age-associated diseases, such as cancer, and extend organismal lifespan. However, stochastic non-deterministic changes in chromatin structure might, over time, also contribute to the break down of nuclear, cell and tissue function, and consequently aging and age-associated diseases. PMID:18423606

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

2008-03-12

353

Aging by epigenetics-A consequence of chromatin damage?  

SciTech Connect

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

Sedivy, John M. [Brown University, Division of Biology and Medicine, Laboratories for Molecular Medicine, 70 Ship Street, Providence, RI 02903 (United States)], E-mail: john_sedivy@brown.edu; Banumathy, Gowrishankar [Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111 (United States); Adams, Peter D. [Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111 (United States)], E-mail: peter.adams@fccc.edu

2008-06-10

354

Computer program predicts condenser cleanliness  

SciTech Connect

Depending on unit size, condenser tube fouling can cost a utility hundreds of thousands of dollars per year in lost efficiency. Because the condenser is the largest heat exchanger in the condensate-feed-water circuit, condenser performance can significantly affect unit heat rates. This paper reports that good condenser monitoring procedures can improve efficiency and avoid tube corrosion and fouling. The primary cause of poor performance in cooling water condensers is tube fouling, a problem which can result from a variety of mechanisms. Two of the most common are mineral deposition on internal tube surfaces, and microbiological growth on the cooling water side of the condenser. In addition, air leakage through the condenser shell or auxiliary equipment can blanket the steam side of the tubes with undissolved gases. Although each of these contaminants has its own characteristics, all of them have the same effect: they reduce heat transfer.

Buecker, B.

1992-06-01

355

Evaluation of estrogenic activities of hydroxylated polycyclic aromatic hydrocarbons in cigarette smoke condensate  

Microsoft Academic Search

Estrogenic activities of cigarette smoke condensates obtained from the extraction of particulate matters from mainstream and sidestream cigarette smoke with benzene\\/ethanol were evaluated by using a yeast two-hybrid assay system expressing human estrogen receptor ? (hER?). To identify the constituents of the cigarette smoke condensate which are responsible for the estrogenic activity, the condensate was fractionated into eleven fractions by

Makiko Kamiya; Akira Toriba; Yu Onoda; Ryoichi Kizu; Kazuichi Hayakawa

2005-01-01

356

Quantitative proteomic analysis of chromatin-associated factors  

Microsoft Academic Search

A method to identify and quantify chromatin-associated proteins has been developed and applied to the analysis of changes\\u000a in chromatin-associated proteins induced by Myc oncoprotein expression in human B lymphocytes. Chromatin-enriched fractions\\u000a were isolated by differential detergent\\/salt extraction and analyzed by ICAT reagent labeling, multi-dimensional chromatography\\u000a and tandem mass spectrometry. Many known chromatin-associated regulatory factors were identified and quantified. The

Yuzuru Shiio; Robert N. Eisenman; Eugene C. Yi; Sam Donohoe; David R. Goodlett; Ruedi Aebersold

2003-01-01

357

Protein-dependent conformational behavior of DNA in chromatin  

SciTech Connect

Information from circular dichroism (CD) and DNA thermal denaturation has been used in concert to study the conformation behavior of DNA in the extended 11-nm fiber of chromatin isolated from HeLa nuclei. The histone-dependent conformational states of the system were investigated by selectively removing the hydrophilic histone domains with trypsin. These were compared to acetylated chromatin from the same source. The integrated intensity of the positive CD band for DNA above 260 nm is found to increase with the content of relatively unstressed B-form DNA. This same increase is observed along the series of whole, H1-stripped, and trypsinized chromatin samples as protein is removed. Hence, the ratio of percent hyperchromicity to integrated CD band intensity of the respective melting transitions provides useful information on the conformational state of DNA in the three principal regions of the chromatin fiber: the central loop and flanking nucleosomal regions and the linker. Results from this study suggest that central loop DNA in both hyperacetylated and control chromatin relaxes as protein is removed. However, hyperacetylated chromatin shows significantly less dependence than control chromatin upon core histone hydrophilic domains in the flanking and linker regions. Thus, histone hyperacetylation evidently relaxes DNA in chromatin with no major overall conformational changes. A possible role of histone hyperacetylation may therefore be to reduce cooperativity in the unfolding transition in chromatin and thus provide for greater localized control of unfolding during transcription.

Riehm, M.R.; Harrington, R.E.

1987-05-19

358

The impact of chromatin regulation on the floral transition.  

PubMed

Transcription in eukaryotes is regulated by many enzymes that influence the nuclear organization of DNA in chromatin. Several of these enzymes regulate histone modifications. These modifications function as a platform for assembly of protein complexes that influence chromatin structure. Dynamic changes between chromatin states that facilitate or inhibit transcription are important in the transcriptional regulation of pathways controlling floral induction in response to environmental and developmental signals. This review focuses on the mechanistic aspects of histone modifications and chromatin changes at flowering-time genes and how these relate to the initiation of flowering. PMID:18708152

Farrona, Sara; Coupland, George; Turck, Franziska

2008-07-30

359

Gas condensates as diesel fuel  

Microsoft Academic Search

The data of Table 1 show that the distillation curve of the gas condensate from the Yangi-Kazgan field matches that of A-72 gasoline; the Gazli condensate corresponds to light TS-I jet fuel; the Urtabulak, Gazli-Neft', and Urengoi condensates correspond to wide-cut jet fuel; the Kultak, Zevardy, l~barek, and Shurtan condensates correspond to wide-cut engine fuel. It will be noted that

A. A. Kukushkin; V. S. Azev; G. N. Gerasimova; V. M. Aprelenko; A. I. Kirsanov

1985-01-01

360

Chromatin architecture defines the glucocorticoid response.  

PubMed

The glucocorticoid receptor (GR) functions to regulate a wide group of physiological processes through hormone inducible interaction with genomic loci and subsequent manipulation of the transcriptional output of target genes. Despite expression in a wide variety of tissues, the GR has diverse roles that are regulated tightly in a cell type specific manner. With the advent of whole genome approaches, the details of that diversity and the mechanisms regulating them are beginning to be elucidated. This review aims describe the recent advances detailing the role chromatin structure plays in dictating GR specificity. PMID:23545159

Burd, Craig J; Archer, Trevor K

2013-03-29

361

Role of chromatin states in transcriptional memory  

PubMed Central

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

Kundu, Sharmistha; Peterson, Craig L.

2009-01-01

362

Facilitated Diffusion of Proteins on Chromatin  

NASA Astrophysics Data System (ADS)

We present a theoretical model of facilitated diffusion of proteins in the cell nucleus. This model, which takes into account the successive binding and unbinding events of proteins to DNA, relies on a fractal description of the chromatin which has been recently evidenced experimentally. Facilitated diffusion is shown quantitatively to be favorable for a fast localization of a target locus by a transcription factor and even to enable the minimization of the search time by tuning the affinity of the transcription factor with DNA. This study shows the robustness of the facilitated diffusion mechanism, invoked so far only for linear conformations of DNA.

Bénichou, O.; Chevalier, C.; Meyer, B.; Voituriez, R.

2011-01-01

363

Chromatin remodeling and cancer, part II: ATP-dependent chromatin remodeling  

Microsoft Academic Search

Connections between perturbations that lie outside of our genome, that is, epigenetic alternations, and tumor- igenesis have become increasingly apparent. Dynamic chromatin remodeling of the fundamental nucleosomal structure (covered in this review) or the covalent marks residing in the histone proteins that make up this struc- ture (covered previously in part I) underlie many funda- mental cellular processes, including transcriptional

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

2007-01-01

364

Condensation Processes in Geothermal Systems  

Microsoft Academic Search

We model condensation processes in geothermal systems to understand how this process changes fluid chemistry. We assume two processes operate in geothermal systems: 1) condensation of a vapor phase derived by boiling an aqueous geothermal fluid into a cool near surface water and 2) condensation of a magmatic vapor by a deep circulating meteoric thermal fluid. It is assumed that

D. I. Norman; J. N. Moore

2005-01-01

365

Modeling of Rapid Direct Contact Condensation.  

National Technical Information Service (NTIS)

The focus of the study is on rapid direct-contact condensation phenomena, that is, direct contact condensation situations characterized by extremely high condensation rates and violent mixing at the liquid-vapor interface. Rapid condensation phenomena ari...

G. B. Wallis H. J. Richter J. A. Valenzuela P. H. Rothe

1985-01-01

366

TFIIIC Binding Sites Function as both Heterochromatin Barriers and Chromatin Insulators in Saccharomyces cerevisiae?  

PubMed Central

Chromosomal sites of RNA polymerase III (Pol III) transcription have been demonstrated to have “extratranscriptional” functions, as the assembled Pol III complex can act as chromatin boundaries or pause sites for replication forks, can alter nucleosome positioning or affect transcription of neighboring genes, and can play a role in sister chromatid cohesion. Several studies have demonstrated that assembled Pol III complexes block the propagation of heterochromatin-mediated gene repression. Here we show that in Saccharomyces cerevisiae tRNA genes (tDNAs) and even partially assembled Pol III complexes containing only the transcription factor TFIIIC can exhibit chromatin boundary functions both as heterochromatin barriers and as insulators to gene activation. Both the TRT2 tDNA and the ETC4 site which binds only the TFIIIC complex prevented an upstream activation sequence from activating the GAL promoters in our assay system, effectively acting as chromatin insulators. Additionally, when placed downstream from the heterochromatic HMR locus, ETC4 blocked the ectopic spread of Sir protein-mediated silencing, thus functioning as a barrier to repression. Finally, we show that TRT2 and the ETC6 site upstream of TFC6 in their natural contexts display potential insulator-like functions, and ETC6 may represent a novel case of a Pol III factor directly regulating a Pol II promoter. The results are discussed in the context of how the TFIIIC transcription factor complex may function to demarcate chromosomal domains in yeast and possibly in other eukaryotes.

Simms, Tiffany A.; Dugas, Sandra L.; Gremillion, Jason C.; Ibos, Megan E.; Dandurand, M. Nicole; Toliver, Tasha T.; Edwards, Daniel J.; Donze, David

2008-01-01

367

TFIIIC binding sites function as both heterochromatin barriers and chromatin insulators in Saccharomyces cerevisiae.  

PubMed

Chromosomal sites of RNA polymerase III (Pol III) transcription have been demonstrated to have "extratranscriptional" functions, as the assembled Pol III complex can act as chromatin boundaries or pause sites for replication forks, can alter nucleosome positioning or affect transcription of neighboring genes, and can play a role in sister chromatid cohesion. Several studies have demonstrated that assembled Pol III complexes block the propagation of heterochromatin-mediated gene repression. Here we show that in Saccharomyces cerevisiae tRNA genes (tDNAs) and even partially assembled Pol III complexes containing only the transcription factor TFIIIC can exhibit chromatin boundary functions both as heterochromatin barriers and as insulators to gene activation. Both the TRT2 tDNA and the ETC4 site which binds only the TFIIIC complex prevented an upstream activation sequence from activating the GAL promoters in our assay system, effectively acting as chromatin insulators. Additionally, when placed downstream from the heterochromatic HMR locus, ETC4 blocked the ectopic spread of Sir protein-mediated silencing, thus functioning as a barrier to repression. Finally, we show that TRT2 and the ETC6 site upstream of TFC6 in their natural contexts display potential insulator-like functions, and ETC6 may represent a novel case of a Pol III factor directly regulating a Pol II promoter. The results are discussed in the context of how the TFIIIC transcription factor complex may function to demarcate chromosomal domains in yeast and possibly in other eukaryotes. PMID:18849469

Simms, Tiffany A; Dugas, Sandra L; Gremillion, Jason C; Ibos, Megan E; Dandurand, M Nicole; Toliver, Tasha T; Edwards, Daniel J; Donze, David

2008-10-10

368

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

PubMed Central

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

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

2009-01-01

369

Chromatin structure of hormono-dependent promoters.  

PubMed

Transient transfections of mutated MMTV LTRs, driving the luciferase reporter gene, have shown the presence of at least one cis-acting element cooperating with the GREs. Studies of the chromatin structure of two glucocorticoid-regulated promoters, the mouse mammary tumor virus (MMTV) long terminal repeat (LTR), a retroviral promoter, and the rat tyrosine aminotransferase (TAT) promoter, demonstrate that both DNAs are organized into precisely positioned nucleosomes. Hormonal activation of transcription is accompanied by structural changes of one (MMTV LTR) or two (TAT promoter) nucleosomes associated with the hormone-response elements (HREs). These changes can be visualized by the appearance of DNasel hypersensitive sites. Association of the hormone-receptor complex with the nucleus is necessary to induce the DNasel hypersensitive site and to maintain transcription, but is not necessary to maintain DNasel hypersensitivity. Anti-hormones, even when able to promote a strong binding of the receptor to the nucleus, are unable to induce the chromatin structural change. Using cell lines containing approx. 200 copies of a MMTV LTR/Hv-ras chimeric construct, we have demonstrated a strong, hormono-independent nuclear matrix interaction of sequences located just upstream and downstream of the ras coding sequences. PMID:1683563

Adom, J; Carr, K D; Gouilleux, F; Marsaud, V; Richard-Foy, H

1991-01-01

370

Topology and Fermionic Condensate  

NASA Astrophysics Data System (ADS)

The purpose of this paper is to investigate an influence of a space-time topology on the formation of fermionic condensate in the model with four-fermion interaction ()2. The value for the space-time with topology of R1 × R1 × S1 is found. Moreover a relation of the value of fermionic condensate to a periodic length is studied. In this connection the possibility of a relation of the topologic deposits to structure of hadrons is discussed.Translated AbstractTopologie und FermikondensatEs wird der Einfluß einer Raum-Zeittopologie auf die Bildung des Fermikondensats in einem Modell mit Vierfermionenwechselwirkung ()2 untersucht. Für eine Raum-Zeit mit der Topologie R1 × R2 × S1 werden die Parameter gegeben. Weiterhin wird die Relation der Größe des Fermikondensats zu einer periodischen Länge untersucht. In diesem Zusammenhang wird die Verbindung des topologischen Depots zur Struktur der Hadronen diskutiert.

Kulikov, I.; Pronin, P.

371

The Bulk Chromatin Structure of a Murine Transgene Does Not Vary with Its Transcriptional or DNA Methylation Status  

Microsoft Academic Search

TheDNAmethylationstatusofHRD,amurinetransgene,canbecontrolledbythegeneticbackgroundupon which it is carried. We found the transgene to be transcribed in competent tissues only when undermethylated. Chromatin structure over the transgene was assayed by nuclear accessibility with DNase I, MspI, and PstI. While the transgene was up tofivefold more resistant toMspI when methylated than when not methylated, we observednosuchdifferencewithDNaseIorPstI.Wesuggestthatmethyl-CpG-bindingproteinsareresponsible for the difference observed with MspI, but that the

ANDREW WENG; PETER ENGLER; ANDURSULA STORB

372

The T-Lineage-affiliated CD2 Gene Lies within an Open Chromatin Environment in Acute Promyelocytic Leukemia Cells1  

Microsoft Academic Search

The nature of hemopoietic progenitors subject to leukemic transforma- tion in acute myeloid leukemia (AML) has not been clearly defined. To address this issue, we have used DNase I hypersensitivity assays to study the chromatin structure surrounding the T-lineage-affiliated CD2 gene in the acute promyelocytic subtype of AML (APL). Upstream and down- stream flanking regions of CD2 were found to

David Grimwade; Susan V. Outram; Rajinder Flora; Stuart J. Ings; Arnold R. Pizzey; Ricardo Morilla; Charles F. Craddock; David C. Linch; Ellen Solomon

2002-01-01

373

ADHM is tachyon condensation  

Microsoft Academic Search

We completely realize the ADHM construction of instantons in D-brane language of tachyon condensations. Every step of the construction is given a physical interpretation in string theory, in a boundary state formalism valid all order in alpha'. Accordingly, equivalence between Yang-Mills configurations on D4-branes and D0-branes inside the D4-branes is proven, which shows that small instanton configurations of the Yang-Mills

Koji Hashimoto; Seiji Terashima

2006-01-01

374

Developmental patterns of chromatin structure and DNA methylation responsible for epigenetic expression of a maize regulatory gene.  

PubMed Central

Epigenetic regulatory mechanisms heritably alter patterns of gene expression without changes in DNA sequence. Epigenetic states are often correlated with developmentally imposed alterations in genomic DNA methylation and local chromatin structure. Pl-Blotched is a stable epigenetic allele of the maize anthocyanin regulatory gene, purple plant1(pl). Pl-Blotched plants display a variegated pattern of pigmentation that contrasts sharply with the uniformly dark purple pigmentation of plants carrying the dominant Pl-Rhoades allele. Previously, we showed that the lower level of pigmentation in Pl-Blotched is correlated with lower pl mRNA levels and increased DNA methylation at some sites. To explore how DNA methylation, chromatin structure, and developmental stage might contribute to the expression of Pl-Blotched, we used methylation-sensitive restriction enzymes and DNaseI sensitivity assays to compare the methylation status and chromatin structure of Pl-Blotched and Pl-Rhoades at different stages in development. Both alleles exhibit developmentally sensitive changes in methylation. In Pl-Blotched, methylation of two diagnostic HpaII/MspI sites increases progressively, coincident with the juvenile-to-adult transition in growth. In seedlings, the chromatin encompassing the coding region of the gene is less sensitive to DNaseI digestion in Pl-Blotched than in Pl-Rhoades. Developmental maturation from seedling to adult is accompanied by expansion of this closed chromatin domain to include the promoter and downstream flanking sequences. We provide evidence to show that chromatin structure, rather than DNA methylation, is the primary epigenetic determinant for the phenotypic differences between Pl-Blotched and Pl-Rhoades.

Hoekenga, O A; Muszynski, M G; Cone, K C

2000-01-01

375

Chromosome condensation induced by geminivirus infection of mature plant cells.  

PubMed

Tomato golden mosaic virus (TGMV) is a geminivirus that replicates its single-stranded DNA genome through double-stranded DNA intermediates in nuclei of differentiated plant cells using host replication machinery. We analyzed the distribution of viral and plant DNA in nuclei of infected leaves using fluorescence in situ hybridization (FISH). TGMV-infected nuclei showed up to a sixfold increase in total volume and displayed a variety of viral DNA accumulation patterns. The most striking viral DNA patterns were bright, discrete intranuclear compartments, but diffuse nuclear localization was also observed. Quantitative and spatial measurements of high resolution 3-dimensional image data revealed that these compartments accounted for 1-18% of the total nuclear volume or 2-45% of the total nuclear FISH signals. In contrast, plant DNA was concentrated around the nuclear periphery. In a significant number of nuclei, the peripheral chromatin was organized as condensed prophase-like fibers. A combination of FISH analysis and indirect immunofluorescence with viral coat protein antibodies revealed that TGMV virions are associated with the viral DNA compartments. However, the coat protein antibodies failed to cross react with some large viral DNA inclusions, suggesting that encapsidation may occur after significant viral DNA accumulation. Infection by a TGMV mutant with a defective coat protein open reading frame resulted in fewer and smaller viral DNA-containing compartments. Nevertheless, nuclei infected with the mutant virus increased in size and in some cases showed chromosome condensation. Together, these results established that geminivirus infection alters nuclear architecture and can induce plant chromatin condensation characteristic of cells arrested in early mitosis. PMID:10704366

Bass, H W; Nagar, S; Hanley-Bowdoin, L; Robertson, D

2000-04-01

376

Chromosome condensation in mitosis and meiosis of rye (Secale cereale L.).  

PubMed

Structural investigation and morphometry of meiotic chromosomes by scanning electron microscopy (in comparison to light microscopy) of all stages of condensation of meiosis I + II show remarkable differences during chromosome condensation in mitosis and meiosis I of rye (Secale cereale) with respect to initiation, mode and degree of condensation. Mitotic chromosomes condense in a linear fashion, shorten in length and increase moderately in diameter. In contrast, in meiosis I, condensation of chromosomes in length and diameter is a sigmoidal process with a retardation in zygotene and pachytene and an acceleration from diplotene to diakinesis. The basic structural components of mitotic chromosomes of rye are "parallel fibers" and "chromomeres" which become highly compacted in metaphase. Although chromosome architecture in early prophase of meiosis seems similar to mitosis in principle, there is no equivalent stage during transition to metaphase I when chromosomes condense to a much higher degree and show a characteristic "smooth" surface. No indication was found for helical winding of chromosomes either in mitosis or in meiosis. Based on measurements, we propose a mechanism for chromosome dynamics in mitosis and meiosis, which involves three individual processes: (i) aggregation of chromatin subdomains into a chromosome filament, (ii) condensation in length, which involves a progressive increase in diameter and (iii) separation of chromatids. PMID:15218269

Zoller, J F; Herrmann, R G; Wanner, G

2004-01-01

377

SWI/SNF protein component BAF250a regulates cardiac progenitor cell differentiation by modulating chromatin accessibility during second heart field development.  

PubMed

ATP-dependent SWI/SNF chromatin remodeling complexes alter the structure of chromatin at specific loci and facilitate tissue-specific gene regulation during development. Several SWI/SNF subunits are required for cardiogenesis. However, the function and mechanisms of SWI/SNF in mediating cardiac progenitor cell (CPC) differentiation during cardiogenesis are not well understood. Our studies of the SWI/SNF chromatin remodeling complex identified that BAF250a, a regulatory subunit of the SWI/SNF, plays a key role in CPC differentiation. BAF250a ablation in mouse second heart field (SHF) led to trabeculation defects in the right ventricle, ventricular septal defect, persistent truncus arteriosus, reduced myocardial proliferation, and embryonic lethality around E13. Using an embryonic stem cell culture system that models the formation and differentiation of SHF CPCs in vivo, we have shown that BAF250a ablation in CPCs specifically inhibits cardiomyocyte formation. Moreover, BAF250a selectively regulates the expression of key cardiac factors Mef2c, Nkx2.5, and Bmp10 in SHF CPCs. Chromatin immunoprecipitation and DNase I digestion assays indicate that BAF250a regulates gene expression by binding selectively to its target gene promoters and recruiting Brg1, the catalytic subunit of SWI/SNF, to modulate chromatin accessibility. Our results thus identify BAF250a-mediated chromatin remodeling as an essential epigenetic mechanism mediating CPC differentiation. PMID:22621927

Lei, Ienglam; Gao, Xiaolin; Sham, Mai Har; Wang, Zhong

2012-05-23

378

Modulation of chromatin organization by RH-3, a preparation of Hippophae rhamnoides, a possible role in radioprotection.  

PubMed

The present study was aimed to understand the mode of action of alcoholic extract of whole berries of Hippophae rhamnoides (RH-3) which has already been reported to render more than 80 % protection against radiation induced mortality in mice. Direct and indirect antioxidant action (free radical scavenging and metal chelating potential) were assayed using 2-deoxy ribose degradation and 2,2'-bipiridyl assays. Effect of RH-3 on radiation and chemical oxidant mediated DNA damage was evaluated using single cell gel electrophoresis (Comet assay) and alkaline halo assay. Ability of RH-3 to bind with calf thymus DNA was assayed through change in melting temperature (Tm) while toxicity was assayed in thymocytes by trypan blue exclusion. RH-3 inhibited 2-deoxy ribose degradation in a dose dependent manner (IC 50 approximately 500 microg/ml). 2,2'-bipiridyl assay revealed the inability of RH-3 to chelate Fe2+ ions. RH-3 inhibited radiation and tertiary butyl hydroperoxide induced DNA strand breaks in a dose dependent manner and at concentrations of 100 and 120 microg/ml the length of comet tail was considerably reduced and became almost similar to that of untreated control. RH-3 at a concentration of 120 pg/ml or more induced a strong compaction of chromatin as was evident from lack of tail and appearance of intensely stained circular bodies. This made the nuclei resistant even to a radiation dose of 1,000 Gy. The compaction of chromatin was not reversed even by relaxation buffer indicating that salt concentration had no role in RH-3 induced chromatin compaction. Alkaline halo assay also corroborated the results of comet assay. Lower DNA-RH-3 concentrations (1:0.5 and 1:1) induced a shift of Tm towards left by 2 and 5 degrees C respectively; however higher concentrations (1:8 and 1:16) shifted the Tm towards right increasing it by 10 and 21 degrees C correspondingly. RH-3, evinced only a mild free radical scavenging activity at concentrations used in the present study, therefore its ability to protect DNA could mainly be attributed to direct modulation of chromatin organization. Further work to unravel these facts would be necessary. PMID:12349895

Kumar, I Prem; Namita, Samanta; Goel, H C

2002-09-01

379

Chromatin structure and the inheritance of epigenetic information  

Microsoft Academic Search

Although it is widely accepted that the regulation of the chromatin landscape is pivotal to conveying the epigenetic program, it is still unclear how a defined chromatin domain is reproduced following DNA replication and transmitted from one cell generation to the next. Here, we review the multiple mechanisms that potentially affect the inheritance of epigenetic information in somatic cells. We

Raphaël Margueron; Danny Reinberg

2010-01-01

380

Quinacrine studies of sex chromatin and nucleoli in human Brain  

Microsoft Academic Search

Quinacrine fluorescent studies of sex chromatin in human neurons indicate that the distal portion of the Y chromosome is constantly seen in association with the nucleolus in the male. The degree of association between the “Y body” and the nucleolus in other cell types is much lower. The degree of association between the X chromatin and the nucleolus in human

Robert J. Iorio; Herman E. Wyandt

1973-01-01

381

Brd4 Shields Chromatin from ATM Kinase Signaling Storms.  

PubMed

Upon activation, ataxia telangiectasia mutated (ATM) kinase rapidly phosphorylates hundreds of proteins, setting off chaotic signaling storms from areas of damaged chromatin. Recent work by Kaidi and Jackson and Floyd et al. advance our knowledge of the mechanisms that initiate or limit ATM kinase signaling storms at chromatin. PMID:24045152

Choi, Serah; Bakkenist, Christopher J

2013-09-17

382

Activators and repressors: making use of chromatin to regulate transcription  

Microsoft Academic Search

Metazoans and yeast use enzymes that modulate histone acetylation and nucleosomal integrity in order to regulate transcription. Repressor complexes deacetylate histones and stabilize nucleosomes. Activator complexes acetylate histones and disrupt nucleosomes. Variation in chromatin structure makes a major contribution to gene regulation. Here we discuss the enzymatic complexes and molecular machines that make use of chromatin to control transcription.

Alan P. Wolffe; Jiemin Wong; Dmitry Pruss

1997-01-01

383

Relationship between abnormal sperm chromatin packing and IVF results  

Microsoft Academic Search

This study was initiated to determine the relationship between the fertilizing potential of spermatozoa and abnormalities in the compact packing of their chromatin which occurs in the final stage of male germ cell differentiation. Chromatin packing involves disulphide bridge covalent cross-linking. The degree of packing was determined from the accessibility of DNA to a fluorescent dye, ethidium bromide, following detergent

M. V. Filatov; E. V. Semenova; O. A. Vorob' eva; O. A. Leont' eva; E. A. Drobchenko

1999-01-01

384

An oestrogen-receptor-alpha-bound human chromatin interactome.  

PubMed

Genomes are organized into high-level three-dimensional structures, and DNA elements separated by long genomic distances can in principle interact functionally. Many transcription factors bind to regulatory DNA elements distant from gene promoters. Although distal binding sites have been shown to regulate transcription by long-range chromatin interactions at a few loci, chromatin interactions and their impact on transcription regulation have not been investigated in a genome-wide manner. Here we describe the development of a new strategy, chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) for the de novo detection of global chromatin interactions, with which we have comprehensively mapped the chromatin interaction network bound by oestrogen receptor alpha (ER-alpha) in the human genome. We found that most high-confidence remote ER-alpha-binding sites are anchored at gene promoters through long-range chromatin interactions, suggesting that ER-alpha functions by extensive chromatin looping to bring genes together for coordinated transcriptional regulation. We propose that chromatin interactions constitute a primary mechanism for regulating transcription in mammalian genomes. PMID:19890323

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

2009-11-01

385

Role of chromatin factors in Arabidopsis root stem cell maintenance  

Microsoft Academic Search

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 features in mammalian stem cells (Chapter 1). The role of chromatin factors in plant stem cell control

N. G. Kornet

2008-01-01

386

Topoisomerase Function during Replication-Independent Chromatin Assembly in Yeast  

Microsoft Academic Search

DNA topoisomerases I and II are the two major nuclear enzymes capable of relieving torsional strain in DNA. Of these enzymes, topoisomerase I plays the dominant role in relieving torsional strain during chromatin assembly in cell extracts from oocytes, eggs, and early embryos. We tested if the topoisomerases are used differentially during chromatin assembly in Saccharomyces cerevisiae by a combined

WENDY I. GARINTHER; MICHAEL C. SCHULTZ

1997-01-01

387

Unusual chromatin structure associated with monoparalogous transcription of the Babesia bovis ves multigene family.  

PubMed

Rapid antigenic variation in Babesia bovis involves the variant erythrocyte surface antigen-1 (VESA1), a heterodimeric protein with subunits encoded by two branches of the ves multigene family. The ves1? and ves1? gene pair encoding VESA1a and 1b, respectively, are transcribed in a monoparalogous manner from a single locus of active ves transcription (LAT), just one of many quasi-palindromic ves loci. To determine whether this organization plays a role in transcriptional regulation, chromatin structure was first assessed. Limited treatment of isolated nuclei with micrococcal nuclease to assay nucleosomal patterning revealed a periodicity of 156-159 bp in both bulk chromatin and specific gene coding regions. This pattern also was maintained in the intergenic regions (IGr) of non-transcribed ves genes. In contrast, the LAT IGr adopts a unique pattern, yielding an apparent cluster of five closely-spaced hypersensitive sites flanked by regions of reduced nucleosomal occupancy. ves loci fall into three patterns of overall sensitivity to micrococcal nuclease or DNase I digestion, with only the LAT being consistently very sensitive. Non-transcribed ves genes are inconsistent in their sensitivity to the two enzymatic probes. Non-linear DNA structure in chromatin was investigated to determine whether unique structure arising as a result of the quasi-palindromic nature of the LAT may effect transcriptional control. The in vitro capacity of ves IGr sequences to adopt stable higher-order DNA structure is demonstrated here, but the presence of such structure in vivo was not supported. Based upon these results a working model is proposed for the chromatin structural remodeling responsible for the sequential expression of ves multigene family members from divergently-organized loci. PMID:23178996

Huang, Yingling; Xiao, Yu-Ping; Allred, David R

2012-11-23

388

Chromatin and epigenetic features of long-range gene regulation.  

PubMed

The precise regulation of gene transcription during metazoan development is controlled by a complex system of interactions between transcription factors, histone modifications and modifying enzymes and chromatin conformation. Developments in chromosome conformation capture technologies have revealed that interactions between regions of chromatin are pervasive and highly cell-type specific. The movement of enhancers and promoters in and out of higher-order chromatin structures within the nucleus are associated with changes in expression and histone modifications. However, the factors responsible for mediating these changes and determining enhancer:promoter specificity are still not completely known. In this review, we summarize what is known about the patterns of epigenetic and chromatin features characteristic of elements involved in long-range interactions. In addition, we review the insights into both local and global patterns of chromatin interactions that have been revealed by the latest experimental and computational methods. PMID:23766291

Harmston, Nathan; Lenhard, Boris

2013-06-13

389

Chromatin modulators as facilitating factors in cellular reprogramming.  

PubMed

In the last few years, cellular reprogramming has emerged as a means to alter cellular identity and generate diverse cell types for disease modeling, drug testing, and potential therapeutic use. Since each cell type is a result of a specific gene expression profile finely regulated by the activity of a repertoire of transcription factors (TFs), reprogramming approaches have, thus far, been relatively inefficient and based largely on the forced expression of selective cell type-specific TFs. TFs function within the confines of chromatin, and the chromatin states can in turn be modulated by TF activity. Therefore, the knowledge of how chromatin remodeling factors alter chromatin structure, control TF activity and gene expression has led to an improved reprogramming efficiency and extended the number of cellular types that can be generated by cellular reprogramming. Here we review recent insights into the role and mechanisms by which chromatin remodeling, histone modifications, and DNA methylation contribute to cellular differentiation and reprogramming. PMID:23993229

Luna-Zurita, Luis; Bruneau, Benoit G

2013-08-28

390

Comprehensive analysis of the chromatin landscape in Drosophila  

PubMed Central

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

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

2011-01-01

391

Closed chromatin loops at the ends of chromosomes  

PubMed Central

The termini of eukaryotic chromosomes contain specialized protective structures, the telomeres, composed of TTAGGG repeats and associated proteins which, together with telomerase, control telomere length. Telomere shortening is associated with senescence and inappropriate telomerase activity may lead to cancer. Little is known about the chromatin context of telomeres, because, in most cells, telomere chromatin is tightly anchored within the nucleus. We now report the successful release of telomere chromatin from chicken erythrocyte and mouse lymphocyte nuclei, both of which have a reduced karyoskeleton. Electron microscopy reveals telomere chromatin fibers in the form of closed terminal loops, which correspond to the “t-loop” structures adopted by telomere DNA. The ability to recognize isolated telomeres in their native chromatin conformation opens the way for detailed structural and compositional studies.

Nikitina, Tatiana; Woodcock, Christopher L.

2004-01-01

392

The chromatin regulatory code: Beyond a histone code  

NASA Astrophysics Data System (ADS)

In this commentary on the contribution by Arndt Benecke in this issue, I discuss why the notion of “chromatin code” introduced and elaborated in this paper is to be preferred to that of “histone code”. Speaking of a code as regards nucleosome conformation and histone tail post-translational modifications only makes sense within the chromatin fiber, where their physico-chemical features can be translated into regulatory programs at the genome level, by means of a complex, multi-level interplay with the fiber architecture and dynamics settled in the course of Evolution. In particular, this chromatin code presumably exploits allosteric transitions of the chromatin fiber. The chromatin structure dependence of its translation suggests two alternative modes of transcription initiation regulation, also proposed in the paper by A. Benecke in this issue for interpreting strikingly bimodal micro-array data.

Lesne, A.

2006-03-01

393

Chromatin and epigenetic features of long-range gene regulation  

PubMed Central

The precise regulation of gene transcription during metazoan development is controlled by a complex system of interactions between transcription factors, histone modifications and modifying enzymes and chromatin conformation. Developments in chromosome conformation capture technologies have revealed that interactions between regions of chromatin are pervasive and highly cell-type specific. The movement of enhancers and promoters in and out of higher-order chromatin structures within the nucleus are associated with changes in expression and histone modifications. However, the factors responsible for mediating these changes and determining enhancer:promoter specificity are still not completely known. In this review, we summarize what is known about the patterns of epigenetic and chromatin features characteristic of elements involved in long-range interactions. In addition, we review the insights into both local and global patterns of chromatin interactions that have been revealed by the latest experimental and computational methods.

Harmston, Nathan; Lenhard, Boris

2013-01-01

394

Revisiting Higher-order and Large-scale Chromatin Organization  

PubMed Central

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

Bian, Qian

2012-01-01

395

Regulation Of Chromatin Structure And Function By HMGN Proteins  

PubMed Central

High Mobility Group Nucleosome-binding (HMGN) proteins are architectural non-histone chromosomal proteins that bind to nucleosomes and modulate the structure and function of chromatin. The interaction of HMGN proteins with nucleosomes is dynamic and the proteins compete with the linker histone H1 chromatin binding sites. HMGNs reduce the H1 mediated compaction of the chromatin fiber and facilitate the targeting of regulatory factors to chromatin. They modulate the cellular epigenetic profile, affect gene expression, and impact the biological processes such as development and the cellular response to environmental and hormonal signals. Here we review the role of HMGN in chromatin structure, the link between HMGN proteins and histone modifications, and discuss the consequence of this link on nuclear processes and cellular phenotype.

Postnikov, Yuri; Bustin, Michael

2009-01-01

396

Higher order chromatin structure: bridging physics and biology  

PubMed Central

Recent advances in microscopy and genomic techniques have provided new insight into spatial chromatin organization inside of the nucleus. In particular, chromosome conformation capture data has highlighted the relevance of polymer physics for high-order chromatin organization. In this context, we review basic polymer states, discuss how an appropriate polymer model can be determined from experimental data, and examine the success and limitations of various polymer models of high-order interphase chromatin organization. By taking into account topological constraints acting on the chromatin fiber, recently-developed polymer models of interphase chromatin can reproduce the observed scaling of distances between genomic loci, chromosomal territories, and probabilities of contacts between loci measured by chromosome conformation capture methods. Polymer models provide a framework for the interpretation of experimental data as ensembles of conformations rather than collections of loops, and will be crucial for untangling functional implications of chromosomal organization.

Fudenberg, Geoffrey; Mirny, Leonid A.

2012-01-01

397

Centromeric heterochromatin assembly in fission yeast--balancing transcription, RNA interference and chromatin modification  

PubMed Central

Distinct regions of the eukaryotic genome are packaged into different types of chromatin, with euchromatin representing gene rich, transcriptionally active regions and heterochromatin more condensed and gene poor. The assembly and maintenance of heterochromatin is important for many aspects of genome control, including silencing of gene transcription, suppression of recombination, and to ensure proper chromosome segregation. The precise mechanisms underlying heterochromatin establishment and maintenance are still unclear, but much progress has been made towards understanding this process during the last few years, particularly from studies performed in fission yeast. In this review, we hope to provide a conceptual model of centromeric heterochromatin in fission yeast that integrates our current understanding of the competing forces of transcription, replication, and RNA decay that influence its assembly and propagation.

Alper, Benjamin J.; Lowe, Brandon R.

2013-01-01

398

Effect of DNA Groove Binder Distamycin A upon Chromatin Structure  

PubMed Central

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

Majumder, Parijat; Dasgupta, Dipak

2011-01-01

399

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

PubMed Central

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

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

2013-01-01

400

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

PubMed

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

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

2013-09-30

401

Effect of heparin on the porcine lymphocyte chromatin--II. Comparative study of sedimentation of chromatin DNA and isolated DNA.  

PubMed

1. Sedimentation of chromatin DNA and isolated deproteinized DNA was compared in neutral and alkaline sucrose density gradients after incubation of chromatin or DNA with various concentrations of heparin. 2. Irrespective of the molecular weight of DNA, an increase in the sedimentation constant of DNA was found with increasing concentration of the polyanion employed. PMID:6862086

Koceva-Chyla, A; Zale?na, G; Strzelecka, E; Leyko, W

1983-01-01

402

Chromatin organisation of transgenes in Dictyostelium.  

PubMed

The introduction of transgenes in Dictyostelium discoideum typically results in the integration of the transformation vector into the genome at one or a few insertion sites as tandem arrays of approximately 100 copies. Exceptions are extrachromosomal vectors, which do not integrate into chromosomes, and vectors containing resistance markers such as blasticidin, which integrate as single copies at one or a few sites. Here we report that low copy number vector inserts display typical euchromatic features while high copy number insertions are enriched for modifications associate with heterochromatin. Interestingly, high copy number insertions also colocalise with heterochromatin, are enriched for the centromeric histone CenH3 and display centromere-like behaviour during mitosis. We also found that the chromatin organisation on extrachromosmal transgenes is different from those integrated into the chromosomes. PMID:23923643

Windhof, I M; Dubin, M J; Nellen, W

2013-07-01

403

RNA Polymerase III Transcription - Regulated by Chromatin Structure and Regulator of Nuclear Chromatin Organization.  

PubMed

RNA polymerase III (Pol III) transcription is regulated by modifications of the chromatin. DNA methylation and post-translational modifications of histones, such as acetylation, phosphorylation and methylation have been linked to Pol III transcriptional activity. In addition to being regulated by modifications of DNA and histones, Pol III genes and its transcription factors have been implicated in the organization of nuclear chromatin in several organisms. In yeast, the ability of the Pol III transcription system to contribute to nuclear organization seems to be dependent on direct interactions of Pol III genes and/or its transcription factors TFIIIC and TFIIIB with the structural maintenance of chromatin (SMC) protein-containing complexes cohesin and condensin. In human cells, Pol III genes and transcription factors have also been shown to colocalize with cohesin and the transcription regulator and genome organizer CCCTC-binding factor (CTCF). Furthermore, chromosomal sites have been identified in yeast and humans that are bound by partial Pol III machineries (extra TFIIIC sites - ETC; chromosome organizing clamps - COC). These ETCs/COC as well as Pol III genes possess the ability to act as boundary elements that restrict spreading of heterochromatin. PMID:23150255

Pascali, Chiara; Teichmann, Martin

2012-01-01

404

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

PubMed Central

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

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

2007-01-01

405

Confinement Contains Condensates  

SciTech Connect

Dynamical chiral symmetry breaking and its connection to the generation of hadron masses has historically been viewed as a vacuum phenomenon. We argue that confinement makes such a position untenable. If quark-hadron duality is a reality in QCD, then condensates, those quantities that have commonly been viewed as constant empirical mass-scales that fill all spacetime, are instead wholly contained within hadrons; i.e., they are a property of hadrons themselves and expressed, e.g., in their Bethe-Salpeter or light-front wave functions. We explain that this paradigm is consistent with empirical evidence, and incidentally expose misconceptions in a recent Comment.

Brodsky, Stanley J.; Roberts, Craig D.; Shrock, Robert; Tandy, Peter C.

2012-03-12

406

Cosmological tachyon condensation  

SciTech Connect

We consider the prospects for dark matter/energy unification in k-essence type cosmologies. General mappings are established between the k-essence scalar field, the hydrodynamic and braneworld descriptions. We develop an extension of the general relativistic dust model that incorporates the effects of both pressure and the associated acoustic horizon. Applying this to a tachyon model, we show that this inhomogeneous 'variable Chaplygin gas' does evolve into a mixed system containing cold dark matter like gravitational condensate in significant quantities. Our methods can be applied to any dark energy model, as well as to mixtures of dark energy and traditional dark matter.

Bilic, Neven; Tupper, Gary B.; Viollier, Raoul D. [Rudjer Boskovic Institute, 10002 Zagreb (Croatia); Centre of Theoretical Physics and Astrophysics, University of Cape Town, Rondebosch 7701 (South Africa)

2009-07-15

407

Confinement contains condensates  

NASA Astrophysics Data System (ADS)

Dynamical chiral symmetry breaking and its connection to the generation of hadron masses has historically been viewed as a vacuum phenomenon. We argue that confinement makes such a position untenable. If quark-hadron duality is a reality in QCD, then condensates, those quantities that have commonly been viewed as constant empirical mass scales that fill all space-time, are instead wholly contained within hadrons; i.e., they are a property of hadrons themselves and expressed, e.g., in their Bethe-Salpeter or light-front wave functions. We explain that this paradigm is consistent with empirical evidence and incidentally expose misconceptions in a recent Comment.

Brodsky, Stanley J.; Roberts, Craig D.; Shrock, Robert; Tandy, Peter C.

2012-06-01

408

TM6, a novel nuclear matrix attachment region, enhances its flanking gene expression through influencing their chromatin structure.  

PubMed

Nuclear matrix attachment regions (MARs) regulate the higher-order organization of chromatin and affect the expression of their flanking genes. In this study, a tobacco MAR, TM6, was isolated and demonstrated to remarkably increase the expression of four different promoters that drive gusA gene and adjacent nptII gene. In turn, this expression enhanced the transformation frequency of transgenic tobacco. Deletion analysis of topoisomerase II-binding site, AT-rich element, and MAR recognition signature (MRS) showed that MRS has the highest contribution (61.7%) to the TM6 sequence-mediated transcription activation. Micrococcal nuclease (MNase) accessibility assay showed that 35S and NOS promoter regions with TM6 are more sensitive than those without TM6. The analysis also revealed that TM6 reduces promoter DNA methylation which can affect the gusA expression. In addition, two tobacco chromatin-associated proteins, NtMBP1 and NtHMGB, isolated using a yeast one-hybrid system, specifically bound to the TM6II-1 region (761 bp to 870 bp) and to the MRS element in the TM6II-2 (934 bp to 1,021 bp) region, respectively. We thus suggested that TM6 mediated its chromatin opening and chromatin accessibility of its flanking promoters with consequent enhancement of transcription. PMID:23852133

Ji, Lusha; Xu, Rui; Lu, Longtao; Zhang, Jiedao; Yang, Guodong; Huang, Jinguang; Wu, Changai; Zheng, Chengchao

2013-07-12

409

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

PubMed Central

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

Baltanas, Fernando C.; Casafont, Inigo; Lafarga, Vanesa; Weruaga, Eduardo; Alonso, Jose R.; Berciano, Maria T.; Lafarga, Miguel

2011-01-01

410

Cytoplasmic Volume Condensation is an Integral Part of Mitosis  

PubMed Central

Cell growth and osmotic volume regulation are undoubtedly linked to the progression of the cell cycle as with each division, a newly generated cell must compensate for loss of half of its volume to its sister cell. The extent to which size influences cell cycle decisions, however, is controversial in mammalian cells. Further, a mechanism by which cells can monitor and therefore regulate their size has not been fully elucidated. Despite an ongoing debate, there have been few studies which directly address the question in single cell real-time experiments. In this study we used fluorescent time-lapse imaging to quantitatively assess volume in individual spontaneously dividing cells throughout the cell cycle. Together with biophysical studies, these establish that the efflux of salt and water brings about a condensation of cytoplasmic volume as glioma cells progress through mitosis. As cells undergo this pre-mitotic condensation (PMC) they approach a preferred cell volume preceding each division. This is functionally linked to chromatin condensation, suggesting that PMC plays an integral role in mitosis.

Habela, Christa W.; Sontheimer, Harald

2007-01-01

411

Cytoplasmic volume condensation is an integral part of mitosis.  

PubMed

Cell growth and osmotic volume regulation are undoubtedly linked to the progression of the cell cycle as with each division, a newly generated cell must compensate for loss of half of its volume to its sister cell. The extent to which size influences cell cycle decisions, however, is controversial in mammalian cells. Further, a mechanism by which cells can monitor and therefore regulate their size has not been fully elucidated. Despite an ongoing debate, there have been few studies which directly address the question in single cell real-time experiments. In this study we used fluorescent time-lapse imaging to quantitatively assess volume in individual spontaneously dividing cells throughout the cell cycle. Together with biophysical studies, these establish that the efflux of salt and water brings about a condensation of cytoplasmic volume as glioma cells progress through mitosis. As cells undergo this pre-mitotic condensation (PMC) they approach a preferred cell volume preceding each division. This is functionally linked to chromatin condensation, suggesting that PMC plays an integral role in mitosis. PMID:17581282

Habela, Christa W; Sontheimer, Harald

2007-04-27

412

Flow Boiling and Condensation Experiment  

NASA Video Gallery

The Flow Boiling and Condensation Experiment is another investigation that examines the flow of a mixture of liquids and the vapors they produce when in contact with hot space system equipment. Cooling hot surfaces in these systems occur when cool liquids vaporize or boil when flowing past the hot surface. Scientists recognize the lack of understanding of the behavior of mixtures of liquids and their vapor flow in condensers and boilers in low gravity. This video shows condensation film in microgravity.

Kristine Rainey

2013-01-11

413

Pion condensation in holographic QCD  

SciTech Connect

We study pion condensation at zero temperature in a hard-wall holographic model of hadrons with isospin chemical potential. We find that the transition from the hadronic phase to the pion condensate phase is first order except in a certain limit of model parameters. Our analysis suggests that immediately across the phase boundary the condensate acts as a stiff medium approaching the Zel'dovich limit of equal energy density and pressure.

Albrecht, Dylan; Erlich, Joshua [Particle Theory Group, Department of Physics, College of William and Mary, Williamsburg, Virginia 23187-8795 (United States)

2010-11-01

414

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

PubMed

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

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

2010-05-19

415

CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA  

PubMed Central

Centromere protein (CENP) B boxes, recognition sequences of CENP-B, appear at regular intervals in human centromeric ?-satellite DNA (alphoid DNA). In this study, to determine whether information carried by the primary sequence of alphoid DNA is involved in assembly of functional human centromeres, we created four kinds of synthetic repetitive sequences: modified alphoid DNA with point mutations in all CENP-B boxes, resulting in loss of all CENP-B binding activity; unmodified alphoid DNA containing functional CENP-B boxes; and nonalphoid repetitive DNA sequences with or without functional CENP-B boxes. These four synthetic repetitive DNAs were introduced into cultured human cells (HT1080), and de novo centromere assembly was assessed using the mammalian artificial chromosome (MAC) formation assay. We found that both the CENP-B box and the alphoid DNA sequence are required for de novo MAC formation and assembly of functional centromere components such as CENP-A, CENP-C, and CENP-E. Using the chromatin immunoprecipitation assay, we found that direct assembly of CENP-A and CENP-B in cells with synthetic alphoid DNA required functional CENP-B boxes. To the best of our knowledge, this is the first reported evidence of a functional molecular link between a centromere-specific DNA sequence and centromeric chromatin assembly in humans.

Ohzeki, Jun-ichirou; Nakano, Megumi; Okada, Teruaki; Masumoto, Hiroshi

2002-01-01

416

Simultaneously defining cell phenotypes, cell cycle, and chromatin modifications at single-cell resolution.  

PubMed

Heterogeneity of cellular phenotypes in asynchronous cell populations placed in the same biochemical and biophysical environment may depend on cell cycle and chromatin modifications; however, no current method can measure these properties at single-cell resolution simultaneously and in situ. Here, we develop, test, and validate a new microscopy assay that rapidly quantifies global acetylation on histone H3 and measures a wide range of cell and nuclear properties, including cell and nuclear morphology descriptors, cell-cycle phase, and F-actin content of thousands of cells simultaneously, without cell detachment from their substrate, at single-cell resolution. These measurements show that isogenic, isotypic cells of identical DNA content and the same cell-cycle phase can still display large variations in H3 acetylation and that these variations predict specific phenotypic variations, in particular, nuclear size and actin cytoskeleton content, but not cell shape. The dependence of cell and nuclear properties on cell-cycle phase is assessed without artifact-prone cell synchronization. To further demonstrate its versatility, this assay is used to quantify the complex interplay among cell cycle, epigenetic modifications, and phenotypic variations following pharmacological treatments affecting DNA integrity, cell cycle, and inhibiting chromatin-modifying enzymes. PMID:23538711

Chambliss, Allison B; Wu, Pei-Hsun; Chen, Wei-Chiang; Sun, Sean X; Wirtz, Denis

2013-03-28

417

Chromatin folding--from biology to polymer models and back.  

PubMed

There is rapidly growing evidence that folding of the chromatin fibre inside the interphase nucleus has an important role in the regulation of gene expression. In particular, the formation of loops mediated by the interaction between specific regulatory elements, for instance enhancers and promoters, is crucial in gene control. Biochemical studies that were based on the chromosome conformation capture (3C) technology have confirmed that eukaryotic genomes are highly looped. Insight into the underlying principles comes from polymer models that explore the properties of the chromatin fibre inside the nucleus. Recent models indicate that chromatin looping can explain various properties of interphase chromatin, including chromatin compaction and compartmentalisation of chromosomes. Entropic effects have a key role in these models. In this Commentary, we give an overview of the recent conjunction of ideas regarding chromatin looping in the fields of biology and polymer physics. Starting from simple linear polymer models, we explain how specific folding properties emerge upon introducing loops and how this explains a variety of experimental observations. We also discuss different polymer models that describe chromatin folding and compare them to experimental data. Experimentally testing the predictions of such polymer models and their subsequent improvement on the basis of measurements provides a solid framework to begin to understand how our genome is folded and how folding relates to function. PMID:21378305

Tark-Dame, Mariliis; van Driel, Roel; Heermann, Dieter W

2011-03-15

418

CTCF-mediated functional chromatin interactome in pluripotent cells.  

PubMed

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

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

2011-06-19

419

An advance in condensable composites.  

PubMed

With the constant advance of technology and the public's increasing awareness of esthetically pleasing restorations, alternatives for amalgam and early composite materials have been brought to the forefront of dentistry. Condensable composites offer characteristics that distinguish them from their traditional hybrid counterparts: decreased wear, packability, and bulk curing with less polymerization shrinkage. Prodigy Condensable expands the first generation of condensables by altering its resin phase to make the material more packable. Through increased filler content and methacryoyloxyethlpolycaprolactonephosphate and rheological control additives, this second-generation condensable composite possesses strength and ease of use comparable to and even exceeding amalgam while demonstrating superior esthetics for all posterior restorations. PMID:12089755

Munoz-Viveros, C A

1999-01-01