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Sperm chromatin structure assay (SCSA®).  


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



Major changes in chromatin condensation suggest the presence of an apoptotic pathway in plant cells.  


A large decrease in fluorescence intensity of propidium iodide (PI)-stained nuclei is observed during senescence of plant cells. The phenomenon reflects a decrease in accessibility of DNA to this fluorochrome and is a consequence of chromatin condensation. This decrease is substantially greater than usually found in animal nuclei whose chromatin undergoes condensation, e.g., during differentiation or quiescence. Chromatin condensation was confirmed by analyses of (i) DNA accessibility to DNase I, (ii) histone disassociation induced by HCl, (iii) saturation of binding sites by the PI fluorochrome (iv), and (v) visual inspection by fluorescence and confocal microscopy. The extent of changes revealed by these assays was used to map progressive changes in chromatin condensation which allowed us to identify different stages in an apoptosis-like pathway in plants. The initial step of chromatin condensation which occurred prior to endonucleolytic DNA degradation was detected by fluorescence and confocal microscopy and confirmed by a variety of assays employing flow cytometry. The initial chromatin condensation appears to be a reversible step in the early stage of apoptosis. The loss of reversibility of chromatin condensation observed subsequently may be a critical point in the cascade of apoptotic events, leading to further irreversible changes during apoptosis in plants. PMID:9633512

O'Brien, I E; Murray, B G; Baguley, B C; Morris, B A; Ferguson, I B



PTEN Interacts with Histone H1 and Controls Chromatin Condensation.  


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

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



Major Changes in Chromatin Condensation Suggest the Presence of an Apoptotic Pathway in Plant Cells  

Microsoft Academic Search

A large decrease in fluorescence intensity of propidium iodide (PI)-stained nuclei is observed during senescence of plant cells. The phenomenon reflects a decrease in accessibility of DNA to this fluorochrome and is a consequence of chromatin condensation. This decrease is substantially greater than usually found in animal nuclei whose chromatin undergoes condensation, e.g., during differentiation or quiescence. Chromatin condensation was

Iona E. W. O'Brien; Brian G. Murray; Bruce C. Baguley; Bret A. M. Morris; Ian B. Ferguson



DNA methyltransferase 3b preferentially associates with condensed chromatin  

PubMed Central

In mammals, DNA methylation is catalyzed by DNA methyltransferases (DNMTs) encoded by Dnmt1, Dnmt3a and Dnmt3b. Since, the mechanisms of regulation of Dnmts are still largely unknown, the physical interaction between Dnmt3b and chromatin was investigated in vivo and in vitro. In embryonic stem cell nuclei, Dnmt3b preferentially associated with histone H1-containing heterochromatin without any significant enrichment of silent-specific histone methylation. Recombinant Dnmt3b preferentially associated with nucleosomal DNA rather than naked DNA. Incorporation of histone H1 into nucleosomal arrays promoted the association of Dnmt3b with chromatin, whereas histone acetylation reduced Dnmt3b binding in vitro. In addition, Dnmt3b associated with histone deacetylase SirT1 in the nuclease resistant chromatin. These findings suggest that Dnmt3b is preferentially recruited into hypoacetylated and condensed chromatin. We propose that Dnmt3b is a ‘reader’ of higher-order chromatin structure leading to gene silencing through DNA methylation. PMID:20923784

Kashiwagi, Katsunobu; Nimura, Keisuke; Ura, Kiyoe; Kaneda, Yasufumi



Easy detection of chromatin binding proteins by the histone association assay  

Microsoft Academic Search

The Histone Association Assay provides an easy approach for detecting proteins that bind chromatin in vivo. This technique\\u000a is based on a chromatin immunoprecipitation protocol using histone H3-specific antibodies to precipitate bulk chromatin from\\u000a crosslinked whole cell extracts. Proteins that co-precipitate with chromatin are subsequently detected by conventional SDS-PAGE\\u000a and Western blot analysis. Unlike techniques that separate chromatin and nonchromatin

Robin M. Ricke; Anja-Katrin Bielinsky



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


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. PMID:17005720

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



Histone deacetylase 2 is required for chromatin condensation and subsequent enucleation of cultured mouse fetal erythroblasts  

E-print Network

Background: During the final stages of differentiation of mammalian erythroid cells, the chromatin is condensed and enucleated. We previously reported that Rac GTPases and their downstream target, mammalian homolog of ...

Ji, Peng


Chromatin condensation and differential sensitivity of mammalian and insect cells to DNA strand breaks induced by bleomycin.  


Bleomycin (BLM) induces DNA damage in living cells. In this report we analyzed the role of chromatin compactness in the differential response of mosquito (ATC-15) and mammalian (CHO) cells to DNA strand breaks induced by BLM. We used cells unexposed and exposed to sodium butyrate (NaB), which induces chromatin decondensation. By nucleoid sedimentation assay and digestions of nuclei with DNAse I, untreated mosquito cells (no BLM; no NaB) were shown to have more chromatin condensation than untreated CHO cells. By alkaline unwinding ATC-15 cells treated with NaB showed more BLM-induced DNA strand breaks than NaB-untreated CHO cells. The time-course of BLM-induced DNA damage to nuclear DNA was similar for NaB-untreated mammalian and insect cells, but with mosquito cells showing less DNA strand breaks, both at physiological temperatures and at 4 degrees C. However, when DNA repair was inhibited by low temperatures and chromatin was decondensed by NaB treatments, differences in BLM-induced DNA damage between these cells lines were no longer observed. In both cell lines, NaB did not affect BLM action on cell growth and viability. On the other hand, the low sensitivity of ATC-15 cells to BLM was reflected in their better growth efficiency. These cells exhibited a satisfactory growth at BLM doses that produced a permanent arrest of growth in CHO cells. The data suggest that mosquito cells might have linker DNAs shorter than those of mammalian cells, which would result in the observed both greater chromatin condensation and greater resistance to DNA damage induced by BLM as compared to CHO cells. PMID:16620879

López-Larraza, Daniel M; Padrón, Juan; Ronci, Natalia E; Vidal Rioja, Lidia A



Purification and Assay of the Human INO80 and SRCAP Chromatin Remodeling Complexes  

PubMed Central

Chromatin modifying and remodeling enzymes play critical roles in many aspects of chromosome biology including transcription, replication, recombination, and repair. Our laboratory recently identified and characterized two multisubunit human chromatin remodeling enzymes designated the INO80 and SRCAP complexes. Mechanistic studies revealed that the human INO80 complex catalyzes nucleosome sliding and the SRCAP complex catalyzes ATP-dependent exchange of histone H2A/H2B dimers containing the histone variant H2A.Z into nucleosomes. Here we describe methods for purification and assay of the INO80 and SRCAP chromatin remodeling complexes. PMID:17101442

Cai, Yong; Jin, Jingji; Gottschalk, Aaron J.; Yao, Tingting; Conaway, Joan W.; Conaway, Ronald C.



The impact of testicular and accessory sex gland function on sperm chromatin integrity as assessed by the sperm chromatin structure assay (SCSA)  

Microsoft Academic Search

BACKGROUND: The sperm chromatin structure assay (SCSA) provides an objective assessment of sperm chromatin integrity, which is essential for normal sperm function. SCSA is valuable as a fertility marker in epidemiological studies and in the clinical situation. Little is known about the impact of testicular and post-testicular function on SCSA parameters. METHODS: Ejaculates from 278 military conscripts of median age

J. Richthoff; M. Spano; Y. L. Giwercman; B. Frohm; K. Jepson; J. Malm; S. Elzanaty; M. Stridsberg; A. Giwercman




PubMed Central

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. PMID:19172406

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.



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.



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. PMID:19794910

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



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



Human heterochromatin protein 1? promotes nucleosome associations that drive chromatin condensation.  


HP1(Hs?)-containing heterochromatin is located near centric regions of chromosomes and regulates DNA-mediated processes such as DNA repair and transcription. The higher-order structure of heterochromatin contributes to this regulation, yet the structure of heterochromatin is not well understood. We took a multidisciplinary approach to determine how HP1(Hs?)-nucleosome interactions contribute to the structure of heterochromatin. We show that HP1(Hs?) preferentially binds histone H3K9Me3-containing nucleosomal arrays in favor of non-methylated nucleosomal arrays and that nonspecific DNA interactions and pre-existing chromatin compaction promote binding. The chromo and chromo shadow domains of HP1(Hs?) play an essential role in HP1(Hs?)-nucleosome interactions, whereas the hinge region appears to have a less significant role. Electron microscopy of HP1(Hs?)-associated nucleosomal arrays showed that HP1(Hs?) caused nucleosome associations within an array, facilitating chromatin condensation. Differential sedimentation of HP1(Hs?)-associated nucleosomal arrays showed that HP1(Hs?) promotes interactions between arrays. These strand-to-strand interactions are supported by in vivo studies where tethering the Drosophila homologue HP1a to specific sites promotes interactions with distant chromosomal sites. Our findings demonstrate that HP1(Hs?)-nucleosome interactions cause chromatin condensation, a process that regulates many chromosome events. PMID:24415761

Azzaz, Abdelhamid M; Vitalini, Michael W; Thomas, Andrew S; Price, Jason P; Blacketer, Melissa J; Cryderman, Diane E; Zirbel, Luka N; Woodcock, Christopher L; Elcock, Adrian H; Wallrath, Lori L; Shogren-Knaak, Michael A



Spermatogenesis and chromatin condensation in male germ cells of sea cucumber Holothuria leucospilota (Clark, 1920).  


Male germ cells in the testis of Holothuria leucospilota can be divided into 12 stages based on ultrastructure and patterns of chromatin condensation. The spermatogonium (Sg) is a spherical-shaped cell with a diameter of about 6.5-7microm. Its nucleus mostly contains euchromatin and small blocks of heterochromatin scattered throughout the nucleus. The nucleolus is prominent. Primary spermatocytes are divided into six stages, i.e., leptotene (LSc), zygotene (ZSc), pachytene (PSc), diplotene (DSc), diakinesis (DiSc) and metaphase (MSc). The early cells are round while in DiSc and in MSc cells are oval in shape. From LSc to MSc, the sizes of cells range from 3.5 to 4microm. LSc contains large blocks of heterochromatin as a result of increasingly condensed 17nm fibers. In ZSc, the nucleus contains prominent synaptonemal complexes but a nucleolus is absent. In PSc, heterochromatin blocks are tightly packed together by 26nm fibers and appeared as large patches in DSc. Heterochromatin patches were enlarged to form chromosomes in DiSc and MSc and then the chromosome are moved to be aligned along equatorial region. The secondary spermatocyte (SSc) is an oval cell about 4.5-5.5microm. Their nuclei contain large clumps of heterochromatin along the nuclear envelope and in the center nuclear region. Spermatids are divided into two stages, i.e., early spermatid (ESt) and late spermatid (LSt). The nuclei decrease in size by a half and become spherical; thus the chromatin fibers condensed into 20nm and are closely packed together leaving only small spaces in LSt. The spermatozoa (Sz), with chromatin tightly packed in the spherical nucleus with a diameter of 2microm and a small acrosome situated at the anterior of the nucleus. The tail consists of a pair of centrioles lying perpendicular to each other and surrounded by a mitochondrial ring, and an axonemal complex, surrounded by a plasma membrane. PMID:18336850

Thongkukiatkul, A; Jungudomjaroen, S; Ratanapahira, C



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

PubMed Central

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



Induction of chromatin condensation by nuclear expression of a novel arginine-rich cationic protein genetically engineered from the enhanced green fluorescent protein  

Microsoft Academic Search

In the interphase nuclei of cultured cells, chromatin is compacted and organized in higher-order structures through the condensation\\u000a and decondensation processes. Chromosomes in the interphase nucleus are known to occupy distinct territories. The chromosome\\u000a territory-interchromatin compartment model premises that the interchromatin compartment is separated from compact higher-order\\u000a chromatin domains and expands in between these chromatin-organized territories. Chromatin in cultured cells

Yukihiro Higashiyama; Akinori Takahashi; Yasunori Fukumoto; Yuji Nakayama; Naoto Yamaguchi



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



Chromatin condensation and terminal differentiation process in embryonic chicken lens in vivo and in vitro.  


During embryonic chick lens differentiation, the epithelial cells become transformed into elongated fibres. Concomitantly, the fibre nuclei undergo degeneration and high molecular weight (HMW) DNA breaks down due to nuclear endodeoxyribonuclease activity. An electronmicroscopic study of lens epithelial and fibre nuclei was made at different stages of chick embryonic development, both in vivo and in vitro. The in vitro conditions are conducive to the expression of endogenous endodeoxyribonuclease activity in fibres. In both conditions we observed condensation of chromatin. The organization of some nuclear material into distinct linear arrays followed by streaming of nuclear material into the cytoplasm is recorded only in vitro. Such a condition may lead to acceleration of the process of aging in lens fibres. PMID:3770096

Sanwal, M; Muel, A S; Chaudun, E; Courtois, Y; Counis, M F



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


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



Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic  

Microsoft Academic Search

The sperm chromatin structure assay (SCSA) was used to measure over 500 human semen samples from two independent studies: Study I, 402 samples from 165 presumably fertile couples wishing to achieve pregnancy over 12 menstrual cycles; Study II, samples from 115 patients seeking fertility counselling. The SCSA measures susceptibility to DNA denaturation in situ in spermatozoa exposed to acid for

D. P. Evenson; L. K. Jost; D. Marshall; M. J. Zinaman; E. Clegg; K. Purvis; P. de Angelis; O. P. Claussen



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



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


The acetylation of core histones has been correlated with the deposition of free histones onto newly replicated DNA, transcriptional activity and the displacement of histones by protamines during spermiogenesis. The aim of the present study was to investigate the immunohistochemical distribution of hyperacetylated H4 during spermatogenesis in Polyodon spathula and to correlate these findings with the pattern of chromatin condensation in spermatids. In immature testis, the Sertoli cells showed more intense immunoreactivity for highly acetylated H4 than that of most primary spermatogonia. The testis of paddlefish at the beginning of spermatogenesis possessed early secondary spermatogonia and late secondary spermatogonia/preleptotene primary spermatocyte with intense nuclear immunoreactivity for highly acetylated H4. In seminiferous tubules in which secondary spermatogonia nuclei were intensely immunostained, Sertoli cell nuclei were unstained. Testes in late spermatogenesis contained tubules in which the immunohistochemical reaction for highly acetylated H4 was positive in the nuclei of preleptotene primary spermatocytes and secondary spermatocytes and in spermatids at the beginning of the elongation process. No immunostaining was found in round spermatids and spermatozoa. During the resting stage, immunostaining was confined to the nuclei of spermatogonia and the cells from the interstitial tissue. Transmission electron microscopy revealed that early spermatids had a round nucleus with filaments of fine chromatin that were dispersed or condensed in clumps. In later stages of maturation, the spermatids had slightly oval nuclei and homogeneous granular chromatin. The chromatin of advanced spermatids was organized into thick fibres. At the end of spermiogenesis, spermatozoan nuclei consisted of homogeneous highly compacted chromatin. PMID:17252243

Zarnescu, Otilia



An Improved Restriction Enzyme Accessibility Assay (REAA) for Analyzing Changes in Chromatin Structure in Samples of Limited Cell Number  

PubMed Central

Studies investigating mechanisms that control gene regulation frequently examine the accessibility of specific DNA sequences to nuclease cleavage. In general, sequences that are sensitive to nuclease cleavage are considered to be in an “open” chromatin conformation that is associated with regulatory factor binding, while sequences resistant to nuclease cleavage are considered to be in a “closed” conformation commonly associated with chromatin that is neither poised for transcription nor being actively transcribed. Changes in nuclease accessibility at specific genomic sequences reflect changes in the local chromatin structure that can occur as a result of signaling cues in the extracellular environment. These changes in chromatin structure usually precede or are coincident with changes in gene expression patterns and are therefore a useful marker of regulatory events controlling transcription. We describe a method to perform restriction enzyme accessibility assays (REAA) that utilizes ligation-mediated polymerase chain reaction (LM-PCR) technology and that permits assessment of samples from any source containing as few as 1,000 cells. Use of this modified REAA protocol will enhance analysis of chromatin structural changes at specific DNA sequences of interest by making it possible to analyze samples where unrestricted amounts of sample are not readily available. PMID:22130859

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



Isolation of Nuclei from Skeletal Muscle Satellite Cells and Myofibers for Use in Chromatin lmmunoprecipitation Assays  

PubMed Central

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; Dacwag Vallaster, Caroline S.; lmbalzano, Anthony N.



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



A Novel Thyroid Hormone Mediated Regulation of HSV-1 Gene Expression and Replication is Specific to Neuronal Cells and Associated with Disruption of Chromatin Condensation  

PubMed Central

Previously we showed that thyroid hormone (T3) regulated the Herpes Simplex Virus Type -1 (HSV-1) gene expression and replication through its nuclear receptor TR via histone modification and chromatin remodeling in a neuroblastoma cell line neuro-2a cells (N2a). This observation suggested that T3 regulation may be neuron-specific and have implication in HSV-1 latency and reactivation. In this study, our in vitro latency/reactivation model demonstrated that removal of T3 can de-repress the HSV-1 replication and favor reactivation. Transfection studies and infection assays indicated that HSV-1 thymidine kinase (TK), a key viral gene during reactivation, was repressed by TR/T3 in cells with neuronal origin but not in non-neuronal cells. Additional studies showed that RCC1 (Regulator of Chromosome Condensation 1) was sequestered but efficiently detected upon viral infection in N2a cells. Western blot analyses indicated that addition of T3 repressed the RCC1 expression upon infection. It is likely that diminution of RCC1 upon infection in neuronal cells under the influence of TR/T3 may lead to repression of viral replication/gene expression thus promote latency. Together these results demonstrated that TR/T3 mediated regulation is specific to neuronal cells and differential chromosome condensation may play a critical role in this process.

Chen, Feng; Palem, Jay; Balish, Matthew; Figliozzi, Robert; Ajavon, Amakoe; Hsia, S Victor



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


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

Agnieszka, Wojtczak



Q 2 ChIP, a Quick and Quantitative Chromatin Immunoprecipitation Assay, Unravels Epigenetic Dynamics of Developmentally Regulated Genes in Human Carcinoma Cells  

Microsoft Academic Search

Chromatin immunoprecipitation (ChIP) is a key technique for studying protein-DNA interactions and mapping epige- netic histone modifications on DNA. Current ChIP protocols require extensive sample handling and large cell numbers. We developed a quick and quantitative (Q 2 )ChIP assay suitable for histone and transcription factor immunoprecipi- tation from chromatin amounts equivalent to as few as 100 cells. DNA-protein cross-linking

John Arne Dahl; Philippe Collas



Identifying and Characterizing Regulatory Sequences in the Human Genome with Chromatin Accessibility Assays  

PubMed Central

After finishing a human genome reference sequence in 2002, the genomics community has turned to the task of interpreting it. A primary focus is to identify and characterize not only protein-coding genes, but all functional elements in the genome. The effort includes both individual investigators and large-scale projects like the Encyclopedia of DNA Elements (ENCODE) project. As part of the ENCODE project, several groups have identified millions of regulatory elements in hundreds of human cell-types using DNase-seq and FAIRE-seq experiments that detect regions of nucleosome-free open chromatin. ChIP-seq experiments have also been used to discover transcription factor binding sites and map histone modifications. Nearly all identified elements are found in non-coding DNA, hypothesizing a function for previously unannotated sequence. In this review, we provide an overview of the ENCODE effort to define regulatory elements, summarize the main results, and discuss implications of the millions of regulatory elements distributed throughout the genome. PMID:24705081

Sheffield, Nathan C.; Furey, Terrence S.




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...


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

SciTech Connect

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

Komura, Jun-ichiro, E-mail: [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan)] [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Ikehata, Hironobu [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan)] [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Mori, Toshio [Radioisotope Research Center, Nara Medical University, Kashihara, Nara 634-8521 (Japan)] [Radioisotope Research Center, Nara Medical University, Kashihara, Nara 634-8521 (Japan); Ono, Tetsuya [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan)] [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan)



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


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

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



Chromatin immunoprecipitation assays revealed CREB and serine 133 phospho-CREB binding to the CART gene proximal promoter  

PubMed Central

Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action. PMID:20451507

Rogge, George A; Shen, Li-Ling; Kuhar, Michael J.



Minor Groove Binder Distamycin Remodels Chromatin but Inhibits Transcription  

PubMed Central

The condensed structure of chromatin limits access of cellular machinery towards template DNA. This in turn represses essential processes like transcription, replication, repair and recombination. The repression is alleviated by a variety of energy dependent processes, collectively known as “chromatin remodeling”. In a eukaryotic cell, a fine balance between condensed and de-condensed states of chromatin helps to maintain an optimum level of gene expression. DNA binding small molecules have the potential to perturb such equilibrium. We present herein the study of an oligopeptide antibiotic distamycin, which binds to the minor groove of B-DNA. Chromatin mobility assays and circular dichroism spectroscopy have been employed to study the effect of distamycin on chromatosomes, isolated from the liver of Sprague-Dawley rats. Our results show that distamycin is capable of remodeling both chromatosomes and reconstituted nucleosomes, and the remodeling takes place in an ATP-independent manner. Binding of distamycin to the linker and nucleosomal DNA culminates in eviction of the linker histone and the formation of a population of off-centered nucleosomes. This hints at a possible corkscrew type motion of the DNA with respect to the histone octamer. Our results indicate that distamycin in spite of remodeling chromatin, inhibits transcription from both DNA and chromatin templates. Therefore, the DNA that is made accessible due to remodeling is either structurally incompetent for transcription, or bound distamycin poses a roadblock for the transcription machinery to advance. PMID:23460895

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



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

SciTech Connect

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

Evenson, Donald P. [HCLD, Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007 (United States) and SCSA Diagnostics, 807 32nd Avenue, Brookings, SD 57007 (United States)]. E-mail:; Wixon, Regina [SCSA Diagnostics, 807 32nd Avenue, Brookings, SD 57007 (United States)



Pyknotic cell death induced by Clostridium difficile?TcdB: chromatin condensation and nuclear blister are induced independently of the glucosyltransferase activity.  


TcdA and TcdB are the main pathogenicity factors of Clostridium difficile-associated diseases. Both toxins inhibit Rho GTPases, and consequently, apoptosis is induced in the affected cells. We found that TcdB at higher concentrations exhibits cytotoxic effects that are independent on Rho glucosylation. TcdB and the glucosyltransferase-deficient mutant TcdB D286/288N induced pyknotic cell death which was associated with chromatin condensation and reduced H3 phosphorylation. Affected cells showed ballooning of the nuclear envelope and loss of the integrity of the plasma membrane. Furthermore, pyknotic cells were positively stained with dihydroethidium indicating production of reactive oxygen species. In line with this, pyknosis was reduced by apocynin, an inhibitor of the NADPH oxidase. Bafilomycin A1 prevented cytotoxic effects showing that the newly observed pyknosis depends on intracellular action of TcdB rather than on a receptor-mediated effect. Blister formation and chromatin condensation was specifically induced by the glucosyltransferase domain of TcdB from strain VPI10473 since neither TcdBF from cdi1470 nor the chimera of TcdB harbouring the glucosyltransferase domain of TcdBF was able to induce these effects. In summary, TcdB induces two different and independent phenotypes: (i) cell rounding due to glucosylation of Rho GTPases and (ii) shrinkage of cells and nuclear blister induced by the high concentrations of TcdB independent of Rho glucosylation. PMID:24898616

Wohlan, Katharina; Goy, Sebastian; Olling, Alexandra; Srivaratharajan, Sangar; Tatge, Helma; Genth, Harald; Gerhard, Ralf



Altered chromatin condensation of heat-stressed spermatozoa perturbs the dynamics of DNA methylation reprogramming in the paternal genome after in vitro fertilisation in cattle.  


Shortly after penetration of the oocyte, sperm DNA is actively demethylated, which is required for totipotent zygotic development. Aberrant DNA methylation is thought to be associated with altered chromatin condensation of spermatozoa. The objectives of this study were to investigate the dynamics of DNA methylation reprogramming in the paternal pronucleus and subsequent fertilisation potential of heat-stressed bull spermatozoa having altered chromatin condensation. Hence, bovine zygotes (n=1239) were collected at three different time points (12, 18 and 24h post insemination, hpi), and stained with an antibody against 5-methylcytosine. Fluorescence intensities of paternal and maternal pronuclei were measured by ImageJ. DNA methylation patterns in paternal pronuclei derived from heat-stressed spermatozoa did not differ between time points (P>0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24hpi, respectively. Moreover, heat-stressed spermatozoa showed a highly reduced (P<0.01) fertilisation rate compared with non-heat-stressed or normal control spermatozoa (53.7% vs 70.2% or 81.5%, respectively). Our data show that the normal pattern of active DNA demethylation followed by de novo methylation in the paternal pronucleus is perturbed when oocytes are fertilised with heat-stressed spermatozoa, which may be responsible for decreased fertilisation potential. PMID:24041366

Rahman, Mohammad Bozlur; Kamal, Md Mostofa; Rijsselaere, Tom; Vandaele, Leen; Shamsuddin, Mohammed; Van Soom, Ann



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)



In Vivo Chromatin Organization of Mouse Rod Photoreceptors Correlates with Histone Modifications  

Microsoft Academic Search

BackgroundThe folding of genetic information into chromatin plays important regulatory roles in many nuclear processes and particularly in gene transcription. Post translational histone modifications are associated with specific chromatin condensation states and with distinct transcriptional activities. The peculiar chromatin organization of rod photoreceptor nuclei, with a large central domain of condensed chromatin surrounded by a thin border of extended chromatin

Caroline Kizilyaprak; Danièle Spehner; Didier Devys; Patrick Schultz; Maurizio C. Capogrossi



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. PMID:17951446

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



Ectopically tethered CP190 induces large-scale chromatin decondensation  

PubMed Central

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

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



Reproducibility of 3D chromatin configuration reconstructions.  


It is widely recognized that the three-dimensional (3D) architecture of eukaryotic chromatin plays an important role in processes such as gene regulation and cancer-driving gene fusions. Observing or inferring this 3D structure at even modest resolutions had been problematic, since genomes are highly condensed and traditional assays are coarse. However, recently devised high-throughput molecular techniques have changed this situation. Notably, the development of a suite of chromatin conformation capture (CCC) assays has enabled elicitation of contacts-spatially close chromosomal loci-which have provided insights into chromatin architecture. Most analysis of CCC data has focused on the contact level, with less effort directed toward obtaining 3D reconstructions and evaluating the accuracy and reproducibility thereof. While questions of accuracy must be addressed experimentally, questions of reproducibility can be addressed statistically-the purpose of this paper. We use a constrained optimization technique to reconstruct chromatin configurations for a number of closely related yeast datasets and assess reproducibility using four metrics that measure the distance between 3D configurations. The first of these, Procrustes fitting, measures configuration closeness after applying reflection, rotation, translation, and scaling-based alignment of the structures. The others base comparisons on the within-configuration inter-point distance matrix. Inferential results for these metrics rely on suitable permutation approaches. Results indicate that distance matrix-based approaches are preferable to Procrustes analysis, not because of the metrics per se but rather on account of the ability to customize permutation schemes to handle within-chromosome contiguity. It has recently been emphasized that the use of constrained optimization approaches to 3D architecture reconstruction are prone to being trapped in local minima. Our methods of reproducibility assessment provide a means for comparing 3D reconstruction solutions so that we can discern between local and global optima by contrasting solutions under perturbed inputs. PMID:24519450

Segal, Mark R; Xiong, Hao; Capurso, Daniel; Vazquez, Mariel; Arsuaga, Javier



Chromatin computation.  


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

Bryant, Barbara



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



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.



Biodosimetry for high dose accidental exposures by drug induced premature chromosome condensation (PCC) assay  

Microsoft Academic Search

The conventional dicentric assay does not provide an accurate dose estimate in the case of accidental exposure to ionizing radiation above 6Gy due to mitotic delay and poor mitotic index. The present study aims to establish a simple and rapid dose assessment technique based on scoring of rings and fragments in PCC spreads of stimulated lymphocytes. Human peripheral blood lymphocytes

Sreedevi Balakrishnan; kapil Shirsath; Nagesh Bhat; Kshiti Anjaria



Measuring Arabidopsis Chromatin Accessibility Using DNase I-Polymerase Chain Reaction and DNase I-Chip Assays1[C][OPEN  

PubMed Central

DNA accessibility is an important layer of regulation of DNA-dependent processes. Methods that measure DNA accessibility at local and genome-wide scales have facilitated a rapid increase in the knowledge of chromatin architecture in animal and yeast systems. In contrast, much less is known about chromatin organization in plants. We developed a robust DNase I-polymerase chain reaction (PCR) protocol for the model plant Arabidopsis (Arabidopsis thaliana). DNA accessibility is probed by digesting nuclei with a gradient of DNase I followed by locus-specific PCR. The reduction in PCR product formation along the gradient of increasing DNase I concentrations is used to determine the accessibility of the chromatin DNA. We explain a strategy to calculate the decay constant of such signal reduction as a function of increasing DNase I concentration. This allows describing DNA accessibility using a single variable: the decay constant. We also used the protocol together with AGRONOMICS1 DNA tiling microarrays to establish genome-wide DNase I sensitivity landscapes. PMID:23739687

Shu, Huan; Gruissem, Wilhelm; Hennig, Lars



2-O-methylisohemigossylic acid lactone, a sesquiterpene, isolated from roots of mokumen (Gossampinus malabarica) induces cell death and morphological change indicative of apoptotic chromatin condensation in human promyelotic leukemia HL-60 cells.  


2-O-methylisohemigossylic acid lactone, a sesquiterpene, was purified from roots of mokumen (Gossampinus malabarica) and identified by Mass, and (1)H- and (13)-NMR. This sesquiterpene displayed strong growth inhibitory effect against human promyelotic leukemia HL-60 cells. Apoptotic morphological change of the nucleus, including chromatin condensation was induced in the HL-60 cells treated with the sesquiterpene. The fragmentation of DNA by the sesquiterpene to oligonucleosomal-sized fragments, a characteristic of apoptosis, was observed to be dose- and time-dependent in the HL-60 cells. Inhibitors of caspases suppressed the DNA fragmentation induced by the sesquiterpene. These findings suggest that growth inhibition by the sesquiterpene of HL-60 cells results from the induction of apoptosis by the sesqui-terpene, and that caspase cascade is involved in the induction of apoptosis by the compound in the HL-60 cells. PMID:15547669

Hibasami, Hiroshige; Saitoh, Kazumi; Katsuzaki, Hirotaka; Imai, Kunio; Aratanechemuge, Yue; Komiya, Takashi



A novel parameter, cell-cycle progression index, for radiation dose absorbed estimation in the premature chromosome condensation assay.  


The calyculin A-induced premature chromosome condensation (PCC) assay is a simple and useful method for assessing the cell-cycle distribution in cells, since calyculin A induces chromosome condensation in various phases of the cell cycle. In this study, a novel parameter, the cell-cycle progression index (CPI), in the PCC assay was validated as a novel biomarker for biodosimetry. Peripheral blood was drawn from healthy donors after informed consent was obtained. CPI was investigated using a human peripheral blood lymphocyte (PBL) ex vivo irradiation ((60)Co-gamma rays: ?0.6 Gy min(-1), or X ray: 1.0 Gy min(-1); 0-10 Gy) model. The calyculin A-induced PCC assay was performed for chromosome preparation. PCC cells were divided into the following five categories according to cell-cycle stage: non-PCC, G1-PCC, S-PCC, G2/M-PCC and M/A-PCC cells. CPI was calculated as the ratio of G2/M-PCC cells to G1-PCC cells. The PCC-stage distribution varied markedly with irradiation doses. The G1-PCC cell fraction was significantly reduced, and the G2/M-PCC cell fraction increased, in 10-Gy-irradiated PBL after 48 h of culture. CPI levels were fitted to an exponential dose-response curve with gamma-ray irradiation [y = 0.6729 + 0.3934 exp(0.5685D), r = 1.0000, p < 0.0001] and X-ray irradiation [y = -0.3743 + 0.9744 exp(0.3321D), r = 0.9999, p < 0.0001]. There were no significant individual (p = 0.853) or gender effects (p = 0.951) on the CPI in the human peripheral blood ex vivo irradiation model. Furthermore, CPI measurements are rapid (< 15 min per case). These results suggest that the CPI is a useful screening tool for the assessment of radiation doses received ranging from 0 to 10 Gy in radiation exposure early after a radiation event, especially after a mass-casualty radiological incident. PMID:24743756

Miura, Tomisato; Nakata, Akifumi; Kasai, Kosuke; Nakano, Manabu; Abe, Yu; Tsushima, Eiki; Ossetrova, Natalia I; Yoshida, Mitsuaki A; Blakely, William F



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

PubMed Central

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

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



Chromatin compaction protects genomic DNA from radiation damage.  


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



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


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



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



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




NET23/STING Promotes Chromatin Compaction from the Nuclear Envelope  

PubMed Central

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

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



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

PubMed Central

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




Chromatin remodelling during development  

Microsoft Academic Search

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

Lena Ho; Gerald R. Crabtree



Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear ( Ursus arctos) spermatozoa  

Microsoft Academic Search

The Cantabrian brown bear (Ursus arctos) is a highly endangered species in Spain and basic studies are necessary in order to bank its germplasm. Sperm heads are mainly made up of chromatin, thus their shape depends partly on chromatin structure. Thawed semen from 10 bears was used to analyze chromatin status by sperm chromatin structure assay (SCSA) and head morphometry

M. Álvarez; V. García-Macías; F. Martínez-Pastor; F. Martínez; S. Borragán; M. Mata; J. Garde; L. Anel; P. De Paz



The role of chromatin structure in cell migration  

PubMed Central

Chromatin dynamics play a major role in regulating genetic processes. Now, accumulating data suggest that chromatin structure may also affect the mechanical properties of the nucleus and cell migration. Global chromatin organization seems to modulate the shape, the size and the stiffness of the nucleus. Directed-cell migration, which often requires nuclear reshaping to allow cellular passage through narrow openings, is dependent not only on changes in cytoskeletal elements, but also on the global chromatin condensation. Conceivably, during cell migration a physical link between the chromatin and the cytoskeleton facilitates coordinated structural changes in these two components. Thus, in addition to regulating genetic processes, we suggest that alterations in chromatin structure may facilitate cellular reorganizations necessary for efficient migration. PMID:20951589

Gerlitz, Gabi; Bustin, Michael




EPA Science Inventory

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


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


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



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. PMID:23408895

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



Nuclear translocation of p19INK4d in response to oxidative DNA damage promotes chromatin relaxation.  


DNA is continuously exposed to damaging agents that can lead to changes in the genetic information with adverse consequences. Nonetheless, eukaryotic cells have mechanisms such as the DNA damage response (DDR) to prevent genomic instability. The DNA of eukaryotic cells is packaged into nucleosomes, which fold the genome into highly condensed chromatin, but relatively little is known about the role of chromatin accessibility in DNA repair. p19INK4d, a cyclin-dependent kinase inhibitor, plays an important role in cell cycle regulation and cellular DDR. Extensive data indicate that p19INK4d is a critical factor in the maintenance of genomic integrity and cell survival. p19INK4d is upregulated by various genotoxics, improving the repair efficiency for a variety of DNA lesions. The evidence of p19INK4d translocation into the nucleus and its low sequence specificity in its interaction with DNA prompted us to hypothesize that p19INK4d plays a role at an early stage of cellular DDR. In the present study, we demonstrate that upon oxidative DNA damage, p19INK4d strongly binds to and relaxes chromatin. Furthermore, in vitro accessibility assays show that DNA is more accessible to a restriction enzyme when a chromatinized plasmid is incubated in the presence of a protein extract with high levels of p19INK4d. Nuclear protein extracts from cells overexpressing p19INK4d are better able to repair a chromatinized and damaged plasmid. These observations support the notion that p19INK4d would act as a chromatin accessibility factor that allows the access of the repair machinery to the DNA damage site. PMID:25204969

Sonzogni, Silvina V; Ogara, María F; Castillo, Daniela S; Sirkin, Pablo F; Radicella, J Pablo; Cánepa, Eduardo T



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. PMID:20959100

Gan, Hin Hark; Schlick, Tamar



The Silent Information Regulator 3 Protein, SIR3p, Binds to Chromatin Fibers and Assembles a Hypercondensed Chromatin Architecture in the Presence of Salt  

Microsoft Academic Search

The telomeres and mating-type loci of budding yeast adopt a condensed, heterochromatin-like state through recruitment of the silent information regulator (SIR) proteins SIR2p, SIR3p, and SIR4p. In this study we characterize the chromatin binding determinants of recombinant SIR3p and identify how SIR3p mediates chromatin fiber condensation in vitro. Purified full-length SIR3p was incubated with naked DNA, nucleosome core particles, or

Steven J. McBryant; Christine Krause; Christopher L. Woodcock; Jeffrey C. Hansen



Isolation of neuronal chromatin from brain tissue  

PubMed Central

Background DNA-protein interactions in mature brain are increasingly recognized as key regulators for behavioral plasticity and neuronal dysfunction in chronic neuropsychiatric disease. However, chromatin assays typically lack single cell resolution, and therefore little is known about chromatin regulation of differentiated neuronal nuclei that reside in brain parenchyma intermingled with various types of non-neuronal cells. Results Here, we describe a protocol to selectively tag neuronal nuclei from adult brain – either by (anti-NeuN) immunolabeling or transgene-derived histone H2B-GFP fusion protein – for subsequent fluorescence-activated sorting and chromatin immunoprecipitation (ChIP). To illustrate an example, we compared histone H3 lysine 4 and 9 methylation marks at select gene promoters in neuronal, non-neuronal and unsorted chromatin from mouse forebrain and human cerebral cortex, and provide evidence for neuron-specific histone methylation signatures. Conclusion With the modifications detailed in this protocol, the method can be used to collect nuclei from specific subtypes of neurons from any brain region for subsequent ChIP with native/un-fixed or crosslinked chromatin preparations. Starting with the harvest of brain tissue, ChIP-ready neuronal nuclei can be obtained within one day. PMID:18442397

Jiang, Yan; Matevossian, Anouch; Huang, Hsien-Sung; Straubhaar, Juerg; Akbarian, Schahram



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



DNA replication and chromatin  

Microsoft Academic Search

The study of DNA replication in eukaryotic chromosomes has revealed a multitude of different regulatory levels. Nuclear and chromosomal location as well as chromatin structure may affect the activity of replication origins and their modulation during development.

Susan A Gerbi; Anja-Katrin Bielinsky



Nucleosome-positioning sequence repeats impact chromatin silencing in yeast minichromosomes.  


Eukaryotic gene expression occurs in the context of structurally distinct chromosomal domains such as the relatively open, gene-rich, and transcriptionally active euchromatin and the condensed and gene-poor heterochromatin where its specific chromatin environment inhibits transcription. To study gene silencing by heterochromatin, we created a minichromosome reporter system where the gene silencer elements were used to repress the URA3 reporter gene. The minichromosome reporters were propagated in yeast Saccharomyces cerevisiae at a stable copy number. Conduction of gene silencing through nucleosome arrays was studied by placing various repeats of clone-601 DNA with high affinity for histones between the silencer and reporter in the yeast minichromosomes. High-resolution chromatin mapping with micrococcal nuclease showed that the clone-601 nucleosome positioning downstream of the HML-E gene silencing element was not significantly altered by chromatin silencing. Using URA3 reporter assays, we observed that gene silencing was conducted through arrays of up to eight nucleosomes. We showed that the shorter nucleosome repeat lengths, typical of yeast (167 and 172 bp), were more efficient in conducting silencing in vivo compared to the longer repeats (207 bp) typical of higher eukaryotes. Both the longer and the shorter repeat lengths were able to conduct silencing in minichromosomes independently of clone-601 nucleosome positioning orientations vs. the silencer element. We suggest that the shorter nucleosome linkers are more suitable for conducting gene silencing than the long repeats in yeast due to their higher propensity to support native-like chromatin higher-order folding. PMID:25189873

Chakraborty, Sangita A; Kazi, Abid A; Khan, Tamreen M; Grigoryev, Sergei A



Chromatin dynamics during spermiogenesis.  


The function of sperm is to safely transport the haploid paternal genome to the egg containing the maternal genome. The subsequent fertilization leads to transmission of a new unique diploid genome to the next generation. Before the sperm can set out on its adventurous journey, remarkable arrangements need to be made during the post-meiotic stages of spermatogenesis. Haploid spermatids undergo extensive morphological changes, including a striking reorganization and compaction of their chromatin. Thereby, the nucleosomal, histone-based structure is nearly completely substituted by a protamine-based structure. This replacement is likely facilitated by incorporation of histone variants, post-translational histone modifications, chromatin-remodeling complexes, as well as transient DNA strand breaks. The consequences of mutations have revealed that a protamine-based chromatin is essential for fertility in mice but not in Drosophila. Nevertheless, loss of protamines in Drosophila increases the sensitivity to X-rays and thus supports the hypothesis that protamines are necessary to protect the paternal genome. Pharmaceutical approaches have provided the first mechanistic insights and have shown that hyperacetylation of histones just before their displacement is vital for progress in chromatin reorganization but is clearly not the sole inducer. In this review, we highlight the current knowledge on post-meiotic chromatin reorganization and reveal for the first time intriguing parallels in this process in Drosophila and mammals. We conclude with a model that illustrates the possible mechanisms that lead from a histone-based chromatin to a mainly protamine-based structure during spermatid differentiation. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development. PMID:24091090

Rathke, Christina; Baarends, Willy M; Awe, Stephan; Renkawitz-Pohl, Renate



When repair meets chromatin  

PubMed Central

In eukaryotic cells, the inheritance of both the DNA sequence and its organization into chromatin is critical to maintain genome stability. This maintenance is challenged by DNA damage. To fully understand how the cell can tolerate genotoxic stress, it is necessary to integrate knowledge of the nature of DNA damage, its detection and its repair within the chromatin environment of a eukaryotic nucleus. The multiplicity of the DNA damage and repair processes, as well as the complex nature of chromatin, have made this issue difficult to tackle. Recent progress in each of these areas enables us to address, both at a molecular and a cellular level, the importance of inter-relationships between them. In this review we revisit the ‘access, repair, restore’ model, which was proposed to explain how the conserved process of nucleotide excision repair operates within chromatin. Recent studies have identified factors potentially involved in this process and permit refinement of the basic model. Drawing on this model, the chromatin alterations likely to be required during other processes of DNA damage repair, particularly double-strand break repair, are discussed and recently identified candidates that might perform such alterations are highlighted. PMID:11799057

Green, Catherine M.; Almouzni, Genevieve



Nucleosome spacing and chromatin higher-order folding.  


Packing of about two meters of the human genome DNA into chromatin occupying a several micron-sized cell nucleus requires a high degree of compaction in a manner that allows the information encoded on DNA to remain easily accessible. This packing is mediated by repeated coiling of DNA double helix around histones to form nucleosome arrays that are further folded into higher-order structures. Relatively straight DNA linkers separate the nucleosomes and the spacing between consecutive nucleosome varies between different cells and between different chromosomal loci. In a recent work ( 1) our group used a biochemically defined in vitro reconstituted system to explore how do various DNA linkers mediate nucleosome array packing into higher-order chromatin structures. For long nucleosome linkers (about 60 bp) we observed a more open chromatin structure and no effect of small linker length alterations (±2-4 bp) on chromatin folding. In striking contrast, for shorter linkers (20-32 bp) we found more compact packing with strong periodical dependence upon the linker DNA lengths. Our data together with high-resolution nucleosome position mapping provide evidence for the natural nucleosome repeats to support a chromatin architecture that, by default, restricts spontaneous folding of nucleosome arrays into compact chromatin fibers. We suggest that incomplete folding of the nucleosome arrays may promote global inter-array interactions that lead to chromatin condensation in metaphase chromosomes and heterochromatin. PMID:22990522

Grigoryev, Sergei A



Chromatin Damage and Male Infertility  

Microsoft Academic Search

There is accumulating evidence linking sperm chromatin damage to poor reproductive outcome. The chromatin damage is associated\\u000a to sperm anomalies that manifest themselves as breaks in the sperm nuclear DNA, aberrant ratios of protamine and histones\\u000a in the chromatin, and the presence of apoptotic marker proteins in the ejaculated spermatozoa. This chapter examines the mechanisms\\u000a involved in generating chromatin damage

Denny Sakkas; Davide Bizzaro; Gian C. Manicardi


Chromatin Structure Regulates Gene  

Microsoft Academic Search

Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors. We found that the immunoglobulin Vk pseudogene

Conversion W. Jason Cummings; Munehisa Yabuki; Ellen C. Ordinario; David W. Bednarski; Simon Quay; Nancy Maizels


Trichomonas vaginalis: chromatin and mitotic spindle during mitosis.  


The mitotic phases and the changes that the chromatin and mitotic microtubules undergo during mitosis in the sexually transmitted parasite Trichomonas vaginalis are described. Parasites arrested in the gap 2 phase of the cell cycle by nutrient starvation were induced to mitosis by addition of fresh whole medium. [(3)H] Thymidine labeling of trichomonad parasites for 24 h showed that parasites have at least four synchronic duplications after mitosis induction. Fixed or live and acridine orange (AO)-stained trichomonads analyzed at different times during mitosis by epifluorescence microscopy showed that mitosis took about 45 min and is divided into five stages: prophase, metaphase, early and late anaphase, early and late telophase, and cytokinesis. The AO-stained nucleus of live trichomonads showed green (DNA) and orange (RNA) fluorescence, and the nucleic acid nature was confirmed by DNase and RNase treatment, respectively. The chromatin appeared partially condensed during interphase. At metaphase, it appeared as six condensed chromosomes, as recently reported, which decondensed at anaphase and migrated to the nuclear poles at telophase. In addition, small bundles of microtubules (as hemispindles) were detected only in metaphase with the polyclonal antibody anti-Entamoeba histolytica alpha-tubulin. This antibody showed that the hemispindle and an atractophore-like structure seem to duplicate and polarize during metaphase. In conclusion, T. vaginalis mitosis involves five mitotic phases in which the chromatin undergoes different degrees of condensation, from chromosomes to decondensed chromatin, and two hemispindles that are observed only in the metaphase stage. PMID:11162363

Gómez-Conde, E; Mena-López, R; Hernández-Jaúregui, P; González-Camacho, M; Arroyo, R



BAF chromatin remodeling complex  

PubMed Central

The multi-subunit chromatin remodeling BAF complex controls different developmental processes. Using cortex-specific conditional knockout and overexpression mouse models, we have recently reported that BAF170, a subunit of the vertebrate BAF chromatin remodeling complex, interacts with transcription factor (TF) Pax6 to control cortical size and volume. The mechanistic basis includes suppression of the expression of Pax6 target genes, which are required for genesis of cortical intermediate progenitors (IPs) and specification of late neuronal subtype identity. In addition, we showed that a dynamic competition between BAF170 and BAF155 subunits within the BAF complex during progression of neurogenesis is a primary event in modulating the size of the mammalian cortex. Here, we present additional insights into the interaction between the BAF complex and TF Pax6 in the genesis of IPs of the developing cortex. Furthermore, we show that such competition between BAF170 and BAF155 is involved as well in the determination of the size of the embryonic body. Our results add new insights into a cell-intrinsic mechanism, mediated by the chromatin remodeling BAF complex that controls vertebrate body shape and size. PMID:23974113

Tuoc, Tran Cong; Narayanan, Ramanathan; Stoykova, Anastassia



Low 17beta-estradiol levels in CNR1 knock-out mice affect spermatid chromatin remodeling by interfering with chromatin reorganization.  


The type 1-cannabinoid receptor, CNR1, regulates differentiation of spermatids. Indeed, we have recently reported that the genetic inactivation of Cnr1 in mice influenced chromatin remodeling of spermatids, by reducing histone displacement and then sperm chromatin quality indices (chromatin condensation and DNA integrity). Herein, we have studied, at both central and testicular levels, the molecular signals potentially involved in histone displacement. In particular, investigation of the neuroendocrine axis involved in estrogen production demonstrated down-regulation of the axis supporting FSH/estrogen secretion in Cnr1-knockout male mice. Conversely, Cnr1-knockout male mice treated with 17beta-estradiol showed a weak increase of pituitary Fsh-beta subunit mRNA levels and a rescue of sperm chromatin quality indices demonstrating that estrogens, possibly in combination with FSH secretion, play an important role in regulating chromatin remodeling of spermatids. PMID:23677985

Cacciola, Giovanna; Chioccarelli, Teresa; Altucci, Lucia; Ledent, Catherine; Mason, J Ian; Fasano, Silvia; Pierantoni, Riccardo; Cobellis, Gilda



Dynamic Simulation of Active/Inactive Chromatin Domains Jens Odenheimer (  

E-print Network

Dynamic Simulation of Active/Inactive Chromatin Domains Jens Odenheimer (j (HMG/SAR, HP1, cohesin, condensin, DNA-DNA interaction...) can be modelled as an effective attractive is relatively insensitive to the segment length. Keywords: Chromatin Structure; Simulation; Condensing

Heermann, Dieter W.


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

PubMed Central

Proper telomeric chromatin configuration is thought to be essential for telomere homeostasis and stability. Previous studies in mouse suggested that loss of heterochromatin marks at telomeres might favor onset of Alternative Lengthening of Telomeres (ALT) pathway, by promoting homologous recombination. However, analysis of chromatin status at human ALT telomeres has never been reported. Here, using isogenic human cell lines and cellular hybrids, which rely either on telomerase or ALT to maintain telomeres, we show that chromatin compaction is reduced at ALT telomeres and this is associated with a global decrease in telomeric H3K9me3. This, subsequently, leads to upregulation of telomere transcription. Accordingly, restoration of a more condensed telomeric chromatin through telomerase-dependent elongation of short ALT telomeres reduces telomere transcription. We further show that loss of ATRX chromatin remodeler function, a frequent characteristic of ALT cells, is not sufficient to decrease chromatin condensation at telomeres nor to increase the expression of telomeric RNA species. These results offer new insight on telomeric chromatin properties in ALT cells and support the hypothesis that telomeric chromatin decondensation is important for ALT pathway. PMID:24500201

Episkopou, Harikleia; Draskovic, Irena; Van Beneden, Amandine; Tilman, Gaelle; Mattiussi, Marina; Gobin, Matthieu; Arnoult, Nausica; Londono-Vallejo, Arturo; Decottignies, Anabelle



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

PubMed Central

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

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



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



Chromatin in situ proximity (ChrISP): single-cell analysis of chromatin proximities at a high resolution.  


Current techniques for analyzing chromatin structures are hampered by either poor resolution at the individual cell level or the need for a large number of cells to obtain higher resolution. This is a major problem as it hampers our understanding of chromatin conformation in single cells and how these respond to environmental cues. Here we describe a new method, chromatin in situ proximity (ChrISP), which reproducibly scores for proximities between two different chromatin fibers in 3-D with a resolution of ~170Å in single cells. The technique is based on the in situ proximity ligation assay (ISPLA), but ChrISP omits the rolling circle amplification step (RCA). Instead, the proximities between chromatin fibers are visualized by a fluorescent connector oligonucleotide DNA, here termed splinter, forming a circular DNA with another circle-forming oligonucleotide, here termed backbone, upon ligation. In contrast to the regular ISPLA technique, our modification enables detection of chromatin fiber proximities independent of steric hindrances from nuclear structures. We use this method to identify higher order structures of individual chromosomes in relation to structural hallmarks of interphase nuclei and beyond the resolution of the light microscope. PMID:24641475

Chen, Xingqi; Shi, Chengxi; Yammine, Samer; Göndör, Anita; Rönnlund, Daniel; Fernandez-Woodbridge, Alejandro; Sumida, Noriyuki; Widengren, Jerker; Ohlsson, Rolf



Supercoiling in DNA and chromatin?  

PubMed Central

Supercoiling is a fundamental property of DNA and chromatin. It is modulated by polymerase and topoisomerase activities and, through regulated constraint, by DNA/chromatin binding proteins. As a non-covalent and elusive topological modification, supercoiling has proved intractable to research despite being a crucial regulator of nuclear structure and function. Recent studies have improved our understanding of the formation, regulation and organisation of supercoiling domains in vivo, and reinforce the prospect that the propagation of supercoiling can influence local and global chromatin structure. However, to further our understanding the development of new experimental tools and models are required to better dissect the mechanics of this key topological regulator. PMID:24584092

Gilbert, Nick; Allan, James



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. PMID:18691602

Cortez, Connie C.; Jones, Peter A.



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. PMID:22345607

Rando, Oliver J.; Winston, Fred



The Fanconi anemia core complex associates with chromatin during S phase.  


Fanconi anemia (FA) is an autosomal recessive disease marked by bone marrow failure, birth defects, and cancer. The FA proteins FANCA, FANCC, FANCE, FANCF, FANCG, and FANCL participate in a core complex. We previously have shown that several members of this complex bind to chromatin until mitosis and that this binding increases after DNA damage. The purpose of the present study was to determine the dynamics of complex movement between cytoplasm and nuclear compartments. Fluorescent-tagged versions of FANCA, FANCC, and FANCG colocalize in cytoplasm and nucleus, chiefly in chromatin. At the G1-S border, the FA core complex exists as foci on chromatin, progressively diffusing and migrating to the nuclear periphery and becoming completely excluded from condensed chromosomes by mitosis. Chromatin fiber analysis shows FA proteins diffusely staining along chromatin fibers during G1-S and S phase. Treatment with the DNA cross-linker mitomycin C results in a diffusion of foci and increased binding of complex proteins to chromatin, as well as diffuse and increased complex binding to chromatin fibers. These data are consistent with the idea that the FA proteins function at the level of chromatin during S phase to regulate and maintain genomic stability. PMID:15256425

Mi, Jun; Kupfer, Gary M



DNA Looping Facilitates Targeting of a Chromatin Remodeling Enzyme  

PubMed Central

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

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



Impaired sperm chromatin integrity in obese mice.  


An increased global prevalence of obesity coincides with an apparent decline in male sperm quality and a possible association between these pathologies has been suggested. In this study, we examined the effects of obesity on sperm chromatin integrity using two mouse models of obesity. In one group of mice, obesity was induced by a high-fat diet (HFD) (diet-induced obesity; DIO model), whereas in the other group, leptin deficiency was used to study the effects of obesity independently of the influence of dietary factors. Sperm chromatin integrity is recognized as an important measure of male infertility, and was analysed by the sperm chromatin structure assay. We found increased sperm DNA fragmentation in both groups of obese mice compared to lean mice, whereas the percentage of immature spermatozoa was not increased by obesity. The DIO model reflects the human condition more closely than the leptin-deficient model and was therefore selected for examination of the transcriptional response of a selection of marker genes in the testis by quantitative real-time PCR. The analysis of transcript levels of the selected testicular marker genes showed moderate, but significant, up-regulation of the Cyp2e1, Cyp19a1, Tnf and Pparg genes in DIO mice compared to lean mice. In conclusion, a clear positive correlation between body mass index and sperm DNA fragmentation was found in two mouse models of obesity. However, the variability in sperm DNA fragmentation within the two groups of obese animals was high. The observed changes in the transcript level of the marker genes suggest that there may be a local response in testicular cells to the HFD regimen with a potential impact on intratesticular signalling and spermatogenesis. PMID:24459046

Duale, N; Steffensen, I-L; Andersen, J; Brevik, A; Brunborg, G; Lindeman, B



Sudemycin E influences alternative splicing and changes chromatin modifications  

PubMed Central

Sudemycin E is an analog of the pre-messenger RNA splicing modulator FR901464 and its derivative spliceostatin A. Sudemycin E causes the death of cancer cells through an unknown mechanism. We found that similar to spliceostatin A, sudemycin E binds to the U2 small nuclear ribonucleoprotein (snRNP) component SF3B1. Native chromatin immunoprecipitations showed that U2 snRNPs physically interact with nucleosomes. Sudemycin E induces a dissociation of the U2 snRNPs and decreases their interaction with nucleosomes. To determine the effect on gene expression, we performed genome-wide array analysis. Sudemycin E first causes a rapid change in alternative pre-messenger RNA splicing, which is later followed by changes in overall gene expression and arrest in the G2 phase of the cell cycle. The changes in alternative exon usage correlate with a loss of the H3K36me3 modification in chromatin encoding these exons. We propose that sudemycin E interferes with the ability of U2 snRNP to maintain an H3K36me3 modification in actively transcribed genes. Thus, in addition to the reversible changes in alternative splicing, sudemycin E causes changes in chromatin modifications that result in chromatin condensation, which is a likely contributing factor to cancer cell death. PMID:24623796

Convertini, Paolo; Shen, Manli; Potter, Philip M.; Palacios, Gustavo; Lagisetti, Chandraiah; de la Grange, Pierre; Horbinski, Craig; Fondufe-Mittendorf, Yvonne N.; Stamm, Stefan



A Physical Model for the Condensation and Decondensation of Eukaryotic Chromosomes  

E-print Network

During the eukaryotic cell cycle, chromatin undergoes several conformational changes, which are believed to play key roles in gene expression regulation during interphase, and in genome replication and division during mitosis. In this paper, we propose a scenario for chromatin structural reorganization during mitosis, which bridges all the different scales involved in chromatin architecture, from nucleosomes to chromatin loops. We build a model for chromatin, based on available data, taking into account both physical and topological constraints DNA has to deal with. Our results suggest that the mitotic chromosome condensation/decondensation process is induced by a structural change at the level of the nucleosome itself.

Julien Mozziconacci; Christophe Lavelle; Maria Barbi; Annick Lesne; Jean-Marc Victor



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


The sugarcane borer Diatraea saccharalis is widely known as the main pest of sugarcane crop, causing increased damage to the entire fields. Measures to control this pest involve the use of chemicals and biological control with Cotesia flavipes wasps. In this study, we evaluated the insecticides fipronil (Frontline; 0.0025%), malathion (Malatol Bio Carb; 0.4%), cipermetrina (Galgotrin; 10%), and neem oil (Natuneem; 100%) and the herbicide nicosulfuron (Sanson 40 SC; 100%) in the posterior region silk glands of 3rd- and 5th-instar D. saccharalis by studying the variation in the critical electrolyte concentration (CEC). Observations of 3rd-instar larvae indicated that malathion, cipermetrina, and neem oil induced increased chromatin condensation that may consequently disable genes. Tests with fipronil showed no alteration in chromatin condensation. With the use of nicosulfuron, there was chromatin and probable gene decompaction. In the 5th-instar larvae, the larval CEC values indicated that malathion and neem oil induced increased chromatin condensation. The CEC values for 5th-instar larvae using cipermetrina, fipronil, and nicosulfuron indicated chromatin unpacking. These observations led us to conclude that the quantity of the pesticide does not affect the mortality of these pests, can change the conformation of complexes of DNA, RNA, and protein from the posterior region of silk gland cells of D. saccharalis, activating or repressing the expression of genes related to the defense mechanism of the insect and contributing to the selection and survival of resistant individuals. PMID:25299111

Santos, S A; Fermino, F; Moreira, B M T; Araujo, K F; Falco, J R P; Ruvolo-Takasusuki, M C C



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



The constantly changing face of chromatin.  


Many recent findings have altered our vision of chromatin and its role in the regulation of cellular functions. Our perspective concerning chromatin has changed to a much more complex, but also more dynamic, view of chromatin as an entity that is intimately involved in the regulation of a variety of cellular functions. In this review, we describe the various types of proteins that alter the structure and, therefore, the function of chromatin and discuss the possible role of chromatin in cell aging. The elucidation of the mechanisms that link chromatin to aging will be one of the most exciting and striking advancements in the coming years PMID:12844523

Vaquero, Alejandro; Loyola, Alejandra; Reinberg, Danny



Highly compacted chromatin formed in vitro reflects the dynamics of transcription activation in vivo.  


High-order chromatin was reconstituted in vitro. This species reflects the criteria associated with transcriptional regulation in vivo. Histone H1 was determinant to formation of condensed structures, with deacetylated histones giving rise to highly compacted chromatin that approximated 30 nm fibers as evidenced by electron microscopy. Using the PEPCK promoter, we validated the integrity of these templates that were refractory to transcription by attaining transcription through the progressive action of the pertinent factors. The retinoic acid receptor binds to highly compacted chromatin, but the NF1 transcription factor binds only after histone acetylation by p300 and SWI/SNF-mediated nucleosome mobilization, reflecting the in vivo case. Mapping studies revealed the same pattern of nucleosomal repositioning on the PEPCK promoter in vitro and in vivo, correlating with NF1 binding and transcription. The reconstitution of such highly compacted "30 nm" chromatin that mimics in vivo characteristics should advance studies of its conversion to a transcriptionally active form. PMID:20385088

Li, Guohong; Margueron, Raphael; Hu, Guobin; Stokes, David; Wang, Yuh-Hwa; Reinberg, Danny



Gene activation and deactivation related changes in the three-dimensional structure of chromatin.  


Chromatin in the interphase nucleus is dynamic, decondensing where genes are activated and condensing where they are silenced. Local chromatin remodelling to a more open structure during gene activation is followed by changes in nucleosome distribution through the action of the transcriptional machinery. This leads to chromatin expansion and looping out of whole genomic regions. Such chromatin loops can extend beyond the chromosome territory. As several studies point to the location of transcription sites inside chromosome territories as well as at their periphery, extraterritorial loops cannot simply be a mechanism for making transcribed genes accessible to the transcriptional machinery and must occur for other reasons. The level of decondensation within an activated region varies greatly and probably depends on the density of activated genes and the number of engaged RNA polymerases. Genes that are silenced during development form a more closed chromatin structure. Specific histone modifications are correlated with gene activation and silencing, and silenced genes may become associated with heterochromatin protein 1 homologues or with polycomb group complexes. Several levels of chromatin packaging are found in the nucleus relating to the different functions of and performed by active genes; euchromatic and heterochromatic regions and the models explaining higher-order chromatin structure are still disputed. PMID:16075283

Wegel, Eva; Shaw, Peter



Global quantitative modeling of chromatin factor interactions.  


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

Zhou, Jian; Troyanskaya, Olga G



Magnetic Tweezers Instrumentation: We have used magnetic tweezers to study chromatin assembly and disassembly and RNA  

E-print Network

Magnetic Tweezers Instrumentation: We have used magnetic tweezers to study chromatin assembly and disassembly and RNA transcription. Magnetic tweezers surface magnetic bead F DNA external magnets F =kBT l/> l F x surface Instrumental set-up video camera beam condenser hollow bearing with magnet 90x oil

Leuba, Sanford


Exposure of male rats to cyclophosphamide alters the chromatin structure and basic proteome in spermatozoa  

Microsoft Academic Search

BACKGROUND: The formation of mature sperm involves the expression of numerous proteins during spermiogen- esis and the replacement of histones with protamines to package the genome. Exposure to cyclophosphamide (CPA), an anticancer alkylating agent, during spermiogenesis may disrupt chromatin condensation with adverse conse- quences to the offspring. METHODS: Adult male rats were given CPA in one of two schedules: (i)

A. M. Codrington; B. F. Hales; B. Robaire



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


The effect of chromatin structure on the extent of radiation damage induced by low doses of 100 KeV X rays was investigated using a fluorescent assay for DNA unwinding. Chromatin was isolated from V-79 Chinese hamster lung fibroblast nuclei by partial digestion with micrococcal nuclease. Gel electrophoresis of the isolated DNA showed the molecular weight of the chromatin preparation to be 10.6 X 10(6) with a size range of 6.6-21.7 X 10(6) Da while a size of 10.2 +/- 0.9 X 10(6) Da was found by sedimenting the DNA in alkaline sucrose gradients. The repeat length of V-79 chromatin was found to be 194 +/- 3 bp. The typical nucleosomal repeat structure of the isolated chromatin and that of intact nuclei was identical. Irradiation with 50 and 100 Gy of 100 KeV X rays and analysis by alkaline sucrose density centrifugation indicated that V-79 chromatin sustained 0.56 +/- 0.19 and 0.69 +/- 0.09 single-strand breaks per 10 Gy per 10(8) Da of DNA, respectively. Irradiation with doses of 0.5-3.0 Gy of 100 KeV X rays and analysis by the fluorometric assay showed that the radiation sensitivity of V-79 chromatin decreases sharply on compaction with MgCl2. Histone H1 depletion, which inhibits compaction and causes chromatin to expand by increasing the linker from 26 to 48 bp, results in a considerable increase in the radiation sensitivity. It is concluded that radiation damage sustained by DNA is greatly influenced by chromatin structure. PMID:3562793

Heussen, C; Nackerdien, Z; Smit, B J; Böhm, L



Drosophila Paf1 modulates chromatin structure at actively transcribed genes.  


The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. We have identified and characterized the Drosophila homologs of three subunits of the yeast Paf1 complex and found striking differences between the yeast and Drosophila Paf1 complexes. We demonstrate that although Drosophila Paf1, Rtf1, and Cdc73 colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure. PMID:16354696

Adelman, Karen; Wei, Wenxiang; Ardehali, M Behfar; Werner, Janis; Zhu, Bing; Reinberg, Danny; Lis, John T



Regulation of DNA transposition by CpG methylation and chromatin structure in human cells  

PubMed Central

Background The activity of transposable elements can be regulated by different means. DNA CpG methylation is known to decrease or inhibit transpositional activity of diverse transposons. However, very surprisingly, it was previously shown that CpG methylation of the Sleeping Beauty (SB) transposon significantly enhanced transposition in mouse embryonic stem cells. Results In order to investigate the unexpected response of SB transposition to CpG methylation, related transposons from the Tc1/mariner superfamily, that is, Tc1, Himar1, Hsmar1, Frog Prince (FP) and Minos were tested to see how transposition was affected by CpG methylation. A significant increase of >20-fold in transposition of SB, FP and Minos was seen, whereas Tc1, Himar1 and Hsmar1 showed no difference in transposition upon CpG-methylation. The terminal inverted repeats (TIRs) of the SB, FP and Minos elements share a common structure, in which each TIR contains two functionally important binding sites for the transposase (termed the IR/DR structure). The group of IR/DR elements showed increased excision after CpG methylation compared to untreated transposon donor plasmids. We found that de novo CpG methylation is not required for transposition. A mutated FP donor plasmid with depleted CpG sites in both TIRs was as efficient in transposition as the wild-type transposon, indicating that CpG sites inside the TIRs are not responsible for altered binding of factors potentially modulating transposition. By using an in vivo one-hybrid DNA-binding assay in cultured human cells we found that CpG methylation had no appreciable effect on the affinity of SB transposase to its binding sites. However, chromatin immunoprecipitation indicated that CpG-methylated transposon donor plasmids are associated with a condensed chromatin structure characterized by trimethylated histone H3K9. Finally, DNA compaction by protamine was found to enhance SB transposition. Conclusions We have shown that DNA CpG methylation upregulates transposition of IR/DR elements in the Tc1/mariner superfamily. CpG methylation provokes the formation of a tight chromatin structure at the transposon DNA, likely aiding the formation of a catalytically active complex by facilitating synapsis of sites bound by the transposase. PMID:23676100



Chromatin Structure Regulates Gene Conversion  

Microsoft Academic Search

Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors. We found that the immunoglobulin V? pseudogene

W. Jason Cummings; Munehisa Yabuki; Ellen C Ordinario; David W Bednarski; Simon Quay; Nancy Maizels



Spectroscopic study of laser irradiated chromatin  

NASA Astrophysics Data System (ADS)

The effects of three UV excimer laser radiations, with wavelengths of 193, 248 and 282 nm respectively, on the structure of chromatin (the complex of deoxyribonucleic acid with proteins that exists in eukaryotic cells nuclei) were investigated. The chromatin was extracted from livers of Winstar rats. The spectroscopic methods used are: fluorescence (Förster) resonance energy transfer (FRET), time resolved fluorescence and steady-state fluorescence. A chromatin deoxyribonucleic acid radiolysis, a chromatin proteins damage and a change of the global chromatin structure on lasers action were indicated by this study. It exists some small differences between the actions of these three laser radiations.

Radu, Liliana; Mihailescu, I.; Gazdaru, Doina; Preoteasa, V.



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


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

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



Chromatin Position Effects Assayed by Thousands of Reporters  

E-print Network

to this work 6Present address: Computer Science and Artificial Intelligence Laboratory, Massachusetts InstituteDivision of Molecular Genetics 2Division of Gene Regulation 3Division of Molecular Carcinogenesis range of aspects of gene regulation. INTRODUCTION Control of gene expression in eukaryotes is a complex

Zhang, Jianzhi


Immunochemical detection of chromosomal protein HMG-17 in chromatin subunits.  


Chromosomal protein HMG-17, purified from calf thymus, has been used to elicit specific antibodies in rabbits. Specific serological reaction between the antigen and the antisera is demonstrated by solid-phase radioimmunoassay and by competitive inhibition assays. The antisera did not cross-react with histones or other chromosomal HMG proteins. The antisera bound specifically to chromatin subunits isolated from HeLa cells, demonstrating that it may be used to study the in situ organization of this chromosomal protein. Chromatin purified from HeLa nuclei was digested with micrococcal nuclease, and the resulting mono- and oligonucleosomes were fractionated on a sucrose gradient. Analyses of the content of chromosomal proteins HMG-1, HMG-17, and H4 in different size nucleosomal particles, by the solid-phase radioimmunoassay, reveal that the distribution of HMG-17 was the same as that of H4, but different from that of HMG-1. PMID:6452161

Tahourdin, C S; Neihart, N K; Isenberg, I; Bustin, M



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


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

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



Cohesinopathy mutations disrupt the subnuclear organization of chromatin  

PubMed Central

In Saccharomyces cerevisiae, chromatin is spatially organized within the nucleus with centromeres clustering near the spindle pole body, telomeres clustering into foci at the nuclear periphery, ribosomal DNA repeats localizing within a single nucleolus, and transfer RNA (tRNA) genes present in an adjacent cluster. Furthermore, certain genes relocalize from the nuclear interior to the periphery upon transcriptional activation. The molecular mechanisms responsible for the organization of the genome are not well understood. We find that evolutionarily conserved proteins in the cohesin network play an important role in the subnuclear organization of chromatin. Mutations that cause human cohesinopathies had little effect on chromosome cohesion, centromere clustering, or viability when expressed in yeast. However, two mutations in particular lead to defects in (a) GAL2 transcription and recruitment to the nuclear periphery, (b) condensation of mitotic chromosomes, (c) nucleolar morphology, and (d) tRNA gene–mediated silencing and clustering of tRNA genes. We propose that the cohesin network affects gene regulation by facilitating the subnuclear organization of chromatin. PMID:19948494

Gard, Scarlett; Light, William; Xiong, Bo; Bose, Tania; McNairn, Adrian J.; Harris, Bethany; Fleharty, Brian; Seidel, Chris; Brickner, Jason H.



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. PMID:23241214



Brain function and chromatin plasticity  

Microsoft Academic Search

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

Catherine Dulac



Characterization of a lipophilic plasmid DNA condensate formed with a cationic peptide fatty acid conjugate.  


In the presence of cationic ligands, DNA molecules can become aggregated into larger particles in a process known as condensation. DNA condensates are of interest as models for the dense packing found in naturally occurring structures such as phage heads and chromatin. They have found extensive application in DNA transfection and also provide convenient models with which to study DNA damage by the direct effect of ionizing radiation. Further, conjugates of cationic peptides with fatty acids may represent a class of attractive ligands for these areas because of their simple synthesis. When plasmid pUC18 is used as the DNA target and N-caproyl-penta-arginine amide (Cap-R(5)-NH(2)) is used as the ligand, the physical properties of the resulting mixtures were characterized using static and dynamic light scattering, sedimentation, dye exclusion, circular dichroism, nanoparticle tracking, and atomic force microscopy. Their chemical properties were assayed using solvent extraction and protection against hydroxyl radical attack and nuclease digestion. Titration of the plasmid with the Cap-R(5)-NH(2) ligand produced sharply defined changes in both chemical and physical properties, which was associated with the formation of condensed DNA particles in the 100-2000 nm size range. The caproyl group at the ligand's N-terminus produced a large increase in the partitioning of the resulting condensate from water into chloroform and in its binding to the neutral detergent Pluronic F-127. Both the physical and chemical data were all consistent with condensation of the plasmid by the ligand where the presence in the ligand of the caproyl group conferred an extensive lipophilic character upon the condensate. PMID:21410151

Do, Trinh T; Tang, Vicky J; Aguilera, Joe A; Perry, Christopher C; Milligan, Jamie R



Immunogenetics. Chromatin state dynamics during blood formation.  


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

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



Proteomics of a fuzzy organelle: interphase chromatin  

PubMed Central

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

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



Proteomics of a fuzzy organelle: interphase chromatin.  


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

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



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. PMID:14615812

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



Human tRNA genes function as chromatin insulators  

PubMed Central

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

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



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


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



Histone H3 phosphorylation - A versatile chromatin modification for different occasions  

PubMed Central

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



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. PMID:17651496

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



Expression-dependent folding of interphase chromatin.  


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. PMID:22649534

Jerabek, Hansjoerg; Heermann, Dieter W



Comparative analysis of metazoan chromatin organization.  


Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal 'arms', and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function. PMID:25164756

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



Genomic and proteomic dissection and characterization of the human sperm chromatin.  


The mammalian spermatozoon has a unique chromatin structure where the majority of DNA is packaged by protamines, while a small fraction (?8%) remains associated with nucleosomes. However, the chromatin affinity and repertoire of the additional proteins constituting the different sperm chromatin fractions have not yet been explored. To address this we have carried out a genomic and proteomic characterization of human sperm samples subjected to chromatin fractionation using either 0.65 M NaCl extraction followed by EcoRI/BamHI DNA restriction enzyme digestion, or micrococcal nuclease digestion. DNA fractions corresponding to the nucleosome-packaged DNA were sequenced, confirming an appropriate dissection of the sperm chromatin. In addition we detected and sequenced a subnucleosomal particle. Although both fractions were highly enriched at gene promoters, some sequences were found to be exclusively associated with one of those. The results of the proteomic analyses demonstrate that there are two distinct sets of sperm proteins which differ in chromatin affinity. Histone variants, transcription factors, chromatin-associated and modifying proteins involved in regulatory roles were identified as weakly attached to the sperm DNA, whereas proteins with structural roles were identified in the condensed fraction. Many factors, such as the histone lysine demethylase PHF8 identified for the first time in the human sperm cell in this study, were identified exclusively in soluble fraction. Our results provide additional support to the possibility that all of these factors may constitute additional layers of sperm epigenetic information or have structural or regulatory roles transmitted by the sperm cell to the oocyte at fertilization. PMID:25193639

Castillo, Judit; Amaral, Alexandra; Azpiazu, Rubén; Vavouri, Tanya; Estanyol, Josep Maria; Ballescà, Josep Lluis; Oliva, Rafael



Overlapping chromatin-remodeling systems collaborate genome wide at dynamic chromatin transitions.  


ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4 and Snf2h. The localization patterns of all three proteins substantially overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome wide and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Many regions require the activity of more than one remodeler to regulate accessibility. These findings provide a dynamic view of chromatin organization and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes. PMID:24317492

Morris, Stephanie A; Baek, Songjoon; Sung, Myong-Hee; John, Sam; Wiench, Malgorzata; Johnson, Thomas A; Schiltz, R Louis; Hager, Gordon L



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

SciTech Connect

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

Tone, Shigenobu [Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 (Japan)], E-mail:; Sugimoto, Kenji [Laboratory of Applied Molecular Biology, Division of Bioscience and Informatics, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531 (Japan); Tanda, Kazue [Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 (Japan); Suda, Taiji; Uehira, Kenzo [Electron Microscope Center, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 (Japan); Kanouchi, Hiroaki [Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 (Japan); Samejima, Kumiko [Wellcome Trust Centre for Cell Biology, ICMB, King's Buildings, The University of Edinburgh, Edinburgh, EH93JR, Scotland (United Kingdom); Minatogawa, Yohsuke [Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 (Japan); Earnshaw, William C. [Wellcome Trust Centre for Cell Biology, ICMB, King's Buildings, The University of Edinburgh, Edinburgh, EH93JR, Scotland (United Kingdom)], E-mail:



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

SciTech Connect

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

Namdar, Mandana [Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS (United Kingdom); Kearsey, Stephen E. [Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS (United Kingdom)]. E-mail:



Adaptive elastic properties of chromatin fiber  

NASA Astrophysics Data System (ADS)

Chromatin is a complex of DNA and specific proteins forming an intermediary level of organization of eukaryotic genomes, between double-stranded DNA and chromosome. Within a generic modeling of chromatin assembly, we investigate the interplay between the mechanical properties of the chromatin fiber and its biological functions. A quantitative step is to relate the mechanics at the DNA level and the mechanics described at the chromatin fiber level. It allows to calculate the complete set of chromatin elastic constants (twist and bend persistence lengths, stretch modulus and twist-stretch coupling constant), in terms of DNA elastic properties and geometric features of the fiber. These elastic constants are strongly sensitive to the local architecture of the fiber and we argue that this tunable elasticity might be a key feature in chromatin functions, for instance in the initiation and regulation of transcription. Moreover, this analysis provides a framework to interpret micromanipulation studies of chromatin fiber and suggests further experiments involving intercalators to scan the tunable elasticity of the fiber.

Ben-Ha??m, Eli; Lesne, Annick; Victor, Jean-Marc



Anti-aging peptide bioregulators induce reactivation of chromatin.  


The effect of synthetic peptide bioregulators (Epitalon, Livagen and Vilon) on structural and facultative heterochromatin of cultivated lymphocytes have been studied among old (75-88yr.) people. The data obtained indicate that epitalon, livagen and vilon: 1) activate synthetic processes, caused by reactivation of ribosomal genes as a result of deheterochromatinization (decondensation) of nucleolus organizer regions; 2) induce unrolling (deheterochromatinization) of total heterochromatin; 3) release genes repressed by heterochromatinization (condensation) of euchromatic regions forming facultative heterochromatin; 4) epitalon and livagen induce deheterochromatinization (decondensation) of pericentromeric structural heterochromatin of the chromosomes1 and 9. However, vilon does not induce deheterochromatinization of pericentromeric structural heterochromatin. These results indicate that peptide bioregulators Epitalon, Livagen and Vilon cause activation (deheterochromatinization) of chromatin in lymphocytes of old individuals. PMID:16705247

Lezhava, T; Monaselidze, J; Kadotani, T; Dvalishvili, N; Buadze, T



Systems proteomics of healthy and diseased chromatin  

PubMed Central

Summary Differences in chromatin-associated proteins allow the same genome to participate in multiple cell types and to respond to an array of stimuli in any given cell. To understand the fundamental properties of chromatin and to reveal its cell- and/or stimulus-specific behaviors, quantitative proteomics is an essential technology. This chapter details the methods for fractionation and quantitative mass spectrometric analysis of chromatin from hearts or isolated adult myocytes, detailing some of the considerations for applications to understanding heart disease. The state-of-the-art methodology for data interpretation and integration through bioinformatics is reviewed. PMID:23606250

Chen, Haodong; Monte, Emma; Vondriska, Thomas M.; Franklin, Sarah



Intracellular manipulation of chromatin using magnetic nanoparticles.  


Magnetic tweezers are widely used for manipulating small magnetic beads inside the cell cytoplasm in order to gain insight into the structural and mechanical properties of the cytoskeleton. Here we discuss the use of magnetic tweezers for the study of nuclear architecture and the mechanical properties of chromatin in living cells. A custom-built, dedicated micro magnetic tweezer set-up is described. We review progress that has been made in applying this technology for the study of chromatin structure and discuss its prospects for the in situ analysis of nuclear architecture and chromatin function. PMID:18461487

Kanger, Johannes S; Subramaniam, Vinod; van Driel, Roel



Chromatin targeting drugs in cancer and immunity  

PubMed Central

Recent advances in the enzymology of transcription and chromatin regulation have led to the discovery of proteins that play a prominent role in cell differentiation and the maintenance of specialized cell functions. Knowledge about post-synthetic DNA and histone modifications as well as information about the rules that guide the formation of multimolecular chromatin-bound complexes have helped to delineate gene-regulating pathways and describe how these pathways are altered in various pathological conditions. The present review focuses on the emerging area of therapeutic interference with chromatin function for the purpose of cancer treatment and immunomodulation. PMID:23964091

Prinjha, Rab; Tarakhovsky, Alexander



Mechanisms for the Inheritance of Chromatin States  

PubMed Central

Studies in eukaryotes ranging from yeast to mammals indicate that specific chromatin structures can be inherited following DNA replication via mechanisms acting in cis. Both the initial establishment of such chromatin structures and their inheritance require sequence-dependent specificity factors and changes in histone posttranslational modifications. Here I propose models for the maintenance of epigenetic information in which DNA silencers or nascent RNA scaffolds act as sensors that work cooperatively with parentally inherited histones to re-establish chromatin states following DNA replication. PMID:21854979

Moazed, Danesh



Chromatin and the genome integrity network  

PubMed Central

The maintenance of genome integrity is essential for organism survival and for the inheritance of traits to offspring. Genomic instability is caused by DNA damage, aberrant DNA replication or uncoordinated cell division, which can lead to chromosomal aberrations and gene mutations. Recently, chromatin regulators that shape the epigenetic landscape have emerged as potential gatekeepers and signalling coordinators for the maintenance of genome integrity. Here, we review chromatin functions during the two major pathways that control genome integrity: namely, repair of DNA damage and DNA replication. We also discuss recent evidence that suggests a novel role for chromatin-remodelling factors in chromosome segregation and in the prevention of aneuploidy. PMID:23247436

Papamichos-Chronakis, Manolis; Peterson, Craig L.



Expanding the roles of chromatin insulators in nuclear architecture, chromatin organization and genome function.  


Of the numerous classes of elements involved in modulating eukaryotic chromosome structure and function, chromatin insulators arguably remain the most poorly understood in their contribution to these processes in vivo. Indeed, our view of chromatin insulators has evolved dramatically since their chromatin boundary and enhancer blocking properties were elucidated roughly a quarter of a century ago as a result of recent genome-wide, high-throughput methods better suited to probing the role of these elements in their native genomic contexts. The overall theme that has emerged from these studies is that chromatin insulators function as general facilitators of higher-order chromatin loop structures that exert both physical and functional constraints on the genome. In this review, we summarize the result of recent work that supports this idea as well as a number of other studies linking these elements to a diverse array of nuclear processes, suggesting that chromatin insulators exert master control over genome organization and behavior. PMID:25012699

Schoborg, Todd; Labrador, Mariano



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


In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation. PMID:24889195

Bigeard, Jean; Rayapuram, Naganand; Pflieger, Delphine; Hirt, Heribert



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

PubMed Central

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



Chromatin modifiers: regulators of cellular differentiation  

PubMed Central

Cellular differentiation, by definition, is epigenetic. Genome-wide profiling of pluripotent cells and differentiated cells suggests global chromatin remodeling during differentiation, resulting in progressive transition from a relatively open chromatin configuration to a more compact state. Genetic studies in mouse models demonstrate major roles for a variety of histone modifiers and chromatin remodelers in key developmental transitions, such as the segregation of embryonic and extraembryonic lineages in blastocyst stage embryos, the formation of the three germ layers during gastrulation, and differentiation of adult stem cells. Furthermore, rather than merely stabilizing the gene expression changes driven by developmental transcription factors, evidence is emerging that chromatin regulators have multifaceted roles in cell fate decisions. PMID:24366184

Chen, Taiping; Dent, Sharon Y. R.



Identifying chromatin interactions at high spatial resolution  

E-print Network

This thesis presents two computational approaches for identifying chromatin interactions at high spatial resolution from ChIA-PET data. We introduce SPROUT which is a hierarchical probabilistic model that discovers high ...

Reeder, Christopher Campbell



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. PMID:20709077

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



Charged Condensation  

E-print Network

We consider Bose-Einstein condensation of massive electrically charged scalars in a uniform background of charged fermions. We focus on the case when the scalar condensate screens the background charge, while the net charge of the system resides on its boundary surface. A distinctive signature of this substance is that the photon acquires a Lorentz-violating mass in the bulk of the condensate. Due to this mass, the transverse and longitudinal gauge modes propagate with different group velocities. We give qualitative arguments that at high enough densities and low temperatures a charged system of electrons and helium-4 nuclei, if held together by laboratory devices or by force of gravity, can form such a substance. We briefly discuss possible manifestations of the charged condensate in compact astrophysical objects.

Gregory Gabadadze; Rachel A. Rosen



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. PMID:21030234

Liang, Yun; Hetzer, Martin W



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


Topoisomerase assays.  


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 enzymes, which make 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 for 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 to examine topoisomerase covalent complexes in vivo, and an assay for measuring DNA cleavage in vitro. PMID:22684721

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



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

PubMed Central

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

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



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


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

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



Pin1 promotes histone H1 dephosphorylation and stabilizes its binding to chromatin  

PubMed Central

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



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

PubMed Central

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

Redi, Carlo Alberto; Zuccotti, Maurizio



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. PMID:18556763

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



Open chromatin profiling in mice livers reveals unique chromatin variations induced by high fat diet.  


Metabolic diseases result from multiple genetic and environmental factors. We report here that one manner in which environmental factors can contribute to metabolic disease progression is through modification to chromatin. We demonstrate that high fat diet leads to chromatin remodeling in the livers of C57BL/6J mice, as compared with mice fed a control diet, and that these chromatin changes are associated with changes in gene expression. We further show that the regions of greatest variation in chromatin accessibility are targeted by liver transcription factors, including HNF4?, CCAAT/enhancer-binding protein ? (CEBP/?), and FOXA1. Repeating the chromatin and gene expression profiling in another mouse strain, DBA/2J, revealed that the regions of greatest chromatin change are largely strain-specific and that integration of chromatin, gene expression, and genetic data can be used to characterize regulatory regions. Our data indicate dramatic changes in the epigenome due to diet and demonstrate strain-specific dynamics in chromatin remodeling. PMID:25006255

Leung, Amy; Parks, Brian W; Du, Juan; Trac, Candi; Setten, Ryan; Chen, Yin; Brown, Kevin; Lusis, Aldons J; Natarajan, Rama; Schones, Dustin E



Cohesin codes - interpreting chromatin architecture and the many facets of cohesin function  

PubMed Central

Summary Sister chromatid tethering is maintained by cohesin complexes that minimally contain Smc1, Smc3, Mcd1 and Scc3. During S-phase, chromatin-associated cohesins are modified by the Eco1/Ctf7 family of acetyltransferases. Eco1 proteins function during S phase in the context of replicated sister chromatids to convert chromatin-bound cohesins to a tethering-competent state, but also during G2 and M phases in response to double-stranded breaks to promote error-free DNA repair. Cohesins regulate transcription and are essential for ribosome biogenesis and complete chromosome condensation. Little is known, however, regarding the mechanisms through which cohesin functions are directed. Recent findings reveal that Eco1-mediated acetylation of different lysine residues in Smc3 during S phase promote either cohesion or condensation. Phosphorylation and SUMOylation additionally impact cohesin functions. Here, we posit the existence of a cohesin code, analogous to the histone code introduced over a decade ago, and speculate that there is a symphony of post-translational modifications that direct cohesins to function across a myriad of cellular processes. We also discuss evidence that outdate the notion that cohesion defects are singularly responsible for cohesion-mutant-cell inviability. We conclude by proposing that cohesion establishment is linked to chromatin formation. PMID:23516328

Rudra, Soumya; Skibbens, Robert V.



The Archaeal Histone-Fold Protein HMf Organizes DNA into Bona Fide Chromatin Fibers  

Microsoft Academic Search

Background: The discovery of histone-like proteins in Archaea urged studies into the possible organization of archaeal genomes in chromatin. Despite recent advances, a variety of structural questions remain unanswered.Results: We have used the atomic force microscope (AFM) with traditional nuclease digestion assays to compare the structure of nucleoprotein complexes reconstituted from tandemly repeated eukaryal nucleosome-positioning sequences and histone octamers, H3\\/H4

Miroslav Tomschik; Mikhail A. Karymov; Jordanka Zlatanova; Sanford H. Leuba



Cohesin Is Required for Higher-Order Chromatin Conformation at the Imprinted IGF2-H19 Locus  

PubMed Central

Cohesin is a chromatin-associated protein complex that mediates sister chromatid cohesion by connecting replicated DNA molecules. Cohesin also has important roles in gene regulation, but the mechanistic basis of this function is poorly understood. In mammalian genomes, cohesin co-localizes with CCCTC binding factor (CTCF), a zinc finger protein implicated in multiple gene regulatory events. At the imprinted IGF2-H19 locus, CTCF plays an important role in organizing allele-specific higher-order chromatin conformation and functions as an enhancer blocking transcriptional insulator. Here we have used chromosome conformation capture (3C) assays and RNAi–mediated depletion of cohesin to address whether cohesin affects higher order chromatin conformation at the IGF2-H19 locus in human cells. Our data show that cohesin has a critical role in maintaining CTCF–mediated chromatin conformation at the locus and that disruption of this conformation coincides with changes in IGF2 expression. We show that the cohesin-dependent, higher-order chromatin conformation of the locus exists in both G1 and G2 phases of the cell cycle and is therefore independent of cohesin's function in sister chromatid cohesion. We propose that cohesin can mediate interactions between DNA molecules in cis to insulate genes through the formation of chromatin loops, analogous to the cohesin mediated interaction with sister chromatids in trans to establish cohesion. PMID:19956766

Ito, Yoko; Huddleston, Joanna E.; Uribe-Lewis, Santiago; Woodfine, Kathryn; Krueger, Christel; Reik, Wolf; Peters, Jan-Michael; Murrell, Adele



Effects of estrogen and progesterone on transcription, chromatin and ovalbumin gene expression in the chick oviduct.  


The effects of estrogen, progesterone and estrogen + progesterone combined on nuclear transcriptional processes in oviducts of immature chicks, previously withdrawn from estrogen, are reported. The responses to the steroids of the endogenous nuclear RNA polymerase activities, both nucleolar (I) and nucleoplasmic (II), the chromatin compositions and template capacities, and the appearance of ovalbumin messenger RNA (mRNA) are compared. When immature chicks (previously treated at 14 days with estrogen) are withdrawn from estrogen treatment, there is a gradual reduction in both polymerase activities. Diurnal variations in polymerase II activties in the oviduct of withdrawn chicks required that subsequent experiments include time-matched controls. The hormones alter RNA polymerase II and II activities in vivo as assayed in isolated nuclei. Progesterone represses the polymerase I and II activities, while estrogen alone and estrogen + progesterone enhance both polymerase activities immediately after injection. Diethylstillbestrol, a synthetic estrogen, causes changes similar to those of estrogen. The effects of these steroids on the polymerases are detected within 15 min of hormone injection. Changes in the capacities of chromatins to serve as template for RNA synthesis in general correlated with changes in polymerase II activities. Interestingly, in the case of estrogen treatment, the acidic chromatin protein (but not histone) levels fluctuate positively with the template capacities of the chromatin. An antagonism between estrogen and progesterone is observed in the responses of both RNA polymerases I and II activities as well as in the chromatin template capacity. Levels of messenger RNA coding for ovalbumin, as detected by hybridization with labeled complementary DNA, increase in oviducts of withdrawn chicks within 2--3 of the injection of estrogen, progesterone or estrogen + progesterone. This rapid accumulation of ovalbumin mRNA is not accompanied in each case by a similar increase in polymerase II activity or chromatin template capacity. PMID:952904

Spelsberg, T C; Cox, R F



Chromatin associations in Arabidopsis interphase nuclei  

PubMed Central

The arrangement of chromatin within interphase nuclei seems to be caused by topological constraints and related to gene expression depending on tissue and developmental stage. In yeast and animals it was found that homologous and heterologous chromatin association are required to realize faithful expression and DNA repair. To test whether such associations are present in plants we analyzed Arabidopsis thaliana interphase nuclei by FISH using probes from different chromosomes. We found that chromatin fiber movement and variable associations, although in general relatively seldom, may occur between euchromatin segments along chromosomes, sometimes even over large distances. The combination of euchromatin segments bearing high or low co-expressing genes did not reveal different association frequencies probably due to adjacent genes of deviating expression patterns. Based on previous data and on FISH analyses presented here, we conclude that the global interphase chromatin organization in A. thaliana is relatively stable, due to the location of its 10 centromeres at the nuclear periphery and of the telomeres mainly at the centrally localized nucleolus. Nevertheless, chromatin movement enables a flexible spatial genome arrangement in plant nuclei.

Schubert, Veit; Rudnik, Radoslaw; Schubert, Ingo



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. PMID:22363642

Larson, Jessica L.; Yuan, Guo-Cheng



Chromatin decouples promoter threshold from dynamic range  

PubMed Central

Chromatin influences gene expression by restricting access of DNA binding proteins to their cognate sites in the genome1–3. Large-scale characterization of nucleosome positioning in Saccharomyces cerevisiae has revealed a stereotyped promoter organization in which a nucleosome-free region (NFR) is present within several hundred base pairs upstream of the translation start site4,5. Many transcription factors bind within NFRs and nucleate chromatin remodelling events which then expose other cis-regulatory elements6–9. However, it is not clear how transcription-factor binding and chromatin influence quantitative attributes of gene expression. Here we show that nucleosomes function largely to decouple the threshold of induction from dynamic range. With a series of variants of one promoter, we establish that the affinity of exposed binding sites is a primary determinant of the level of physiological stimulus necessary for substantial gene activation, and sites located within nucleosomal regions serve to scale expression once chromatin is remodelled. Furthermore, we find that the S. cerevisiae phosphate response (PHO) pathway exploits these promoter designs to tailor gene expression to different environmental phosphate levels. Our results suggest that the interplay of chromatin and binding-site affinity provides a mechanism for fine-tuning responses to the same cellular state. Moreover, these findings may be a starting point for more detailed models of eukaryotic transcriptional control. PMID:18418379

Lam, Felix H.; Steger, David J.; O'Shea, Erin K.



New Mitotic Regulators Released from Chromatin  

PubMed Central

Faithful action of the mitotic spindle segregates duplicated chromosomes into daughter cells. Perturbations of this process result in chromosome mis-segregation, leading to chromosomal instability and cancer development. Chromosomes are not simply passengers segregated by spindle microtubules but rather play a major active role in spindle assembly. The GTP bound form of the Ran GTPase (RanGTP), produced around chromosomes, locally activates spindle assembly factors. Recent studies have uncovered that chromosomes organize mitosis beyond spindle formation. They distinctly regulate other mitotic events, such as spindle maintenance in anaphase, which is essential for chromosome segregation. Furthermore, the direct function of chromosomes is not only to produce RanGTP but, in addition, to release key mitotic regulators from chromatin. Chromatin-remodeling factors and nuclear pore complex proteins, which have established functions on chromatin in interphase, dissociate from mitotic chromatin and function in spindle assembly or maintenance. Thus, chromosomes actively organize their own segregation using chromatin-releasing mitotic regulators as well as RanGTP. PMID:24380075

Yokoyama, Hideki; Gruss, Oliver J.



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. PMID:23724067

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



A Genetic Screen to Discover Pathways Affecting Cohesin Function in Schizosaccharomyces pombe Identifies Chromatin Effectors  

PubMed Central

Cohesion, the force that holds sister chromatids together from the time of DNA replication until separation at the metaphase to anaphase transition, is mediated by the cohesin complex. This complex is also involved in DNA damage repair, chromosomes condensation, and gene regulation. To learn more about the cellular functions of cohesin, we conducted a genetic screen in Schizosaccharomyces pombe with two different cohesin mutants (eso1-G799D and mis4-242). We found synthetic negative interactions with deletions of genes involved in DNA replication and heterochromatin formation. We also found a few gene deletions that rescued the growth of eso1-G799D at the nonpermissive temperature, and these genes partially rescue the lagging chromosome phenotype. These genes are all chromatin effectors. Overall, our screen revealed an intimate association between cohesin and chromatin. PMID:23050226

Chen, Zhiming; McCroskey, Scott; Guo, Weichao; Li, Hua; Gerton, Jennifer L.



Structure of RCC1 chromatin factor bound to the nucleosome core particle  

SciTech Connect

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

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



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. PMID:17014129

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



Autism genes keep turning up chromatin  

PubMed Central

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

LaSalle, Janine M.



Chromatin Remodeling, DNA Damage Repair and Aging  

PubMed Central

Cells are constantly exposed to a variety of environmental and endogenous conditions causing DNA damage, which is detected and repaired by conserved DNA repair pathways to maintain genomic integrity. Chromatin remodeling is critical in this process, as the organization of eukaryotic DNA into compact chromatin presents a natural barrier to all DNA-related events. Studies on human premature aging syndromes together with normal aging have suggested that accumulated damages might lead to exhaustion of resources that are required for physiological functions and thus accelerate aging. In this manuscript, combining the present understandings and latest findings, we focus mainly on discussing the role of chromatin remodeling in the repair of DNA double-strand breaks (DSBs) and regulation of aging. PMID:23633913

Liu, Baohua; Yip, Raymond KH; Zhou, Zhongjun



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



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. PMID:22223048

Murawska, Magdalena



High-pressure freezing of spermiogenic nuclei supports a dynamic chromatin model for the histone-to-protamine transition.  


In this study, we present for the first time a description of the dynamic chromatin changes that occur during spermiogenesis in the internally fertilizing caenogastropod mollusc Nucella lamellosa. Chromatin condensation in developing sperm cells in some animals, such as the model biological system used here, involves the histone-to-protamine transition and proceeds through a patterning stage from granules to fibers to lamellae. This may be due to the physicochemical phenomenon of phase separation by spinodal decomposition, a dynamic mechanism known to generate pattern. This hypothesis is based entirely on published transmission electron microscopy photomicrographs using conventional fixation technology. We now report that spermatid nuclear patterning and subsequent condensation in testis of Nucella lamellosa fixed by high-pressure freezing and freeze substitution (HPF/FS) is similar to that in glutaraldehyde-fixed testis, and can be related to the processing of sperm nuclear basic proteins (SNBPs). PMID:19830786

Martens, Garnet; Humphrey, Elaine C; Harrison, Lionel G; Silva-Moreno, Begonia; Ausió, Juan; Kasinsky, Harold E



Chromatin Degradation in Differentiating Fiber Cells of the Eye Lens  

PubMed Central

During development, the lens of the eye becomes transparent, in part because of the elimination of nuclei and other organelles from the central lens fiber cells by an apoptotic-like mechanism. Using confocal microscopy we showed that, at the border of the organelle-free zone (OFZ), fiber cell nuclei became suddenly irregular in shape, with marginalized chromatin. Subsequently, holes appeared in the nuclear envelope and underlying laminae, and the nuclei collapsed into condensed, spherical structures. Nuclear remnants, containing DNA, histones, lamin B2, and fragments of nuclear membrane, were detected deep in the OFZ. We used in situ electrophoresis to demonstrate that fragmented DNA was present only in cells bordering the OFZ. Confocal microscopy of terminal deoxynucleotidyl transferase (TdT)–labeled lens slices confirmed that DNA fragmentation was a relatively late event in fiber differentiation, occurring after the loss of the nuclear membrane. DNA fragments with 3?-OH or 3?-PO4 ends were not observed elsewhere in the lens under normal conditions, although they could be produced by pretreatment with DNase I or micrococcal nuclease, respectively. Dual labeling with TdT and an antibody against protein disulfide isomerase, an ER-resident protein, revealed a distinct spatial and temporal gap between the disappearance of ER and nuclear membranes and the onset of DNA degradation. Thus, fiber cell chromatin disassembly differs significantly from classical apoptosis, in both the sequence of events and the time course of the process. The fact that DNA degradation occurs only after the disappearance of mitochondrial, ER, and nuclear membranes suggests that damage to intracellular membranes may be an initiating event in nuclear breakdown. PMID:9105035

Bassnett, Steven; Mataic, Danijela



Comprehensive analysis of the chromatin landscape in Drosophila melanogaster  

E-print Network

Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin ...

Ernst, Jason M.


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

PubMed Central

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

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



An autonomous chromatin/DNA-PK mechanism for localized DNA damage signaling in mammalian cells  

PubMed Central

Rapid phosphorylation of histone variant H2AX proximal to DNA breaks is an initiating event and a hallmark of eukaryotic DNA damage responses. Three mammalian kinases are known to phosphorylate H2AX in response to DNA damage. However, the mechanism(s) for damage-localized phosphorylation remains incompletely understood. The DNA-dependent protein kinase (DNA-PK) is the most abundant H2AX-modifying kinases and uniquely activated by binding DNA termini. Here, we have developed a novel approach to examine enzyme activity and substrate properties by executing biochemical assays on intact cellular structures. We apply this approach to examine the mechanisms of localized protein modification in chromatin within fixed cells. DNA-PK retains substrate specificity and independently generates break-localized ?H2AX foci in chromatin. In situ DNA-PK activity recapitulates localization and intensity of in vivo H2AX phosphorylation and requires no active cellular processes. Nuclease treatments or addition of exogenous DNA resulted in genome-wide H2AX phosphorylation, showing that DNA termini dictated the locality of H2AX phosphorylation in situ. DNA-PK also reconstituted focal phosphorylation of structural maintenance of chromatin protein 1, but not activating transcription factor 2. Allosteric regulation of DNA-PK by DNA termini protruding from chromatin constitutes an autonomous mechanism for break-localized protein phosphorylation that generates sub-nuclear foci. We discuss generalized implications of this mechanism in localizing mammalian DNA damage responses. PMID:23325849

Muñoz, Denise P.; Kawahara, Misako; Yannone, Steven M.



Models incorporating chromatin modification data identify functionally important p53 binding sites.  


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. PMID:23599002

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



The Roles of Chromatin Remodelling Factors in Replication  

Microsoft Academic Search

Dynamic changes of chromatin structure control DNA-dependent events, including DNA replication.\\u000a Along with DNA, chromatin organization must be replicated to maintain genetic and epigenetic information\\u000a through cell generations. Chromatin remodelling is important for several steps in replication: determination\\u000a and activation of origins of replication, replication machinery progression, chromatin assembly and\\u000a DNA repair. Histone chaperones such as the FACT complex assist

Ana Neves-Costa; Patrick Varga-Weisz


UT MD Anderson scientists discover secret life of chromatin:

Chromatin--the intertwined histone proteins and DNA that make up chromosomes--constantly receives messages that pour in from a cell’s intricate signaling networks... But chromatin also talks back, scientists at The University of Texas M.D. Anderson Cancer Center report today in the journal Cell, issuing orders affecting a protein that has nothing to do with chromatin’s central role in gene transcription--the first step in protein formation.


GENE EXPRESSION & METABOLISM Chromatin and Transcription in Yeast  

E-print Network

for H2A.Z in transcription 360 Chromatin-Remodeling Factors 361 Identification of the Swi/Snf and RSC complexes 361 Swi/Snf complexes have chromatin-remodeling activity 362 Regulation of transcription by Swi/Snf 362 RSC plays broad roles in gene expression and chromatin structure 363 Bromodomains in Swi/Snf

Winston, Fred


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



Interplay between mismatch repair and chromatin assembly  

PubMed Central

Single strand nicks and gaps in DNA have been reported to increase the efficiency of nucleosome loading mediated by chromatin assembly factor 1 (CAF-1). However, on mismatch-containing substrates, these strand discontinuities are utilized by the mismatch repair (MMR) system as loading sites for exonuclease 1, at which degradation of the error-containing strand commences. Because packaging of DNA into chromatin might inhibit MMR, we were interested to learn whether chromatin assembly is differentially regulated on heteroduplex and homoduplex substrates. We now show that the presence of a mismatch in a nicked plasmid substrate delays nucleosome loading in human cell extracts. Our data also suggest that, once the mismatch is removed, repair of the single-stranded gap is accompanied by efficient nucleosome loading. We postulated that the balance between MMR and chromatin assembly might be governed by proliferating cell nuclear antigen (PCNA), the processivity factor of replicative DNA polymerases, which is loaded at DNA termini and which interacts with the MSH6 subunit of the mismatch recognition factor MutS?, as well as with CAF-1. We now show that this regulation might be more complex; MutS? and CAF-1 interact not only with PCNA, but also with each other. In vivo this interaction increases during S-phase and may be controlled by the phosphorylation status of the p150 subunit of CAF-1. PMID:22232658

Schöpf, Barbara; Bregenhorn, Stephanie; Quivy, Jean-Pierre; Kadyrov, Farid A.; Almouzni, Genevieve; Jiricny, Josef



Establishment of Histone Modifications after Chromatin Assembly  

PubMed Central

Every cell has to duplicate its entire genome during S-phase of the cell cycle. After replication, the newly synthesized DNA is rapidly assembled into chromatin. The newly assembled chromatin ‘matures’ and adopts a variety of different conformations. This differential packaging of DNA plays an important role for the maintenance of gene expression patterns and has to be reliably copied in each cell division. Posttranslational histone modifications are prime candidates for the regulation of the chromatin structure. In order to understand the maintenance of chromatin structures, it is crucial to understand the replication of histone modification patterns. To study the kinetics of histone modifications in vivo, we have pulse-labeled synchronized cells with an isotopically labeled arginine (15N4) that is 4 Da heavier than the naturally occurring 14N4 isoform. As most of the histone synthesis is coupled with replication, the cells were arrested at the G1/S boundary, released into S-phase and simultaneously incubated in the medium containing heavy arginine, thus labeling all newly synthesized proteins. This method allows a comparison of modification patterns on parental versus newly deposited histones. Experiments using various pulse/chase times show that particular modifications have considerably different kinetics until they have acquired a modification pattern indistinguishable from the parental histones. PMID:19541851

Scharf, Annette N. D.; Barth, Teresa K.; Imhof, Axel



Structure of interphase nuclei in relation to the cell cycle. Chromatin organization in mouse L cells temperature-sensitive for DNA replication  

PubMed Central

Mutant lines of mouse L cells, TS A1S9, and TS C1, show temperature- sensitive (TS) DNA synthesis and cell division when shifted from 34 degrees to 38.5 degrees C. With TS A1S9 the decline in DNA synthesis begins after 6-8 h at 38.5 degrees C and is most marked at about 24 h. Most cells in S, G2, or M at temperature upshift complete one mitosis and accumulate in the subsequent interphase at G1 or early S as a result of expression of a primary defect, failure of elongation of newly made small DNA fragments. Heat inactivation of TS C1 cells is more rapid; they fail to complete the interphase in progress at temperature upshift and accumulate at late S or G2. Inhibition of both cell types is reversible on return to 34 degrees C. Cell and nuclear growth continues during inhibition of replication. Expression of both TS mutations leads to a marked change in gross organization of chromatin as revealed by electron microscopy. Nuclei of wild-type cells at 34 degrees and 38.5 degrees C and mutant cells at 34 degrees C show a range of aggregation of condensed chromatin from small dispersed bodies to large discrete clumps, with the majority in an intermediate state. In TS cells at 38.5 degrees C, condensed chromatin bodies in the central nuclear region become disaggregated into small clumps dispersed through the nucleus. Morphometric estimation of volume of condensed chromatin indicates that this process is not due to complete decondensation of chromatin fibrils, but rather involves dispersal of large condensed chromatin bodies into finer aggregates and loosening of fibrils within the aggregates. The dispersed condition is reversed in nuclei which resume DNA synthesis when TS cells are downshifted from 38.5 degrees to 34 degrees C. The morphological observations are consistent with the hypothesis that condensed chromatin normally undergoes an ordered cycle of transient, localized disaggregation and reaggregation associated with replication. In temperature-inactivated mutants, normal progressive disaggregation presumably occurs, but subsequent lack of chromatin replication prevents reaggregation. PMID:659512



Factors affecting chromatin stability of bovine spermatozoa.  


The structural stability of transcriptionally inert paternal chromatin is of vital importance for the fertilization process and early embryonic development. Accordingly, a series of eight experiments were conducted during a 7-month period to investigate: (1) effects of bull breed, individuality, successive ejaculations, semen quality characteristics (SQC), semen dilution rates and hypothermic storage of semen in a Tris-egg yolk extender on incidence of sperm nuclear chromatin instability (NCI), and (2) effects of the interaction between variation of NCI within a frozen ejaculate and variation of oocytes quality due to maturation time and/or season on the efficiency of in vitro embryo production (IVEP). Semen samples were collected once a week from six bulls using an AV and only ejaculates (n=220) of >0.30x10(9) sperm/ml and >or=60% motility were used. NCI was measured by: (1) detection of lysine-rich histones in sperm chromatin using aniline blue staining, (2) sperm susceptibility to acid-induced nuclear DNA denaturation in situ using acridine orange test, and (3) sperm susceptibility to nuclear chromatin decondensation (NCD). Bovine oocytes (n=695) were matured in vitro for 18 or 24 h, fertilized after sperm selection through a swim-up procedure and cultured for 72 h. The results showed that the 2nd ejaculates were superior to the 1st ones with respect to chromatin stability. Dilution of semen to 49.67+/-8.56x10(6) sperm/ml (1:19) decreased resistance of sperm to NCD. Cooling of semen had no significant effect on chromatin stability. Cryopreservation of semen augmented sperm vulnerability to DNA denaturation. Improvement of SQC (semen volume, sperm motility, velocity, viability and morphological normalcy) was generally concomitant with increase of sperm resistance to NCI. While Blonde d'Aquitaine bulls had a resistance to NCD higher than Limousine bulls in fresh semen, the former showed a greater susceptibility to DNA denaturation than the latter in cooled semen. Individuality significantly influenced NCI. The variability of NCI within a frozen ejaculate affected efficiency of IVEP. Significant negative correlations were observed between incidence of NCI and both fertilization rate and developmental capacity of embryos after maturation of oocytes for 18 h. The significant variation in IVEP traits due to season was independent of the effect of sperm chromatin instability. PMID:17398042

Khalifa, T A A; Rekkas, C A; Lymberopoulos, A G; Sioga, A; Dimitriadis, I; Papanikolaou, Th



Recruitment of Phosphorylated Chromatin Assembly Factor 1to Chromatin after UV Irradiation of Human Cells  

Microsoft Academic Search

The subcellular distribution and posttransla- tional modification of human chromatin assembly fac- tor 1 (CAF-1) have been investigated after UV irradia- tion of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-asso- ciated fraction. This fraction is most abundant during S phase in

Emmanuelle Martini; Danièle M. J. Roche; Kathrin Marheineke; Alain Verreault; Geneviève Almouzni



Chromatin modifying enzymes as modulators of reprogramming  

PubMed Central

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodeling1. While several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming2,3, the role of specific chromatin modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used shRNAs to target genes in DNA and histone methylation pathways, and have identified positive and negative modulators of iPSC generation. While inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase Ezh2, reduced reprogramming efficiency, suppression of SUV39H1, YY1, and Dot1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase Dot1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for Klf4 and c-Myc. Inhibition of Dot1L early in the reprogramming process is associated with a marked increase in two alternative factors, Nanog and Lin28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. Dot1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors. PMID:22388813

Onder, Tamer T.; Kara, Nergis; Cherry, Anne; Sinha, Amit U.; Zhu, Nan; Bernt, Kathrin M.; Cahan, Patrick; Mancarci, Ogan. B.; Unternaehrer, Juli; Gupta, Piyush B.; Lander, Eric S.; Armstrong, Scott A.; Daley, George Q.




PubMed Central

Methylation of specific amino acids in histone tails is responsible for packaging DNA into condensed, repressed chromatin, and into open chromatin that is accessible to the transcription machinery. Monomethylation and trimethylation of histone H4-lysine 20 (H4-K20) control the formation of repressed chromatin. Using antibodies that specifically recognize the three methyl marks of histone H4-K20, we characterized their regulation during the cell cycle and throughout development. We find free mono- and trimethylated histone H4-K20 in unfertilized Drosophila eggs and in S2 tissue culture cells. Soluble mono-. di-, and trimethylated H4-K20 are also found in HeLa cells. These soluble modified histones may represent a pool of free histones that can rapidly be incorporated into chromatin. The three methyl marks are each regulated differentially during development and their detection on western blots does not overlap with their detection on chromosomes. Monomethylated H4-K20 is detected on condensed chromosomes throughout development, while di-, and trimethylated H4-K20 is detected on metaphase chromosomes at specific stages. Our results suggest that the detection of methylated H4-K20 on chromosomes may reveal chromatin packaging rather than the distribution of the methyl marks. PMID:17229421

Karachentsev, Dmitry; Druzhinina, Marina; Steward, Ruth



The PHD and chromo domains regulate the ATPase activity of the human chromatin remodeler CHD4.  


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. PMID:22575888

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



Chromatin dynamics in DNA double-strand break repair  

PubMed Central

DNA double-strand breaks (DSBs) occur in the context of a highly organized chromatin environment and are, thus, a significant threat to the epigenomic integrity of eukaryotic cells. Changes in break-proximal chromatin structure are thought to be a prerequisite for efficient DNA repair and may help protect the structural integrity of the nucleus. Unlike most bona fide DNA repair factors, chromatin influences the repair process at several levels: the existing chromatin context at the site of damage directly affects the access and kinetics of the repair machinery; DSB induced chromatin modifications influence the choice of repair factors, thereby modulating repair outcome; lastly, DNA damage can have a significant impact on chromatin beyond the site of damage. We will discuss recent findings that highlight both the complexity and importance of dynamic and tightly orchestrated chromatin reorganization to ensure efficient DSB repair and nuclear integrity. PMID:22285574

Shi, Lei; Oberdoerffer, Philipp



Regulating the chromatin landscape: structural and mechanistic perspectives.  


A large family of chromatin remodelers that noncovalently modify chromatin is crucial in cell development and differentiation. They are often the targets of cancer, neurological disorders, and other human diseases. These complexes alter nucleosome positioning, higher-order chromatin structure, and nuclear organization. They also assemble chromatin, exchange out histone variants, and disassemble chromatin at defined locations. We review aspects of the structural organization of these complexes, the functional properties of their protein domains, and variation between complexes. We also address the mechanistic details of these complexes in mobilizing nucleosomes and altering chromatin structure. A better understanding of these issues will be vital for further analyses of subunits of these chromatin remodelers, which are being identified as targets in human diseases by NGS (next-generation sequencing). PMID:24606138

Bartholomew, Blaine



Minireview: Conversing with chromatin: the language of nuclear receptors.  


Nuclear receptors are transcription factors that are activated by physiological stimuli to bind DNA in the context of chromatin and regulate complex biological pathways. Major advances in nuclear receptor biology have been aided by genome scale examinations of receptor interactions with chromatin. In this review, we summarize the roles of the chromatin landscape in regulating nuclear receptor function. Chromatin acts as a central integrator in the nuclear receptor-signaling axis, operating in distinct temporal modalities. Chromatin effects nuclear receptor action by specifying its genomic localization and interactions with regulatory elements. On receptor binding, changes in chromatin operate as an effector of receptor signaling to modulate transcriptional events. Chromatin is therefore an integral component of the pathways that guide nuclear receptor action in cell-type-specific and cell state-dependent manners. PMID:24196351

Biddie, Simon C; John, Sam



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


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

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



Direct Evidence for Pitavastatin Induced Chromatin Structure Change in the KLF4 Gene in Endothelial Cells  

PubMed Central

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

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



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


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



Changes in chromatin and the phosphorylation of nuclear proteins during heat shock of Achlya ambisexualis.  

PubMed Central

Heat shock led to marked changes in the apparent levels of phosphorylation of nuclear proteins in the fungus Achlya ambisexualis. We characterized these heat shock-induced changes in nuclear proteins on two types of two-dimensional polyacrylamide gel systems. We report here that one of two Achlya H3 histones (H3.1) and also the oomycete histone alpha appear to be highly phosphorylated with heat shock. Additional changes observed in acid-soluble nuclear proteins included an apparent increase in the 32P labeling of a 43,000-molecular-weight protein and the dephosphorylation of a major group of Achlya phosphoproteins in the 30,000-to-32,000-molecular-weight range. The changes in protein phosphorylation were accompanied by striking changes in the morphology of Achlya nuclei. Nuclei in the heat-shocked cells, but not in control cells, exhibited marked chromatin condensation and contained bundles of filaments which were approximately 4 nm in diameter. Concomitantly, the bulk of chromatin from heat-shocked nuclei showed a decreased sensitivity to digestion with the enzyme DNase I relative to chromatin from control cells. Images PMID:6504045

Pekkala, D; Heath, B; Silver, J C



Involvement of the SATB1/F-actin complex in chromatin reorganization during active cell death  

PubMed Central

Over the past years, confirmations on the presence of actin and/or its polymerized form, F-actin, in the cell nucleus are progressively accumulating. Nevertheless, the function and localization of F-actin in the nucleus is still not fully characterized. Thus, the aim of the present study was to evaluate the association between F-actin and sequence-binding protein 1 (SATB1) and their involvement in chromatin remodeling associated with active cell death. Both SATB1 and F-actin were colocalized in the transcriptional active regions of the cell nucleus and a functional interaction was observed between SATB1 and higher-organized nuclear F-actin structures at the border between condensed and decondensed chromatin. These results extend the knowledge on the role of SATB1 and nuclear F-actin in three-dimensional chromatin organization and their functions during active cell death. Additionally, this study opens the discussion on the involvement of the SATB1/F-actin functional complex in active cell death; further studies are required to fully elucidate these issues. PMID:24676287




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. PMID:24098838

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



On the topology of chromatin fibres.  


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

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



Intracellular manipulation of chromatin using magnetic nanoparticles  

Microsoft Academic Search

Magnetic tweezers are widely used for manipulating small magnetic beads inside the cell cytoplasm in order to gain insight\\u000a into the structural and mechanical properties of the cytoskeleton. Here we discuss the use of magnetic tweezers for the study\\u000a of nuclear architecture and the mechanical properties of chromatin in living cells. A custom-built, dedicated micro magnetic\\u000a tweezer set-up is described.

Johannes S. Kanger; Vinod Subramaniam; Roel van Driel



MYST-family histone acetyltransferases: beyond chromatin  

Microsoft Academic Search

Covalently modifying a protein has proven to be a powerful mechanism of functional regulation. N-epsilon acetylation of lysine\\u000a residues was initially discovered on histones and has been studied extensively in the context of chromatin and DNA metabolism,\\u000a such as transcription, replication and repair. However, recent research shows that acetylation is more widespread than initially\\u000a thought and that it regulates various

Vasileia Sapountzi; Jacques Côté



Chromatin signature of widespread monoallelic expression  

PubMed Central

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

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



Genes for embryo development are packaged in blocks of multivalent chromatin in zebrafish sperm  

PubMed Central

In mature human sperm, genes of importance for embryo development (i.e., transcription factors) lack DNA methylation and bear nucleosomes with distinctive histone modifications, suggesting the specialized packaging of these developmental genes in the germline. Here, we explored the tractable zebrafish model and found conceptual conservation as well as several new features. Biochemical and mass spectrometric approaches reveal the zebrafish sperm genome packaged in nucleosomes and histone variants (and not protamine), and we find linker histones high and H4K16ac absent, key factors that may contribute to genome condensation. We examined several activating (H3K4me2/3, H3K14ac, H2AFV) and repressing (H3K27me3, H3K36me3, H3K9me3, hypoacetylation) modifications/compositions genome-wide and find developmental genes packaged in large blocks of chromatin with coincident activating and repressing marks and DNA hypomethylation, revealing complex “multivalent” chromatin. Notably, genes that acquire DNA methylation in the soma (muscle) are enriched in transcription factors for alternative cell fates. Remarkably, whereas H3K36me3 is located in the 3? coding region of heavily transcribed genes in somatic cells, H3K36me3 is present in the promoters of “silent” developmental regulators in sperm, suggesting different rules for H3K36me3 in the germline and soma. We also reveal the chromatin patterns of transposons, rDNA, and tDNAs. Finally, high levels of H3K4me3 and H3K14ac in sperm are correlated with genes activated in embryos prior to the mid-blastula transition (MBT), whereas multivalent genes are correlated with activation at or after MBT. Taken together, gene sets with particular functions in the embryo are packaged by distinctive types of complex and often atypical chromatin in sperm. PMID:21383318

Wu, Shan-Fu; Zhang, Haiying; Cairns, Bradley R.



The Arabidopsis CAP-D proteins are required for correct chromatin organisation, growth and fertility.  


In plants as in other eukaryotes, the structural maintenance of chromosome (SMC) protein complexes cohesin, condensin and SMC5/6 are essential for sister chromatid cohesion, chromosome condensation, DNA repair and recombination. The presence of paralogous genes for various components of the different SMC complexes suggests the diversification of their biological functions during the evolution of higher plants. In Arabidopsis thaliana, we identified two candidate genes (Cap-D2 and Cap-D3) which may express conserved proteins presumably associated with condensin. In silico analyses using public databases suggest that both genes are controlled by factors acting in a cell cycle-dependent manner. Cap-D2 is essential because homozygous T-DNA insertion mutants were not viable. The heterozygous mutant showed wild-type growth habit but reduced fertility. For Cap-D3, we selected two homozygous mutants expressing truncated transcripts which are obviously not fully functional. Both mutants show reduced pollen fertility and seed set (one of them also reduced plant vigour), a lower chromatin density and frequent (peri)centromere association in interphase nuclei. Sister chromatid cohesion was impaired compared to wild-type in the cap-D3 mutants but not in the heterozygous cap-D2 mutant. At superresolution (Structured Illumination Microscopy), we found no alteration of chromatin substructure for both cap-D mutants. Chromosome-associated polypeptide (CAP)-D3 and the cohesin subunit SMC3 form similar but positionally non-overlapping reticulate structures in 2C-16C nuclei, suggesting their importance for interphase chromatin architecture in differentiated nuclei. Thus, we presume that CAP-D proteins are required for fertility, growth, chromatin organisation, sister chromatid cohesion and in a process preventing the association of centromeric repeats. PMID:23929493

Schubert, Veit; Lermontova, Inna; Schubert, Ingo



Chromatin structure in situ: the contribution of DNA ultrastructural cytochemistry.  


Ultrastructural studies conducted in situ using conventional transmission electron microscopy have had relatively little impact on defining the structural organization of chromatin. This is due to the fact that in routine transmission electron microscopy, together with the deoxyribonucleoprotein, many different intermingled substances are contrasted, masking the ultrastructure of chromatin. By selective staining of DNA in thin sections, using the Feulgen-like osmium-ammine reaction, these drawbacks have been overcome and worthwhile data have been obtained both on the gross morphology and the ultrastructural-functional organization of chromatin in situ. In the present study these results are reviewed and discussed in light of recent achievements in both interphase nuclear chromatin compartmentalization in interphase nuclei and in the structural organization of chromatin fibers in transcriptionally active and inactive chromatin. PMID:24704998

Derenzini, M; Olins, A L; Olins, D E



Chromatin Structure in Situ: The Contribution of DNA Ultrastructural Cytochemistry  

PubMed Central

Ultrastructural studies conducted in situ using conventional transmission electron microscopy have had relatively little impact on defining the structural organization of chromatin. This is due to the fact that in routine transmission electron microscopy, together with the deoxyribonucleoprotein, many different intermingled substances are contrasted, masking the ultrastructure of chromatin. By selective staining of DNA in thin sections, using the Feulgen-like osmium-ammine reaction, these drawbacks have been overcome and worthwhile data have been obtained both on the gross morphology and the ultrastructural-functional organization of chromatin in situ. In the present study these results are reviewed and discussed in light of recent achievements in both interphase nuclear chromatin compartmentalization in interphase nuclei and in the structural organization of chromatin fibers in transcriptionally active and inactive chromatin. PMID:24704998

Derenzini, M.; Olins, A.L.; Olins, D.E.



Depletion of the Chromatin Looping Proteins CTCF and Cohesin Causes Chromatin Compaction: Insight into Chromatin Folding by Polymer Modelling  

PubMed Central

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

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



Depletion of the Chromatin Looping Proteins CTCF and Cohesin Causes Chromatin Compaction: Insight into Chromatin Folding by Polymer Modelling.  


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

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



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


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



The Centromere: Chromatin Foundation for the Kinetochore Machinery  

PubMed Central

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

Fukagawa, Tatsuo; Earnshaw, William C.



Myogenic Differential Methylation: Diverse Associations with Chromatin Structure  

PubMed Central

Employing a new algorithm for identifying differentially methylated regions (DMRs) from reduced representation bisulfite sequencing profiles, we identified 1972 hypermethylated and 3250 hypomethylated myogenic DMRs in a comparison of myoblasts (Mb) and myotubes (Mt) with 16 types of nonmuscle cell cultures. DMRs co-localized with a variety of chromatin structures, as deduced from ENCODE whole-genome profiles. Myogenic hypomethylation was highly associated with both weak and strong enhancer-type chromatin, while hypermethylation was infrequently associated with enhancer-type chromatin. Both myogenic hypermethylation and hypomethylation often overlapped weak transcription-type chromatin and Polycomb-repressed-type chromatin. For representative genes, we illustrate relationships between DNA methylation, the local chromatin state, DNaseI hypersensitivity, and gene expression. For example, MARVELD2 exhibited myogenic hypermethylation in transcription-type chromatin that overlapped a silenced promoter in Mb and Mt while TEAD4 had myogenic hypomethylation in intronic subregions displaying enhancer-type or transcription-type chromatin in these cells. For LSP1, alternative promoter usage and active promoter-type chromatin were linked to highly specific myogenic or lymphogenic hypomethylated DMRs. Lastly, despite its myogenesis-associated expression, TBX15 had multiple hypermethylated myogenic DMRs framing its promoter region. This could help explain why TBX15 was previously reported to be underexpressed and, unexpectedly, its promoter undermethylated in placentas exhibiting vascular intrauterine growth restriction. PMID:24949935

Chandra, Sruti; Baribault, Carl; Lacey, Michelle; Ehrlich, Melanie



The centromere: chromatin foundation for the kinetochore machinery.  


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

Fukagawa, Tatsuo; Earnshaw, William C



Snapshots: Chromatin control of viral infection David M. Knipe a  

E-print Network

Adenovirus Papillomavirus Human Immunodeficiency Virus Influenza virus a b s t r a c t Like their cellular . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Association of papillomavirus genomes with host chromatin . . . . . . . . . . . . . . . . . . . .

Knipe, David M.


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

NASA Technical Reports Server (NTRS)

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

Zhang, Ye; Hada, Megumi; Wu, Honglu



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

PubMed Central

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

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



Condensation model for the ESBWR passive condensers  

SciTech Connect

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

Revankar, S. T. [Pohang Univ. of Science and Technology, 400 Central Drive, West Lafayette, IN 47906 (United States); Zhou, W.; Wolf, B.; Oh, S. [Purdue Univ., West Lafayette, IN 47906 (United States)



Histone acetyltransferase-dependent chromatin remodeling and the vascular clock.  


Rhythmic gene expression is central to the circadian control of physiology in mammals. Transcriptional activation of Per and Cry genes by heterodimeric bHLH-PAS proteins is a key event in the feedback loop that drives rhythmicity; however, the mechanism is not clearly understood. Here we show the transcriptional coactivators and histone acetyltransferases, p300/CBP, PCAF, and ACTR associate with the bHLH-PAS proteins, CLOCK and NPAS2, to regulate positively clock gene expression. Furthermore, Cry2 mediated repression of NPAS2:BMAL1 is overcome by overexpression of p300 in transactivation assays. Accordingly, p300 exhibits a circadian time-dependent association with NPAS2 in the vasculature, which precedes peak expression of target genes. In addition, a rhythm in core histone H3 acetylation on the mPer1 promoter in vivo correlates with the cyclical expression of their mRNAs. Temporal coactivator recruitment and HAT-dependent chromatin remodeling on the promoter of clock controlled genes in the vasculature permits the mammalian clock to orchestrate circadian gene expression. PMID:14645221

Curtis, Anne M; Seo, Sang-beom; Westgate, Elizabeth J; Rudic, Radu Daniel; Smyth, Emer M; Chakravarti, Debabrata; FitzGerald, Garret A; McNamara, Peter



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



Geothermal steam condensate reinjection  

Microsoft Academic Search

Geothermal electric generating plants which use condensing turbines and generate and excess of condensed steam which must be disposed of are discussed. At the Geysers, California, the largest geothermal development in the world, this steam condensate has been reinjected into the steam reservoir since 1968. A total of 3,150,000,000 gallons of steam condensate has been reinjected since that time with

A. J. Chasteen



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


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



Tau promotes neurodegeneration through global chromatin relaxation  

PubMed Central

The microtubule-associated protein tau is involved in a number of neurodegenerative disorders, including Alzheimer’s disease (AD). Previous studies link oxidative stress and subsequent DNA damage to neuronal death in AD and related tauopathies. Since DNA damage can significantly alter chromatin structure, we examined epigenetic changes in tau-induced neurodegeneration. We have found widespread loss of heterochromatin in tau transgenic Drosophila and mice, and in human AD. Importantly, genetic rescue of tau-induced heterochromatin loss substantially reduced neurodegeneration in Drosophila. We identified oxidative stress and subsequent DNA damage as a mechanistic link between transgenic tau expression and heterochromatin relaxation, and found that heterochromatin loss permits aberrant gene expression in tauopathies. Furthermore, large-scale analyses from human AD brains revealed a widespread transcriptional increase in genes that are heterochromatically silenced in controls. Our results establish heterochromatin loss as a toxic effector of tau-induced neurodegeneration, and identify chromatin structure as a potential therapeutic target in AD. PMID:24464041

Frost, Bess; Hemberg, Martin; Lewis, Jada; Feany, Mel B.



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



CHD chromatin remodelling enzymes and the DNA damage response.  


The protein and DNA complex known as chromatin is a dynamic structure, adapting to alter the spatial arrangement of genetic information within the nucleus to meet the ever changing demands of life. Following decades of research, a dizzying array of regulatory factors is now known to control the architecture of chromatin at nearly every level. Amongst these, ATP-dependent chromatin remodelling enzymes play a key role, required for the establishment, maintenance and re-organization of chromatin through their ability to adjust the contact points between DNA and histones, the spacing between individual nucleosomes and the over-arching chromatin superstructure. Utilizing energy from ATP hydrolysis, these enzymes serve as the gatekeepers of genomic access and are essential for transcriptional regulation, DNA replication and cell division. In recent years, a vital role in DNA Double Strand Break (DSB) repair has emerged, particularly within complex chromatin environments such as heterochromatin, or regions undergoing energetic transactions such as transcription or DNA replication. Here, we will provide an overview of what is understood about ATP-dependent chromatin remodelling enzymes in the context of the DNA damage response. We will first touch upon all four major chromatin remodelling enzyme families and then focus chiefly on the nine members of the Chromodomain, Helicase, DNA-binding (CHD) family, particularly CHD3, CHD4, CHD5 and CHD6. These four proteins have established and emerging roles in DNA repair, the oxidative stress response, the maintenance of genomic stability and/or cancer prevention. PMID:23954449

Stanley, Fintan K T; Moore, Shaun; Goodarzi, Aaron A



Cooperation between Complexes that Regulate Chromatin Structure and Transcription  

Microsoft Academic Search

Chromatin structure creates barriers for each step in eukaryotic transcription. Here we discuss how the activities of two major classes of chromatin-modifying complexes, ATP-dependent remodeling complexes and HAT or HDAC complexes, might be coordinated to create a DNA template that is accessible to the general transcription apparatus.

Geeta J. Narlikar; Hua-Ying Fan; Robert E. Kingston



Chromatin Immunoprecipitation (ChIPs) Protocol (Farnham Lab)  

E-print Network

and transfer to a new tube. Preclear chromatin by adding blocked Staph A cells. Use 10-15 uls of preblocked tube and divide equally among your samples. Be sure to include a "no antibody" sample. We also include a "mock" samples which contains 1X dialysis buffer instead of chromatin (no antibody and mock are critical

Gozani, Or


Chromatin remodelling: the industrial revolution of DNA around histones  

Microsoft Academic Search

Chromatin remodellers are specialized multi-protein machines that enable access to nucleosomal DNA by altering the structure, composition and positioning of nucleosomes. All remodellers have a catalytic ATPase subunit that is similar to known DNA-translocating motor proteins, suggesting DNA translocation as a unifying aspect of their mechanism. Here, we explore the diversity and specialization of chromatin remodellers, discuss how nucleosome modifications

Anjanabha Saha; Jacqueline Wittmeyer; Bradley R. Cairns



Histone H4 acetylation is essential to proceed from a histone- to a protamine-based chromatin structure in spermatid nuclei of Drosophila melanogaster.  


In humans, other mammals, and also in Drosophila, the paternal genome in the sperm is highly condensed and organized mainly in a protamine-based chromatin structure. However, the timing and mechanism of the switch from a histone- to the protamine-based chromatin configuration is still poorly understood. We therefore established Drosophila in vitro cultures of cysts with 64 synchronously developing spermatids genetically marked with histone H2AvD-RFP and ProtamineB-eGFP. Live cell imaging showed that the switch from H2AvD-RFP to Protamine-eGFP chromatin takes approximately five hours, with a short but clear overlap of the presence of both histones and protamines. Moreover, cultured pupal testes showed H4 hyperacetylation at the canoe stage shortly before histone removal; a feature previously observed in the intact animal. We then used TSA to inhibit histone deacetylation and found that premature hyperacetylation was already induced at the round nuclei stage of spermatids. However, this premature hyperacetylation did not lead to a premature switch to the protamine-based chromatin structure, showing that histone hyperacetylation is not the sole inducer of the histone to protamine switch. Importantly, we observed that inactivation of histone acetyltransferases by anacardic acid blocks further differentiation and thus prevents the degradation of histones and the switch to a protamine-based chromatin. Thus, we conclude that H4 hyperacetylation is an essential feature but not the sole inducer of the histone to protamine switch during spermiogenesis. PMID:20170286

Awe, Stephan; Renkawitz-Pohl, Renate



TM6, a novel nuclear matrix attachment region, enhances its flanking gene expression through influencing their chromatin structure.  


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



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. PMID:22459407

Bian, Qian



Connecting Chromatin Modifying Factors to DNA Damage Response  

PubMed Central

Cells are constantly damaged by factors that can induce DNA damage. Eukaryotic cells must rapidly load DNA repair proteins onto damaged chromatin during the DNA damage response (DDR). Chromatin-remodeling complexes use the energy from ATP hydrolysis to remodel nucleosomes and have well-established functions in transcription. Emerging lines of evidence indicate that chromatin-remodeling complexes are important and may remodel nucleosomes during DNA damage repair. New studies also reveal that ATP-dependent chromatin remodeling is involved in cell cycle progression, signal transduction pathways, and interaction and modification of DDR-related proteins that are specifically and intimately connected with the process of DNA damage. This article summarizes the recent advances in our understanding of the interplay between chromatin remodeling and DNA damage response. PMID:23348929

Lai, Weiwei; Li, Hongde; Liu, Shuang; Tao, Yongguang



Chromatin regulators of the AID induced DNA damage response  

PubMed Central

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

Daniel, Jeremy A.; Nussenzweig, Andre



Condensation system for power plant  

Microsoft Academic Search

A condensation system for use in a power plant including a steam generator and steam turbine comprises at least one side stream condenser. The side stream condenser defines therein first and second hot wells with the first hot well receiving therein condensate produced by condensing steam exhausted from the steam turbine. A condensate pump device forcibly delivers the condensate through

H. Ishimaru; T. Masuda; Y. Nagai



Chromatin Composition Is Changed by Poly(ADP-ribosyl)ation during Chromatin Immunoprecipitation  

PubMed Central

Chromatin-immunoprecipitation (ChIP) employs generally a mild formaldehyde cross-linking step, which is followed by isolation of specific protein-DNA complexes and subsequent PCR testing, to analyze DNA-protein interactions. Poly(ADP-ribosyl)ation, a posttranslational modification involved in diverse cellular functions like repair, replication, transcription, and cell death regulation, is most prominent after DNA damage. Poly(ADP-ribose)polymerase-1 is activated upon binding to DNA strand-breaks and coordinates repair by recruitment or displacement of proteins. Several proteins involved in different nuclear pathways are directly modified or contain poly(ADP-ribose)-interaction motifs. Thus, poly(ADP-ribose) regulates chromatin composition. In immunofluorescence experiments, we noticed artificial polymer-formation after formaldehyde-fixation of undamaged cells. Therefore, we analyzed if the formaldehyde applied during ChIP also induces poly(ADP-ribosyl)ation and its impact on chromatin composition. We observed massive polymer-formation in three different ChIP-protocols tested independent on the cell line. This was due to induction of DNA damage signaling as monitored by ?H2AX formation. To abrogate poly(ADP-ribose) synthesis, we inhibited this enzymatic reaction either pharmacologically or by increased formaldehyde concentration. Both approaches changed ChIP-efficiency. Additionally, we detected specific differences in promoter-occupancy of tested transcription factors as well as the in the presence of histone H1 at the respective sites. In summary, we show here that standard ChIP is flawed by artificial formation of poly(ADP-ribose) and suppression of this enzymatic activity improves ChIP-efficiency in general. Also, we detected specific changes in promoter-occupancy dependent on poly(ADP-ribose). By preventing polymer synthesis with the proposed modifications in standard ChIP protocols it is now possible to analyze the natural chromatin-composition. PMID:22479348

Beneke, Sascha; Meyer, Kirstin; Holtz, Anja; Huttner, Katharina; Burkle, Alexander



[Cytophotometric study of changes in the structure of nuclear chromatin induced by the oncogenic adenovirus SA7 (C8)].  


Infection of primary murine embryonic cell cultures by adenovirus SA7 (C8) results in an increase in chromatin condensation Average optical density of Feulgen stained nuclei 24 h following virus absorption increased for G0/1, S, and G2 cells by 16.1, 11.3 and 13.1%, respectively. This phenomenon is associated with the stimulation of proliferation, with an increase of the S cell amount by 50% of the control values and a decrease of average cell nuclei areas in all phases of cell cycle. PMID:6616062

Kotel'nikova, I N; Ageenko, A I; Khristenko, A V



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



SATB1-mediated Functional Packaging of Chromatin into Loops  

PubMed Central

Mammalian genomes are organized into multiple layers of higher-order chromatin structure, and in this organization chromatin looping is a striking and crucial feature that brings together distal genomic loci into close spatial proximity. Such three-dimensional organization of chromatin has been suggested to be functionally important in gene regulation. Many important questions need to be addressed, such as what types of nuclear proteins are responsible for folding chromatin into loops, whether there are any genomic marks that serve as the core sites of chromatin folding events, how distal genomic sites are brought together, and what are the biological consequences for interactions between distal genomic loci. In order to address these fundamental questions, it is essential to devise and employ methods that can capture higher-order structures formed by specific nuclear proteins at high resolution. In this article, in order to describe methods of analyzing protein-mediated chromatin interactions, we will use as an example a global genome-organizer protein, SATB1, which mediates chromatin looping. PMID:22782115

Kohwi-Shigematsu, Terumi; Kohwi, Yoshinori; Takahashi, Keiko; Richards, Hunter W.; Ayers, Stephen D.; Han, Hye-Jung; Cai, Shutao



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

PubMed Central

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

Mathe, Csaba; M-Hamvas, Marta; Vasas, Gabor



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



Beware of condenser fouling  

Microsoft Academic Search

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




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. PMID:22733402

Alper, Benjamin J.; Lowe, Brandon R.



Evidence supporting a biological role for aluminum in chromatin compaction and epigenetics.  


Averaging 8.1% (w/w) of the earth's crust, aluminum is the most highly abundant metal in our biosphere, yet has long been thought to serve no essential biological function. In aqueous solutions, aluminum salts and hydroxides are exceptionally potent aggregators of biological molecules, often coalescing molecular species to the point that they precipitate out of solution. A biological function for aluminum is proposed in which this abundant, high charge density metal cation has a significant role in biomolecular compaction. Sometimes, molecules ectopically aggregated by aluminum are associated with pathological conditions. The data further suggests that a specific consequence of 'aluminum biocompaction' may be particularly important in the condensation of A+T-rich chromatin domains, and in silencing the expression of specific kinds of genetic information. PMID:20684046

Lukiw, Walter J



Evaluation of genetic damage in open-cast coal mine workers using the buccal micronucleus cytome assay.  


Coal is the largest fossil fuel source used for the generation of energy. However, coal extraction and its use constitute important pollution factors; thus, risk characterization and estimation are extremely important for the safety of coal workers and the environment. Candiota is located to the southeast of the state of Rio Grande do Sul and has the largest coal reserves in Brazil, and the largest thermal power complex in the state. In the open-cast mines, the coal miners are constantly exposed to coal dust. The human buccal micronucleus cytome (BMCyt) assay has been used widely to investigate biomarkers for DNA damage, cell death, and basal cell frequency in buccal cells. The aim of this study was to assess whether prolonged exposure to coal dust could lead to an increase in genomic instability, cell death, and frequency of basal cells using the BMCyt assay. In the analysis of epithelial cells, the exposed group (n = 41) presented with a significantly higher frequency of basal cells, micronuclei in basal and differentiated cells, and binucleated cells compared to the non-exposed group (n = 29). The exposed group showed a significantly lower frequency of condensed chromatin cells than the non-exposed group. However, we found no correlation between DNA damage and metal concentration in the blood of mine workers. DNA damage observed in the mine workers may be a consequence of oxidative damage resulting from exposure to coal residue mixtures. In addition, our findings confirm that the BMCyt assay can be used to identify occupational risk. PMID:23055270

Rohr, Paula; da Silva, Juliana; da Silva, Fernanda R; Sarmento, Merielen; Porto, Carem; Debastiani, Rafaela; Dos Santos, Carla E I; Dias, Johnny F; Kvitko, Kátia



FXR mediates a chromatin looping in the GR promoter thus promoting the resolution of colitis in rodents.  


Glucocorticoids (GCs) are important endocrine regulators of a wide range of physiological processes ranging from immune function to glucose and lipid metabolism. For decades, synthetic glucocorticoids such as dexamethasone have been the cornerstone for the clinical treatment of inflammatory bowel diseases (IBD). A previous study has shown that farnesoid X receptor (FXR) enhances the transcription of NR3C1 gene, which encodes for human GR, by binding to a conserved FXR response element (FXRE) in the distal promoter of this gene. In the present study we demonstrate that FXR promotes the resolution of colitis in rodents by enhancing Gr gene transcription. We used the chromatin conformation capture (3C) assay to demonstrate that this FXRE is functional in mediating a head-to-tail chromatin looping, thus increasing Gr transcription efficiency. These findings underscore the importance of FXR/GR axis in the control of intestinal inflammation. PMID:24004655

Renga, Barbara; D'Amore, Claudio; Cipriani, Sabrina; Mencarelli, Andrea; Carino, Adriana; Sepe, Valentina; Zampella, Angela; Distrutti, Eleonora; Fiorucci, Stefano



Chromatin is a Complex of whose Structure is Only Visible During Mitosis in Eukaryotic Cells  

E-print Network

Chromatin Chromatin is a Complex of whose Structure is Only Visible During Mitosis in Eukaryotic Cells Chromatin is Comprised of Tightly which are Uncoiled at the End of Mitosis and Disappear During Chromatin During Mitosis is the Result of Contraction of in Length of the Chromosomal DNA The Organization

Cutler, Chris


Mechanisms and Functions of ATP-Dependent Chromatin-Remodeling Enzymes  

PubMed Central

Chromatin provides both a means to accommodate a large amount of genetic material in a small space and a means to package the same genetic material in different chromatin states. Transitions between chromatin states are enabled by chromatin-remodeling ATPases, which catalyze a diverse range of structural transformations. Biochemical evidence over the last two decades suggests that chromatin-remodeling activities may have emerged by adaptation of ancient DNA translocases to respond to specific features of chromatin. Here, we discuss such evidence and also relate mechanistic insights to our understanding of how chromatin-remodeling enzymes enable different in vivo processes. PMID:23911317

Narlikar, Geeta J.; Sundaramoorthy, Ramasubramanian; Owen-Hughes, Tom



p53 and the PWWP domain containing effector proteins in chromatin damage repair  

PubMed Central

In eukaryotic cells, DNA damage repair occurs on a template DNA that is organized with histones to form nucleosomes and chromatin structures. As such, chromatin plays an important role in DNA damage repair. In this review, we will use “chromatin damage repair” as a framework and highlight recent progress in understanding the role of chromatin, chromatin modifiers, chromatin binding effectors (e.g., the PWWP domain proteins), and the p53 tumor suppressor. We view chromatin as an active participant during DNA damage repair.

Hu, Jing; Wang, Yanming



Evaluation of DNA fragmentation in llama (Lama glama) sperm using the sperm chromatin dispersion test.  


The integrity of sperm chromatin is now viewed as an important factor in male fertility and in early embryonic development. The objectives of this study were: (1) adapt the simple and inexpensive sperm chromatin dispersion (SCD) test to evaluate DNA fragmentation in llama sperm and establish the halo patterns observed in this species, (2) determine an effective and reliable positive control for this technique and (3) evaluate correlation between the SCD test and the toluidine blue (TB) stain. To adapt the SCD test, three different mercaptoethanol (ME) concentrations were assayed (2.5%, 5% and 10% ME). To determine an effective positive control, three treatments (incubation at 100 °C for 30 min, incubation with 0.3 M NaOH for 30 min at room temperature and exposure to UV light for 2h) were assayed. The concentration selected to use in the SCD test was 5% ME, because it produced the largest halo while still conserving the structure of the core. Four DNA dispersion patterns were clearly observed: (I) nuclei with large DNA dispersion halos; (II) nuclei with medium halos; (III) nuclei with very small halos and (IV) nuclei with no halo. All treatments used as positive controls were effective in producing DNA fragmentation. A high correlation (r=0.84, P=0.03) was observed between spermatozoa without halos and TB positive cells. To conclude, SCD patterns in llama sperm have been established as well as a repeatable positive control for the assay. The SCD test and TB stain are simple and inexpensive techniques that can be used to evaluate DNA damage in llama sperm. PMID:22437148

Carretero, M I; Lombardo, D; Arraztoa, C C; Giuliano, S M; Gambarotta, M C; Neild, D M



Integrative annotation of chromatin elements from ENCODE data  

E-print Network

The ENCODE Project has generated a wealth of experimental information mapping diverse chromatin properties in several human cell lines. Although each such data track is independently informative toward the annotation of ...

Ernst, Jason


Insights into Chromatin Structure and Dynamics in Plants  

PubMed Central

The packaging of chromatin into the nucleus of a eukaryotic cell requires an extraordinary degree of compaction and physical organization. In recent years, it has been shown that this organization is dynamically orchestrated to regulate responses to exogenous stimuli as well as to guide complex cell-type-specific developmental programs. Gene expression is regulated by the compartmentalization of functional domains within the nucleus, by distinct nucleosome compositions accomplished via differential modifications on the histone tails and through the replacement of core histones by histone variants. In this review, we focus on these aspects of chromatin organization and discuss novel approaches such as live cell imaging and photobleaching as important tools likely to give significant insights into our understanding of the very dynamic nature of chromatin and chromatin regulatory processes. We highlight the contribution plant studies have made in this area showing the potential advantages of plants as models in understanding this fundamental aspect of biology. PMID:24833230

Rosa, Stefanie; Shaw, Peter



Chromatin remodeler mutations in human cancers: epigenetic implications.  


Chromatin remodeler complexes exhibit the ability to alter nucleosome composition and positions, with seemingly divergent roles in the regulation of chromatin architecture and gene expression. The outcome is directed by subunit variation and interactions with accessory factors. Recent studies have revealed that subunits of chromatin remodelers display an unexpectedly high mutation rate and/or are inactivated in a number of cancers. Consequently, a repertoire of epigenetic processes are likely to be affected, including interactions with histone modifying factors, as well as the ability to precisely modulate nucleosome positions, DNA methylation patterns and potentially, higher-order genome structure. However, the true significance of chromatin remodeler genetic aberrations in promoting a cascade of epigenetic changes, particularly during initiation and progression of cancer, remains largely unknown. PMID:25333849

Skulte, Katherine A; Phan, Lisa; Clark, Susan J; Taberlay, Phillippa C



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


The centromere is a specialized chromosomal region that serves as the assembly site of the kinetochore. At the centromere, CENP-A nucleosomes form part of a chromatin landscape termed centrochromatin. This chromatin environment conveys epigenetic marks regulating kinetochore formation. Recent work sheds light on the intricate relationship between centrochromatin state, the CENP-A assembly pathway and the maintenance of centromere function. Here, we review the emerging picture of how chromatin affects mammalian kinetochore formation. We place particular emphasis on data obtained from Human Artificial Chromosome (HAC) biology and the targeted engineering of centrochromatin using synthetic HACs. We discuss implications of these findings, which indicate that a delicate balance of histone modifications and chromatin state dictates both de novo centromere formation and the maintenance of centromere identity in dividing cell populations. PMID:22825423

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



Insights into chromatin structure and dynamics in plants.  


The packaging of chromatin into the nucleus of a eukaryotic cell requires an extraordinary degree of compaction and physical organization. In recent years, it has been shown that this organization is dynamically orchestrated to regulate responses to exogenous stimuli as well as to guide complex cell-type-specific developmental programs. Gene expression is regulated by the compartmentalization of functional domains within the nucleus, by distinct nucleosome compositions accomplished via differential modifications on the histone tails and through the replacement of core histones by histone variants. In this review, we focus on these aspects of chromatin organization and discuss novel approaches such as live cell imaging and photobleaching as important tools likely to give significant insights into our understanding of the very dynamic nature of chromatin and chromatin regulatory processes. We highlight the contribution plant studies have made in this area showing the potential advantages of plants as models in understanding this fundamental aspect of biology. PMID:24833230

Rosa, Stefanie; Shaw, Peter



KAT5 tyrosine phosphorylation couples chromatin sensing to ATM signalling.  


The detection of DNA lesions within chromatin represents a critical step in cellular responses to DNA damage. However, the regulatory mechanisms that couple chromatin sensing to DNA-damage signalling in mammalian cells are not well understood. Here we show that tyrosine phosphorylation of the protein acetyltransferase KAT5 (also known as TIP60) increases after DNA damage in a manner that promotes KAT5 binding to the histone mark H3K9me3. This triggers KAT5-mediated acetylation of the ATM kinase, promoting DNA-damage-checkpoint activation and cell survival. We also establish that chromatin alterations can themselves enhance KAT5 tyrosine phosphorylation and ATM-dependent signalling, and identify the proto-oncogene c-Abl as a mediator of this modification. These findings define KAT5 tyrosine phosphorylation as a key event in the sensing of genomic and chromatin perturbations, and highlight a key role for c-Abl in such processes. PMID:23708966

Kaidi, Abderrahmane; Jackson, Stephen P



From neural development to cognition: unexpected roles for chromatin  

PubMed Central

Recent genome-sequencing studies in human neurodevelopmental and psychiatric disorders have uncovered mutations in many chromatin regulators. These human genetic studies, along with studies in model organisms, are providing insight into chromatin regulatory mechanisms in neural development and how alterations to these mechanisms can cause cognitive deficits, such as intellectual disability. We discuss several implicated chromatin regulators, including BAF (also known as SWI/SNF) and CHD8 chromatin remodellers, HDAC4 and the Polycomb component EZH2. Interestingly, mutations in EZH2 and certain BAF complex components have roles in both neurodevelopmental disorders and cancer, and overlapping point mutations are suggesting functionally important residues and domains. We speculate on the contribution of these similar mutations to disparate disorders. PMID:23568486

Ronan, Jehnna L.; Wu, Wei



ChIP-less analysis of chromatin states  

PubMed Central

Background Histone post-translational modifications (PTMs) are key epigenetic regulators in chromatin-based processes. Increasing evidence suggests that vast combinations of PTMs exist within chromatin histones. These complex patterns, rather than individual PTMs, are thought to define functional chromatin states. However, the ability to interrogate combinatorial histone PTM patterns at the nucleosome level has been limited by the lack of direct molecular tools. Results Here we demonstrate an efficient, quantitative, antibody-free, chromatin immunoprecipitation-less (ChIP-less) method for interrogating diverse epigenetic states. At the heart of the workflow are recombinant chromatin reader domains, which target distinct chromatin states with combinatorial PTM patterns. Utilizing a newly designed combinatorial histone peptide microarray, we showed that three reader domains (ATRX-ADD, ING2-PHD and AIRE-PHD) displayed greater specificity towards combinatorial PTM patterns than corresponding commercial histone antibodies. Such specific recognitions were employed to develop a chromatin reader-based affinity enrichment platform (matrix-assisted reader chromatin capture, or MARCC). We successfully applied the reader-based platform to capture unique chromatin states, which were quantitatively profiled by mass spectrometry to reveal interconnections between nucleosomal histone PTMs. Specifically, a highly enriched signature that harbored H3K4me0, H3K9me2/3, H3K79me0 and H4K20me2/3 within the same nucleosome was identified from chromatin enriched by ATRX-ADD. This newly reported PTM combination was enriched in heterochromatin, as revealed by the associated DNA. Conclusions Our results suggest the broad utility of recombinant reader domains as an enrichment tool specific to combinatorial PTM patterns, which are difficult to probe directly by antibody-based approaches. The reader affinity platform is compatible with several downstream analyses to investigate the physical coexistence of nucleosomal PTM states associated with specific genomic loci. Collectively, the reader-based workflow will greatly facilitate our understanding of how distinct chromatin states and reader domains function in gene regulatory mechanisms. PMID:24872844



HMG Nuclear Proteins: Linking Chromatin Structure to Cellular Phenotype  

PubMed Central

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

Reeves, Raymond



Understanding chromatin and chromosomes: from static views to dynamic thinking  

PubMed Central

The 106th Boehringer Ingelheim Fonds International Titisee Conference, ‘Reconstituting Chromatin: From Self-assembly to Self-organization', took place in October 2012. The organizers, Andrea Musacchio and Tom Muir, brought together biologists, chemists and physicists to discuss the principles of chromosome assembly and organization. Topics of discussion ranged from new insights gained from the static views provided by crystal structures to analyses of chromatin dynamics inside living cells. PMID:23318629

Haering, Christian H; Losada, Ana



Chromatin dynamics at the Saccharomyces cerevisiae PHO5 promoter  

E-print Network

Neighboring UASp1 Persists at Extended Times of Activation??????????????... 112 4-5 Chromatin Remodeling of the Wild-Type PHO5 Promoter Spreads from UASp1??????????????????????... 115 4-6 Activation Localizes SWI/SNF Preferentially at the UAS... propagation of chromatin remodeling from enhancer-bound activators, and (ii) that interaction of SWI/SNF with the promoter localizes to the enhancer region. SIGNIFICANCE Early studies of the mechanisms by which activator proteins stimulate transcription...

Jessen, Walter Joseph



Clinical Assay Development Program (CADP)

Skip to Content Search this site Clinical Assay Development Program (CADP) Do you need: Advice on further development of a cancer diagnostics assay? Assay optimization? Design or implementation of assay controls, assay standards or assay calibrators? Determination


Chromatin modifications and the DNA damage response to ionizing radiation  

PubMed Central

In order to survive, cells have evolved highly effective repair mechanisms to deal with the potentially lethal DNA damage produced by exposure to endogenous as well as exogenous agents. Ionizing radiation exposure induces highly lethal DNA damage, especially DNA double-strand breaks (DSBs), that is sensed by the cellular machinery and then subsequently repaired by either of two different DSB repair mechanisms: (1) non-homologous end joining, which re-ligates the broken ends of the DNA and (2) homologous recombination, that employs an undamaged identical DNA sequence as a template, to maintain the fidelity of DNA repair. Repair of DSBs must occur within the natural context of the cellular DNA which, along with specific proteins, is organized to form chromatin, the overall structure of which can impede DNA damage site access by repair proteins. The chromatin complex is a dynamic structure and is known to change as required for ongoing cellular processes such as gene transcription or DNA replication. Similarly, during the process of DNA damage sensing and repair, chromatin needs to undergo several changes in order to facilitate accessibility of the repair machinery. Cells utilize several factors to modify the chromatin in order to locally open up the structure to reveal the underlying DNA sequence but post-translational modification of the histone components is one of the primary mechanisms. In this review, we will summarize chromatin modifications by the respective chromatin modifying factors that occur during the DNA damage response. PMID:23346550

Kumar, Rakesh; Horikoshi, Nobuo; Singh, Mayank; Gupta, Arun; Misra, Hari S.; Albuquerque, Kevin; Hunt, Clayton R.; Pandita, Tej K.



A chromatin remodelling complex that loads cohesin onto human chromosomes  

NASA Astrophysics Data System (ADS)

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

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



Environmental-stress-induced Chromatin Regulation and its Heritability  

PubMed Central

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

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



Deficiency of the multi-copy mouse Y gene Sly causes sperm DNA damage and abnormal chromatin packaging  

PubMed Central

Summary In mouse and man Y chromosome deletions are frequently associated with spermatogenic defects. Mice with extensive deletions of non-pairing Y chromosome long arm (NPYq) are infertile and produce sperm with grossly misshapen heads, abnormal chromatin packaging and DNA damage. The NPYq-encoded multi-copy gene Sly controls the expression of sex chromosome genes after meiosis and Sly deficiency results in a remarkable upregulation of sex chromosome genes. Sly deficiency has been shown to be the underlying cause of the sperm head anomalies and infertility associated with NPYq gene loss, but it was not known whether it recapitulates sperm DNA damage phenotype. We produced and examined mice with transgenically (RNAi) silenced Sly and demonstrated that these mice have increased incidence of sperm with DNA damage and poorly condensed and insufficiently protaminated chromatin. We also investigated the contribution of each of the two Sly-encoded transcript variants and noted that the phenotype was only observed when both variants were knocked down, and that the phenotype was intermediate in severity compared with mice with severe NPYq deficiency. Our data demonstrate that Sly deficiency is responsible for the sperm DNA damage/chromatin packaging defects observed in mice with NPYq deletions and point to SLY proteins involvement in chromatin reprogramming during spermiogenesis, probably through their effect on the post-meiotic expression of spermiogenic genes. Considering the importance of the sperm epigenome for embryonic and fetal development and the possibility of its inter-generational transmission, our results are important for future investigations of the molecular mechanisms of this biologically and clinically important process. PMID:23178944

Riel, Jonathan M.; Yamauchi, Yasuhiro; Sugawara, Atsushi; Li, Ho Yan J.; Ruthig, Victor; Stoytcheva, Zoia; Ellis, Peter J. I.; Cocquet, Julie; Ward, Monika A.




EPA Science Inventory

Home semen collection kits allow men to collect a sample at their convenience and send it via overnight mail to the laboratory. Benefits of this approach include facilitated sample collection from different geographic locations, minimized variability through analysis by a central...


Epigenetics and cancer: altered chromatin remodeling via Snf5 loss leads to aberrant cell cycle regulation.  


The term 'epigenetics' refers to heritable changes in gene function that occur in the absence of any change in DNA sequence. Perturbations of epigenetic gene regulation may play a critical role in the genesis of most, if not all, cancers. These alterations include changes in covalent modifications of DNA and histones as well as non covalent changes in nucleosome positioning. Covalent epigenetic modifications have been the main focus of cancer investigation, perhaps because they are more readily assayed and understood than non covalent modifications. Recently, evidence has emerged demonstrating that perturbation of complexes that remodel the structure of chromatin by mobilizing nucleosomes may have a key role in tumor suppression and oncogenic transformation. For example, Snf5 (Ini1/Baf47/Smarcb1), a core component of the Swi/Snf ATPase chromatin remodeling complex, is a potent tumor suppressor that is specifically inactivated in lethal childhood cancers. Notably, these cancers may serve as a paradigm for epigenetic cancers as, despite their extremely aggressive nature, the majority have an entirely normal karyotype with only microdeletions at the Snf5 locus. Recent investigations have shed light upon the mechanistic basis of Snf5 function by demonstrating that Snf5 and the Swi/Snf complex regulate the cell cycle and cooperate with p53 to prevent oncogenic transformation. PMID:16582616

Sansam, Courtney G; Roberts, Charles W M



Direct study of DNA-protein interactions in repressed and active chromatin in living cells.  

PubMed Central

Current methods for analysis of chromatin architecture are invasive, utilizing chemicals or nucleases that damage DNA, making detection of labile constituents and conclusions about true in vivo structure problematic. We describe a sensitive assay of chromatin structure which is performed in intact, living yeast. The approach utilizes expression of SssI DNA methyltransferase (MTase) in Saccharomyces cerevisiae to provide an order-of-magnitude increase in resolution over previously introduced MTases. Combining this resolution increase with the novel application of a PCR-based, positive chemical display of modified cytosines provides a significant advance in the direct study of DNA-protein interactions in growing cells that enables quantitative footprinting. The validity and efficacy of the strategy are demonstrated in mini-chromosomes, where positioned nucleosomes and a labile, operator-bound repressor are detected. Also, using a heterologous system to study gene activation, we show that in vivo hormone occupancy of the estrogen receptor is required for maximal site-specific DNA binding, whereas, at very high receptor-expression levels, hormone-independent partial occupancy of an estrogen-responsive element was observed. Receptor binding to a palindromic estrogen-responsive element leads to a footprint with strand-specific asymmetry, which is explicable by known structural information. Images PMID:8947052

Kladde, M P; Xu, M; Simpson, R T



Developmentally stable chromatin structure in the human. beta. -globin gene cluster  

SciTech Connect

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

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



Screening for Inhibitors of an Essential Chromatin Remodeler in Mouse Embryonic Stem Cells by Monitoring Transcriptional Regulation  

PubMed Central

The SWI/SNF-like adenosine triphosphate (ATP)–dependent chromatin remodeling complex, esBAF, is both necessary and, in some contexts, sufficient to induce the pluripotent state. Furthermore, mutations in various BAF subunits are associated with cancer. Little is known regarding the precise mechanism(s) by which this complex exerts its activities. Thus, it is unclear which protein interactions would be important to disrupt to isolate a relevant readout of mechanism. To address this, we developed a gene expression–based assay to identify inhibitors of the native esBAF complex. Specifically, a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay was developed in mouse embryonic stem (ES) cells to monitor expression of Bmi1, a developmentally important gene repressed by the esBAF complex. The assay was miniaturized to a 384-well format and used to screen a diverse collection of compounds, including novel products of diversity-oriented synthesis (DOS). Confirmed hits were validated using a knock-in ES cell reporter line in which luciferase is inserted into the Bmi1 locus. Several of the validated hits regulate a panel of target genes in a manner similar to the BAF chromatin-remodeling complex. Together these data indicate that expression-based screening using qRT-PCR is a successful approach to identify compounds targeting the regulation of key developmental genes in ES cells. PMID:22853929

Dykhuizen, Emily C.; Carmody, Leigh C.; Tolliday, Nicola; Crabtree, Gerald R.; Palmer, Michelle A. J.



Economical Condensing Turbines?  

E-print Network

Economical Condensing Turbines? by J.E.Dean, P.E. Steam turbines have long been used at utilities and in industry to generate power. There are three basic types of steam turbines: condensing, letdown 1 and extraction/condensing. ? Letdown... turbines reduce the pressure of the incoming steam to one or more pressures and generate power very efficiently, assuming that all the letdown steam has a use. Two caveats: ? Letdown turbines produce power based upon steam requirements and not based upon...

Dean, J. E.


Quark Condensates: Flavour Dependence  

E-print Network

We determine the q-bar q condensate for quark masses from zero up to that of the strange quark within a phenomenologically successful modelling of continuum QCD by solving the quark Schwinger-Dyson equation. The existence of multiple solutions to this equation is the key to an accurate and reliable extraction of this condensate using the operator product expansion. We explain why alternative definitions fail to give the physical condensate.

R. Williams; C. S. Fischer; M. R. Pennington



Fibronectin matrix assembly is essential for cell condensation during chondrogenesis.  


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

Singh, Purva; Schwarzbauer, Jean E



The NuRD Chromatin-Remodeling Enzyme CHD4 Promotes Embryonic Vascular Integrity by Transcriptionally Regulating Extracellular Matrix Proteolysis  

PubMed Central

The extracellular matrix (ECM) supports vascular integrity during embryonic development. Proteolytic degradation of ECM components is required for angiogenesis, but excessive ECM proteolysis causes blood vessel fragility and hemorrhage. Little is understood about how ECM proteolysis is transcriptionally regulated during embryonic vascular development. We now show that the NuRD ATP-dependent chromatin-remodeling complex promotes vascular integrity by preventing excessive ECM proteolysis in vivo. Mice lacking endothelial CHD4—a catalytic subunit of NuRD complexes—died at midgestation from vascular rupture. ECM components surrounding rupture-prone vessels in Chd4 mutants were significantly downregulated prior to embryonic lethality. Using qPCR arrays, we found two critical mediators of ECM stability misregulated in mutant endothelial cells: the urokinase-type plasminogen activator receptor (uPAR or Plaur) was upregulated, and thrombospondin-1 (Thbs1) was downregulated. Chromatin immunoprecipitation assays showed that CHD4-containing NuRD complexes directly bound the promoters of these genes in endothelial cells. uPAR and THBS1 respectively promote and inhibit activation of the potent ECM protease plasmin, and we detected increased plasmin activity around rupture-prone vessels in Chd4 mutants. We rescued ECM components and vascular rupture in Chd4 mutants by genetically reducing urokinase (uPA or Plau), which cooperates with uPAR to activate plasmin. Our findings provide a novel mechanism by which a chromatin-remodeling enzyme regulates ECM stability to maintain vascular integrity during embryonic development. PMID:24348274

Ingram, Kyle G.; Curtis, Carol D.; Silasi-Mansat, Robert; Lupu, Florea; Griffin, Courtney T.



The NuRD chromatin-remodeling enzyme CHD4 promotes embryonic vascular integrity by transcriptionally regulating extracellular matrix proteolysis.  


The extracellular matrix (ECM) supports vascular integrity during embryonic development. Proteolytic degradation of ECM components is required for angiogenesis, but excessive ECM proteolysis causes blood vessel fragility and hemorrhage. Little is understood about how ECM proteolysis is transcriptionally regulated during embryonic vascular development. We now show that the NuRD ATP-dependent chromatin-remodeling complex promotes vascular integrity by preventing excessive ECM proteolysis in vivo. Mice lacking endothelial CHD4--a catalytic subunit of NuRD complexes--died at midgestation from vascular rupture. ECM components surrounding rupture-prone vessels in Chd4 mutants were significantly downregulated prior to embryonic lethality. Using qPCR arrays, we found two critical mediators of ECM stability misregulated in mutant endothelial cells: the urokinase-type plasminogen activator receptor (uPAR or Plaur) was upregulated, and thrombospondin-1 (Thbs1) was downregulated. Chromatin immunoprecipitation assays showed that CHD4-containing NuRD complexes directly bound the promoters of these genes in endothelial cells. uPAR and THBS1 respectively promote and inhibit activation of the potent ECM protease plasmin, and we detected increased plasmin activity around rupture-prone vessels in Chd4 mutants. We rescued ECM components and vascular rupture in Chd4 mutants by genetically reducing urokinase (uPA or Plau), which cooperates with uPAR to activate plasmin. Our findings provide a novel mechanism by which a chromatin-remodeling enzyme regulates ECM stability to maintain vascular integrity during embryonic development. PMID:24348274

Ingram, Kyle G; Curtis, Carol D; Silasi-Mansat, Robert; Lupu, Florea; Griffin, Courtney T



Caspase-activated DNase is necessary and sufficient for oligonucleosomal DNA breakdown, but not for chromatin disassembly during caspase-dependent apoptosis of LN-18 glioblastoma cells.  


Caspase-dependent apoptosis is a controlled type of cell death characterized by oligonucleosomal DNA breakdown and major nuclear morphological alterations. Other kinds of cell death do not share these highly distinctive traits because caspase-activated DNase (DFF40/CAD) remains inactive. Here, we report that human glioblastoma multiforme-derived LN-18 cells do not hydrolyze DNA into oligonucleosomal fragments after apoptotic insult. Furthermore, their chromatin remains packaged into a single mass, with no signs of nuclear fragmentation. However, ultrastructural analysis reveals that nuclear disassembly occurs, although compacted chromatin does not localize into apoptotic nuclear bodies. Caspases become properly activated, and ICAD, the inhibitor of DFF40/CAD, is correctly processed. Using cell-free in vitro assays, we show that chromatin from isolated nuclei of LN-18 cells is suitable for hydrolysis into oligonuclesomal fragments by staurosporine-pretreated SH-SY5Y cytoplasms. However, staurosporine-pretreated LN-18 cytoplasms do not induce DNA laddering in isolated nuclei from either LN-18 or SH-SY5Y cells because LN-18 cells express lower amounts of DFF40/CAD. DFF40/CAD overexpression makes LN-18 cells fully competent to degrade their DNA into oligonucleosome-sized fragments, and yet they remain unable to arrange their chromatin into nuclear clumps after apoptotic insult. Indeed, isolated nuclei from LN-18 cells were resistant to undergoing apoptotic nuclear morphology in vitro. The use of LN-18 cells has uncovered a previously unsuspected cellular model, whereby a caspase-dependent chromatin package is DFF40/CAD-independent, and DFF40/CAD-mediated double-strand DNA fragmentation does not warrant the distribution of the chromatin into apoptotic nuclear bodies. The studies highlight a not-yet reported DFF40/CAD-independent mechanism driving conformational nuclear changes during caspase-dependent cell death. PMID:24838313

Sánchez-Osuna, María; Garcia-Belinchón, Mercè; Iglesias-Guimarais, Victoria; Gil-Guiñón, Estel; Casanelles, Elisenda; Yuste, Victor J



Condensation Energy of a Spacetime Condensate  

E-print Network

Starting from an analogy between the Planck-Einstein scale and the dual length scales in Ginzburg-Landau theory of superconductivity, and assuming that space-time is a condensate of neutral fermionic particles with Planck mass, we derive the baryonic mass of the universe. In that theoretical framework baryonic matter appears to be associated with the condensation energy gained by spacetime in the transition from its normal (symetric) to its (less symetric) superconducting-like phase. It is shown however that the critical transition temperature cannot be the Planck temperature. Thus leaving open the enigma of the microscopic description of spacetime at quantum level.

Clovis Jacinto de Matos; Pavol Valko



Chromatin is an ancient innovation conserved between Archaea and Eukarya  

PubMed Central

The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps ?147 bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have historically been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-depleted regions at transcriptional start sites and conserved ?1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient. DOI: PMID:23240084

Ammar, Ron; Torti, Dax; Tsui, Kyle; Gebbia, Marinella; Durbic, Tanja; Bader, Gary D; Giaever, Guri; Nislow, Corey



Chromatin modification and remodeling during early seed development.  


Seed development starts at double fertilization when two sperms fuse with a female gamete, the egg and central cell, giving rise to the embryo and endosperm, respectively. Uniting two parental genomes into one, unique, zygotic genome is certainly the first event requiring large-scale chromatin modifications and remodeling. Although little is known about the molecular mechanisms, recent progress was made allowing live imaging of parental chromatin dynamics at fertilization. Further growth and patterning processes will shape the future plant seedling and its surrounding nurse tissue. Studies over the last decade identified key chromatin modifiers involved in these processes. However, the dynamics of these modifications mediated, in particular, by the Polycomb group complexes only start to be understood. The precise molecular mechanisms altering chromatin state in relation to early seed development remains difficult to address because of the relative inaccessibility of the fertilization products. Nonetheless, with the emergence of in vivo imaging techniques, laser-capture dissection, and genome-wide chromatin modification profiling, the future promises new, exciting discoveries. PMID:18029170

Baroux, Célia; Pien, Stéphane; Grossniklaus, Ueli



Recovering ensembles of chromatin conformations from contact probabilities  

PubMed Central

The 3D higher order organization of chromatin within the nucleus of eukaryotic cells has so far remained elusive. A wealth of relevant information, however, is increasingly becoming available from chromosome conformation capture (3C) and related experimental techniques, which measure the probabilities of contact between large numbers of genomic sites in fixed cells. Such contact probabilities (CPs) can in principle be used to deduce the 3D spatial organization of chromatin. Here, we propose a computational method to recover an ensemble of chromatin conformations consistent with a set of given CPs. Compared with existing alternatives, this method does not require conversion of CPs to mean spatial distances. Instead, we estimate CPs by simulating a physically realistic, bead-chain polymer model of the 30-nm chromatin fiber. We then use an approach from adaptive filter theory to iteratively adjust the parameters of this polymer model until the estimated CPs match the given CPs. We have validated this method against reference data sets obtained from simulations of test systems with up to 45 beads and 4 loops. With additional testing against experiments and with further algorithmic refinements, our approach could become a valuable tool for researchers examining the higher order organization of chromatin. PMID:23143266

Meluzzi, Dario; Arya, Gaurav



Dissecting the chromatin interactome of microRNA genes.  


Our knowledge of the role of higher-order chromatin structures in transcription of microRNA genes (MIRs) is evolving rapidly. Here we investigate the effect of 3D architecture of chromatin on the transcriptional regulation of MIRs. We demonstrate that MIRs have transcriptional features that are similar to protein-coding genes. RNA polymerase II-associated ChIA-PET data reveal that many groups of MIRs and protein-coding genes are organized into functionally compartmentalized chromatin communities and undergo coordinated expression when their genomic loci are spatially colocated. We observe that MIRs display widespread communication in those transcriptionally active communities. Moreover, miRNA-target interactions are significantly enriched among communities with functional homogeneity while depleted from the same community from which they originated, suggesting MIRs coordinating function-related pathways at posttranscriptional level. Further investigation demonstrates the existence of spatial MIR-MIR chromatin interacting networks. We show that groups of spatially coordinated MIRs are frequently from the same family and involved in the same disease category. The spatial interaction network possesses both common and cell-specific subnetwork modules that result from the spatial organization of chromatin within different cell types. Together, our study unveils an entirely unexplored layer of MIR regulation throughout the human genome that links the spatial coordination of MIRs to their co-expression and function. PMID:24357409

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



Chromatin modifications and DNA repair: beyond double-strand breaks  

PubMed Central

DNA repair must take place in the context of chromatin, and chromatin modifications and DNA repair are intimately linked. The study of double-strand break repair has revealed numerous histone modifications that occur after induction of a DSB, and modification of the repair factors themselves can also occur. In some cases the function of the modification is at least partially understood, but in many cases it is not yet clear. Although DSB repair is a crucial activity for cell survival, DSBs account for only a small percentage of the DNA lesions that occur over the lifetime of a cell. Repair of single-strand gaps, nicks, stalled forks, alternative DNA structures, and base lesions must also occur in a chromatin context. There is increasing evidence that these repair pathways are also regulated by histone modifications and chromatin remodeling. In this review, we will summarize the current state of knowledge of chromatin modifications that occur during non-DSB repair, highlighting similarities and differences to DSB repair as well as remaining questions. PMID:25250043

House, Nealia C. M.; Koch, Melissa R.; Freudenreich, Catherine H.



Histone modifications and chromatin dynamics: a focus on filamentous fungi  

PubMed Central

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

Brosch, Gerald; Loidl, Peter; Graessle, Stefan



Chromatin: a tunable spring at work inside chromosomes  

E-print Network

This paper focuses on mechanical aspects of chromatin biological functioning. Within a basic geometric modeling of the chromatin assembly, we give for the first time the complete set of elastic constants (twist and bend persistence lengths, stretch modulus and twist-stretch coupling constant) of the so-called 30-nm chromatin fiber, in terms of DNA elastic properties and geometric properties of the fiber assembly. The computation naturally embeds the fiber within a current analytical model known as the ``extensible worm-like rope'', allowing a straightforward prediction of the force-extension curves. We show that these elastic constants are strongly sensitive to the linker length, up to 1 bp, or equivalently to its twist, and might locally reach very low values, yielding a highly flexible and extensible domain in the fiber. In particular, the twist-stretch coupling constant, reflecting the chirality of the chromatin fiber, exhibits steep variations and sign changes when the linker length is varied. We argue that this tunable elasticity might be a key feature for chromatin function, for instance in the initiation and regulation of transcription.

Eli Ben-Haïm; Annick Lesne; Jean-Marc Victor



Dissecting the chromatin interactome of microRNA genes  

PubMed Central

Our knowledge of the role of higher-order chromatin structures in transcription of microRNA genes (MIRs) is evolving rapidly. Here we investigate the effect of 3D architecture of chromatin on the transcriptional regulation of MIRs. We demonstrate that MIRs have transcriptional features that are similar to protein-coding genes. RNA polymerase II–associated ChIA-PET data reveal that many groups of MIRs and protein-coding genes are organized into functionally compartmentalized chromatin communities and undergo coordinated expression when their genomic loci are spatially colocated. We observe that MIRs display widespread communication in those transcriptionally active communities. Moreover, miRNA–target interactions are significantly enriched among communities with functional homogeneity while depleted from the same community from which they originated, suggesting MIRs coordinating function-related pathways at posttranscriptional level. Further investigation demonstrates the existence of spatial MIR–MIR chromatin interacting networks. We show that groups of spatially coordinated MIRs are frequently from the same family and involved in the same disease category. The spatial interaction network possesses both common and cell-specific subnetwork modules that result from the spatial organization of chromatin within different cell types. Together, our study unveils an entirely unexplored layer of MIR regulation throughout the human genome that links the spatial coordination of MIRs to their co-expression and function. PMID:24357409

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



Proceedings: Condenser Technology Conference  

Microsoft Academic Search

Steam surface condenser and associated systems performance strongly affects availability and. heat rate in nuclear and fossil power plants. Thirty-one papers presented at the 1993 conference discuss research results, industry experience, and case histories of condenser problems and solutions. These papers have been cataloged separately elsewhere.




Chromatin insulator factors involved in long-range DNA interactions and their role in the folding of the Drosophila genome.  


Chromatin insulators are genetic elements implicated in the organization of chromatin and the regulation of transcription. In Drosophila, different insulator types were characterized by their locus-specific composition of insulator proteins and co-factors. Insulators mediate specific long-range DNA contacts required for the three dimensional organization of the interphase nucleus and for transcription regulation, but the mechanisms underlying the formation of these contacts is currently unknown. Here, we investigate the molecular associations between different components of insulator complexes (BEAF32, CP190 and Chromator) by biochemical and biophysical means, and develop a novel single-molecule assay to determine what factors are necessary and essential for the formation of long-range DNA interactions. We show that BEAF32 is able to bind DNA specifically and with high affinity, but not to bridge long-range interactions (LRI). In contrast, we show that CP190 and Chromator are able to mediate LRI between specifically-bound BEAF32 nucleoprotein complexes in vitro. This ability of CP190 and Chromator to establish LRI requires specific contacts between BEAF32 and their C-terminal domains, and dimerization through their N-terminal domains. In particular, the BTB/POZ domains of CP190 form a strict homodimer, and its C-terminal domain interacts with several insulator binding proteins. We propose a general model for insulator function in which BEAF32/dCTCF/Su(HW) provide DNA specificity (first layer proteins) whereas CP190/Chromator are responsible for the physical interactions required for long-range contacts (second layer). This network of organized, multi-layer interactions could explain the different activities of insulators as chromatin barriers, enhancer blockers, and transcriptional regulators, and suggest a general mechanism for how insulators may shape the organization of higher-order chromatin during cell division. PMID:25165871

Vogelmann, Jutta; Le Gall, Antoine; Dejardin, Stephanie; Allemand, Frederic; Gamot, Adrien; Labesse, Gilles; Cuvier, Olivier; Nègre, Nicolas; Cohen-Gonsaud, Martin; Margeat, Emmanuel; Nöllmann, Marcelo



Chromatin-remodeling complex specificity and embryonic vascular development  

PubMed Central

Vascular development is a dynamic process that relies on the coordinated expression of numerous genes, but the factors that regulate gene expression during blood vessel development are not well defined. ATP-dependent chromatin-remodeling complexes are gaining attention for their specific temporal and spatial effects on gene expression during vascular development. Genetic mutations in chromatin-remodeling complex subunits are revealing roles for the complexes in vascular signaling pathways at discrete developmental time points. Phenotypic analysis of these models at various stages of vascular development will continue to expand our understanding of how chromatin remodeling impacts new blood vessel growth. Such research could also provide novel therapeutic targets for the treatment of vascular pathologies. PMID:22618247

Curtis, Carol D.; Davis, Reema B.; Ingram, Kyle G.; Griffin, Courtney T.



Structural plasticity of single chromatin fibers revealed by torsional manipulation  

E-print Network

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

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



Footprint traversal by adenosine-triphosphate-dependent chromatin remodeler motor  

NASA Astrophysics Data System (ADS)

Adenosine-triphosphate (ATP)-dependent chromatin remodeling enzymes (CREs) are biomolecular motors in eukaryotic cells. These are driven by a chemical fuel, namely, ATP. CREs actively participate in many cellular processes that require accessibility of specific segments of DNA which are packaged as chromatin. The basic unit of chromatin is a nucleosome where 146 bp ˜ 50 nm of a double-stranded DNA (dsDNA) is wrapped around a spool formed by histone proteins. The helical path of histone-DNA contact on a nucleosome is also called “footprint.” We investigate the mechanism of footprint traversal by a CRE that translocates along the dsDNA. Our two-state model of a CRE captures effectively two distinct chemical (or conformational) states in the mechanochemical cycle of each ATP-dependent CRE. We calculate the mean time of traversal. Our predictions on the ATP dependence of the mean traversal time can be tested by carrying out in vitro experiments on mononucleosomes.

Garai, Ashok; Mani, Jesrael; Chowdhury, Debashish



Chromatin fractionation analysis of licensing factors in mammalian cells.  


ORC, Cdc6, Cdt1, and MCM2-7 are replication-licensing factors, which play a central role in the once-per-cell cycle control of DNA replication. ORC, Cdc6, and Cdt1 collaborate to load MCM2-7 onto replication origins in order to license them for replication. MCM2-7 is a DNA helicase directly involved in DNA replication and dissociates from DNA as S phase progresses and each replicon is replicated. In the cell cycle, the loading of MCM2-7 is restricted during the end of mitosis and the G1 phase. Thus, the levels of chromatin-bound MCM2-7 and its loaders oscillate during the cell cycle. Chromatin association of these factors can be analyzed by separating a cell lysate into soluble and chromatin-enriched insoluble fractions in mammalian cells. PMID:24906333

Nishitani, Hideo; Morino, Masayuki; Murakami, Yusuke; Maeda, Takeshi; Shiomi, Yasushi



Modification of enhancer chromatin: what, how and why?  

PubMed Central

Emergence of form and function during embryogenesis arises in large part through cell type- and cell state- specific variation in gene expression patterns, mediated by specialized cis-regulatory elements called enhancers. Recent large-scale epigenomic mapping revealed unexpected complexity and dynamics of enhancer utilization patterns, with 400,000 putative human enhancers annotated by the ENCODE project alone. These large-scale efforts were largely enabled through understanding that enhancers share certain stereotypical chromatin features. However, an important question still lingers: What is the functional significance of enhancer chromatin modification? Here we give an overview of enhancer-associated modifications of histones and DNA, and discuss enzymatic activities involved in their dynamic deposition and removal. We describe potential downstream effectors of these marks and propose models for exploring functions of chromatin modification in regulating enhancer activity during development. PMID:23473601

Calo, Eliezer; Wysocka, Joanna



Statistical physics of nucleosome positioning and chromatin structure  

NASA Astrophysics Data System (ADS)

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

Morozov, Alexandre



Measurement of local chromatin compaction by spectral precision distance microscopy  

NASA Astrophysics Data System (ADS)

Fluorescence in situ hybridization (FISH) offers an appropriate technique to specifically label any given chromatin region by multi spectrally labeled, specific DNA probes. Using confocal laser scanning microscopy, quantitative measurements on the spatial distribution of labeling sites can be performed in 3D conserved cell nuclei. Recently, 'Spectral Precision Distance Microscopy' has been developed that allows 3D distance measurements between point-like fluorescence objects of different spectral signatures far beyond the diffraction limited resolution. In a well characterized and sequenced DNA region, the Prader- Willi/Angelman region q11-13 on chromosome 15, geometric distances between the fluorescence intensity bary centers of four different 'point-like' labeling sites were measured. More than 300 cell nuclei were evaluated with a 3D resolution equivalent better than 100 nm. The geometric bary center distances in nanometers were compared with the genomic bary center distance in kilobases (kb). A direct correlation, for instance linear correlation between geometric and genomic distances was not observed. From the measured values, a local compaction factor for the high order chromatin folding in the analyzed genome region was calculated. Along the 1000 kb chromatin segment analyzed, which spans nearly the compete Prader-Willi/Angelman region, different compaction factors were found. The compaction factor 40 typical for a straight 30 nm chromatin fiber was not observed. This shows that chromatin folding and compaction in intact nuclei may be more complex. With SPDM, however, a microscopical technique is available that can sensitively analyze chromatin organization in the 100 nm range in 3D conserved cell nuclei.

Rauch, Joachim; Hausmann, Michael; Solovei, Irina; Horsthemke, Bernhard; Cremer, Thomas; Cremer, Christoph G.



Chromatin dynamics during interphase explored by single particle tracking  

PubMed Central

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

Levi, Valeria; Gratton, Enrico



Chromatin fiber polymorphism triggered by variations of DNA linker lengths.  


Deciphering the factors that control chromatin fiber structure is key to understanding fundamental chromosomal processes. Although details remain unknown, it is becoming clear that chromatin is polymorphic depending on internal and external factors. In particular, different lengths of the linker DNAs joining successive nucleosomes (measured in nucleosome-repeat lengths or NRLs) that characterize different cell types and cell cycle stages produce different structures. NRL is also nonuniform within single fibers, but how this diversity affects chromatin fiber structure is not clear. Here we perform Monte Carlo simulations of a coarse-grained oligonucleosome model to help interpret fiber structure subject to intrafiber NRL variations, as relevant to proliferating cells of interphase chromatin, fibers subject to remodeling factors, and regulatory DNA sequences. We find that intrafiber NRL variations have a profound impact on chromatin structure, with a wide range of different architectures emerging (highly bent narrow forms, canonical and irregular zigzag fibers, and polymorphic conformations), depending on the NRLs mixed. This stabilization of a wide range of fiber forms might allow NRL variations to regulate both fiber compaction and selective DNA exposure. The polymorphic forms spanning canonical to sharply bent structures, like hairpins and loops, arise from large NRL variations and are surprisingly more compact than uniform NRL structures. They are distinguished by tail-mediated far-nucleosome interactions, in addition to the near-nucleosome interactions of canonical 30-nm fibers. Polymorphism is consistent with chromatin's diverse biological functions and heterogeneous constituents. Intrafiber NRL variations, in particular, may contribute to fiber bending and looping and thus to distant communication in associated regulatory processes. PMID:24847063

Collepardo-Guevara, Rosana; Schlick, Tamar



Dicer Is Associated with Ribosomal DNA Chromatin in Mammalian Cells  

PubMed Central

Background RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear. Methodology/Principal Findings Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA) repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES) cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer?/? ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines. Conclusion/Significance We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity. However, localization of Dicer to the transcribed region suggests that transcription may contribute to the Dicer deposition at rDNA chromatin. We hypothesize that Dicer functions in maintaining integrity of rDNA arrays. PMID:20730047

Filipowicz, Witold; Svoboda, Petr



Rapid genome-scale mapping of chromatin accessibility in tissue  

PubMed Central

Background The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on large amounts of purified nuclei as starting material. This complicates analysis of trace clinical tissue samples that are often stored frozen. We have developed an alternative nuclease based procedure to bypass nuclear preparation to interrogate nuclease accessible regions in frozen tissue samples. Results Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh). The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergence between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases and accordingly accessibility of CpG islands is probed more efficiently using TACh. Conclusion The TACh methodology identifies accessible chromatin derived from frozen tissue samples. We propose that this simple, robust approach can be applied across a broad range of clinically relevant samples to allow demarcation of regulatory elements of considerable prognostic significance. PMID:22734930



Modeling gene expression using chromatin features in various cellular contexts  

PubMed Central

Background Previous work has demonstrated that chromatin feature levels correlate with gene expression. The ENCODE project enables us to further explore this relationship using an unprecedented volume of data. Expression levels from more than 100,000 promoters were measured using a variety of high-throughput techniques applied to RNA extracted by different protocols from different cellular compartments of several human cell lines. ENCODE also generated the genome-wide mapping of eleven histone marks, one histone variant, and DNase I hypersensitivity sites in seven cell lines. Results We built a novel quantitative model to study the relationship between chromatin features and expression levels. Our study not only confirms that the general relationships found in previous studies hold across various cell lines, but also makes new suggestions about the relationship between chromatin features and gene expression levels. We found that expression status and expression levels can be predicted by different groups of chromatin features, both with high accuracy. We also found that expression levels measured by CAGE are better predicted than by RNA-PET or RNA-Seq, and different categories of chromatin features are the most predictive of expression for different RNA measurement methods. Additionally, PolyA+ RNA is overall more predictable than PolyA- RNA among different cell compartments, and PolyA+ cytosolic RNA measured with RNA-Seq is more predictable than PolyA+ nuclear RNA, while the opposite is true for PolyA- RNA. Conclusions Our study provides new insights into transcriptional regulation by analyzing chromatin features in different cellular contexts. PMID:22950368



Screening assays for epigenetic targets using native histones as substrates.  


In the past years, a lot of attention has been given to the identification and characterization of selective and potent inhibitors of chromatin-modifying enzymes to better understand their specific role in transcriptional regulation. As aberrant histone methylation is involved in different pathological processes, the search for methyltransferase and demethylase inhibitors has emerged as a crucial issue in current medicinal chemistry research. High-throughput in vitro assays are important tools for the identification of new methyltransferase or demethylase inhibitors. These usually use oligopeptide substrates derived from histone sequences, although in many cases, they are not good substrates for these enzymes. Here, the authors report about the setup and establishment of in vitro assays that use native core histones as substrates, enabling an assay environment that better resembles native conditions. They have applied these substrates for the known formaldehyde dehydrogenase assay for the histone demethylase LSD1 and have established two new antibody-based assays. For LSD1, a heterogeneous assay format was set up, and a homogeneous assay was used for the characterization of the arginine methyltransferase PRMT1. Validation of the system was achieved with reference inhibitors in each case. PMID:21965113

Hauser, Alexander-Thomas; Bissinger, Elisabeth-Maria; Metzger, Eric; Repenning, Antje; Bauer, Uta-Maria; Mai, Antonello; Schüle, Roland; Jung, Manfred



Chromatin Structure and the Inheritance of Epigenetic Information  

PubMed Central

Although it is widely accepted that the regulation of the chromatin landscape is pivotal to conveying epigenetic phenomena, it is still unclear how a defined chromatin domain is reproduced following replication and transmitted from one generation to another. Here we review multiple mechanisms that contribute to the inheritance of epigenetic information with emphasis on the recycling of old histones following replication, the requirement for a positive feedback loop, long-range gene interactions, and the complex network of trans-acting factors. PMID:20300089

Margueron, Raphael; Reinberg, Danny



Phosphoinositides as Regulators of Protein-Chromatin Interactions  

NSDL National Science Digital Library

The molecular function of phospholipids in the nucleus has been only partially elucidated. The upsurge of epigenetic research has contributed to increased interest in nuclear phospholipids, such as phosphoinositides, and their involvement in gene transcription. However, the mechanisms by which phosphoinositides regulate transcription is still unknown at the molecular level. Certain phosphoinositide species can regulate protein-chromatin and protein–nucleic acid interactions, and specific nuclear target proteins link nuclear signaling lipids to gene expression. We propose that a phosphoinositide-mediated detachment of proteins from chromatin is a general biological mechanism that partly underlies the signaling effects of nuclear phosphoinositides.

Keijo Viiri (School of Medicine and Tampere University Hospital;University of Tampere REV); Markku Maki (School of Medicine and Tampere University Hospital;University of Tampere REV); Olli Lohi (School of Medicine and Tampere University Hospital;University of Tampere REV)



Schultheiss, Schiepe, & Rawolle Hormone assays 1 Running head: HORMONE ASSAYS  

E-print Network

Schultheiss, Schiepe, & Rawolle Hormone assays 1 Running head: HORMONE ASSAYS Hormone assays Oliver: Schultheiss, O. C., Schiepe, A., & Rawolle, M. (2012). Hormone assays. In H. Cooper, P. M. Camic, D. L. Long Association. #12;Schultheiss, Schiepe, & Rawolle Hormone assays 2 Hormone assays Hormones can be assayed from

Schultheiss, Oliver C.


Interplay of Ribosomal DNA Loci in Nucleolar Dominance: Dominant NORs Are Up-Regulated by Chromatin Dynamics in the Wheat-Rye System  

PubMed Central

Background Chromatin organizational and topological plasticity, and its functions in gene expression regulation, have been strongly revealed by the analysis of nucleolar dominance in hybrids and polyploids where one parental set of ribosomal RNA (rDNA) genes that are clustered in nucleolar organizing regions (NORs), is rendered silent by epigenetic pathways and heterochromatization. However, information on the behaviour of dominant NORs is very sparse and needed for an integrative knowledge of differential gene transcription levels and chromatin specific domain interactions. Methodology/Principal Findings Using molecular and cytological approaches in a wheat-rye addition line (wheat genome plus the rye nucleolar chromosome pair 1R), we investigated transcriptional activity and chromatin topology of the wheat dominant NORs in a nucleolar dominance situation. Herein we report dominant NORs up-regulation in the addition line through quantitative real-time PCR and silver-staining technique. Accompanying this modification in wheat rDNA trascription level, we also disclose that perinucleolar knobs of ribosomal chromatin are almost transcriptionally silent due to the residual detection of BrUTP incorporation in these domains, contrary to the marked labelling of intranucleolar condensed rDNA. Further, by comparative confocal analysis of nuclei probed to wheat and rye NORs, we found that in the wheat-rye addition line there is a significant decrease in the number of wheat-origin perinucleolar rDNA knobs, corresponding to a diminution of the rDNA heterochromatic fraction of the dominant (wheat) NORs. Conclusions/Significance We demonstrate that inter-specific interactions leading to wheat-origin NOR dominance results not only on the silencing of rye origin NOR loci, but dominant NORs are also modified in their transcriptional activity and interphase organization. The results show a cross-talk between wheat and rye NORs, mediated by ribosomal chromatin dynamics, revealing a conceptual shift from differential amphiplasty to ‘mutual amphiplasty’ in the nucleolar dominance process. PMID:19048103

Silva, Manuela; Pereira, H. Sofia; Bento, Miguel; Santos, Ana Paula; Shaw, Peter; Delgado, Margarida; Neves, Nuno; Viegas, Wanda



How chromatin looping and nuclear envelope attachment affect genome organization in eukaryotic cell nuclei.  


To understand how interphase chromatin is organized in eukaryotic cell nuclei, it is essential to understand what kind of interactions influence the nuclear architecture and to what extent. Using a mesoscale model that incorporates chromatin-chromatin interactions as well as binding of chromatin to the nuclear envelope, we can show that chromatin loops and envelope bonds are major players in genome organization because they largely affect the entropy of the chromatin fibres. The model allows us to consistently reproduce multiple characteristic chromatin parameters in agreement with experimental data. We focus on the question of how an inversion of the nuclear architecture, in the course of which the highly active euchromatin changes its preferential position from the nuclear center to the periphery, can be achieved. We find that the transition between the common and inverted organization is driven by the strength of the envelope interaction and the nuclear chromatin density. PMID:24380599

Jerabek, Hansjoerg; Heermann, Dieter W



The Chromatin "Landscape" of a Murine Adult ?-Globin Gene Is Unaffected by Deletion of Either the Gene Promoter or a Downstream Enhancer  

PubMed Central

In mammals, the complex tissue- and developmental-specific expression of genes within the ?-globin cluster is known to be subject to control by the gene promoters, by a locus control region (LCR) located upstream of the cluster, and by sequence elements located across the intergenic regions. Despite extensive investigation, however, the complement of sequences that is required for normal regulation of chromatin structure and gene expression within the cluster is not fully defined. To further elucidate regulation of the adult ?-globin genes, we investigate the effects of two deletions engineered within the endogenous murine ?-globin locus. First, we find that deletion of the ?2-globin gene promoter, while eliminating ?2-globin gene expression, results in no additional effects on chromatin structure or gene expression within the cluster. Notably, our observations are not consistent with competition among the ?-globin genes for LCR activity. Second, we characterize a novel enhancer located 3? of the ?2-globin gene, but find that deletion of this sequence has no effect whatsoever on gene expression or chromatin structure. This observation highlights the difficulty in assigning function to enhancer sequences identified by the chromatin “landscape” or even by functional assays. PMID:24817273

Cadiz-Rivera, Brenda; Fromm, George; de Vries, Christina; Fields, Jennifer; McGrath, Kathleen E.; Fiering, Steven; Bulger, Michael



Regulation of the dynamic chromatin architecture of the epidermal differentiation complex is mediated by a c-Jun/AP-1-modulated enhancer.  


The epidermal differentiation complex (EDC) locus comprises a syntenic and linear cluster of genes whose concomitant expression is a hallmark feature of differentiation in the developing skin epidermis. Many of the EDC proteins are cross-linked together to form the cornified envelope, an essential and discrete unit of the mammalian skin barrier. The mechanism underlying coordinate transcriptional activation of the EDC is unknown. Within the human EDC, we identified an epidermal-specific regulatory enhancer, 923, which responded to the developmental and spatiotemporal cues at the onset of epidermal differentiation in the mouse embryo. Comparative chromosomal conformation capture assays in proliferating and differentiated primary mouse keratinocytes revealed multiple physiologically sensitive chromatin interactions between the 923 enhancer and EDC gene promoters, thus depicting the dynamic chromatin topology of the EDC. We elucidate a mechanistic link between c-Jun/AP-1 and 923, whereby AP-1- and 923-mediated EDC chromatin remodeling are required for functional EDC gene activation. Thus, we identify a critical enhancer/transcription factor axis governing the dynamic regulation of the EDC chromatin architecture and gene expression and provide a framework for future studies toward understanding gene regulation in cutaneous diseases. PMID:24468747

Oh, Inez Y; Albea, Danielle M; Goodwin, Zane A; Quiggle, Ashley M; Baker, Breeana P; Guggisberg, Ann M; Geahlen, Jessica H; Kroner, Grace M; Strong, Cristina de Guzman



The Tudor protein survival motor neuron (SMN) is a chromatin-binding protein that interacts with methylated lysine 79 of histone H3.  


Spinal muscular atrophy (SMA) is a muscular disease characterized by the death of motoneurons, and is a major genetic cause of infant mortality. Mutations in the SMN1 gene, which encodes the protein survival motor neuron (SMN), are responsible for the disease. SMN belongs to the Tudor domain protein family, whose members are known to interact with methylated arginine (R) or lysine (K) residues. SMN has well-defined roles in the metabolism of small non-coding ribonucleoproteins (snRNPs) and spliceosome activity. We previously showed that SMN relocated to damaged interphase centromeres, together with the Cajal-body-associated proteins coilin and fibrillarin, during the so-called interphase centromere damage response (iCDR). Here we reveal that SMN is a chromatin-binding protein that specifically interacts with methylated histone H3K79, a gene expression- and splicing-associated histone modification. SMN relocation to damaged centromeres requires its functional Tudor domain and activity of the H3K79 methyltransferase DOT1L. In vitro pulldown assays showed that SMN interacts with H3K79me1,2 at its functional Tudor domain. Chromatin immunoprecipitation confirmed that SMN binds to H3K79me1,2-containing chromatin in iCDR-induced cells. These data reveal a novel SMN property in the detection of specific chromatin modifications, and shed new light on the involvement of a putative epigenetic dimension to the occurrence of SMA. PMID:23750013

Sabra, Mirna; Texier, Pascale; El Maalouf, Jhony; Lomonte, Patrick



Discovery of MLL1 binding units, their localization to CpG Islands, and their potential function in mitotic chromatin  

PubMed Central

Background Mixed Lineage Leukemia 1 (MLL1) is a mammalian ortholog of the Drosophila Trithorax. In Drosophila, Trithorax complexes transmit the memory of active genes to daughter cells through interactions with Trithorax Response Elements (TREs). However, despite their functional importance, nothing is known about sequence features that may act as TREs in mammalian genomic DNA. Results By analyzing results of reported DNA binding assays, we identified several CpG rich motifs as potential MLL1 binding units (defined as morphemes). We find that these morphemes are dispersed within a relatively large collection of human promoter sequences and appear densely packed near transcription start sites of protein-coding genes. Genome wide analyses localized frequent morpheme occurrences to CpG islands. In the human HOX loci, the morphemes are spread across CpG islands and in some cases tail into the surrounding shores and shelves of the islands. By analyzing results of chromatin immunoprecipitation assays, we found a connection between morpheme occurrences, CpG islands, and chromatin segments reported to be associated with MLL1. Furthermore, we found a correspondence of reported MLL1-driven “bookmarked” regions in chromatin to frequent occurrences of MLL1 morphemes in CpG islands. Conclusion Our results implicate the MLL1 morphemes in sequence-features that define the mammalian TREs and provide a novel function for CpG islands. Apparently, our findings offer the first evidence for existence of potential TREs in mammalian genomic DNA and the first evidence for a connection between CpG islands and gene-bookmarking by MLL1 to transmit the memory of highly active genes during mitosis. Our results further suggest a role for overlapping morphemes in producing closely packed and multiple MLL1 binding events in genomic DNA so that MLL1 molecules could interact and reside simultaneously on extended potential transcriptional maintenance elements in human chromosomes to transmit the memory of highly active genes during mitosis. PMID:24373511



Steam condenser performance  

SciTech Connect

To understand condenser performance, it is necessary to perceive it as a whole machine and not just a single tube or tube bundle. A new heat-transfer rate is defined based on overall tube-bundle performance. This condenser performance standard has achieved its intended mission. Overall tube-bundle heat-transfer rates ensure that users can rely on the results in their plant design. It also assures users that they can purchase the performance from competent manufacturers. The paper discusses condenser operation and design, tube bundle operation and design, and heat transfer rate.




Electrolyte vapor condenser  


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. 3 figs.

Sederquist, R.A.; Szydlowski, D.F.; Sawyer, R.D.



Electrolyte vapor condenser  


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)



Polariton Condensate Transistor Switch  

E-print Network

A polariton condensate transistor switch is realized through optical excitation of a microcavity ridge with two beams. The ballistically ejected polaritons from a condensate formed at the source are gated using the 20 times weaker second beam to switch on and off the flux of polaritons. In the absence of the gate beam the small built-in detuning creates potential landscape in which ejected polaritons are channelled toward the end of the ridge where they condense. The low loss photon-like propagation combined with strong nonlinearities associated with their excitonic component makes polariton based transistors particularly attractive for the implementation of all-optical integrated circuits.

Gao, T; Liew, T C H; Tsintzos, S I; Stavrinidis, G; Deligeorgis, G; Hatzopoulos, Z; Savvidis, P G



Identification of p100 target promoters by chromatin immunoprecipitation-guided ligation and selection (ChIP-GLAS)  

PubMed Central

The multifunctional protein p100 is a vital transcriptional regulator that increases gene transcription by forming a physical bridge between promoter-specific transcription factors and the basal transcription machinery. To identify potential signal transduction pathways in which human p100 acts as a coregulator and to find target promoter regions that may interact with p100, we performed a promoter microarray assay called chromatin immunoprecipitation-guided ligation and selection (ChIP-GLAS). From this assay, we determined that a set of promoter fragments, including several factors in the transforming growth factor beta (TGF-?) signaling pathway, exhibited interaction with p100. The ChIP-GLAS data were validated by RT-PCR assessing the mRNA expression of various factors in the TGF-? signaling pathway in cell lines. PMID:20921938

Liu, Xin; Dong, Lijie; Zhang, Xuejun; Wang, Baoya; Wang, Xinting; Li, Hu; He, Jinyan; Ge, Lin; Jing, Xiang; Yao, Zhi; Yang, Jie



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

PubMed Central

DNA repair in eukaryotic cells takes place in the context of chromatin, where DNA, including damaged DNA, is tightly packed into nucleosomes and higher order chromatin structures. Chromatin intrinsically restricts accessibility of DNA repair proteins to the damaged DNA and impacts upon the overall rate of DNA repair. Chromatin is highly responsive to DNA damage and undergoes specific remodeling to facilitate DNA repair. How damaged DNA is accessed, repaired and restored to the original chromatin state, and how chromatin remodeling coordinates these processes in vivo, remains largely unknown. ATP-dependent chromatin remodelers (ACRs) are the master regulators of chromatin structure and dynamics. Conserved from yeast to humans, ACRs utilize the energy of ATP to reorganize packing of chromatin and control DNA accessibility by sliding, ejecting or restructuring nucleosomes. Several studies have demonstrated that ATP-dependent remodeling activity of ACRs plays important roles in coordination of spatio-temporal steps of different DNA repair pathways in chromatin. This review focuses on the role of ACRs in regulation of various aspects of nucleotide excision repair (NER) in the context of chromatin. We discuss current understanding of ATP-dependent chromatin remodeling by various subfamilies of remodelers and regulation of the NER pathway in vivo. PMID:23109894

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



Biochemical characterization of chromatin fractions isolated from induced and uninduced friend erythroleukemia cells  

Microsoft Academic Search

Chromatin fractions from Friend erythroleukemia cells after induction of differentiation by dimethylsulfoxide (DMSO) were compared in their biochemical characteristics to fractions from uninduced cells. Fractions were prepared by extracting chromatin from nuclei after mild micrococcal nuclease treatment with increasing concentrations of NaCl according to Sanders [1]. This procedure has been found to release chromatin containing hyperacetylated histones preferentially [2]. The

Olga Knosp; Bernhard Redl; Bernd Puschendorf



KEVIN STRUHL GENE EXPRESSION Chromatin and transcription factors : who's on first?  

E-print Network

in chromatin structure are very likely to af feet the access and/or the function of transcription fac- tors DNA templates in vitro, leading some to view chromatin structure as simply a mech- anism long enough to permit HNF5 access to DNA. Chromatin structural changes often pre- cede transcriptional


The INO80 chromatin remodeling complex is involved in the nucleotide excision repair pathway  

Microsoft Academic Search

The genomic DNA of eukaryotic cells is well organized into chromatin structures. However, these repressed structures present barriers that block the access of regulatory factors to the genome during various nuclear events. To overcome the obstacle, two major cellular processes, post-modification of histone tails and ATP-dependent chromatin remodeling, are involved in reconfiguring chromatin structure and creating accessible DNA. Despite the

Yingjun Jiang



Ghost Condensate Busting  

E-print Network

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




SciTech Connect

The Color Glass Condensate is a state of high density gluonic matter which controls the high energy limit of hadronic interactions. Its properties are important for the initial conditions for matter produced at RHIC.




Ghost condensate busting  

SciTech Connect

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.

Bilic, Neven [Rudjer Boskovic Institute, 10002 Zagreb (Croatia)] [Rudjer Boskovic Institute, 10002 Zagreb (Croatia); Tupper, Gary B; Viollier, Raoul D, E-mail:, E-mail:, E-mail: [Centre of Theoretical Physics and Astrophysics, University of Cape Town, Rondebosch 7701 (South Africa)



Key condenser failure mechanisms  

SciTech Connect

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

Buecker, B.



From Macroscopic to Mesoscopic Models of Chromatin Folding Tamar Schlick  

E-print Network

is the protein/nucleic acid fiber that stores the genetic material in higher organisms. Many biological questions- tions, the effects of variations in linker DNA length and of histone variants on chromatin structure in viruses, heart muscle motion, RNA translation, or fruit fly circadian rhythm. For these applications

Schlick, Tamar


Sperm-derived histones contribute to zygotic chromatin in humans  

Microsoft Academic Search

BACKGROUND: about 15% to 30% of the DNA in human sperm is packed in nucleosomes and transmission of this fraction to the embryo potentially serves as a mechanism to facilitate paternal epigenetic programs during embryonic development. However, hitherto it has not been established whether these nucleosomes are removed like the protamines or indeed contribute to paternal zygotic chromatin, thereby potentially

Godfried W van der Heijden; Liliana Ramos; Esther B Baart; Ilse M van den Berg; Alwin AHA Derijck; Johan van der Vlag; Elena Martini; Peter de Boer



A Chromatin-Mediated Reversible Drug-Tolerant State  

E-print Network

A Chromatin-Mediated Reversible Drug-Tolerant State in Cancer Cell Subpopulations Sreenath V to stressful exposures, including drug treatments. While modeling the acute response to various anti- cancer of resistance to cancer drugs remains a key obstacle to successful cancer therapy. Substan- tial efforts

Oregon, University of


Heterochromatin, Position Effects, and the Genetic Dissection of Chromatin  

Microsoft Academic Search

The partitioning of the eukaryotic chromosome regions into heterochromatin and euchromatin reflects fundamental differences in the packaging of DNA. The observation that chromosome rearrangments juxtaposing euchromatic sequences with heterochromatic regions usually result in silencing of nearby euchromatic genes (termed “position effect silencing”) led to genetic screens for position effect modifiers. These screens uncovered key proteins involved in chromatin assembly, including

Joel C Eissenberg; Lori L Wallrath



Cytochemical studies on the chromatin elimination in solenobia (Lepidoptera)  

Microsoft Academic Search

Summary The chemical nature of the elimination chromatin in the first maturation division ofSolenobia was investigated with the Feulgen reaction, by staining with methyl green-pyronin. with toluidine blue before and after treatment with ribonuclease and cold perchloric acid, with the Hotchkiss periodic acid-Schiff test, and the Millon reaction.

Hans Ris; Ruth Kleinfeld



A role for chromatin remodellers in replication of damaged DNA.  


In eukaryotic cells, replication past damaged sites in DNA is regulated by the ubiquitination of proliferating cell nuclear antigen (PCNA). Little is known about how this process is affected by chromatin structure. There are two isoforms of the Remodels the Structure of Chromatin (RSC) remodelling complex in yeast. We show that deletion of RSC2 results in a dramatic reduction in the level of PCNA ubiquitination after DNA-damaging treatments, whereas no such effect was observed after deletion of RSC1. Similarly, depletion of the BAF180 component of the corresponding PBAF (Polybromo BRG1 (Brahma-Related Gene 1) Associated Factor) complex in human cells led to a similar reduction in PCNA ubiquitination. Remarkably, we found that depletion of BAF180 resulted after UV-irradiation, in a reduction not only of ubiquitinated PCNA but also of chromatin-associated unmodified PCNA and Rad18 (the E3 ligase that ubiquitinates PCNA). This was accompanied by a modest decrease in fork progression. We propose a model to account for these findings that postulates an involvement of PBAF in repriming of replication downstream from replication forks blocked at sites of DNA damage. In support of this model, chromatin immunoprecipitation data show that the RSC complex in yeast is present in the vicinity of the replication forks, and by extrapolation, this is also likely to be the case for the PBAF complex in human cells. PMID:22638582

Niimi, Atsuko; Chambers, Anna L; Downs, Jessica A; Lehmann, Alan R



Reverse-engineering Chromatin Folding via The Gaussian Polymer Model  

NASA Astrophysics Data System (ADS)

Recent technological advancements in techniques exploring chromatin conformation, such as 3C and its derivatives, provide us with information about contact frequency between chromatin segments. There is an urgent need for systematic methods of reconstructing folding patterns of the chromatin from such data. Dekker et al have previously summarized this experimental data in the form of a so-called `spatially averaged' conformation around which fluctuations occur. This would be accurate for regions where the chromatin is mostly frozen around one structure, but rather misleading for more dynamic euchromatin. To address the ill-posed problem of reconstruction of probability distribution from pairwise contact data, we propose a minimum relative entropy approach, which reduces to finding an interacting polymer model that reproduces the contact strengths, and allows for both very dynamic as well as frozen structures depending upon the indicated degree of interaction. While comparing contact frequencies computed from this model to observed data, we introduce the minimal number of interactions required as determined by criteria controlling model complexity. This technique allows us to reproduce known interactions for several biologically important regions like the beta-globin locus.

Apratim, Manjul; Mukhopadhyay, Swagatam; Sengupta, Anirvan



Chromatin-regulating proteins as targets for cancer therapy.  


Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium butyrate and valproic acid) have been shown to sensitize cancer cells to ionizing irradiation in preclinical models, and some of these molecules are being tested in clinical trials, either alone or in combination with radiotherapy. Meanwhile, recent large-scale genome analyses have identified frequent mutations in genes encoding chromatin-regulating proteins, especially in those encoding subunits of the SWI/SNF chromatin-remodeling complex, in various human cancers. These observations have driven researchers toward development of targeted therapies against cancers carrying these mutations. DOT1L inhibition in MLL-rearranged leukemia, EZH2 inhibition in EZH2-mutant or MLL-rearranged hematologic malignancies and SNF5-deficient tumors, BRD4 inhibition in various hematologic malignancies, and BRM inhibition in BRG1-deficient tumors have demonstrated promising anti-tumor effects in preclinical models, and these strategies are currently awaiting clinical application. Overall, the data collected so far suggest that targeting chromatin-regulating proteins is a promising strategy for tomorrow's cancer therapy, including radiotherapy and molecularly targeted chemotherapy. PMID:24522270

Oike, Takahiro; Ogiwara, Hideaki; Amornwichet, Napapat; Nakano, Takashi; Kohno, Takashi



Mortality ratios and life expectancy in X chromatin positive males  

Microsoft Academic Search

In a prospective study of 466 X chromatin positive males an increase in mortality of about 50% has been observed. The increase is associated with a loss of about five years in life span. There is no convincing evidence that the increase is concentrated at any particular age group but this possibility could not be excluded. No effect of mode

W H Price; J F Clayton; S Collyer; R de Mey



Regulation of chromatin structure by poly(ADP-ribosyl)ation  

PubMed Central

The interaction of DNA with proteins in the context of chromatin has to be tightly regulated to achieve so different tasks as packaging, transcription, replication and repair. The very rapid and transient post-translational modification of proteins by poly(ADP-ribose) has been shown to take part in all four. Originally identified as immediate cellular answer to a variety of genotoxic stresses, already early data indicated the ability of this highly charged nucleic acid-like polymer to modulate nucleosome structure, the basic unit of chromatin. At the same time the enzyme responsible for synthesizing poly(ADP-ribose), the zinc-finger protein poly(ADP-ribose) polymerase-1 (PARP1), was shown to control transcription initiation as basic factor TFIIC within the RNA-polymerase II machinery. Later research focused more on PARP-mediated regulation of DNA repair and cell death, but in the last few years, transcription as well as chromatin modulation has re-appeared on the scene. This review will discuss the impact of PARP1 on transcription and transcription factors, its implication in chromatin remodeling for DNA repair and probably also replication, and its role in controlling epigenetic events such as DNA methylation and the functionality of the insulator protein CCCTC-binding factor. PMID:22969794

Beneke, Sascha



Evolution of histone 2A for chromatin compaction in eukaryotes  

PubMed Central

During eukaryotic evolution, genome size has increased disproportionately to nuclear volume, necessitating greater degrees of chromatin compaction in higher eukaryotes, which have evolved several mechanisms for genome compaction. However, it is unknown whether histones themselves have evolved to regulate chromatin compaction. Analysis of histone sequences from 160 eukaryotes revealed that the H2A N-terminus has systematically acquired arginines as genomes expanded. Insertion of arginines into their evolutionarily conserved position in H2A of a small-genome organism increased linear compaction by as much as 40%, while their absence markedly diminished compaction in cells with large genomes. This effect was recapitulated in vitro with nucleosomal arrays using unmodified histones, indicating that the H2A N-terminus directly modulates the chromatin fiber likely through intra- and inter-nucleosomal arginine–DNA contacts to enable tighter nucleosomal packing. Our findings reveal a novel evolutionary mechanism for regulation of chromatin compaction and may explain the frequent mutations of the H2A N-terminus in cancer. DOI: PMID:24939988

Macadangdang, Benjamin R; Oberai, Amit; Spektor, Tanya; Campos, Oscar A; Sheng, Fang; Carey, Michael F; Vogelauer, Maria; Kurdistani, Siavash K



Measure Guideline: Evaporative Condensers  

SciTech Connect

The purpose of this measure guideline on evaporative condensers is to provide information on a cost-effective solution for energy and demand savings in homes with cooling loads. This is a prescriptive approach that outlines selection criteria, design and installation procedures, and operation and maintenance best practices. This document has been prepared to provide a process for properly designing, installing, and maintaining evaporative condenser systems as well as understanding the benefits, costs, and tradeoffs.

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



Condensed tannins act against cattle nematodes.  


The use of natural plant anthelmintics was suggested as a possible alternative control of gastrointestinal nematodes (GIN) in ruminants. Direct anthelmintic effects of tannin-containing plants have already been shown in sheep and goat GIN. These anthelmintic properties are mainly associated with condensed tannins. In the present study, we evaluated possible in vitro effects of three tannin-containing plants against bovine GIN. Effects of Onobrychis viciifolia, Lotus pedunculatus and Lotus corniculatus condensed tannin (CT) extracts on Cooperia oncophora and Ostertagia ostertagi were determined by a larval feeding inhibition assay (LFIA) and a larval exsheathment assay (LEA). In the LFIA, all three plant extracts significantly inhibited larval feeding behaviour of both C. oncophora and O. ostertagi first stage larvae in a dose-dependent manner. The L. pedunculatus extract, based on EC(50) (effective concentration for 50% inhibition), was the most effective against both nematodes, followed by O. viciifolia and L. corniculatus. The effect of CT extracts upon larval feeding behaviour correlates with CT content and procyanidin/prodelphidin ratio. Larval exsheathment of C. oncophora and O. ostertagi L3 larvae (third stage larvae) was also affected by CT extracts from all three plants. In both in vitro assays, extracts with added polyvinylpolypyrrolidone, an inhibitor of tannins, generated almost the same values as the negative control; this confirms the role of CT in the anthelmintic effect of these plant extracts. Our results, therefore, indicated that tannin-containing plants could act against cattle nematodes. PMID:21726942

Novobilský, Adam; Mueller-Harvey, Irene; Thamsborg, Stig Milan



Open Problems in $?$ Particle Condensation  

E-print Network

$\\alpha$ particle condensation is a novel state in nuclear systems. We briefly review the present status on the study of $\\alpha$ particle condensation and address the open problems in this research field: $\\alpha$ particle condensation in heavier systems other than the Hoyle state, linear chain and $\\alpha$ particle rings, Hoyle-analogue states with extra neutrons, $\\alpha$ particle condensation related to astrophysics, etc.

Y. Funaki; M. Girod; H. Horiuchi; G. Roepke; P. Schuck; A. Tohsaki; T. Yamada



Condensate dark matter stars  

E-print Network

We investigate the structure and stability properties of compact astrophysical objects that may be formed from the Bose-Einstein condensation of dark matter. Once the critical temperature of a boson gas is less than the critical temperature, a Bose-Einstein Condensation process can always take place during the cosmic history of the universe. Therefore we model the dark matter inside the star as a Bose-Einstein condensate. In the condensate dark matter star model, the dark matter equation of state can be described by a polytropic equation of state, with polytropic index equal to one. We derive the basic general relativistic equations describing the equilibrium structure of the condensate dark matter star with spherically symmetric static geometry. The structure equations of the condensate dark matter stars are studied numerically. The critical mass and radius of the dark matter star are given by $M_{crit}\\approx 2(l_a/1fm)^{1/2}(m_{\\chi}/1\\;{\\rm GeV})^{-3/2}M_{\\odot}$ and $R_{crit}\\approx 1.1 \\times 10^6(l_a/1\\;{\\rm fm})^{1/2}(m_{\\chi}/1\\;{\\rm GeV})^{-3/2}$ cm respectively, where $l_a$ and $m_{\\chi}$ are the scattering length and the mass of dark matter particle, respectively.

X. Y. Li; T. Harko; K. S. Cheng



Changing the DNA landscape: putting a SPN on chromatin.  


In eukaryotic cells, transcription and replication each occur on DNA templates that are incorporated into nucleosomes. Formation of chromatin generally limits accessibility of specific DNA sequences and inhibits progression of polymerases as they copy information from the DNA. The processes that select sites for initiating either transcription or replication are therefore strongly influenced by factors that modulate the properties of chromatin proteins. Further, in order to elongate their products, both DNA and RNA polymerases must be able to overcome the inhibition presented by chromatin (Lipford and Bell 2001; Workman and Kingston 1998). One way to adjust the properties of chromatin proteins is to covalently modify them by adding or removing chemical moieties. Both histone and non-histone chromatin proteins are altered by acetylation, methylation, and other changes, and the 'nucleosome modifying' complexes that perform these reactions are important components of pathways of transcriptional regulation (Cote 2002; Orphanides and Reinberg 2000; Roth et al. 2001; Strahl and Allis 2000; Workman and Kingston 1998). Another way to alter the effects of nucleosomes is to change the position of the histone octamers relative to specific DNA sequences (Orphanides and Reinberg 2000; Verrijzer 2002; Wang 2002; Workman and Kingston 1998). Since the ability of a sequence to be bound by specific proteins can vary significantly whether the sequence is in the linkers between nucleosomes or at various positions within a nucleosome, 'nucleosome remodeling' complexes that rearrange nucleosome positioning are also important regulators of transcription. Since the DNA replication machinery has to encounter many of the same challenges posed by chromatin, it seems likely that modifying and remodeling complexes also act during duplication of the genome, but most of the current information on these factors relates to regulation of transcription. This chapter describes the factor known variously as FACT in humans, where it promotes elongation of RNA polymerase II on nucleosomal templates in vitro (Orphanides et al. 1998, 1999), DUF in frogs, where it is needed for DNA replication in oocyte extracts (Okuhara et al. 1999), and CP or SPN in yeast, where it is linked in vivo to both transcription and replication (Brewster et al. 2001; Formosa et al. 2001). Like the nucleosome modifying and remodeling complexes, it is broadly conserved among eukaryotes, affects a wide range of processes that utilize chromatin, and directly alters the properties of nucleosomes. However, it does not have nucleosome modifying or standard ATP-dependent remodeling activity, and therefore represents a third class of chromatin modulating factors. It is also presently unique in the extensive connections it displays with both transcription and replication: FACT/DUF/CP/SPN appears to modify nucleosomes in a way that is directly important for the efficient functioning of both RNA polymerases and DNA polymerases. While less is known about the mechanisms it uses to promote its functions than for other factors that affect chromatin, it is clearly an essential part of the complex mixture of activities that modulate access to DNA within chromatin. Physical and genetic interactions suggest that FACT/DUF/CP/SPN affects multiple pathways within replication and transcription as a member of several distinct complexes. Some of the interactions are easy to assimilate into models for replication or transcription, such as direct binding to DNA polymerase alpha (Wittmeyer and Formosa 1997; Wittmeyer et al. 1999), association with nucleosome modifying complexes (John et al. 2000), and interaction with factors that participate in elongation of RNA Polymerase II (Gavin et al. 2002; Squazzo et al. 2002). Others are more surprising such as an association with the 19S complex that regulates the function of the 20S proteasome (Ferdous et al. 2001; Xu et al. 1995), and the indication that FACT/DUF/CP/SPN can act as a specificity factor for casein kinase II (Keller et al. 2001). This chapter reviews the

Formosa, T



CPF-Associated Phosphatase Activity Opposes Condensin-Mediated Chromosome Condensation  

PubMed Central

Functional links connecting gene transcription and condensin-mediated chromosome condensation have been established in species ranging from prokaryotes to vertebrates. However, the exact nature of these links remains misunderstood. Here we show in fission yeast that the 3? end RNA processing factor Swd2.2, a component of the Cleavage and Polyadenylation Factor (CPF), is a negative regulator of condensin-mediated chromosome condensation. Lack of Swd2.2 does not affect the assembly of the CPF but reduces its association with chromatin. This causes only limited, context-dependent effects on gene expression and transcription termination. However, CPF-associated Swd2.2 is required for the association of Protein Phosphatase 1 PP1Dis2 with chromatin, through an interaction with Ppn1, a protein that we identify as the fission yeast homologue of vertebrate PNUTS. We demonstrate that Swd2.2, Ppn1 and PP1Dis2 form an independent module within the CPF, which provides an essential function in the absence of the CPF-associated Ssu72 phosphatase. We show that Ppn1 and Ssu72, like Swd2.2, are also negative regulators of condensin-mediated chromosome condensation. We conclude that Swd2.2 opposes condensin-mediated chromosome condensation by facilitating the function of the two CPF-associated phosphatases PP1 and Ssu72. PMID:24945319

Vanoosthuyse, Vincent; Legros, Penelope; van der Sar, Sjaak J. A.; Yvert, Gael; Toda, Kenji; Le Bihan, Thierry; Watanabe, Yoshinori; Hardwick, Kevin; Bernard, Pascal



Hajjoul/Mathon et al. Chromatin dynamics in living yeasts High throughput chromatin motion tracking in living yeast reveals the  

E-print Network

,3 Keywords: chromatin / live cell imaging / high-throughput particle tracking / nuclear architecture-throughput single particle tracking we record the movements of 9 fluorescently labeled chromosome loci located to technological breakthroughs in imaging and in molecular biology ranging from painting single chromosomes

Boyer, Edmond


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

NASA Technical Reports Server (NTRS)

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

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



Absolute nuclear material assay  


A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

Prasad, Manoj K. (Pleasanton, CA); Snyderman, Neal J. (Berkeley, CA); Rowland, Mark S. (Alamo, CA)



An ikaros-containing chromatin-remodeling complex in adult-type erythroid cells.  


We have previously described a SWI/SNF-related protein complex (PYR complex) that is restricted to definitive (adult-type) hematopoietic cells and that specifically binds DNA sequences containing long stretches of pyrimidines. Deletion of an intergenic DNA-binding site for this complex from a human beta-globin locus construct results in delayed human gamma- to beta-globin switching in transgenic mice, suggesting that the PYR complex acts to facilitate the switch. We now show that PYR complex DNA-binding activity also copurifies with subunits of a second type of chromatin-remodeling complex, nucleosome-remodeling deacetylase (NuRD), that has been shown to have both nucleosome-remodeling and histone deacetylase activities. Gel supershift assays using antibodies to the ATPase-helicase subunit of the NuRD complex, Mi-2 (CHD4), confirm that Mi-2 is a component of the PYR complex. In addition, we show that the hematopoietic cell-restricted zinc finger protein Ikaros copurifies with PYR complex DNA-binding activity and that antibodies to Ikaros also supershift the complex. We also show that NuRD and SWI/SNF components coimmunopurify with each other as well as with Ikaros. Competition gel shift experiments using partially purified PYR complex and recombinant Ikaros protein indicate that Ikaros functions as a DNA-binding subunit of the PYR complex. Our results suggest that Ikaros targets two types of chromatin-remodeling factors-activators (SWI/SNF) and repressors (NuRD)-in a single complex (PYR complex) to the beta-globin locus in adult erythroid cells. At the time of the switch from fetal to adult globin production, the PYR complex is assembled and may function to repress gamma-globin gene expression and facilitate gamma- to beta-globin switching. PMID:11003653

O'Neill, D W; Schoetz, S S; Lopez, R A; Castle, M; Rabinowitz, L; Shor, E; Krawchuk, D; Goll, M G; Renz, M; Seelig, H P; Han, S; Seong, R H; Park, S D; Agalioti, T; Munshi, N; Thanos, D; Erdjument-Bromage, H; Tempst, P; Bank, A



Establishing a hematopoietic genetic network through locus-specific integration of chromatin regulators  

PubMed Central

The establishment and maintenance of cell type-specific transcriptional programs require an ensemble of broadly expressed chromatin remodeling and modifying enzymes. Many questions remain unanswered regarding the contributions of these enzymes to specialized genetic networks that control critical processes, such as lineage commitment and cellular differentiation. We have been addressing this problem in the context of erythrocyte development driven by the transcription factor GATA-1 and its coregulator Friend of GATA-1 (FOG-1). As certain GATA-1 target genes have little to no FOG-1 requirement for expression, presumably additional coregulators can mediate GATA-1 function. Using a genetic complementation assay and RNA interference in GATA-1–null cells, we demonstrate a vital link between GATA-1 and the histone H4 lysine 20 methyltransferase PR-Set7/SetD8 (SetD8). GATA-1 selectively induced H4 monomethylated lysine 20 at repressed, but not activated, loci, and endogenous SetD8 mediated GATA-1–dependent repression of a cohort of its target genes. GATA-1 used different combinations of SetD8, FOG-1, and the FOG-1–interacting nucleosome remodeling and deacetylase complex component Mi2? to repress distinct target genes. Implicating SetD8 as a context-dependent GATA-1 corepressor expands the repertoire of coregulators mediating establishment/maintenance of the erythroid cell genetic network, and provides a biological framework for dissecting the cell type-specific functions of this important coregulator. We propose a coregulator matrix model in which distinct combinations of chromatin regulators are required at different GATA-1 target genes, and the unique attributes of the target loci mandate these combinations. PMID:23959865

DeVilbiss, Andrew W.; Boyer, Meghan E.; Bresnick, Emery H.



Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle  

PubMed Central

In yeast, the Origin Recognition Complex (ORC) is bound to replication origins throughout the cell-cycle, but in animal cells, there are conflicting data as to whether and when ORC is removed from chromatin. We find ORC1, 2 and ORC4 to be metabolically stable proteins that co-fractionate with chromatin throughout the cell-cycle in Chinese hamster fibroblasts. Since cellular extraction methods cannot directly examine the chromatin binding properties of proteins in vivo, we examined ORC:chromatin interactions in living cells. Fluorescence loss in photobleaching (FLIP) studies revealed ORC1 and ORC4 to be highly dynamic proteins during the cell-cycle with exchange kinetics similar to other regulatory chromatin proteins. In vivo interaction with chromatin was not significantly altered throughout the cell-cycle, including S-phase. These data support a model in which ORC subunits dynamically interact with chromatin throughout the cell-cycle. PMID:15950218

McNairn, Adrian J.; Okuno, Yukiko; Misteli, Tom; Gilbert, David M.



[The structural reorganization of chromatin as a process of its self-organization in eukaryotic cells and DNA repair problem].  


Below were demonstrated the differences in the cell reaction of the chromosome loci transference induced by adaptive doses X-radiation in the cells nucleus in norm cells and in cells with repair process defect of the oncological patients and patients with hereditary disease. It was supposed that the transference of the homologous chromosome loci is necessary for the realization of the correct DNA's double strand repair. The chromosome loci transference in normal cells could be induced by different factors such as X-radiation, RNA-polymerize II repression by alpha-amanitin and possible by other factors. In cells with BSCA1 and BRCA2 genes mutations the chromosome loci transference could not be induced by the X-radiation, but it could be induced by the RNA-polymerize II repression by alpha-amanitin. The defect of the chromosome loci transference condition on genetics or the another determinatives and this defect could be the one of the important reasons of the genome instability. There was suggested the new conception of the mechanisms of the cell differentiation and cell chronological senescence. The vector of the cell differentiation mechanisms is the progressive chromatin condensation determined by the physical mechanisms, i.e., transformation from less probable (the stem cells) to more probable cell system state (the differentiated cells) and as a result of it changing of the genes transcription. The continued chromatin condensation (most pronounced in the old cells) decreases the probability of the realization of the DNA repair as the reason of the chromosome loci transference limitation. In this work are presented experimental and theoretical information that proves our conception. PMID:16304766

Spitkovski?, D M; Ve?ko, N N; Moiseeva, O S; Ermakov, A V; Terekhov, S M



Method for modification of a synthetically generated assay using measured whole crude properties  

US Patent & Trademark Office Database

The present invention is a method for modifying any synthetically generated assay of a whole crude oil or a portion of a whole crude, such as a condensate or resid material, by using measured crude properties. These measured properties are used to adjust the synthesized assay values to obtain a more accurate representation of the unknown hydrocarbon material.



A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors  

PubMed Central

To clarify the role of a number of mRNA processing factors in transcription elongation, we developed an in vivo assay for direct analysis of elongation on chromatin. The assay relies on two substrates containing two G-less cassettes separated by either a long and GC-rich or a short and GC-poor DNA sequence (G-less-based run-on (GLRO) assay). We demonstrate that PAF, THSC/TREX-2, SAGA, the exosome component Rrp6 and two subunits of cleavage factor IA (Rna14 and Rna15) are required for efficient transcription elongation, in contrast to some results obtained using other assays. Next, we undertook a mutant screen and found out that the Nup84 nucleoporin complex is also required for transcription elongation, as confirmed by the GLRO assay and RNA polymerase II chromatin immunoprecipitations. Therefore, in addition to showing that the GLRO assay is a sensitive and reliable method for the analysis of elongation in vivo, this study provides evidence for a new role of the Nup84 complex and a number of mRNA processing factors in transcription elongation that supports a connection of pre-mRNA processing and nuclear export with transcription elongation. PMID:21478823

Tous, Cristina; Rondon, Ana G; Garcia-Rubio, Maria; Gonzalez-Aguilera, Cristina; Luna, Rosa; Aguilera, Andres



The effect of cilostamide on gap junction communication dynamics, chromatin remodeling, and competence acquisition in pig oocytes following parthenogenetic activation and nuclear transfer.  


In the pig, the efficiency of in vitro embryo production and somatic cell nuclear transfer (SCNT) procedures remains limited. It has been suggested that prematuration treatments (pre-IVM) based on the prolongation of a patent, bidirectional crosstalk between the oocyte and the cumulus cells through gap junction mediate communication (GJC), with the maintenance of a proper level of cAMP, could improve the developmental capability of oocytes. The aim of this study was to assess: 1) dose-dependent effects of cilostamide on nuclear maturation kinetics, 2) the relationship between treatments on GJC functionality and large-scale chromatin configuration changes, and 3) the impact of treatments on developmental competence acquisition after parthenogenetic activation (PA) and SCNT. Accordingly, cumulus-oocyte complexes were collected from 3- to 6-mm antral follicles and cultured for 24 h in defined culture medium with or without 1 ?M cilostamide. GJC functionality was assessed by Lucifer yellow microinjection, while chromatin configuration was evaluated by fluorescence microscopy after nuclear staining. Cilostamide administration sustained functional coupling for up to 24 h of culture and delayed meiotic resumption, as only 25.6% of cilostamide-treated oocytes reached the pro-metaphase I stage compared to the control (69.7%; P < 0.05). Moreover, progressive chromatin condensation was delayed before meiotic resumption based upon G2/M biomarker phosphoprotein epitope acquisition using immunolocalization. Importantly, cilostamide treatment under these conditions improved oocyte developmental competence, as reflected in higher blastocyst quality after both parthenogenetic activation and SCNT. PMID:23926281

Dieci, Cecilia; Lodde, Valentina; Franciosi, Federica; Lagutina, Irina; Tessaro, Irene; Modina, Silvia C; Albertini, David F; Lazzari, Giovanna; Galli, Cesare; Luciano, Alberto M



Mass condensation on networks  

NASA Astrophysics Data System (ADS)

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

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



Immunochromatographic assay on thread.  


Lateral-flow immunochromatographic assays are low-cost, simple-to-use, rapid tests for point-of-care screening of infectious diseases, drugs of abuse, and pregnancy. However, lateral flow assays are generally not quantitative, give a yes/no answer, and lack multiplexing. Threads have recently been proposed as a support for transporting and mixing liquids in lateral-flow immunochromatographic assays, but their use for quantitative high-sensitivity immunoassays has yet to be demonstrated. Here, we introduce the immunochromatographic assay on thread (ICAT) in a cartridge format that is suitable for multiplexing. The ICAT is a sandwich assay performed on a cotton thread knotted to a nylon fiber bundle, both of which are precoated with recognition antibodies against one target analyte. Upon sample application, the assay results become visible to the eye within a few minutes and are quantified using a flatbed scanner. Assay conditions were optimized, the binding curves for C-reactive protein (CRP) in buffer and diluted serum were established and a limit of detection of 377 pM was obtained. The possibility of multiplexing was demonstrated using three knotted threads coated with antibodies against CRP, osteopontin, and leptin proteins. The performance of the ICAT was compared with that of the paper-based and conventional assays. The results suggest that thread is a suitable support for making low-cost, sensitive, simple-to-use, and multiplexed diagnostic tests. PMID:22889381

Zhou, Gina; Mao, Xun; Juncker, David



Condensation phenomena in plasmonics  

E-print Network

We study arrays of plasmonic nanoparticles combined with quantum emitters, quantum plasmonic lattices, as a platform for room temperature studies of quantum many-body physics. We outline a theory to describe surface plasmon polariton distributions when they are coupled to externally pumped molecules. The possibility of tailoring the dispersion in plasmonic lattices allows realization of a variety of distributions, including the Bose-Einstein distribution as in photon condensation. We show that the presence of losses can relax some of the standard dimensionality restrictions for condensation.

Martikainen, J -P; Törmä, P



Galaxies as condensates  

E-print Network

A novel interpretation of MOND is presented. For galactic data, in addition to Newtonian acceleration, there is an attractive acceleration peaking at Milgrom's parameter a_0. The peak lies within experimental error where a_0 = cH_0/2\\pi and H_0 is the present-time value of the Hubble constant. This peaking may be understood in terms of quantum mechanical mixing between Newtonian gravitation and the condensation mechanism. There are five pointers towards galaxies being Fermi-Dirac condensates.

D. V. Bugg



Frequent mutations in chromatin-remodeling genes in pulmonary carcinoids  

PubMed Central

Pulmonary carcinoids are rare neuroendocrine tumors of the lung. The molecular alterations underlying the pathogenesis of these tumors have not been systematically studied so far. Here we perform gene copy number analysis (n=54), genome/exome (n=44) and transcriptome (n=69) sequencing of pulmonary carcinoids and observe frequent mutations in chromatin-remodeling genes. Covalent histone modifiers and subunits of the SWI/SNF complex are mutated in 40% and 22.2% of the cases respectively, with MEN1, PSIP1 and ARID1A being recurrently affected. In contrast to small-cell lung cancer and large-cell neuroendocrine tumors, TP53 and RB1 mutations are rare events, suggesting that pulmonary carcinoids are not early progenitor lesions of the highly aggressive lung neuroendocrine tumors but arise through independent cellular mechanisms. These data also suggest that inactivation of chromatin remodeling genes is sufficient to drive transformation in pulmonary carcinoids. PMID:24670920

Lu, Xin; Sun, Ruping; Ozretic, Luka; Seidal, Danila; Zander, Thomas; Leenders, Frauke; George, Julie; Muller, Christian; Dahmen, Ilona; Pinther, Berit; Bosco, Graziella; Konrad, Kathryn; Altmuller, Janine; Nurnberg, Peter; Achter, Viktor; Lang, Ulrich; Schneider, Peter M; Bogus, Magdalena; Soltermann, Alex; Brustugun, Odd Terje; Helland, Aslaug; Solberg, Steinar; Lund-Iversen, Marius; Ansen, Sascha; Stoelben, Erich; Wright, Gavin M.; Russell, Prudence; Wainer, Zoe; Solomon, Benjamin; Field, John K; Hyde, Russell; Davies, Michael PA.; Heukamp, Lukas C; Petersen, Iver; Perner, Sven; Lovly, Christine; Cappuzzo, Federico; Travis, William D; Wolf, Jurgen; Vingron, Martin; Brambilla, Elisabeth; Haas, Stefan A.; Buettner, Reinhard; Thomas, Roman K



Barrier Proteins Remodel and Modify Chromatin To Restrict Silenced Domains†  

PubMed Central

Transcriptionally active and inactive domains are frequently found adjacent to one another in the eukaryotic nucleus. To better understand the underlying mechanisms by which domains maintain opposing transcription patterns, we performed a systematic genomewide screen for proteins that may block the spread of silencing in yeast. This analysis identified numerous proteins with efficient silencing blocking activities, and some of these have previously been shown to be involved in chromatin dynamics. We isolated subunits of Swi/Snf, mediator, and TFIID, as well as subunits of the Sas-I, SAGA, NuA3, NuA4, Spt10p, Rad6p, and Dot1p complexes, as barrier proteins. We demonstrate that histone acetylation and chromatin remodeling occurred at the barrier and correlated with a block to the spread of silencing. Our data suggest that multiple overlapping mechanisms were involved in delimiting silenced and active domains in vivo. PMID:14966276

Oki, Masaya; Valenzuela, Lourdes; Chiba, Tomoko; Ito, Takashi; Kamakaka, Rohinton T.



Androgen receptor-driven chromatin looping in prostate cancer  

PubMed Central

Androgen receptor (AR) is important for prostate cancer development and progression. Genome-wide mapping of AR binding sites in prostate cancer have found that the majority of AR binding sites are located within non-promoter regions. These distal AR binding regions regulate AR target genes (e.g. UBE2C) involved in prostate cancer growth through chromatin looping. In addition to long-distance gene regulation, looping has been shown to induce spatial proximity of two genes otherwise located far away along the genomic sequence and the formation of double strand DNA breaks, resulting in aberrant gene fusions (e.g. TMPRSS2-ERG) that also contribute to prostate tumorigenesis. Elucidating the mechanisms of AR-driven chromatin looping will increase our understanding of prostate carcinogenesis and may lead to the identification of new therapeutic targets. PMID:21889355

Wu, Dayong; Zhang, Chunpeng; Shen, Yanping; Nephew, Kenneth P.; Wang, Qianben



Radiation-induced DNA damage and chromatin structure  

NASA Technical Reports Server (NTRS)

DNA lesions induced by ionizing radiation in cells are clustered and not randomly distributed. For low linear energy transfer (LET) radiation this clustering occurs mainly on the small scales of DNA molecules and nucleosomes. For example, experimental evidence suggests that both strands of DNA on the nucleosomal surface can be damaged in single events and that this damage occurs with a 10-bp modulation because of protection by histones. For high LET radiation, clustering also occurs on a larger scale and depends on chromatin organization. A particularly significant clustering occurs when an ionizing particle traverses the 30 nm chromatin fiber with generation of heavily damaged DNA regions with an average size of about 2 kbp. On an even larger scale, high LET radiation can produce several DNA double-strand breaks in closer proximity than expected from randomness. It is suggested that this increases the probability of misrejoining of DNA ends and generation of lethal chromosome aberrations.

Rydberg, B.; Chatterjee, A. (Principal Investigator)



Chromatin structure and transposable elements in organismal aging  

PubMed Central

Epigenetic regulatory mechanisms are increasingly appreciated as central to a diverse array of biological processes, including aging. An association between heterochromatic silencing and longevity has long been recognized in yeast, and in more recent years evidence has accumulated of age-related chromatin changes in Caenorhabditis elegans, Drosophila, and mouse model systems, as well as in the tissue culture-based replicative senescence model of cell aging. In addition, a number of studies have linked expression of transposable elements (TEs), as well as changes in the RNAi pathways that cells use to combat TEs, to the aging process. This review summarizes the recent evidence linking chromatin structure and function to aging, with a particular focus on the relationship of heterochromatin structure to organismal aging. PMID:24363663

Wood, Jason G.; Helfand, Stephen L.



piRNA clusters and open chromatin structure  

PubMed Central

Transposable elements (TEs) are major structural components of eukaryotic genomes; however, mobilization of TEs generally has negative effects on the host genome. To counteract this threat, host cells have evolved genetic and epigenetic mechanisms that keep TEs silenced. One such mechanism involves the Piwi-piRNA complex, which represses TEs in animal gonads either by cleaving TE transcripts in the cytoplasm or by directing specific chromatin modifications at TE loci in the nucleus. Most Piwi-interacting RNAs (piRNAs) are derived from genomic piRNA clusters. There has been remarkable progress in our understanding of the mechanisms underlying piRNA biogenesis. However, little is known about how a specific locus in the genome is converted into a piRNA-producing site. In this review, we will discuss a possible link between chromatin boundaries and piRNA cluster formation. PMID:25126116



Chromatin Immunoprecipitation to Characterize the Epigenetic Profiles of Imprinted Domains  

PubMed Central

Summary Imprinted genes are marked by parental allele specific DNA methylation and histone modifications which regulate their monoallelic expression. Chromatin immunoprecipitation (ChIP) is the technique of choice to characterize the histones associated with either the maternal or paternal chromosomes. To study allele-specific chromatin composition at imprinted regions, the method has to be efficient to work on limiting amount of starting material, and specific enough to recognize one of the parental alleles. We optimized the commonly used ChIP technique for efficient recovery of one parental allele from small number of cells. We provide examples to show that this ChIP protocol can specifically distinguish between parental alleles in mouse embryo fibroblasts carrying maternal and paternal duplication of mouse distal Chr7 and also in normal mouse embryo fibroblasts carrying single nucleotide polymorphism at imprinted regions. PMID:22907496

Singh, Purnima; Szabo, Piroska E.



Epigenetic control of hematopoiesis: the PU.1 chromatin connection.  


Purine-rich box1 (PU.1) is a transcription factor that not only has a key role in the development of most hematopoietic cell lineages but also in the suppression of leukemia. To exert these functions, PU.1 can cross-talk with multiple different proteins by forming complexes in order to activate or repress transcription. Among its protein partners are chromatin remodelers, DNA methyltransferases, and a number of other transcription factors with important roles in hematopoiesis. While a great deal of knowledge has been acquired about PU.1 function over the years, it was the development of novel genome-wide technologies, which boosted our understanding of how PU.1 acts on the chromatin to drive its repertoire of target genes. This review summarizes current knowledge and ideas of molecular mechanisms by which PU.1 controls hematopoiesis and suppresses leukemia. PMID:25205721

van Riel, Boet; Rosenbauer, Frank



AGING: Aging, Chromatin, and Food Restriction--Connecting the Dots  

NSDL National Science Digital Library

Access to the article is free, however registration and sign-in are required. Model organisms such as yeast have proved exceptionally valuable for revealing new information about the molecular pathways involved in the aging of cells. In her Perspective, Campisi comments on new work showing that caloric restriction increases longevity in yeast by activating the SIR2 gene, which alters the compactness of chromatin and thus regulates gene expression (Lin et al.).

Judith Campisi (Lawrence Berkeley National Laboratory;Life Sciences Division)



Chromatin Architecture and the Generation of Antigen Receptor Diversity  

PubMed Central

The adaptive immune system generates a specific response to a vast spectrum of antigens. This remarkable property is achieved by lymphocytes that each express single and unique antigen receptors. During lymphocyte development, antigen receptor coding elements are assembled from widely dispersed gene segments. The assembly of antigen receptors is controlled at multiple levels, including epigenetic marking, nuclear location, and chromatin topology. Here we review recently uncovered mechanisms that underpin long-range genomic interactions and the generation of antigen receptor diversity. PMID:19665968

Jhunjhunwala, Suchit; van Zelm, Menno C.; Peak, Mandy; Murre, Cornelis



Chromatin structure analyses identify miRNA promoters  

Microsoft Academic Search

Although microRNAs (miRNAs) are key regulators of gene expression in normal human physiology and disease, transcriptional regulation of miRNAs is poorly understood, because most miRNA promoters have not yet been characterized. We identified the proximal promoters of 175 human miRNAs by combining nucleosome mapping with chromatin signatures for promoters. We observe that one-third of intronic miRNAs have transcription initiation regions

Fatih Ozsolak; Laura L. Poling; Zhengxin Wang; Hui Liu; X. Shirley Liu; Robert G. Roeder; Xinmin Zhang; Jun S. Song; David E. Fisher



Chromatin dynamics and the preservation of genetic information  

Microsoft Academic Search

The integrity of the genome is frequently challenged by double-strand breaks in the DNA. Defects in the cellular response to double-strand breaks are a major cause of cancer and other age-related pathologies; therefore, much effort has been directed at understanding the enzymatic mechanisms involved in recognizing, signalling and repairing double-strand breaks. Recent work indicates that chromatin — the fibres into

Jessica A. Downs; Michel C. Nussenzweig; André Nussenzweig



Functional Domains of the Ubiquitous Chromatin Protein DEK  

Microsoft Academic Search

DEK was originally described as a proto-oncogene protein and is now known to be a major component of metazoan chromatin. DEK is able to modify the structure of DNA by introducing supercoils. In order to find interaction partners and functional domains of DEK, we performed yeast two-hybrid screens and mutational analyses. Two-hybrid screening yielded C-terminal fragments of DEK, suggesting that

Ferdinand Kappes; Ingo Scholten; Nicole Richter; Claudia Gruss; Tanja Waldmann



Brain neuronal chromatin responses in acute soman intoxicated rats  

Microsoft Academic Search

Male Sprague-Dawley rats (200 g) were injected subcutaneously with soman, a potent neuronal acetylcholinesterase (AChE) inhibitor, at doses of 0.5, 0.8 and 1.0 LD50 (1 LD50=135 µg\\/kg) before decapitation at 1 and 24 h post-exposure. Correlative data were obtained on the severity of brain AChE inactivation and physicochemical changes in nuclear chromatin of cerebrocortical (layer V) and striatal neurons using

L. J. Martin; J. A. Doebler; T. J. Wall; T. M. Shih; A. Anthony



Preservation of large-scale chromatin structure in FISH experiments  

PubMed Central

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

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



Combinatorial epigenetic patterns as quantitative predictors of chromatin biology  

PubMed Central

Background Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is the most widely used method for characterizing the epigenetic states of chromatin on a genomic scale. With the recent availability of large genome-wide data sets, often comprising several epigenetic marks, novel approaches are required to explore functionally relevant interactions between histone modifications. Computational discovery of "chromatin states" defined by such combinatorial interactions enabled descriptive annotations of genomes, but more quantitative approaches are needed to progress towards predictive models. Results We propose non-negative matrix factorization (NMF) as a new unsupervised method to discover combinatorial patterns of epigenetic marks that frequently co-occur in subsets of genomic regions. We show that this small set of combinatorial "codes" can be effectively displayed and interpreted. NMF codes enable dimensionality reduction and have desirable statistical properties for regression and classification tasks. We demonstrate the utility of codes in the quantitative prediction of Pol2-binding and the discrimination between Pol2-bound promoters and enhancers. Finally, we show that specific codes can be linked to molecular pathways and targets of pluripotency genes during differentiation. Conclusions We have introduced and evaluated a new computational approach to represent combinatorial patterns of epigenetic marks as quantitative variables suitable for predictive modeling and supervised machine learning. To foster widespread adoption of this method we make it available as an open-source software-package – epicode at PMID:24472558



The accessible chromatin landscape of the human genome.  


DNase?I hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ?2.9 million DHSs that encompass virtually all known experimentally validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. We connect ?580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is organized with dozens to hundreds of co-activated elements, and the transcellular DNase?I sensitivity pattern at a given region can predict cell-type-specific functional behaviours. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation. PMID:22955617

Thurman, Robert E; Rynes, Eric; Humbert, Richard; Vierstra, Jeff; Maurano, Matthew T; Haugen, Eric; Sheffield, Nathan C; Stergachis, Andrew B; Wang, Hao; Vernot, Benjamin; Garg, Kavita; John, Sam; Sandstrom, Richard; Bates, Daniel; Boatman, Lisa; Canfield, Theresa K; Diegel, Morgan; Dunn, Douglas; Ebersol, Abigail K; Frum, Tristan; Giste, Erika; Johnson, Audra K; Johnson, Ericka M; Kutyavin, Tanya; Lajoie, Bryan; Lee, Bum-Kyu; Lee, Kristen; London, Darin; Lotakis, Dimitra; Neph, Shane; Neri, Fidencio; Nguyen, Eric D; Qu, Hongzhu; Reynolds, Alex P; Roach, Vaughn; Safi, Alexias; Sanchez, Minerva E; Sanyal, Amartya; Shafer, Anthony; Simon, Jeremy M; Song, Lingyun; Vong, Shinny; Weaver, Molly; Yan, Yongqi; Zhang, Zhancheng; Zhang, Zhuzhu; Lenhard, Boris; Tewari, Muneesh; Dorschner, Michael O; Hansen, R Scott; Navas, Patrick A; Stamatoyannopoulos, George; Iyer, Vishwanath R; Lieb, Jason D; Sunyaev, Shamil R; Akey, Joshua M; Sabo, Peter J; Kaul, Rajinder; Furey, Terrence S; Dekker, Job; Crawford, Gregory E; Stamatoyannopoulos, John A



Epigenetics and chromatin plasticity in embryonic stem cells  

PubMed Central

The study of embryonic stem cells is in the spotlight in many laboratories that study the structure and function of chromatin and epigenetic processes. The key properties of embryonic stem cells are their capacity for self-renewal and their pluripotency. Pluripotent stem cells are able to differentiate into the cells of all three germ layers, and because of this property they represent a promising therapeutic tool in the treatment of diseases such as Parkinson’s disease and diabetes, or in the healing of lesions after heart attack. As the basic nuclear unit, chromatin is responsible for the regulation of the functional status of cells, including pluripotency and differentiation. Therefore, in this review we discuss the functional changes in chromatin during differentiation and the correlation between epigenetics events and the differentiation potential of embryonic stem cells. In particular we focus on post-translational histone modification, DNA methylation and the heterochromatin protein HP1 and its unique function in mouse and human embryonic stem cells. PMID:23951389

Prikrylova, Terezia; Pachernik, Jiri; Kozubek, Stanislav; Bartova, Eva



Lessons from Anaplasma phagocytophilum: Chromatin Remodeling by Bacterial Effectors  

PubMed Central

Bacterial pathogens can alter global host gene expression via histone modifications and chromatin remodeling in order to subvert host responses, including those involved with innate immunity, allowing for bacterial survival. Shigella flexneri, Listeria monocytogenes, Chlamydia trachomatis, and Anaplasma phagocytophilum express effector proteins that modify host histones and chromatin structure. A. phagocytophilum modulates granulocyte respiratory burst in part by dampening transcription of several key phagocyte oxidase genes. The A. phagocytophilum protein AnkA localizes to the myeloid cell nucleus where it binds AT-rich regions in the CYBB promoter and decreases its transcription. AT-rich regions of DNA are characteristic of matrix attachment regions (MARs) which are critical for chromatin structure and transcription. MAR-binding proteins, such as SATB1, interact with histone modifying enzymes resulting in altered gene expression. With A. phagocytophilum infection, histone deacetylase 1 (HDAC1) expression is increased and histone H3 acetylation is decreased at the CYBB promoter, suggesting a role for AnkA in altering host epigenetics and modulating gene transcription, at this, and perhaps other loci. This review will focus on how bacterial pathogens alter host epigenetics, by specifically examining A. phagocytophilum AnkA cis-regulation of CYBB transcription and epigenetic changes associated with infection. PMID:23082961

Rennoll-Bankert, Kristen E; Dumler, J Stephen



The accessible chromatin landscape of the human genome  

PubMed Central

DNaseI hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers, and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ~2.9 million DHSs that encompass virtually all known experimentally-validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation, and regulatory factor occupancy patterns. We connect ~580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is choreographed with dozens to hundreds of co-activated elements, and the trans-cellular DNaseI sensitivity pattern at a given region can predict cell type-specific functional behaviors. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation. PMID:22955617

Thurman, Robert E.; Rynes, Eric; Humbert, Richard; Vierstra, Jeff; Maurano, Matthew T.; Haugen, Eric; Sheffield, Nathan C.; Stergachis, Andrew B.; Wang, Hao; Vernot, Benjamin; Garg, Kavita; Sandstrom, Richard; Bates, Daniel; Canfield, Theresa K.; Diegel, Morgan; Dunn, Douglas; Ebersol, Abigail K.; Frum, Tristan; Giste, Erika; Harding, Lisa; Johnson, Audra K.; Johnson, Ericka M.; Kutyavin, Tanya; Lajoie, Bryan; Lee, Bum-Kyu; Lee, Kristen; London, Darin; Lotakis, Dimitra; Neph, Shane; Neri, Fidencio; Nguyen, Eric D.; Reynolds, Alex P.; Roach, Vaughn; Safi, Alexias; Sanchez, Minerva E.; Sanyal, Amartya; Shafer, Anthony; Simon, Jeremy M.; Song, Lingyun; Vong, Shinny; Weaver, Molly; Zhang, Zhancheng; Zhang, Zhuzhu; Lenhard, Boris; Tewari, Muneesh; Dorschner, Michael O.; Hansen, R. Scott; Navas, Patrick A.; Stamatoyannopoulos, George; Iyer, Vishwanath R.; Lieb, Jason D.; Sunyaev, Shamil R.; Akey, Joshua M.; Sabo, Peter J.; Kaul, Rajinder; Furey, Terrence S.; Dekker, Job; Crawford, Gregory E.; Stamatoyannopoulos, John A.



Histone modifications and lamin A regulate chromatin protein dynamics in early embryonic stem cell differentiation  

PubMed Central

Embryonic stem cells are characterized by unique epigenetic features including decondensed chromatin and hyperdynamic association of chromatin proteins with chromatin. Here we investigate the potential mechanisms that regulate chromatin plasticity in embryonic stem cells. Using epigenetic drugs and mutant embryonic stem cells lacking various chromatin proteins, we find that histone acetylation, G9a-mediated histone H3 lysine 9 (H3K9) methylation and lamin A expression, all affect chromatin protein dynamics. Histone acetylation controls, almost exclusively, euchromatin protein dynamics; lamin A expression regulates heterochromatin protein dynamics, and G9a regulates both euchromatin and heterochromatin protein dynamics. In contrast, we find that DNA methylation and nucleosome repeat length have little or no effect on chromatin-binding protein dynamics in embryonic stem cells. Altered chromatin dynamics associates with perturbed embryonic stem cell differentiation. Together, these data provide mechanistic insights into the epigenetic pathways that are responsible for chromatin plasticity in embryonic stem cells, and indicate that the genome’s epigenetic state modulates chromatin plasticity and differentiation potential of embryonic stem cells. PMID:22713752

Melcer, Shai; Hezroni, Hadas; Rand, Eyal; Nissim-Rafinia, Malka; Skoultchi, Arthur; Stewart, Colin L.; Bustin, Michael; Meshorer, Eran



Exciton-polariton condensates  

NASA Astrophysics Data System (ADS)

Recently a new type of system exhibiting spontaneous coherence has emerged--the exciton-polariton condensate. Exciton-polaritons (or polaritons for short) are bosonic quasiparticles that exist inside semiconductor microcavities, consisting of a superposition of an exciton and a cavity photon. Above a threshold density the polaritons macroscopically occupy the same quantum state, forming a condensate. The polaritons have a lifetime that is typically comparable to or shorter than thermalization times, giving them an inherently non-equilibrium nature. Nevertheless, they exhibit many of the features that would be expected of equilibrium Bose-Einstein condensates (BECs). The non-equilibrium nature of the system raises fundamental questions as to what it means for a system to be a BEC, and introduces new physics beyond that seen in other macroscopically coherent systems. In this review we focus on several physical phenomena exhibited by exciton-polariton condensates. In particular, we examine topics such as the difference between a polariton BEC, a polariton laser and a photon laser, as well as physical phenomena such as superfluidity, vortex formation, and Berezinskii-Kosterlitz-Thouless and Bardeen-Cooper-Schrieffer physics. We also discuss the physics and applications of engineered polariton structures.

Byrnes, Tim; Kim, Na Young; Yamamoto, Yoshihisa



Enhanced condensation heat transfer  

SciTech Connect

For the past four years, work has been in progress at ORNL to develop improved condensers for geothermal binary power cycles. The work has centered on optimizing the design variables associated with fluted surfaces on vertical tubes and comparing the tube performance with available enhanced tubes either for vertical or horizontal operation. Data with seven fluids including a hydrocarbon, fluorocarbons, and ammonia condensing on up to 30 different tubes have been obtained. Data for tubes of different effective lengths (1/2 to 4 ft) and inclination have also been obtained. The primary conclusion from this work is that the best fluted tubes can provide an enhancement in condensation coefficient by a factor of approximately 6 over smooth vertical tube performance and a factor of approximately 2 over the best enhanced commercial tubes either operating vertically or horizontally. These data, together with field test data have formed the basis for designing two prototype condensers, one for the 60 kWe Raft River, Idaho, pilot plant and one for the 500 kWe East Mesa, California, direct-contact demonstration plant.

Michel, J.W.; Murphy, R.W.



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



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



Condensed-Matter Physics.  

ERIC Educational Resources Information Center

Discusses ways computers are being used in condensed-matter physics by experimenters and theorists. Experimenters use them to control experiments and to gather and analyze data. Theorists use them for detailed predictions based on realistic models and for studies on systems not realizable in practice. (JN)

Hirsch, Jorge E.; Scalapino, Douglas J.



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



Condenser biofouling control  

SciTech Connect

A compilation of most of the papers presented at the March 1979 symposium in Atlanta, GA on condensor biofouling control is presented. The following aspects of power plant condenser biofouling are discussed: effects on power plant equipment and operations; cost impact; biofilm formation; corrosive effects; control practices using chlorine or bromine chlorides, and the dechlorination of discharges of chlorinated coolants. (LCL)

Garey, J.F.; Jorden, R.M.; Aitken, A.H.; Burton, D.T.; Gray, R.H. (eds.)



Top Quark Condensate Revisited  

Microsoft Academic Search

In the light of recent discovery of a very heavy top quark, we reexamine the top quark condensate model proposed by Miransky, Tanabashi and Yamawaki (MTY) and by Nambu. We first review the original MTY formulation based on the ladder Schwinger-Dyson equation and the Pagels-Stokar formula. It is particularly emphasized that the critical phenomenon gives a simple reason why the

Koichi Yamawaki



The Color Glass Condensate  

E-print Network

We provide a broad overview of the theoretical status and phenomenological applications of the Color Glass Condensate effective field theory describing universal properties of saturated gluons in hadron wavefunctions that are extracted from deeply inelastic scattering and hadron-hadron collision experiments at high energies.

F. Gelis; E. Iancu; J. Jalilian-Marian; R. Venugopalan




Microsoft Academic Search

This article provides data on enhanced tubes developed especially for application to steam condensers. Data are provided for different types of tube-side (cooling water) and shell-side (steam) enhancements. The first test series involved measuring the condensing coefficient at a 327 K saturation temperature. Average enhancement levels (defined as the ratio of the condensing coefficient of an enhanced tube to that

M. Hassib Jaber; Ralph L. Webb



Steam condensate corrosion; mechanism; prevention  

Microsoft Academic Search

Steam condensate is a potential corrosion problem in most industries, and because of the tremendous cost involved to protect piping and condensate recovery systems, the problem is greatly increased. The various stages of steam condensate are discussed from its beginning as raw water, through its conversion to steam in a boiler, and to its disposal to a steam consumer, such



Physics 232 Condensed Matter Physics  

E-print Network

1 Physics 232 Condensed Matter Physics Instructor: Peter Young Office: 212 ISB Telephone: 459:// TOPICS This course on condensed matter physics will cover three areas: · magnetism, · optical physics such as Condensed Matter Physics by M. Mardar Solid State Physics by N. Ashroft and N. D. Mermin

Young, A. Peter


Condenser procurement guidelines. Final report  

Microsoft Academic Search

Steam surface condensers have a major impact on power plant availability and efficiency. Since supplying condensers to the utility industry is a very competitive buisness, it is essential that all of the requirements, all of the operating and design conditions, and any off standard conditions which can affect the condenser design and performance be clearly communicated to potential suppliers. These




Principles of Condensed Matter Physics  

Microsoft Academic Search

Although there are many books on solid state physics and condensed matter, I suspect very few cover the same content as that found in {\\\\it Principles of Condensed Matter Physics} by Chaikin and Lubensky. The title is rather misleading as it suggests a survey of the important concepts in condensed matter. In spite of this there is much to commend

C C Matthai



Non-iterative condensation modeling for steam condensation with non-condensable gas in a vertical tube  

Microsoft Academic Search

Based on a heat and mass transfer analogy, an iterative condensation model for steam condensation in the presence of a non-condensable gas in a vertical tube is proposed including the high mass transfer effect, entrance effect, and interfacial waviness effect on condensation. A non-iterative condensation model is proposed for easy engineering application using the iterative condensation model and the assumption

Hee Cheon No; Hyun Sik Park



The Binding Sites for the Chromatin Insulator Protein CTCF Map to DNA Methylation-Free Domains Genome-Wide  

PubMed Central

All known vertebrate chromatin insulators interact with the highly conserved, multivalent 11-zinc finger nuclear factor CTCF to demarcate expression domains by blocking enhancer or silencer signals in a position-dependent manner. Recent observations document that the properties of CTCF include reading and propagating the epigenetic state of the differentially methylated H19 imprinting control region. To assess whether these findings may reflect a universal role for CTCF targets, we identified more than 200 new CTCF target sites by generating DNA microarrays of clones derived from chromatin-immunopurified (ChIP) DNA followed by ChIP-on-chip hybridization analysis. Target sites include not only known loci involved in multiple cellular functions, such as metabolism, neurogenesis, growth, apoptosis, and signalling, but potentially also heterochromatic sequences. Using a novel insulator trapping assay, we also show that the majority of these targets manifest insulator functions with a continuous distribution of stringency. As these targets are generally DNA methylation-free as determined by antibodies against 5-methylcytidine and a methyl-binding protein (MBD2), a CTCF-based network correlates with genome-wide epigenetic states. PMID:15256511

Mukhopadhyay, Rituparna; Yu, WenQiang; Whitehead, Joanne; Xu, JunWang; Lezcano, Magda; Pack, Svetlana; Kanduri, Chandrasekhar; Kanduri, Meena; Ginjala, Vasudeva; Vostrov, Alexander; Quitschke, Wolfgang; Chernukhin, Igor; Klenova, Elena; Lobanenkov, Victor; Ohlsson, Rolf



In vivo effects of Trichostatin A - A histone deacetylase inhibitor - On chromatin remodeling during Triturus cristatus spermatogenesis.  


A major challenge in developmental biology field is to decipher the molecular mechanisms involved in cellular differentiation and to understand the processes that control and regulate genes expression. The study of nuclear molecular architecture during gametogenesis represents one approach toward deciphering the molecular organization and function of the eukaryotic chromatin. As spermatogenesis progresses, there is a widespread reorganization of the haploid genome followed by extensive DNA compaction. It is becoming increasingly evident that the dynamic composition of chromatin plays an important role in the activities of enzymes and in the processes that act upon it. As the information in the existing literature regarding the epigenetic modifications occurring in the advanced stages of spermatogenesis of crested newt is still scarce, we have investigated the effect of a Histone Deacetylase (HDAC) inhibitor, Trichostatin A (TSA), at the cytological level (by transmission electron microscopy - TEM, immunohistochemistry technique, fluorescence microscopy) and at the molecular level (AUT-PAGE and ChIP assay) on Triturus cristatus spermatogenesis. Our results have revealed an important role for regulation of histone deacetylase activity in controlling histone hyperacetylation and the replacement with sperm nuclear basic proteins during spermiogenesis. PMID:24100069

Burliba?a, Liliana; Zarnescu, Otilia



The Proteasome Inhibitor Bortezomib Induces an Inhibitory Chromatin Environment at a Distal Enhancer of the Estrogen Receptor-? Gene  

PubMed Central

Expression of the estrogen receptor-? (ER?) gene, ESR1, is a clinical biomarker used to predict therapeutic outcome of breast cancer. Hence, there is significant interest in understanding the mechanisms regulating ESR1 gene expression. Proteasome activity is increased in cancer and we previously showed that proteasome inhibition leads to loss of ESR1 gene expression in breast cancer cells. Expression of ESR1 mRNA in breast cancer cells is controlled predominantly through a proximal promoter within ?400 base pair (bp) of the transcription start site (TSS). Here, we show that loss of ESR1 gene expression induced by the proteasome inhibitor bortezomib is associated with inactivation of a distal enhancer located 150 kilobases (kb) from the TSS. Chromatin immunoprecipitation assays reveal several bortezomib-induced changes at the distal site including decreased occupancy of three critical transcription factors, GATA3, FOXA1, and AP2?. Bortezomib treatment also resulted in decreased histone H3 and H4 acetylation and decreased occupancy of histone acetyltransferase, p300. These data suggest a mechanism to explain proteasome inhibitor-induced loss of ESR1 mRNA expression that highlights the importance of the chromatin environment at the ?150 kb distal enhancer in regulation of basal expression of ESR1 in breast cancer cells. PMID:24339902

Powers, Ginny L.; Rajbhandari, Prashant; Solodin, Natalia M.; Bickford, Brant; Alarid, Elaine T.



Characterisation of a subpopulation of sperm with massive nuclear damage, as recognised with the sperm chromatin dispersion test.  


Assessment of human sperm DNA fragmentation by the sperm chromatin dispersion (SCD) test is based on the detection of haloes of spreading DNA loops after sequential DNA denaturing and protamine removal. After the SCD test, sperm without DNA fragmentation show chromatin haloes emerging from the central nuclear core, while sperm containing fragmented DNA present small or no haloes. The nuclear degraded sperm are recognised as a differentiated category within the sperm with fragmented DNA, whose cores appear irregularly and/or faintly stained. This subpopulation is more prevalent in patients with varicocele. Protein staining with 2.7-dibrom-4-hydroxy-mercury-fluorescein demonstrated that degraded sperm intensely lose nuclear core proteins after the SCD processing. Moreover, degraded sperm are 65% more faintly labelled for DNA breaks after in situ nick translation (ISNT) on average, due to extensive DNA loss. A two-dimensional comet assay under sequential neutral and alkaline conditions demonstrated that degraded sperm contain both massive double- and single-strand DNA breaks. The degraded sperm appear as a subpopulation with stronger nuclear damage, affecting both DNA and protein fractions, possibly due to intense intratesticular oxidative stress, what could explain its higher proportion in patients with varicocele. PMID:23710631

Gosálvez, J; Rodríguez-Predreira, M; Mosquera, A; López-Fernández, C; Esteves, S C; Agarwal, A; Fernández, J L



Assays without Borders

CPTAC researchers partner with international labs to demonstrate the ability of Targeted mass spectrometry–based assays to reproducibly quantify Human proteins across labs, countries and continents in a recently published journal article.


Genomic response to Wnt signalling is highly context-dependent - Evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets  

SciTech Connect

Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-{beta}-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of {beta}-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays of gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.

Railo, Antti [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland)] [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland); Pajunen, Antti [Department of Biochemistry, University of Oulu (Finland)] [Department of Biochemistry, University of Oulu (Finland); Itaeranta, Petri; Naillat, Florence; Vuoristo, Jussi; Kilpelaeinen, Pekka [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland)] [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland); Vainio, Seppo, E-mail: [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland)] [Oulu Centre for Cell Matrix Research, Biocenter Oulu, Laboratory of Developmental Biology and Department of Medical Biochemistry and Molecular Biology, FIN-90014, University of Oulu, P. O. Box 5000 (Finland)



Microfluidic DNA hybridization assays  

Microsoft Academic Search

DNA hybridization is one of the most powerful techniques applied in diagnostic assays. Microfluidics provides a promising\\u000a means to analyse small sample volumes, reduce reagent consumption and cost, shorten processing time as well as develop fast,\\u000a sensitive and portable diagnostic tools. By coupling with the microfluidic technology, DNA hybridization assay can achieve\\u000a high sensitivity, enhance hybridization kinetics and decrease the

Xuan WengHai; Hai Jiang; Dongqing Li


Doped colorimetric assay liposomes  


The present invention provides compositions comprising colorimetric assay liposomes. The present invention also provides methods for producing colorimetric liposomes and calorimetric liposome assay systems. In preferred embodiments, these calorimetric liposome systems provide high levels of sensitivity through the use of dopant molecules. As these dopants allow the controlled destabilization of the liposome structure, upon exposure of the doped liposomes to analyte(s) of interest, the indicator color change is facilitated and more easily recognized.

Charych, Deborah (Albany, CA); Stevens, Raymond C. (Albany, CA)



Direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep: in vitro and in vivo studies  

Microsoft Academic Search

In vitro and in vivo studies were conducted to determine possible direct anthelmintic effects of condensed tannins towards different ovine gastrointestinal nematodes. A larval development\\/viability assay was used to investigate the effect of a condensed tannin extract (Quebracho) towards larvae of Haemonchuscontortus, Teladorsagiacircumcincta and Trichostrongylusvitrinus. The development to infective larvae and their viability was assessed in all three species and

S Athanasiadou; I Kyriazakis; F Jackson; R. L Coop



Interference of condensed tannin in lignin analyses of dry bean and forage crops.  


Legumes with high concentrations of condensed tannin (pinto bean [Phaseolus vulgaris L.], sainfoin [Onobrychis viciifolia Scop.], and big trefoil [Lotus uliginosus Hoff.]), were compared to a selection of forages, with low or zero condensed tannin (smooth bromegrass [ Bromus inermis Leyss], Lotus japonicus [Regel] K. Larsen, and alfalfa [Medicago sativa L.]), using four methods to estimate fiber or lignin. Protocols were validated by using semipurified condensed tannin polymers in adulteration assays that tested low-lignin tissue with polyphenolic-enriched samples. The effect on lignin assay methods by condensed tannin concentration was interpreted using a multivariate analysis. There was an overestimation of fiber or lignin in the presence of condensed tannin in the acid detergent fiber (ADF) and Klason lignin (KL) assays compared to that in the thioglycolic acid (TGA) and acid detergent lignin (ADL) methods. Sulfite reagents (present in TGA lignin method) or sequential acidic digests at high temperatures (ADF followed by ADL) were required to eliminate condensed tannin. The ADF (alone) and KL protocols are not recommended to screen nonwoody plants, such as forages, where condensed tannin has accumulated in the tissue. PMID:18841900

Marles, M A Susan; Coulman, Bruce E; Bett, Kirstin E



Low molecular weight peptides controlling transcription are present in the calf thymus chromatin structure  

Microsoft Academic Search

A calf thymus peptide fraction controlling DNA and chromatin template has been purified by DNA-cellulose and Dowex 50 WX2 chromatography and its amino acid composition determined. The active peptide fraction can be extracted in high pH buffer from calf thymus native chromatin previously deproteinized by chloroform-isoamylalcohol and phenol. These data demonstrate that the thymic peptide(s) is (are) a chromatin protein

G. L. Gianfranceschi; L. Guglielmi; D. Amici; F. Bossa; D. Barra; R. Petruzzelli



Probing the W chromosome of the codling moth, Cydia pomonella , with sequences from microdissected sex chromatin  

Microsoft Academic Search

The W chromosome of the codling moth, Cydia pomonella, like that of most Lepidoptera species, is heterochromatic and forms a female-specific sex chromatin body in somatic cells.\\u000a We collected chromatin samples by laser microdissection from euchromatin and W-chromatin bodies. DNA from the samples was\\u000a amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) and used to prepare painting probes and\\u000a start

Iva Fuková; Walther Traut; Magda Vítková; Petr Nguyen; Svatava Kubí?ková; František Marec



Condenser performance monitoring and cleaning  

SciTech Connect

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

Walden, J.V. [Rochester Gas and Electric, Ontario, NY (United States)



Pax6 Interactions with Chromatin and Identification of Its Novel Direct Target Genes in Lens and Forebrain  

PubMed Central

Pax6 encodes a specific DNA-binding transcription factor that regulates the development of multiple organs, including the eye, brain and pancreas. Previous studies have shown that Pax6 regulates the entire process of ocular lens development. In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific populations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification in telencephalon. In the pancreas, Pax6 is essential for the differentiation of ?-, ?- and ?-islet cells. To elucidate molecular roles of Pax6, chromatin immunoprecipitation experiments combined with high-density oligonucleotide array hybridizations (ChIP-chip) were performed using three distinct sources of chromatin (lens, forebrain and ?-cells). ChIP-chip studies, performed as biological triplicates, identified a total of 5,260 promoters occupied by Pax6. 1,001 (133) of these promoter regions were shared between at least two (three) distinct chromatin sources, respectively. In lens chromatin, 2,335 promoters were bound by Pax6. RNA expression profiling from Pax6+/? lenses combined with in vivo Pax6-binding data yielded 76 putative Pax6-direct targets, including the Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca genes. RNA and ChIP data were validated for all these genes. In lens cells, reporter assays established Kib1b and Snca as Pax6 activated and repressed genes, respectively. In situ hybridization revealed reduced expression of these genes in E14 cerebral cortex. Moreover, we examined differentially expressed transcripts between E9.5 wild type and Pax6?/? lens placodes that suggested Efnb2, Fat4, Has2, Nav1, and Trpm3 as novel Pax6-direct targets. Collectively, the present studies, through the identification of Pax6-direct target genes, provide novel insights into the molecular mechanisms of Pax6 gene control during mouse embryonic development. In addition, the present data demonstrate that Pax6 interacts preferentially with promoter regions in a tissue-specific fashion. Nevertheless, nearly 20% of the regions identified are accessible to Pax6 in multiple tissues. PMID:23342162

Huang, Jie; Ninkovic, Jovica; Walcher, Tessa; Wolf, Louise; Vitenzon, Ariel; Zheng, Deyou; Gotz, Magdalena; Beebe, David C.; Zavadil, Jiri; Cvekl, Ales



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.


Small RNA in the nucleus: the RNA-chromatin ping-pong  

PubMed Central

Eukaryotes use several classes of small RNA molecules to guide diverse protein machineries to target messenger RNA. The role of small RNA in post-transcriptional regulation of mRNA stability and translation is now well established. Small RNAs can also guide sequence-specific modification of chromatin structure and thus contribute to establishment and maintenance of distinct chromatin domains. In this review we summarize the model for the inter-dependent interaction between small RNA and chromatin that has emerged from studies on fission yeast and plants. We focus on recent results that link a distinct class of small RNAs, the piRNAs, to chromatin regulation in animals. PMID:22349141

Olovnikov, Ivan; Aravin, Alexei A.; Toth, Katalin Fejes



Transcriptional Regulation During Adipocyte Differentiation: A Role for SWI/SNF Chromatin Remodeling Enzymes: A Dissertation.  

E-print Network

??Chromatin has a compact organization in which most DNA sequences are structurally inaccessible and functionally inactive. Reconfiguration of thechromatir required to activate transcription. This reconfiguration… (more)

Salma, Nunciada



Identifying estrogen receptor ? target genes using integrated computational genomics and chromatin immunoprecipitation microarray  

PubMed Central

The estrogen receptor ? (ER?) regulates gene expression by either direct binding to estrogen response elements or indirect tethering to other transcription factors on promoter targets. To identify these promoter sequences, we conducted a genome-wide screening with a novel microarray technique called ChIP-on-chip. A set of 70 candidate ER? loci were identified and the corresponding promoter sequences were analyzed by statistical pattern recognition and comparative genomics approaches. We found mouse counterparts for 63 of these loci and classified 42 (67%) as direct ER? targets using classification and regression tree (CART) statistical model, which involves position weight matrix and human-mouse sequence similarity scores as model parameters. The remaining genes were considered to be indirect targets. To validate this computational prediction, we conducted an additional ChIP-on-chip assay that identified acetylated chromatin components in active ER? promoters. Of the 27 loci upregulated in an ER?-positive breast cancer cell line, 20 having mouse counterparts were correctly predicted by CART. This integrated approach, therefore, sets a paradigm in which the iterative process of model refinement and experimental verification will continue until an accurate prediction of promoter target sequences is derived. PMID:15608294

Jin, Victor X.; Leu, Yu-Wei; Liyanarachchi, Sandya; Sun, Hao; Fan, Meiyun; Nephew, Kenneth P.; Huang, Tim H.-M.; Davuluri, Ramana V.



Chromatin architecture near a potential 3' end of the igh locus involves modular regulation of histone modifications during B-Cell development and in vivo occupancy at CTCF sites.  


The murine Igh locus has a 3' regulatory region (3' RR) containing four enhancers (hs3A, hs1,2, hs3B, and hs4) at DNase I-hypersensitive sites. The 3' RR exerts long-range effects on class switch recombination (CSR) to several isotypes through its control of germ line transcription. By measuring levels of acetylated histones H3 and H4 and of dimethylated H3 (K4) with chromatin immunoprecipitation assays, we found that early in B-cell development, chromatin encompassing the enhancers of the 3' RR began to attain stepwise modifications typical of an open conformation. The hs4 enhancer was associated with active chromatin initially in pro- and pre-B cells and then together with hs3A, hs1,2, and hs3B in B and plasma cells. Histone modifications were similar in resting splenic B cells and in splenic B cells induced by lipopolysaccharide to undergo CSR. From the pro-B-cell stage onward, the approximately 11-kb region immediately downstream of hs4 displayed H3 and H4 modifications indicative of open chromatin. This region contained newly identified DNase I-hypersensitive sites and several CTCF target sites, some of which were occupied in vivo in a developmentally regulated manner. The open chromatin environment of the extended 3' RR in mature B cells was flanked by regions associated with dimethylated K9 of histone H3. Together, these data suggest that 3' RR elements are located within a specific chromatin subdomain that contains CTCF binding sites and developmentally regulated modules. PMID:15684400

Garrett, Francine E; Emelyanov, Alexander V; Sepulveda, Manuel A; Flanagan, Patrick; Volpi, Sabrina; Li, Fubin; Loukinov, Dmitry; Eckhardt, Laurel A; Lobanenkov, Victor V; Birshtein, Barbara K



Chromatin structure of transcriptionally competent and repressed genes.  

PubMed Central

We have compared transcriptionally competent and repressed genes with respect to their linker histone content and their ability to fold into higher-order structures. Histones were cross-linked covalently to DNA in chicken erythrocyte and oviduct nuclei by UV irradiation, and the DNA that was immunoprecipitated with anti-H1 and (for erythrocytes) anti-H5 antibodies was analysed for particular DNA sequences. None of the sequences investigated was free of H1 (H5). However, in mature erythrocytes the tissue-specific adult beta-globin gene (beta A) appears to be partially depleted of H5, and both the beta-globin gene and the H5 gene (also tissue-specific), as well as the 'housekeeping' beta-actin gene, appear to be partially depleted of H1 relative to inactive genes; in oviduct slight H1-depletion is detected on the ovalbumin gene relative to genes that are inactive in this tissue and the actin gene. Transcriptionally competent erythrocyte chromatin fragments, in contrast to inactive fragments, are unable to self-associate into 'pseudo-higher-order structures'. This is likely to be a consequence of the partial depletion of H5 and/or H1 in active chromatin, resulting in the breakdown of (probably cooperative) interactions between H5 and/or H1 molecules that otherwise mediate the assembly of pseudo-higher-order structures in vitro and a stable 30 nm chromatin filament in vivo. Images Fig. 2. Fig. 3. Fig. 6. Fig. 7. Fig. 8. PMID:2249661

Kamakaka, R T; Thomas, J O



Chromatin immunoprecipitation: advancing analysis of nuclear hormone signaling.  


Recent decades have been filled with groundbreaking research in the field of endocrine hormone signaling. Pivotal events like the isolation and purification of the estrogen receptor, the cloning of glucocorticoid receptor cDNA, or dissemination of nuclear hormone receptor (NHR) DNA binding sequences are well recognized for their contributions. However, the novel genome-wide and gene-specific information obtained over the last decade describing NHR association with chromatin, cofactors, and epigenetic modifications, as well as their role in gene regulation, has been largely facilitated by the adaptation of the chromatin immunoprecipitation (ChIP) technique. Use of ChIP-based technologies has taken the field of hormone signaling from speculating about the transcription-enabling properties of acetylated chromatin and putative transcription (co-)factor genomic occupancy to demonstrating the detailed, stepwise mechanisms of factor binding and transcriptional initiation; from treating hormone-induced transcription as a steady-state event to understanding its dynamic and cyclic nature; from looking at the DNA sequences recognized by various DNA-binding domains in vitro to analyzing the cell-specific genome-wide pattern of nuclear receptor binding and interpreting its physiological implications. Not only have these events propelled hormone research, but, as some of the pioneering studies, have also contributed tremendously to the field of molecular endocrinology as a whole. In this review, we give a brief summary of some of the most important discoveries in hormone signaling using ChIP and other derivative techniques and speculate on what the future may hold. PMID:22872135

Vinckevicius, Aurimas; Chakravarti, Debabrata



Low-angle neutron scattering from chromatin subunit particles.  

PubMed Central

Monomer chromatin particles containing 140 base pairs of DNA and eight histone molecules have been studied by neutron scattering. From measurements in various H2O/D2O mixtures, radii of gyration and the average scattering density of the particle were determined. The radius of gyration under conditions when scattering from the DNA dominates is 50A, and when scattering from the protein dominates, 30A. Consequently the core of the particle is largely occupied by the histones while the outer shell consists of DNA together with some of the histone. PMID:1052536

Pardon, J F; Worcester, D L; Wooley, J C; Tatchell, K; Van Holde, K E; Richards, B M



Centromeric Chromatin and the Pathway that Drives Its Propagation  

PubMed Central

The centromere is the locus that directs chromosomal inheritance at cell division. While centromeres in diverse eukaryotes are commonly found at sites of repetitive DNA, their location is epigenetically specified. The histone H3 variant CENP-A is the prime candidate for epigenetically marking the centromere, and recent work has uncovered several additional proteins that play key roles in centromere assembly and maintenance. We describe advances in the identification and characterization of proteins that form the centromere, and focus on recent findings that have advanced our understanding of the assembly of functional centromeric chromatin. PMID:22154124

Falk, Samantha J.; Black, Ben E.



Environmental sensing by chromatin: an epigenetic contribution to evolutionary change.  


Chromatin structure and function are regulated by families of protein-modifying enzymes that are sensitive to a variety of metabolic and environmental agents. These enzymes, and proteins that read the modifications they maintain, constitute a system by which environmental agents, such as chemical toxins and dietary components, can directly regulate patterns of gene expression. This review describes this environmental sensing system from an evolutionary perspective. It is proposed that persistent environmentally-induced changes in gene expression patterns can cause changes in phenotype that are acted upon by natural selection, and that epigenetic processes can potentially play central roles in evolution. PMID:21115008

Turner, Bryan M



The chromatin structure of endogenous MTV proviruses in Mus musculus  

E-print Network

mapped from the internal Eco Rl site, it was possible to perform alternate genomic digests to specifically analyze the 5' half of unit III. Southern blot bands generated by Hind III digestion of purified DNA and hypersensitive cleavage of chromatin... within the 5' half of the provirus would be 1200 bps smaller if generated by unit III rather than units II or XI. Also, on Hind III digests of purified genomic DNA, the 3' half of unit III was probed for nuclease hypersensitivity with RNA synthesized...

Marich, James Edward



Transcriptional regulation and chromatin remodeling mechanisms at PHO5  

E-print Network

in nucleosome remodeling. The most widely studied complex is the Swi-Snf complex in yeast, named for its role in mating- type switching as well as sucrose fermentation (reviewed in Martens and Winston, 2003). Defects in the human Swi-Snf complex are linked... with permission from Archana Dhasarathy. 12 An earlier study showed that yeast genes could be classified into three distinct classes based on their requirement for the chromatin remodelers Swi- Snf and SAGA: 1) those genes which require both Swi-Snf...

Carvin, Christopher Dumas



Meiotic events at the centromeric heterochromatin: histone H3 phosphorylation, topoisomerase II alpha localization and chromosome condensation.  


Mechanisms of chromosome condensation and segregation during the first meiotic division are not well understood. Resolution of recombination events to form chiasmata is important, for it is chiasmata that hold homologous chromosomes together for their oppositional orientation on the meiotic metaphase spindle, thus ensuring their accurate segregation during anaphase I. Events at the centromere are also important in bringing about proper attachment to the spindle apparatus. This study was designed to correlate the presence and activity of two proteins at the centromeric heterochromatin, topoisomerase II alpha (TOP2A) and histone H3, with the processes of chromosome condensation and individualization of chiasmate bivalents in murine spermatocytes. We tested the hypothesis that phosphorylation of histone H3 is a key event instigating localization of TOP2A to the centromeric heterochromatin and condensation of chromosomes as spermatocytes exit prophase and progress to metaphase. Activity of topoisomerase II is required for condensation of chromatin at the end of meiotic prophase. Histone H3 becomes phosphorylated at the end of prophase, beginning with its phosphorylation at the centromeric heterochromatin in the diplotene stage. However, it cannot be involved in localization of TOP2A, since TOP2A is localized to the centromeric heterochromatin throughout most of meiotic prophase. This observation suggests a meiotic function for TOP2A in addition to its role in chromatin condensation. The use of kinase inhibitors demonstrates that phosphorylation of histone H3 can be uncoupled from meiotic chromosome condensation; therefore other proteins, such as those constituting metaphase-promoting factor, must be involved. These results define the timing of important meiotic events at the centromeric heterochromatin and provide insight into mechanisms of chromosome condensation for meiotic metaphase. PMID:10654080

Cobb, J; Miyaike, M; Kikuchi, A; Handel, M A



Effect of spontaneous condensation on condensation heat transfer in the presence of non-condensable gases  

SciTech Connect

The presence of non condensable gases like nitrogen or air reduces the condensation heat transfer during condensation of binary steam mixtures. The non condensable gas accumulates in the vapor phase boundary layer and causes a high heat transfer resistance. Especially with high pressures and low water temperatures spontaneous condensation reduces heat transfer additionally. Fog forms within the steam-nitrogen boundary layer and the steam condenses on the water droplets of the fog layer. The convective mass transfer to the cooling water interface diminishes. Raman spectroscopy and film theory are used to quantify this effect locally. The calculation of overall condensation rates in large steam nitrogen systems requires to use three dimensional CFD codes. The paper presents equations to predict fog formation in the boundary layer which can be implemented in CFD codes.

Karl, J.; Hein, D.



Interpreting clinical assays for histone deacetylase inhibitors  

PubMed Central

As opposed to genetics, dealing with gene expressions by direct DNA sequence modifications, the term epigenetics applies to all the external influences that target the chromatin structure of cells with impact on gene expression unrelated to the sequence coding of DNA itself. In normal cells, epigenetics modulates gene expression through all development steps. When “imprinted” early by the environment, epigenetic changes influence the organism at an early stage and can be transmitted to the progeny. Together with DNA sequence alterations, DNA aberrant cytosine methylation and microRNA deregulation, epigenetic modifications participate in the malignant transformation of cells. Their reversible nature has led to the emergence of the promising field of epigenetic therapy. The efforts made to inhibit in particular the epigenetic enzyme family called histone deacetylases (HDACs) are described. HDAC inhibitors (HDACi) have been proposed as a viable clinical therapeutic approach for the treatment of leukemia and solid tumors, but also to a lesser degree for noncancerous diseases. Three epigenetic drugs are already arriving at the patient’s bedside, and more than 100 clinical assays for HDACi are registered on the National Cancer Institute website. They explore the eventual additive benefits of combined therapies. In the context of the pleiotropic effects of HDAC isoforms, more specific HDACi and more informative screening tests are being developed for the benefit of the patients. PMID:21625397

Martinet, Nadine; Bertrand, Philippe



Lateral flow strip assay  


A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

Miles, Robin R. (Danville, CA); Benett, William J. (Livermore, CA); Coleman, Matthew A. (Oakland, CA); Pearson, Francesca S. (Livermore, CA); Nasarabadi, Shanavaz L. (Livermore, CA)



Gibberellin-induced change in the structure of chromatin in wheat sprouts: decrease in the accessibility of DNA in preparations of soluble chromatin to the action of EcoRII methylase  

SciTech Connect

A method has been perfected for producing soluble chromatin from whole wheat sprouts at low ionic strength. The chromatin preparations isolated possess a native structure: they have a nucleosome organization. Under identical conditions the soluble wheat chromatin undergoes more profound degradation by DNase I and staphylococcal nuclease than the chromatin from the rat liver. The DNA contained in the isolated chromatin is capable of accepting CHnumber groups from S-(methyl-/sup 3/H)-adenosylmethionine during incubation with DNA methylase EcoRII; not all the CC A/T GG sequences in DNA are methylated in vivo. Chromatin from gibberellin A/sub 3/-treated wheat sprout DNA accepts 40% fewer CH/sub 3/ groups than that from the control sprouts, which is probably due to the greater compactness of the chromatin. In the case of longer incubation, the level of methylation of the chromatin falls, which may be associated with the presence of DNA-demethylating activity.

Noskov, V.A.; Kintsurashvili, L.N.; Smirnova, T.A.; Manamsh'yan, T.A.; Kir'yanov, G.I.; Vanyushin, B.F.



Transcription in the maintenance of centromere chromatin identity  

PubMed Central

Recent evidence has shown that transcription is permissible through the purportedly repressive centromere domain, and that this transcriptional activity is of functional consequence. The best-studied example is transcription of the pericentric DNA repeats in the generation of siRNAs required for pericentric heterochromatin assembly in yeast. However, non-siRNA transcripts emanating from both pericentric and centromere core domains have also been detected in a cell cycle and cellular differentiation-dependent manner. Elevated levels of centromeric transcripts have also been detected in some cancers; however, it is still unclear how high levels of centromere transcripts may contribute towards disease progression. More recent studies have demonstrated that careful regulation of the histone modifications and transcription level at the centromere is vital for the recruitment of key centromere proteins and assembly of CENP-A domain. Here, we compare the transcriptional dynamics and function of various transcripts derived from pericentromeric and centromere core regions. We also propose a model in which the chromatin remodelling activity of transcription, and the resultant transcripts, contribute synergistically to perpetuate centromere chromatin identity. PMID:23066104

Chan, F. Lyn; Wong, Lee H.



The genomic landscape of cohesin-associated chromatin interactions  

PubMed Central

Cohesin is implicated in establishing tissue-specific DNA loops that target enhancers to promoters, and also localizes to sites bound by the insulator protein CTCF, which blocks enhancer-promoter communication. However, cohesin-associated interactions have not been characterized on a genome-wide scale. Here we performed chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) of the cohesin subunit SMC1A in developing mouse limb. We identified 2264 SMC1A interactions, of which 1491 (65%) involved sites co-occupied by CTCF. SMC1A participates in tissue-specific enhancer-promoter interactions and interactions that demarcate regions of correlated regulatory output. In contrast to previous studies, we also identified interactions between promoters and distal sites that are maintained in multiple tissues but are poised in embryonic stem cells and resolve to tissue-specific activated or repressed chromatin states in the mouse embryo. Our results reveal the diversity of cohesin-associated interactions in the genome and highlight their role in establishing the regulatory architecture of development. PMID:23704192

DeMare, Laura E.; Leng, Jing; Cotney, Justin; Reilly, Steven K.; Yin, Jun; Sarro, Richard; Noonan, James P.



Chromatin Loops as Allosteric Modulators of Enhancer-Promoter Interactions  

PubMed Central

The classic model of eukaryotic gene expression requires direct spatial contact between a distal enhancer and a proximal promoter. Recent Chromosome Conformation Capture (3C) studies show that enhancers and promoters are embedded in a complex network of looping interactions. Here we use a polymer model of chromatin fiber to investigate whether, and to what extent, looping interactions between elements in the vicinity of an enhancer-promoter pair can influence their contact frequency. Our equilibrium polymer simulations show that a chromatin loop, formed by elements flanking either an enhancer or a promoter, suppresses enhancer-promoter interactions, working as an insulator. A loop formed by elements located in the region between an enhancer and a promoter, on the contrary, facilitates their interactions. We find that different mechanisms underlie insulation and facilitation; insulation occurs due to steric exclusion by the loop, and is a global effect, while facilitation occurs due to an effective shortening of the enhancer-promoter genomic distance, and is a local effect. Consistently, we find that these effects manifest quite differently for in silico 3C and microscopy. Our results show that looping interactions that do not directly involve an enhancer-promoter pair can nevertheless significantly modulate their interactions. This phenomenon is analogous to allosteric regulation in proteins, where a conformational change triggered by binding of a regulatory molecule to one site affects the state of another site. PMID:25340767

Doyle, Boryana; Fudenberg, Geoffrey; Imakaev, Maxim; Mirny, Leonid A.



Stacking the DEK: From chromatin topology to cancer stem cells  

PubMed Central

Stem cells are essential for development and tissue maintenance and display molecular markers and functions distinct from those of differentiated cell types in a given tissue. Malignant cells that exhibit stem cell-like activities have been detected in many types of cancers and have been implicated in cancer recurrence and drug resistance. Normal stem cells and cancer stem cells have striking commonalities, including shared cell surface markers and signal transduction pathways responsible for regulating quiescence vs. proliferation, self-renewal, pluripotency and differentiation. As the search continues for markers that distinguish between stem cells, progenitor cells and cancer stem cells, growing evidence suggests that a unique chromatin-associated protein called DEK may confer stem cell-like qualities. Here, we briefly describe current knowledge regarding stem and progenitor cells. We then focus on new findings that implicate DEK as a regulator of stem and progenitor cell qualities, potentially through its unusual functions in the regulation of local or global chromatin organization. PMID:23255114

Privette Vinnedge, Lisa M.; Kappes, Ferdinand; Nassar, Nicolas; Wells, Susanne I.



Molecular basis for chromatin binding and regulation of MLL5.  


The human mixed-lineage leukemia 5 (MLL5) protein mediates hematopoietic cell homeostasis, cell cycle, and survival; however, the molecular basis underlying MLL5 activities remains unknown. Here, we show that MLL5 is recruited to gene-rich euchromatic regions via the interaction of its plant homeodomain finger with the histone mark H3K4me3. The 1.48-Å resolution crystal structure of MLL5 plant homeodomain in complex with the H3K4me3 peptide reveals a noncanonical binding mechanism, whereby K4me3 is recognized through a single aromatic residue and an aspartate. The binding induces a unique His-Asp swapping rearrangement mediated by a C-terminal ?-helix. Phosphorylation of H3T3 and H3T6 abrogates the association with H3K4me3 in vitro and in vivo, releasing MLL5 from chromatin in mitosis. This regulatory switch is conserved in the Drosophila ortholog of MLL5, UpSET, and suggests the developmental control for targeting of H3K4me3. Together, our findings provide first insights into the molecular basis for the recruitment, exclusion, and regulation of MLL5 at chromatin. PMID:23798402

Ali, Muzaffar; Rincón-Arano, Héctor; Zhao, Wei; Rothbart, Scott B; Tong, Qiong; Parkhurst, Susan M; Strahl, Brian D; Deng, Lih-Wen; Groudine, Mark; Kutateladze, Tatiana G