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Genome size and chromatin condensation in vertebrates  

Microsoft Academic Search

Cell membrane-dependent chromatin condensation was studied by flow cytometry in erythrocytes of 36 species from six classes of vertebrates. A positive relationship was found between the degree of condensation and genome size. The distribution of variances among taxonomic levels is similar for both parameters. However, chromatin condensation varied relatively more at the lower taxonomic levels, which suggests that the degree

Alexander E. Vinogradov



Osmotic Challenge Drives Rapid and Reversible Chromatin Condensation in Chondrocytes  

PubMed Central

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

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



Transcriptional Coactivator PC4, a Chromatin-Associated Protein, Induces Chromatin Condensation? †  

PubMed Central

Human transcriptional coactivator PC4 is a highly abundant multifunctional protein which plays diverse important roles in cellular processes, including transcription, replication, and repair. It is also a unique activator of p53 function. Here we report that PC4 is a bona fide component of chromatin with distinct chromatin organization ability. PC4 is predominantly associated with the chromatin throughout the stages of cell cycle and is broadly distributed on the mitotic chromosome arms in a punctate manner except for the centromere. It selectively interacts with core histones H3 and H2B; this interaction is essential for PC4-mediated chromatin condensation, as demonstrated by micrococcal nuclease (MNase) accessibility assays, circular dichroism spectroscopy, and atomic force microscopy (AFM). The AFM images show that PC4 compacts the 100-kb reconstituted chromatin distinctly compared to the results seen with the linker histone H1. Silencing of PC4 expression in HeLa cells results in chromatin decompaction, as evidenced by the increase in MNase accessibility. Knocking down of PC4 up-regulates several genes, leading to the G2/M checkpoint arrest of cell cycle, which suggests its physiological role as a chromatin-compacting protein. These results establish PC4 as a new member of chromatin-associated protein family, which plays an important role in chromatin organization.

Das, Chandrima; Hizume, Kohji; Batta, Kiran; Kumar, B. R. Prashanth; Gadad, Shrikanth S.; Ganguly, Semanti; Lorain, Stephanie; Verreault, Alain; Sadhale, Parag P.; Takeyasu, Kunio; Kundu, Tapas K.



Quantification of chromatin condensation level by image processing.  


The level of chromatin condensation is related to the silencing/activation of chromosomal territories and therefore impacts on gene expression. Chromatin condensation changes during cell cycle, progression and differentiation, and is influenced by various physicochemical and epigenetic factors. This study describes a validated experimental technique to quantify chromatin condensation. A novel image processing procedure is developed using Sobel edge detection to quantify the level of chromatin condensation from nuclei images taken by confocal microscopy. The algorithm was developed in MATLAB and used to quantify different levels of chromatin condensation in chondrocyte nuclei achieved through alteration in osmotic pressure. The resulting chromatin condensation parameter (CCP) is in good agreement with independent multi-observer qualitative visual assessment. This image processing technique thereby provides a validated unbiased parameter for rapid and highly reproducible quantification of the level of chromatin condensation. PMID:24099693

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



Incomplete Chromatin Condensation in Enlarged Rat Myelocytic Leukemia Cells  

PubMed Central

The distinguishable morphologic features of nuclei of acute myelogenous leukemia cells with enlarged size and finely distributed nuclear chromatin indicate incomplete chromosome condensation that can be related to elevated gene expression. To confirm this, interphase chromosome structures were studied in exponentially growing rat myelomonocytic leukemia 1 cells isolated at the University of Debrecen (My1/De cells). This cell line was established from primary rat leukemia chemically induced by 7,12-dimethylbenz[a]anthracene treatment. The enlarged nuclei of My1/De cells allowed improved fluorescent visualization of chromosomal structures. Increased resolution revealed major interphase intermediates consisting of (1) veil-like chromatin, (2) chromatin ribbon, (3) chromatin funnel, (4) chromatin bodies, (5) elongated prechromosomes, (6) seal-ring, spiral shaped, and circular chromosomal subunits, (7) elongated, bent, u- and v-shaped prechromosomes, and (8) metaphase chromosomes. Results confirmed the existence of the chromatin funnel, the first visible interphase chromosome generated by the supercoiling of the chromatin ribbon. Other intermediates not seen previously included the spiral subunits that are involved in the chromonemic folding of metaphase chromosomes. The existence of spiral subunits favors the helical coil model of chromosome condensation. Incomplete chromatin condensation in leukemia cells throughout the cell cycle is an indication of euchromatization contributing to enhanced gene expression and is regarded as a leukemic factor.

Trencsenyi, Gyorgy; Nagy, Gabor; Bako, Fruzsina; Kertai, Pal



A cascade of histone modifications induces chromatin condensation in mitosis.  


Metaphase chromosomes are visible hallmarks of mitosis, yet our understanding of their structure and of the forces shaping them is rudimentary. Phosphorylation of histone H3 serine 10 (H3 S10) by Aurora B kinase is a signature event of mitosis, but its function in chromatin condensation is unclear. Using genetically encoded ultraviolet light-inducible cross-linkers, we monitored protein-protein interactions with spatiotemporal resolution in living yeast to identify the molecular details of the pathway downstream of H3 S10 phosphorylation. This modification leads to the recruitment of the histone deacetylase Hst2p that subsequently removes an acetyl group from histone H4 lysine 16, freeing the H4 tail to interact with the surface of neighboring nucleosomes and promoting fiber condensation. This cascade of events provides a condensin-independent driving force of chromatin hypercondensation during mitosis. PMID:24385627

Wilkins, Bryan J; Rall, Nils A; Ostwal, Yogesh; Kruitwagen, Tom; Hiragami-Hamada, Kyoko; Winkler, Marco; Barral, Yves; Fischle, Wolfgang; Neumann, Heinz



Proteome analysis of nuclear matrix proteins during apoptotic chromatin condensation.  


The nuclear matrix (NM) is considered a proteinaceous scaffold spatially organizing the interphase nucleus, the integrity of which is affected during apoptosis. Caspase-mediated degradation of NM proteins, such as nuclear lamins, precedes apoptotic chromatin condensation (ACC). Nevertheless, other NM proteins remain unaffected, which most likely maintain a remaining nuclear structure devoid of chromatin. We, therefore, screened various types of apoptotic cells for changes of the nuclear matrix proteome during the process of apoptotic ACC. Expectedly, we observed fundamental alterations of known chromatin-associated proteins, comprising both degradation and translocation to the cytosol. Importantly, a consistent set of abundant NM proteins, some (e.g. hNMP 200) of which displaying structural features, remained unaffected during apoptosis and might therefore represent constituents of an elementary scaffold. In addition, proteins involved in DNA replication and DNA repair were found accumulated in the NM fraction before cells became irreversibly committed to ACC, a time point characterized in detail by inhibitor studies with orthovanadate. In general, protein alterations of a consistent set of NM proteins (67 of which were identified), were reproducibly detectable in Fas-induced Jurkat cells, in UV-light treated U937 cells and also in staurosporine-treated HeLa cells. Our data indicate that substantial alterations of proteins linking chromatin to an elementary nuclear protein scaffold might play an intriguing role for the process of ACC. PMID:12032676

Gerner, C; Gotzmann, J; Fröhwein, U; Schamberger, C; Ellinger, A; Sauermann, G



Active cytoskeletal force and chromatin condensation independently modulate intranuclear network fluctuations.  


Chromatin remodeling, including the movement of genes and regulatory factors, precedes or accompanies stimulated changes in gene expression. Here we quantify chromatin fluctuations in primary human cells using particle-tracking microrheology and determine the physical mechanisms which influence chromatin reorganization. We find that intranuclear movements are enhanced beyond thermal motion by active force generation from cytoskeletal motor activity propagated through the LINC complex; intranuclear movements are also dependent on the viscoelasticity of the DNA-protein polymer network. Chromatin movements were dramatically altered by modulation of chromatin condensation state, which we independently verified using fluorescence lifetime imaging microscopy (FLIM). These findings suggest that chromatin condensation and cytoskeletal force generation play distinct functional roles in regulating intranuclear movements, and these effects are decoupled as measured by particle tracking. We further utilize this approach in identifying the nuclear responsiveness of primary human endothelial cells to vascular endothelial growth factor (VEGF): early in the response chromatin movements increase and are dominated by cytoskeletal force, which transitions at later times to a chromatin decondensation event. Given the hierarchical genome organization in primary cells, our work generally suggests an important role for force generation and chromatin mechanics in altered gene expression kinetics. PMID:24619297

Spagnol, Stephen T; Noel Dahl, Kris



Dynamic condensation of linker histone C-terminal domain regulates chromatin structure.  


The basic and intrinsically disordered C-terminal domain (CTD) of the linker histone (LH) is essential for chromatin compaction. However, its conformation upon nucleosome binding and its impact on chromatin organization remain unknown. Our mesoscale chromatin model with a flexible LH CTD captures a dynamic, salt-dependent condensation mechanism driven by charge neutralization between the LH and linker DNA. Namely, at low salt concentration, CTD condenses, but LH only interacts with the nucleosome and one linker DNA, resulting in a semi-open nucleosome configuration; at higher salt, LH interacts with the nucleosome and two linker DNAs, promoting stem formation and chromatin compaction. CTD charge reduction unfolds the domain and decondenses chromatin, a mechanism in consonance with reduced counterion screening in vitro and phosphorylated LH in vivo. Divalent ions counteract this decondensation effect by maintaining nucleosome stems and expelling the CTDs to the fiber exterior. Additionally, we explain that the CTD folding depends on the chromatin fiber size, and we show that the asymmetric structure of the LH globular head is responsible for the uneven interaction observed between the LH and the linker DNAs. All these mechanisms may impact epigenetic regulation and higher levels of chromatin folding. PMID:24906881

Luque, Antoni; Collepardo-Guevara, Rosana; Grigoryev, Sergei; Schlick, Tamar



Dynamic condensation of linker histone C-terminal domain regulates chromatin structure  

PubMed Central

The basic and intrinsically disordered C-terminal domain (CTD) of the linker histone (LH) is essential for chromatin compaction. However, its conformation upon nucleosome binding and its impact on chromatin organization remain unknown. Our mesoscale chromatin model with a flexible LH CTD captures a dynamic, salt-dependent condensation mechanism driven by charge neutralization between the LH and linker DNA. Namely, at low salt concentration, CTD condenses, but LH only interacts with the nucleosome and one linker DNA, resulting in a semi-open nucleosome configuration; at higher salt, LH interacts with the nucleosome and two linker DNAs, promoting stem formation and chromatin compaction. CTD charge reduction unfolds the domain and decondenses chromatin, a mechanism in consonance with reduced counterion screening in vitro and phosphorylated LH in vivo. Divalent ions counteract this decondensation effect by maintaining nucleosome stems and expelling the CTDs to the fiber exterior. Additionally, we explain that the CTD folding depends on the chromatin fiber size, and we show that the asymmetric structure of the LH globular head is responsible for the uneven interaction observed between the LH and the linker DNAs. All these mechanisms may impact epigenetic regulation and higher levels of chromatin folding.

Luque, Antoni; Collepardo-Guevara, Rosana; Grigoryev, Sergei; Schlick, Tamar



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

PubMed Central

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 mitosis, the lamina structure is broken down as a result of lamin solubilization and it is possible that a similar process is happening in apoptotic cells. The experiments described in this study have used confluent cultures of an embryonic fibroblast cell line which can be induced to undergo either apoptosis at low serum conditions or mitosis. Solubilization of lamin A+B was analyzed by immunoblotting and indirect immunofluorescence. These studies showed that in mitotic cells lamina breakdown is accompanied by lamin solubilization. In apoptotic cells, a small amount of lamin is solubilized before the onset of apoptosis, thereafter, chromatin condensation is accompanied by degradation of lamin A+B to a 46-kD fragment. Analysis of cellular lysates by probing blots with anti- PSTAIR followed by anti-phosphotyrosine showed that in contrast to mitosis, dephosphorylation on tyrosine residues did not occur in apoptotic cells. At all timepoints after the onset of apoptosis there was no significant increase in the activation of p34cdc2 as determined in the histone H1 kinase assay. Coinduction of apoptosis and mitosis after release of cells from aphidicolin block showed that apoptosis could be induced in parallel with S-phase. The sudden breakdown of chromatin structure may be the result of detachment of the chromatin loops from their anchorage at the nuclear matrix, as bands of 50 kbp and corresponding multimers were detectable by field inversion gel electrophoresis (FIGE). In apoptotic cells all of the DNA was fragmented, but only 14% of the DNA was smaller than 50 kbp. DNA strand breaks were detected at the periphery of the condensed chromatin by in situ tailing (ISTAIL). Chromatin condensation during apoptosis appears to be due to a rapid proteolysis of nuclear matrix proteins which does not involve the p34cdc2 kinase.



Proteomics and the genetics of sperm chromatin condensation.  


Spermatogenesis involves extremely marked cellular, genetic and chromatin changes resulting in the generation of the highly specialized sperm cell. Proteomics allows the identification of the proteins that compose the spermatogenic cells and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and to study the sperm proteins. Catalogs of thousands of testis and spermatozoan proteins in human and different model species are becoming available, setting up the basis for subsequent research, diagnostic applications and possibly the future development of specific treatments. The present review intends to summarize the key genetic and chromatin changes at the different stages of spermatogenesis and in the mature sperm cell and to comment on the presently available proteomic studies. PMID:21042303

Oliva, Rafael; Castillo, Judit



The arrangement of H5 molecules in extended and condensed chicken erythrocyte chromatin.  

PubMed Central

Chemical cross-linking with dithiobis(succinimidyl propionate) has been used to investigate the relative disposition of neighbouring H5 (H1) molecules in chicken erythrocyte chromatin in the extended (nucleosome filament) and condensed (300 A filament) states; in this chromatin H5 and H1 are interspersed along the nucleosome filament, rather than segregated into blocks, as shown by the nature of the cross-linked dimers and their relative amounts. Detailed analysis of the cross-linked H5 homopolymers from extended chromatin and condensed nuclear chromatin indicates which domains of H5 are in contact (or close proximity) in the two states. Two results suggest a polar, head-to-tail arrangement of H5 molecules along the nucleosome filament. This arrangement persists when chromatin adopts higher-order structure but in the folded state neighbouring basic C-terminal domains, in particular, are more closely juxtaposed than they are in extended chromatin. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. Fig. 6. Fig. 7.

Lennard, A C; Thomas, J O



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

Microsoft Academic Search

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

John C Chapman; Sandra D Michael



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




EPA Science Inventory

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


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



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


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

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



Structurally divergent histone H1 variants in chromosomes containing highly condensed interphase chromatin  

PubMed Central

Condensed and late-replicating interphase chromatin in the Dipertan insect Chironomus contains a divergent type of histone H1 with an inserted KAP-KAP repeat that is conserved in single H1 variants of Caenorhabditis elegans and Volvox carteri. H1 peptides comprising the insertion interact specifically with DNA. The Chironomid Glyptotendipes exhibits a corresponding correlation between the presence of condensed chromosome sections and the appearance of a divergent H1 subtype. The centromere regions and other sections of Glyptotendipes barbipes chromosomes are inaccessible to immunodecoration by anti-H2B and anti- H1 antibodies one of which is known to recognize nine different epitopes in all domains of the H1 molecule. Microelectrophoresis of the histones from manually isolated unfixed centromeres revealed the presence of H1 and core histones. H1 genes of G. barpipes were sequenced and found to belong to two groups. H1 II and H1 III are rather similar but differ remarkably from H1 I. About 30% of the deduced amino acid residues were found to be unique to H1 I. Most conspicuous is the insertion, SPAKSPGR, in H1 I that is lacking in H1 II and H1 III and at its position gives rise to the sequence repeat SPAKSPAKSPGR. The homologous H1 I gene in Glyptotendipes salinus encodes the very similar repeat TPAKSPAKSPGR. Both sequences are structurally related to the KAPKAP repeat in H1 I-1 specific for condensed chromosome sites in Chironomus and to the SPKKSPKK repeat in sea urchin sperm H1, lie at almost the same distance from the central globular domain, and could interact with linker DNA in packaging condensed chromatin.



An in vitro assay to study the recruitment and substrate specificity of chromatin modifying enzymes  

Microsoft Academic Search

Post-translational modifications of core histones play an important role in regulating fundamental biological processes such\\u000a as DNA repair, transcription and replication. In this paper, we describe a novel assay that allows sequential targeting of\\u000a distinct histone modifying enzymes to immobilized nucleosomal templates using recombinant chimeric targeting molecules. The\\u000a assay can be used to study the histone substrate specificity of chromatin

Michiel Vermeulen; Hendrik G. Stunnenberg



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



EGFR-Mediated Chromatin Condensation Protects KRAS-Mutant Cancer Cells against Ionizing Radiation.  


Therapeutics that target the epidermal growth factor receptor (EGFR) can enhance the cytotoxic effects of ionizing radiation (IR). However, predictive genomic biomarkers of this radiosensitization have remained elusive. By screening 40 non-small cell lung cancer cell (NSCLC) lines, we established a surprising positive correlation between the presence of a KRAS mutation and radiosensitization by the EGFR inhibitors erlotinib and cetuximab. EGFR signaling in KRAS-mutant NSCLC cells promotes chromatin condensation in vitro and in vivo, thereby restricting the number of DNA double-strand breaks (DSB) produced by a given dose of IR. Chromatin condensation in interphase cells is characterized by an unexpected mitosis-like colocalization of serine 10 phosphorylation and lysine 9 trimethylation on histone H3. Aurora B promotes this process in a manner that is codependent upon EGFR and protein kinase C ? (PKC?). PKC?, in addition to MEK/ERK signaling, is required for the suppression of DSB-inducible premature senescence by EGFR. Blockade of autophagy results in a mutant KRAS-dependent senescence-to-apoptosis switch in cancer cells treated with IR and erlotinib. In conclusion, we identify EGFR as a molecular target to overcome a novel mechanism of radioresistance in KRAS-mutant tumor cells, which stands in contrast to the unresponsiveness of KRAS-mutant cancers to EGFR-directed agents in monotherapy. Our findings may reposition EGFR-targeted agents for combination with DSB-inducing therapies in KRAS-mutant NSCLC. Cancer Res; 74(10); 2825-34. ©2014 AACR. PMID:24648348

Wang, Meng; Kern, Ashley M; Hülskötter, Marieke; Greninger, Patricia; Singh, Anurag; Pan, Yunfeng; Chowdhury, Dipanjan; Krause, Mechthild; Baumann, Michael; Benes, Cyril H; Efstathiou, Jason A; Settleman, Jeff; Willers, Henning



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

SciTech Connect

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

Hittelman, W.N.; Pollard, M.



The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI  

Microsoft Academic Search

INTRODUCTION: Sperm chromatin integrity assessment has been suggested as a fertility predictor. The aim of this study was to examine the relationship between the results of sperm chromatin structure assay (SCSA) and the outcome of IVF, ICSI and intrauterine insemination (IUI). METHODS: A total of 306 consecutive couples under- going assisted reproduction were included. IUI was performed in 131, IVF

M. Bungum; P. Humaidan; M. Spano; K. Jepson; L. Bungum; A. Giwercman



X-ray crystal structure of MENT: evidence for functional loop-sheet polymers in chromatin condensation  

PubMed Central

Most serpins are associated with protease inhibition, and their ability to form loop–sheet polymers is linked to conformational disease and the human serpinopathies. Here we describe the structural and functional dissection of how a unique serpin, the non-histone architectural protein, MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein), participates in DNA and chromatin condensation. Our data suggest that MENT contains at least two distinct DNA-binding sites, consistent with its simultaneous binding to the two closely juxtaposed linker DNA segments on a nucleosome. Remarkably, our studies suggest that the reactive centre loop, a region of the MENT molecule essential for chromatin bridging in vivo and in vitro, is able to mediate formation of a loop–sheet oligomer. These data provide mechanistic insight into chromatin compaction by a non-histone architectural protein and suggest how the structural plasticity of serpins has adapted to mediate physiological, rather than pathogenic, loop–sheet linkages.

McGowan, Sheena; Buckle, Ashley M; Irving, James A; Ong, Poh Chee; Bashtannyk-Puhalovich, Tanya A; Kan, Wan-Ting; Henderson, Kate N; Bulynko, Yaroslava A; Popova, Evgenya Y; Smith, A Ian; Bottomley, Stephen P; Rossjohn, Jamie; Grigoryev, Sergei A; Pike, Robert N; Whisstock, James C



Cytotoxicity of eight cigarette smoke condensates in three test systems: comparisons between assays and condensates.  


Cytotoxic properties of tobacco smoke are associated with chronic tobacco-related diseases. The cytotoxicity of tobacco smoke can be tested with short-term predictive assays. In this study, we compare eight mainstream cigarette smoke condensates (CSCs) from commercial and experimental cigarettes in three different cytotoxicity assays with unique and overlapping endpoints. The CSCs demonstrated cytotoxicity in all assays. In the multiple cytotoxicity endpoint (MCE) assay with TK-6 cells, the cigarette varieties that had the highest EC50s for reduced cell growth also showed a positive dose-response relationship for necrotic cells. In the IdMOC multiple cell-type co-culture (MCTCC) system, all CSCs reduced the viability of the cells. Low concentrations of some CSCs had a stimulatory effect in lung microvascular endothelial cells and small airway epithelial cells. In the neutral dye assay (NDA), except for a 100% flue-cured tobacco CSC, there was little consistency between CSCs producing morphological evidence of moderate or greater toxicity and the CSCs with the lowest EC50s in the MCE or MCTCC assays. Overall, cigarettes made with flue-cured tobacco were the most cytotoxic across the assays. When results were expressed on a per-mg of nicotine basis, lower tar cigarettes were the most cytotoxic in primary human respiratory cells. PMID:20719243

Richter, Patricia A; Li, Albert P; Polzin, Gregory; Roy, Shambhu K



Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays  

Microsoft Academic Search

BACKGROUND: Sperm DNA integrity (SDI) is an important factor in the prognosis of male fertility. Here we compare the toluidine blue (TB) image cytometry test, recently proposed by us for SDI assessment, with two other tests—the sperm chromatin structure assay (SCSA) and the terminal nick-end labelling (TUNEL) assay. METHODS: Sperm samples from 35 men were evaluated for standard sperm parameters

J. Erenpreiss; K. Jepson; A. Giwercman; I. Tsarev; M. Spano



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


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. PMID:22314931

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



Assessment of Chromatin Maturity in Human Spermatozoa: Useful Aniline Blue Assay for Routine Diagnosis of Male Infertility  

PubMed Central

During spermatogenesis, sperm chromatin undergoes structural changes and results in a high condensation. This nuclear compaction would be useful as a predictor of sperm fertilization capacity and pregnancy outcome. We purpose to evaluate firstly the relationship among chromatin maturity assessed by aniline blue staining (AB) and the semen parameters in infertile men. Secondly, we analyzed whether the sperm gradient density centrifugation is effective to select mature spermatozoa. Fifty-one ejaculates were investigated by semen analysis and stained for chromatin condensation with AB to distinguish between unstained mature sperm and stained immature sperm. AB was applied also on 12 ejaculates which proceeded by density gradient centrifugation to compare the rates of immature sperm before and after selection. Neat semen were divided into two groups: G1 (n = 31): immature sperm <20% and G2 (n = 20): immature sperm ?20%. No significant differences were detected in sperm concentration, motility, and normal morphology between G1 and G2. However, the rates of some morphology abnormalities were higher in G2: head abnormalities (P = 0.01) and microcephalic sperm (P = 0.02). We founded significant correlation between sperm immaturity and acrosome abnormalities (r = 0.292; P = 0.03). Sperm selection has significantly reduced the rates of immature sperm. A better understanding of chromatin structure and its impact on the sperm potential is needed to explore male infertility.

Chakroun, Nozha; Ben Zarrouk, Soumaya; Sellami, Hanen; Kebaili, Sahbi; Rebai, Tarek; Keskes, Leila



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



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



Sequential Recruitment of HAT and SWI\\/SNF Components to Condensed Chromatin by VP16  

Microsoft Academic Search

Eukaryotic transcription initiation requires the complex dynamics of hundreds of proteins, many of which are found in large multisubunit complexes [1]. Recent experiments have suggested stepwise recruitment of preassembled complexes, including chromatin remodeling, general transcription factor, mediator, and polymerase complexes [1], in which the actual order of recruitment may vary for different promoters [2]. How do these complexes access target

Sevinci Memedula; Andrew S Belmont



Correlation between phosphorylated H1 histone and condensed chromatin in Planococcus citri.  


Histones of the mealybug Planococcus citri have been isolated and characterised. Although no major differences were observed between the core histones of male and female insects, the pattern of H1 histones was significantly different between the sexes. A specific hyperphosphorylated H1 was found to be present only in male mealybugs, where the paternal chromosomes are condensed and transcriptionally inactive. PMID:6628682

Karnik, P S



Sperm chromatin structure assay (scsa®) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles  

Microsoft Academic Search

ObjectiveTo determine the relationship between sperm chromatin structure assay (SCSA) parameters (DNA fragmentation index [DFI] and high DNA stainability [HDS]), and conventional IVF and IVF\\/intracytoplasmic sperm injection (ICSI) outcomes.

Michael R Virro; Kjersten L Larson-Cook; Donald P Evenson



Sperm Chromatin Integrity: Etiologies and Mechanisms of Abnormality, Assays, Clinical Importance, Preventing and Repairing Damage  

PubMed Central

The standard semen analysis is the first line and the most popular laboratory test in the diagnosis of male fertility. It evaluates sperm concentration, motility, morphology and their vitality. However, it is well-known that normal results of semen analysis can not exclude men from the causes of couples? infertility. One of the most important parameters of sperm in its fertilizing potential is “Sperm chromatin integrity” that has direct positive correlation with Assisted Reproductive Techniques (ART) outcomes including; fertilization rate, embryo quality, pregnancy and successful delivery rate. It seems that sperm DNA chromatin integrity provides better diagnostic and prognostic approaches than standard semen parameters. For these reasons under-standing the sperm chromatin structure, etiology of sperm chromatin abnormality, identification factors that disturbs sperm chromatin integrity and the mechanism of their action can help in recognizing the causes of couples? infertility. Various methods of its evaluation, its importance in male fertility, clinical relevance in the outcomes of ART and application of laboratory and medical protocols to improve this integrity have valuable position in diagnosis and treatment of male infertility. There has recently been interest in the subject and its application in the field of andrology. Therefore, with regard to the above mentioned importance of sperm chromatin integrity, this review article describes details of the useful information pertaining to sperm DNA damage including the origins, assessments, etiologies, clinical aspects, and prevention of it.

Hekmatdoost, Azita; Lakpour, Niknam; Sadeghi, Mohammad Reza




EPA Science Inventory

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


Effect of drying method and assay methodology on detergent fiber analysis in plants containing condensed tannins  

Microsoft Academic Search

Laboratory evaluation of fiber concentration and nutritive value of plants containing condensed tannins (CT) can be complicated by the formation of strong complexes between CT and other molecules as affected by sample preparation and assay methodology. This study looked at fiber and acid detergent insoluble nitrogen (ADIN) concentrations of 10 herbaceous and brushy species with and without naturally occurring CT.

Suzika Pagán; Richard M. Wolfe; Thomas H. Terrill; James P. Muir



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

PubMed Central

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

Gertych, Arkadiusz; Tajbakhsh, Jian



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)




NSDL National Science Digital Library

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

Kessler, James H.; Galvan, Patricia M.



Assessment of sperm chromatin condensation and ploidy status using flow cytometry correlates to fertilization, embryo quality and pregnancy following in vitro fertilization  

Microsoft Academic Search

Purpose  Sperm flow cytometry (SFC) was used to evaluate the association of sperm chromatin condensation and ploidy with fertilization,\\u000a embryo development, pregnancy and abortion rates following IVF.\\u000a \\u000a \\u000a \\u000a \\u000a Methods  Conventional semen analysis was performed in one hundred fifty men, as well as SFC analysis, after acridine orange and propidium\\u000a iodide staining, for the evaluation of sperm maturity and ploidy respectively. Conventional IVF was

Leandros A. Lazaros; Georgios A. Vartholomatos; Elissavet G. Hatzi; Apostolos I. Kaponis; Georgios V. Makrydimas; Sophia N. Kalantaridou; Nikolaos V. Sofikitis; Theodoros Ioannis Stefos; Konstantinos A. Zikopoulos; Ioannis A. Georgiou


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

Microsoft Academic Search

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

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



Etoposide-induced apoptosis in lymphoblastoid leukaemic MOLT-4 cells: evidence that chromatin condensation, loss of phosphatidylserine asymmetry and apoptotic body formation can occur independently.  


Apoptosis is characterised by a series of typical morphological features, such as nuclear and cellular convolution, chromatin condensation and the final disintegration of the cell into membrane-bound apoptotic bodies, which are phagocytosed, by neighbouring cells. Relocation of phosphatidylserine residues from the inner leaflet of the cellular membrane to being exposed on the cell surface is a necessary event for the phagocytic elimination of apoptotic cell debris. Using the MOLT-4 lymphoblastoid leukaemic cell line we investigated whether the formation of apoptotic bodies and loss of phosphatidylserine asymmetry were causally related. We have previously demonstrated that classical apoptotic morphology, including production of apoptotic bodies, was only possible in etoposide-treated MOLT-4 cells when administered in the presence of non-cytotoxic doses (200 microM) of aurin tricarboxylic acid (ATA). Electron microscopic analysis, followed by the quantitation of the ultrastructural morphological features of apoptotic MOLT-4 cells, demonstrated that the etoposide and ATA co-treatment, which caused the cellular fragmentation into apoptotic bodies, was closely associated with extensive chromatin condensation in individual cells. In this model however, the addition of ATA to frank cytotoxic doses of etoposide (50 microM), which we confirmed lead to formation of apoptotic bodies, caused no further increase in externalisation of phosphatidylserine moieties as determined by staining with fluorescence labelled annexin V. Consequently, in MOLT-4 cells undergoing etoposide-induced apoptosis, the mole-cular mechanisms leading to loss of phosphatidylserine asymmetry and the formation of apoptotic bodies are not causally related. PMID:11773706

Ramirez, C D; Catchpoole, D R




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...


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.

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



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

Microsoft Academic Search

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

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



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.

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



Glom Is a Novel Mitochondrial DNA Packaging Protein in Physarum polycephalum and Causes Intense Chromatin Condensation without Suppressing DNA Functions  

PubMed Central

Mitochondrial DNA (mtDNA) is packed into highly organized structures called mitochondrial nucleoids (mt-nucleoids). To understand the organization of mtDNA and the overall regulation of its genetic activity within the mt-nucleoids, we identified and characterized a novel mtDNA packaging protein, termed Glom (a protein inducing agglomeration of mitochondrial chromosome), from highly condensed mt-nucleoids of the true slime mold, Physarum polycephalum. This protein could bind to the entire mtDNA and package mtDNA into a highly condensed state in vitro. Immunostaining analysis showed that Glom specifically localized throughout the mt-nucleoid. Deduced amino acid sequence revealed that Glom has a lysine-rich region with proline-rich domain in the N-terminal half and two HMG boxes in C-terminal half. Deletion analysis of Glom revealed that the lysine-rich region was sufficient for the intense mtDNA condensation in vitro. When the recombinant Glom proteins containing the lysine-rich region were expressed in Escherichia coli, the condensed nucleoid structures were observed in E. coli. Such in vivo condensation did not interfere with transcription or replication of E. coli chromosome and the proline-rich domain was essential to keep those genetic activities. The expression of Glom also complemented the E. coli mutant lacking the bacterial histone-like protein HU and the HMG-boxes region of Glom was important for the complementation. Our results suggest that Glom is a new mitochondrial histone-like protein having a property to cause intense DNA condensation without suppressing DNA functions.

Sasaki, Narie; Kuroiwa, Haruko; Nishitani, Chikako; Takano, Hiroyoshi; Higashiyama, Tetsuya; Kobayashi, Tamaki; Shirai, Yuki; Sakai, Atsushi; Kawano, Shigeyuki; Murakami-Murofushi, Kimiko; Kuroiwa, Tsuneyoshi



Phenotypic and metabolic aspects of prostatic epithelial cells in aged gerbils after antisteroidal therapy: turnover in the state of chromatin condensation and androgen-independent cell replacement.  


The gerbil is a rodent considered a good model for studies of prostatic morphophysiology under different experimental conditions. Studies involving castration and steroidal blockers of aged gerbils showed that the glandular epithelium persists after long-term therapy, preventing the organ atrophy. Thus, the objective of this study was to evaluate the phenotypic characteristics and behavior of prostatic epithelial cells that remained after different periods of hormone ablation in aged gerbils. The identification of elements that influenced the survival of this cell type was performed by morphometric, nuclear phenotypes, ultrastructural and immune histochemical analysis. The most significant responses to treatment, by analyzing morphometric features, were observed during the first three time points (day 1, day 3, and day 7), after which there appeared to be an adjustment of the gland to the hormone ablation. All treatments led to changes in the state of chromatin condensation, DNA methylation pattern and phenotypic changes indicated cell senescence. Additionally, an increase in the basal cells seemed to guarantee self-renewal properties to the epithelium. These data indicate that changes occur at many levels, including gene expression and nuclear architecture in the epithelial cells, when aging and steroidal blockade are associated. These aspects are important when considering castration-resistant prostate cancer, a malignant tumor posing difficult therapeutic intervention. PMID:23942056

Campos, Silvana G P; Gonçalves, Bianca F; Scarano, Wellerson R; Góes, Rejane M; Taboga, Sebastião R



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)



Phosphorylation of H2AX histones in response to double-strand breaks and induction of premature chromatin condensation in hydroxyurea-treated root meristem cells of Raphanus sativus, Vicia faba, and Allium porrum.  


Histone H2A variant H2AX is rapidly phosphorylated on the induction of DNA double-strand breaks by ionizing radiation and hydroxyurea-mediated replication arrest, resulting in the formation of gamma-H2AX foci along megabase chromatin domains nearby the sites of incurred DNA damage. In an attempt to establish a relationship between species-specific nuclear architecture and H2AX phosphorylation in S/G(2) phase-arrested root meristem cells, immunocytochemical comparisons using an antibody raised against human gamma-H2AX were made among three plants differing with respect to DNA contents: Allium porrum, representing a reticulate type of DNA package, Vicia faba, having semireticulate cell nuclei, and Raphanus sativus, characterised by a chromocentric type of chromatin. Another approach was aimed at determining possible correlations between the extent of hydroxyurea-induced phosphorylation of H2AX histones and the quantities of root meristem cells induced by caffeine to enter aberrant mitotic division (premature chromosome condensation). It was concluded that the higher-order structure of chromatin may contribute to the accessibility of molecular factors engaged in the recognition and repair of genetic lesions. Consequently, in contrast to A. porrum and V. faba, a diffuse chromatin in chromocentric cell nuclei of R. sativus may become more vulnerable both to generate DNA double-strand breaks and to recruit molecular elements needed to arrange the cell cycle checkpoint functions, and thus, more resistant to factors which allow the cells to enter premature chromosome condensation spontaneously. On the other hand, however, caffeine-mediated overriding of the S-M checkpoint control system resulted in the typical appearance of premature chromosome condensation, irrespective of the genomic content of DNA. PMID:17111099

Rybaczek, Dorota; Maszewski, Janusz



Assay and digestion of 14C-labelled condensed tannins in the gastrointestinal tract of sheep.  


Three experiments were conducted to determine the fate of condensed tannins (CT) during digestion in sheep. CT were measured as extractable, protein-bound and fibre-bound fractions using the butanol-HCl procedure. In Expt 1, purified CT were added to digesta from different parts of the digestive tract obtained from a pasture-fed sheep. Recoveries of CT after 0 and 4 h of anaerobic incubation at 39 degrees averaged: rumen 78.9 and 57.5%; abomasum 50.9 and 49.0%; duodenum 64.4 and 46.0% and ileum 43.4 and 38.8%. In Expt 2, [14C]CT was given per abomasum over a 6.5 h period at 15 min intervals to a sheep previously fed on Lotus pedunculatus (which contains CT). The sheep was killed at the end of the period and 92.4% of the label was recovered. Virtually all of the label was in the digesta, and none was detected in the blood, so that the CT-carbon appeared not to be absorbed from the small intestine. In Expt 3, rumen, abomasal and ileal digesta and faeces samples from sheep fed on Lotus pedunculatus were analysed for CT and CT flow along the digestive tract calculated from reference to indigestible markers. Values were low in all digesta samples, indicating disappearance of CT across the rumen and small intestine, and CT recovery in faeces was only about 15% of intake. However, the 14C results from Expt 2 suggested that little if any CT-carbon was absorbed and the low recoveries in Expt 1 are considered to be a consequence of either conformational changes to the CT molecule such that it is no longer detectable by colorimetric methods, an inability of the analytical method to release bound CT for the butanol-HCl assay, or interference from other digesta constituents. It is concluded that the butanol-HCl method of CT analysis is appropriate for quantifying CT in herbages but not in digesta or faeces, and that a substantial part of CT released during protein digestion in the small intestine may not be detectable by normal CT analytical methods. PMID:7947660

Terrill, T H; Waghorn, G C; Woolley, D J; McNabb, W C; Barry, T N



Identification of genes directly regulated by the oncogene ZNF217 using chromatin immunoprecipitation (ChIP)-chip assays.  


It has been proposed that ZNF217, which is amplified at 20q13 in various tumors, plays a key role during neoplastic transformation. ZNF217 has been purified in complexes that contain repressor proteins such as CtBP2, suggesting that it acts as a transcriptional repressor. However, the function of ZNF217 has not been well characterized due to a lack of known target genes. Using a global chromatin immunoprecipitation (ChIP)-chip approach, we identified thousands of ZNF217 binding sites in three tumor cell lines (MCF7, SW480, and Ntera2). Further analysis of ZNF217 in Ntera2 cells showed that many promoters are bound by ZNF217 and CtBP2 and that a subset of these promoters are activated upon removal of ZNF217. Thus, our in vivo studies corroborate the in vitro biochemical analyses of ZNF217-containing complexes and support the hypothesis that ZNF217 functions as a transcriptional repressor. Gene ontology analysis showed that ZNF217 targets in Ntera2 cells are involved in organ development, suggesting that one function of ZNF217 may be to repress differentiation. Accordingly we show that differentiation of Ntera2 cells with retinoic acid led to down-regulation of ZNF217. Our identification of thousands of ZNF217 target genes will enable further studies of the consequences of aberrant expression of ZNF217 during neoplastic transformation. PMID:17259635

Krig, Sheryl R; Jin, Victor X; Bieda, Mark C; O'Geen, Henriette; Yaswen, Paul; Green, Roland; Farnham, Peggy J



Premature chromosome condensation (PCC) assay for dose assessment in mass casualty accidents.  


The study was undertaken to establish a dose calibration curve for a practical PCC ring assay and to apply it in a simulated mass casualty accident. The PCC assay was validated against the conventional dicentric assay. A linear relationship was established for PCC rings after (60)Co gamma irradiation with doses up to 20 Gy. In the simulated accident experiment, 62 blood samples were analyzed with both the PCC ring assay and the conventional dicentric assay, applying a triage approach. Samples received various uniform and non-uniform (10-40% partial-body) irradiations up to doses of 13 Gy. The results indicated that both assays yielded good dose estimates for the whole-body exposure scenario, although in the lower-dose range (0-6 Gy) dicentric scoring resulted in more accurate whole-body estimates, whereas PCC rings were better in the high-dose range (>6 Gy). Neither assay was successful in identifying partial-body exposures, most likely due to the low numbers of cells scored in the triage mode. In conclusion, the study confirmed that the PCC ring assay is suitable for use as a biodosimeter after whole-body exposure to high doses of radiation. However, there are limitations for its use in the triage of people exposed to high, partial-body doses. PMID:20041761

Lindholm, Carita; Stricklin, Daniela; Jaworska, Alicja; Koivistoinen, Armi; Paile, Wendla; Arvidsson, Eva; Deperas-Standylo, Joanna; Wojcik, Andrzej



Replacement by Drosophila melanogaster Protamines and Mst77F of Histones during Chromatin Condensation in Late Spermatids and Role of Sesame in the Removal of These Proteins from the Male Pronucleus†  

PubMed Central

Chromatin condensation is a typical feature of sperm cells. During mammalian spermiogenesis, histones are first replaced by transition proteins and then by protamines, while little is known for Drosophila melanogaster. Here we characterize three genes in the fly genome, Mst35Ba, Mst35Bb, and Mst77F. The results indicate that Mst35Ba and Mst35Bb encode dProtA and dProtB, respectively. These are considerably larger than mammalian protamines, but, as in mammals, both protamines contain typical cysteine/arginine clusters. Mst77F encodes a linker histone-like protein showing significant similarity to mammalian HILS1 protein. ProtamineA-enhanced green fluorescent protein (eGFP), ProtamineB-eGFP, and Mst77F-eGFP carrying Drosophila lines show that these proteins become the important chromosomal protein components of elongating spermatids, and His2AvDGFP vanishes. Mst77F mutants [ms(3)nc3] are characterized by small round nuclei and are sterile as males. These data suggest the major features of chromatin condensation in Drosophila spermatogenesis correspond to those in mammals. During early fertilization steps, the paternal pronucleus still contains protamines and Mst77F but regains a nucleosomal conformation before zygote formation. In eggs laid by sesame-deficient females, the paternal pronucleus remains in a protamine-based chromatin status but Mst77F-eGFP is removed, suggesting that the sesame gene product is essential for removal of protamines while Mst77F removal is independent of Sesame.

Jayaramaiah Raja, Sunil; Renkawitz-Pohl, Renate



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



Poly(ADP-ribose) polymerases PARP1 and PARP2 modulate topoisomerase II beta (TOP2B) function during chromatin condensation in mouse spermiogenesis.  


To achieve the specialized nuclear structure in sperm necessary for fertilization, dramatic chromatin reorganization steps in developing spermatids are required where histones are largely replaced first by transition proteins and then by protamines. This entails the transient formation of DNA strand breaks to allow for, first, DNA relaxation and then chromatin compaction. However, the nature and origin of these breaks are not well understood. We previously reported that these DNA strand breaks trigger the activation of poly(ADP-ribose) (PAR) polymerases PARP1 and PARP2 and that interference with PARP activation causes poor chromatin integrity with abnormal retention of histones in mature sperm and impaired embryonic survival. Here we show that the activity of topoisomerase II beta (TOP2B), an enzyme involved in DNA strand break formation in elongating spermatids, is strongly inhibited by the activity of PARP1 and PARP2 in vitro, and this is in turn counteracted by the PAR-degrading activity of PAR glycohydrolase. Moreover, genetic and pharmacological PARP inhibition both lead to increased TOP2B activity in murine spermatids in vivo as measured by covalent binding of TOP2B to the DNA. In summary, the available data suggest a functional relationship between the DNA strand break-generating activity of TOP2B and the DNA strand break-dependent activation of PARP enzymes that in turn inhibit TOP2B. Because PARP activity also facilitates histone H1 linker removal and local chromatin decondensation, cycles of PAR formation and degradation may be necessary to coordinate TOP2B-dependent DNA relaxation with histone-to-protamine exchange necessary for spermatid chromatin remodeling. PMID:21228215

Meyer-Ficca, Mirella L; Lonchar, Julia D; Ihara, Motomasa; Meistrich, Marvin L; Austin, Caroline A; Meyer, Ralph G



Chromatin hydrodynamics.  


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

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



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



Chromatin configuration during meiosis I prophase of spermatogenesis.  


During the pachytene stage of meiotic prophase in male mammals, the X and Y chromosomes become transcriptionally inactive and establish a chromatin domain, the sex body, that is visually distinct from the transcriptionally active autosomes. We used objective criteria to assess these chromatin differences by DNase I sensitivity (DS) of sex chromosome and autosomal sequences at both the cytological and molecular levels. For cytological studies, in situ nick translation techniques were used on air-dried preparations of testicular cells. For molecular studies, nuclei from pachytene spermatocytes were subjected to nuclease sensitivity assays. Both sex-linked and autosomal sequences were assessed, including some gene sequences that are expressed and some that are not expressed in pachytene spermatocytes. There was a wide range of DS in different genomic sequences; however, the sex-linked sequences generally were less nuclease sensitive than were autosomal sequences. Interestingly, a hot spot of recombination (within the Eb gene) showed a high level of nuclease sensitivity, while a cold spot of recombination (centromeric satellite region) exhibited lower sensitivity, more similar to that of sex-linked sequences. We also examined the nuclease sensitivity of a tyrosinase transgene insert, TyBS. In one line of mice, the transgene insert is X-linked, whereas in another, it is autosomal. The transgene was less nuclease sensitive when X-linked than as an autosomal insert. These results support the hypothesis that in pachytene spermatocytes the XY chromosome pair is more condensed and inaccessible to enzymatic digest, whereas the autosomal chromatin is in a more open configuration. In addition, we examined the nuclease sensitivity of some of the same genes in the earlier leptotene/zygotene prophase stage, when the sex chromatin is not maximally condensed. We found that while autosomal gene nuclease sensitivity was equivalent to that at the pachytene stage, X-linked sequences were more nuclease sensitive. Overall, these differences in chromatin nuclease sensitivity correlate with differences in meiotic recombination activity and may be mechanistically related. PMID:9406197

Wiltshire, T; Park, C; Handel, M A



Mitotic Phosphorylation Prevents the Binding of HMGN Proteins to Chromatin  

PubMed Central

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

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



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.



Chromatin dynamics during lytic infection with herpes simplex virus 1.  


Latent HSV-1 genomes are chromatinized with silencing marks. Since 2004, however, there has been an apparent inconsistency in the studies of the chromatinization of the HSV-1 genomes in lytically infected cells. Nuclease protection and chromatin immunoprecipitation assays suggested that the genomes were not regularly chromatinized, having only low histone occupancy. However, the chromatin modifications associated with transcribed and non-transcribed HSV-1 genes were those associated with active or repressed transcription, respectively. Moreover, the three critical HSV-1 transcriptional activators all had the capability to induce chromatin remodelling, and interacted with critical chromatin modifying enzymes. Depletion or overexpression of some, but not all, chromatin modifying proteins affected HSV-1 transcription, but often in unexpected manners. Since 2010, it has become clear that both cellular and HSV-1 chromatins are highly dynamic in infected cells. These dynamics reconcile the weak interactions between HSV-1 genomes and chromatin proteins, detected by nuclease protection and chromatin immunoprecipitation, with the proposed regulation of HSV-1 gene expression by chromatin, supported by the marks in the chromatin in the viral genomes and the abilities of the HSV-1 transcription activators to modulate chromatin. It also explains the sometimes unexpected results of interventions to modulate chromatin remodelling activities in infected cells. PMID:23863878

Conn, Kristen L; Schang, Luis M



Chromatin Dynamics during Lytic Infection with Herpes Simplex Virus 1  

PubMed Central

Latent HSV-1 genomes are chromatinized with silencing marks. Since 2004, however, there has been an apparent inconsistency in the studies of the chromatinization of the HSV-1 genomes in lytically infected cells. Nuclease protection and chromatin immunoprecipitation assays suggested that the genomes were not regularly chromatinized, having only low histone occupancy. However, the chromatin modifications associated with transcribed and non-transcribed HSV-1 genes were those associated with active or repressed transcription, respectively. Moreover, the three critical HSV-1 transcriptional activators all had the capability to induce chromatin remodelling, and interacted with critical chromatin modifying enzymes. Depletion or overexpression of some, but not all, chromatin modifying proteins affected HSV-1 transcription, but often in unexpected manners. Since 2010, it has become clear that both cellular and HSV-1 chromatins are highly dynamic in infected cells. These dynamics reconcile the weak interactions between HSV-1 genomes and chromatin proteins, detected by nuclease protection and chromatin immunoprecipitation, with the proposed regulation of HSV-1 gene expression by chromatin, supported by the marks in the chromatin in the viral genomes and the abilities of the HSV-1 transcription activators to modulate chromatin. It also explains the sometimes unexpected results of interventions to modulate chromatin remodelling activities in infected cells.

Conn, Kristen L.; Schang, Luis M.



The Drosophila MI-2 chromatin-remodeling factor regulates higher-order chromatin structure and cohesin dynamics in vivo.  


dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. Here we report an unanticipated role for dMi-2 in the regulation of higher-order chromatin structure in Drosophila. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells. PMID:22912596

Fasulo, Barbara; Deuring, Renate; Murawska, Magdalena; Gause, Maria; Dorighi, Kristel M; Schaaf, Cheri A; Dorsett, Dale; Brehm, Alexander; Tamkun, John W



Cell-Type-Specific Profiling of Gene Expression and Chromatin Binding without Cell Isolation: Assaying RNA Pol II Occupancy in Neural Stem Cells  

PubMed Central

Summary 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.

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




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...


Chromatin remodeling and cancer, part I: covalent histone modifications  

Microsoft Academic Search

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

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



Chromatin Higher-order Structure and Dynamics  

PubMed Central

The primary role of the nucleus as an information storage, retrieval, and replication site requires the physical organization and compaction of meters of DNA. Although it has been clear for many years that nucleosomes constitute the first level of chromatin compaction, this contributes a relatively small fraction of the condensation needed to fit the typical genome into an interphase nucleus or set of metaphase chromosomes, indicating that there are additional “higher order” levels of chromatin condensation. Identifying these levels, their interrelationships, and the principles that govern their occurrence has been a challenging and much discussed problem. In this article, we focus on recent experimental advances and the emerging evidence indicating that structural plasticity and chromatin dynamics play dominant roles in genome organization. We also discuss novel approaches likely to yield important insights in the near future, and suggest research areas that merit further study.

Woodcock, Christopher L.; Ghosh, Rajarshi P.



Use of chromatin stability assay, mitochondrial stain JC-1, and fluorometric assessment of plasma membrane to evaluate frozen-thawed ram semen  

Microsoft Academic Search

Cryopreservation of semen imposes deleterious effects on spermatozoa, either killing a certain proportion of cells or causing subtle damages on sperm function in the surviving population, changes not easily revealed by conventional assays. We have tested three functional assessment techniques in frozen-thawed ram semen from six adult rams, cryopreserved following eight different protocols (four extenders, and glycerol being added at

F Martinez-Pastor; A Johannisson; J Gil; M Kaabi; L Anel; P Paz; H Rodriguez-Martinez



Fixated on fixation: using ChIP to interrogate the dynamics of chromatin interactions  

PubMed Central

A new study exploits the time-dependence of formaldehyde cross-linking in the commonly used chromatin immunoprecipitation (ChIP) assay to infer the on and off rates for site-specific chromatin interactions.



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



A tripartite DNA-binding element, comprised of the nuclear localization signal and two AT-hook motifs, mediates the association of LEDGF/p75 with chromatin in vivo  

PubMed Central

Lens epithelium-derived growth factor p75 (LEDGF/p75) is a DNA-binding, transcriptional co-activator that participates in HIV-1 integration site targeting. Using complementary approaches, we determined the mechanisms of LEDGF/p75 DNA-binding in vitro and chromatin-association in living cells. The binding of highly-purified, recombinant protein was assayed by surface plasmon resonance (SPR) and electrophoretic mobility gel shift. Neither assay revealed evidence for sequence-specific DNA-binding. Residues 146–197 spanning the nuclear localization signal (NLS) and two AT-hook motifs mediated non-specific DNA-binding, and DNA-binding deficient mutants retained the ability to efficiently stimulate HIV-1 integrase activity in vitro. Chromatin-association was assessed by visualizing the localization of EGFP fusion proteins in interphase and mitotic cells. Although a conserved N-terminal PWWP domain was not required for binding to condensed mitotic chromosomes, its deletion subtly affected the nucleoplasmic distribution of the protein during interphase. A dual AT-hook mutant associated normally with chromatin, yet when the mutations were combined with NLS changes or deletion of the PWWP domain, chromatin-binding function was lost. As the PWWP domain did not readily bind free DNA in vitro, our results indicate that chromatin-association is primarily affected through DNA-binding, with the PWWP domain likely contributing a protein interaction to the overall affinity of LEDGF/p75 for human chromatin.

Turlure, Fanny; Maertens, Goedele; Rahman, Shaila; Cherepanov, Peter; Engelman, Alan



Chromatin remodelling during development  

PubMed Central

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

Ho, Lena; Crabtree, Gerald R.



Chromatin meets its organizers.  


Chromatin organization and gene-gene interactions are critical components of carrying out developmental programs. Phillips-Cremins et al. identify a series of unexpected architectural proteins that work in a combinatorial manner to functionally organize chromatin in a cell-type-specific manner at the submegabase-length scale. PMID:23746835

Bodnar, Megan S; Spector, David L



Relationship Between Chromatin Structure and Sensitivity to Molecularly Targeted Auger Electron Radiation Therapy  

SciTech Connect

Purpose: The open structure of euchromatin renders it susceptible to DNA damage by ionizing radiation (IR) compared with compact heterochromatin. The effect of chromatin configuration on the efficacy of Auger electron radiotherapy was investigated. Methods and Materials: Chromatin structure was altered in MDA-MB-468 and 231-H2N human breast cancer cells by suberoylanilide hydroxamic acid (SAHA), 5-aza-2-deoxycytidine, or hypertonic treatment. The extent and duration of chromatin structural changes were evaluated using the micrococcal nuclease assay. DNA damage ({gamma}H2AX assay) and clonogenic survival were evaluated after exposure to {sup 111}In-DTPA-hEGF, an Auger electron-emitting radiopharmaceutical, or IR. The intracellular distribution of {sup 111}In-DTPA-hEGF after chromatin modification was investigated in cell fractionation experiments. Results: Chromatin remained condensed for up to 20 minutes after NaCl and in a relaxed state 24 hours after SAHA treatment. The number of {gamma}H2AX foci per cell was greater in MDA-MB-468 and 231-H2N cells after IR (0.5 Gy) plus SAHA (1 {mu}M) compared with IR alone (16 {+-} 0.6 and 14 {+-} 0.3 vs. 12 {+-} 0.4 and 11 {+-} 0.2, respectively). More {gamma}H2AX foci were observed in MDA-MB-468 and 231-H2N cells exposed to {sup 111}In-DTPA-hEGF (6 MBq/{mu}g) plus SAHA vs. {sup 111}In-DTPA-hEGF alone (11 {+-} 0.3 and 12 {+-} 0.7 vs. 9 {+-} 0.4 and 7 {+-} 0.3, respectively). 5-aza-2-deoxycytidine enhanced the DNA damage caused by IR and {sup 111}In-DTPA-hEGF. Clonogenic survival was reduced in MDA-MB-468 and 231-H2N cells after IR (6 Gy) plus SAHA (1 {mu}M) vs. IR alone (0.6% {+-} 0.01 and 0.3% {+-} 0.2 vs. 5.8% {+-} 0.2 and 2% {+-} 0.1, respectively) and after {sup 111}In-DTPA-hEGF plus SAHA compared to {sup 111}In-DTPA-hEGF alone (21% {+-} 0.4% and 19% {+-} 4.6 vs. 33% {+-} 2.3 and 32% {+-} 3.7). SAHA did not affect {sup 111}In-DTPA-hEGF nuclear localization. Hypertonic treatment resulted in fewer {gamma}H2AX foci per cell after IR and {sup 111}In-DTPA-hEGF compared to controls but did not significantly alter clonogenic survival. Conclusions: Chromatin structure affects DNA damage and cell survival after exposure to Auger electron radiation.

Terry, Samantha Y.A. [CR-UK/MRC Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Oxford (United Kingdom); Vallis, Katherine A., E-mail: [CR-UK/MRC Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Oxford (United Kingdom)



Where splicing joins chromatin  

PubMed Central

There are numerous data suggesting that two key steps in gene expression—transcription and splicing influence each other closely. For a long time it was known that chromatin modifications regulate transcription, but only recently it was shown that chromatin and histone modifications play a significant role in pre-mRNA splicing. Here we summarize interactions between splicing machinery and chromatin and discuss their potential functional significance. We focus mainly on histone acetylation and methylation and potential mechanisms of their role in splicing. It seems that whereas histone acetylation acts mainly by alterating the transcription rate, histone methylation can also influence splicing directly by recruiting various splicing components.

Hnilicova, Jarmila



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



SUMO and Chromatin Remodelling  

Microsoft Academic Search

\\u000a Many of the increasing number of known SUMO substrates are nuclear proteins, which regulate gene expression and chromatin\\u000a dynamics. Sumoylation, in general, appears to correlate with decreased transcriptional activity, and in many cases modulation\\u000a of the chromatin template is implicated. Sumoylation of the core histones is associated with transcriptional silencing, and\\u000a transcription factor sumoylation can decrease gene expression by promoting

David Wotton; Jacqueline C. Merrill


[Chromatin and transcription regulation].  


Recent years are marked by drastic increase of interest in the role of chromatin in regulation of gene activity. In the seventies of the last century many studies were undertaken in order to identify different forms of histones involved in regulation on transcription. The results of these studies were conflicting. Determination of primary structures of the main forms of histones demonstrated the extreme conservativity of these proteins. Once the nucleosomes were discovered and their organization was studied, it became clear that nucleosome as a basic unit of chromatin is also highly conservative. This conception gradually changed in recent years. Many variant forms of nucleosomal core histones encoded by separate genes were discovered. In addition it was demonstrated that both canonical and variant forms of histones may by modified post-translationally in different ways. As a result, a possibility to assemble a number of different nucleosomal particles became evident. Furthermore, a clear correlation between certain modification of histones and DNA packaging in either active or inactive chromatin was established. Similarly, a correlation between formation of active (inactive) chromatin and incorporation of particular histone variants into nucleosomes was observed. To integrate all the above findings into the existing model of chromatin organization and functioning, the hypothesis of "histone code" was proposed. In this review the present state of our knowledge about chromatin organization and the role of this organization in transcription regulation will be discussed. PMID:17685218

Razin, S V



Chromatin Modifications in DNA Repair  

Microsoft Academic Search

A requirement of nuclear processes that use DNA as a substrate is the manipulation\\u000a of chromatin in which the DNA is packaged. Chromatin modifications cause alterations of histones\\u000a and DNA, and result in a permissive chromatin environment for these nuclear processes. Recent\\u000a advances in the fields of DNA repair and chromatin reveal that both histone modifications and chromatin-remodeling\\u000a complexes are essential for the

Ashby J. Morrison; Xuetong Shen


The Ino80 chromatin-remodeling complex restores chromatin structure during UV DNA damage repair  

PubMed Central

Chromatin structure is modulated during deoxyribonucleic acid excision repair, but how this is achieved is unclear. Loss of the yeast Ino80 chromatin-remodeling complex (Ino80-C) moderately sensitizes cells to ultraviolet (UV) light. In this paper, we show that INO80 acts in the same genetic pathway as nucleotide excision repair (NER) and that the Ino80-C contributes to efficient UV photoproduct removal in a region of high nucleosome occupancy. Moreover, Ino80 interacts with the early NER damage recognition complex Rad4–Rad23 and is recruited to chromatin by Rad4 in a UV damage–dependent manner. Using a modified chromatin immunoprecipitation assay, we find that chromatin disruption during UV lesion repair is normal, whereas the restoration of nucleosome structure is defective in ino80 mutant cells. Collectively, our work suggests that Ino80 is recruited to sites of UV lesion repair through interactions with the NER apparatus and is required for the restoration of chromatin structure after repair.

Sarkar, Sovan; Kiely, Rhian



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



Mitotic checkpoint control and chromatin remodeling.  


In order to maintain chromosomal stability during cell division, eukaryotic cells have evolved a number of surveillance mechanisms termed checkpoints. These checkpoints monitor the completion of essential molecular and cellular processes of one stage before entering another. The spindle checkpoint watches the bi-orientation attachment of spindle microtubules to all condensed chromosomes before initiation of nuclear division during mitosis. Histones are subject to a number of post-translational modifications during the cell cycle, which may in turn modify or facilitate cell cycle progression. Recent studies suggest that mitotic proteins including Bub1 and Sgo1 that are involved in the spindle checkpoint also play a major role in the regulation of histone modifications and chromatin remodeling. This mini-review summarizes emerging information about the new role of spindle checkpoint proteins in chromatin remodeling. PMID:22201785

Yao, Yixin; Dai, Wei



Chromatin structure in globozoospermia: a case report.  


Sperm nuclear abnormalities in patients with globozoospermia have not been well characterized and may lead to the high rates of fertilization failure and embryo loss reported in patients with this form of teratozoospermia. This study used transmission electron microscopy (TEM), the sperm chromatin structure assay (SCSA), and single cell gel eletrophoresis assay (COMET) to assess if globozoospermia is associated with sperm chromatin structure abnormalities, DNA fragmentation, or both. The flow cytometric SCSA measures abnormal chromatin structure based on the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ. COMET measures DNA fragmentation in individual sperm nuclei based upon gel electrophoretic patterns. Although sperm concentration (113 million/mL) and motility (66%) were normal in the patient, there was complete acrosome deficiency. TEM and SCSA data confirmed light microscopic examination that showed that sperm populations included a mixture of round and elongated sperm heads. Even though 100% of sperm had abnormal head morphology, only 13% demonstrated DNA denaturation (COMPalpha(t)), which is below our threshold of 15% COMPalpha(t), and consistent with high-fertility patients. Of interest, 13% of the sperm were also positive in the COMET assay, supporting our previous observations that SCSA-positive cells are also positive for DNA fragmentation. It was unexpected but of great interest that a human sperm population with 100% sperm morphology abnormalities had a chromatin integrity at the molecular level that is equivalent to sperm populations shown in previous studies to be highly fertile. These data are the first reported using SCSA and COMET assays to evaluate a patient with globozoospermia and support previous reports that intracytoplasmic sperm injection of globozoospermia may result in fertility/pregnancy. Lower success rates seen in some patients may be due to unrelated factors. PMID:11330642

Larson, K L; Brannian, J D; Singh, N P; Burbach, J A; Jost, L K; Hansen, K P; Kreger, D O; Evenson, D P



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


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

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



Signaling chromatin to make muscle  

Microsoft Academic Search

Several findings published within the past year have further established key roles for chromatin-modifying enzymes in the control of muscle gene expression, and have thus refined our thinking of how chromatin structure influences muscle differentiation, hypertrophy and fiber type determination. We discuss the interface between chromatin-modifying enzymes and myogenic transcription factors, signaling mechanisms that impinge on these transcriptional complexes, and

Timothy A McKinsey; Chun Li Zhang; Eric N Olson



Chromatin Organization and Radio Resistance in the Bacterium Gemmata obscuriglobus? †  

PubMed Central

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

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



The Telomere Binding Protein TRF2 Induces Chromatin Compaction  

PubMed Central

Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

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



Estrogen-receptor-alpha exchange and chromatin dynamics are ligand- and domain-dependent.  


We report a mammalian-based promoter chromosomal array system developed for single-cell studies of transcription-factor function. Designed after the prolactin promoter-enhancer, it allows for the direct visualization of estrogen receptor alpha (ERalpha) and/or Pit-1 interactions at a physiologically regulated transcription locus. ERalpha- and ligand-dependent cofactor recruitment, large-scale chromatin modifications and transcriptional activity identified a distinct fingerprint of responses for each condition. Ligand-dependent transcription (more than threefold activation compared with vehicle, or complete repression by mRNA fluorescent in situ hybridization) at the array correlated with its state of condensation, which was assayed using a novel high throughput microscopy approach. In support of the nuclear receptor hit-and-run model, photobleaching studies provided direct evidence of very transient ER-array interactions, and revealed ligand-dependent changes in k(off). ERalpha-truncation mutants indicated that helix-12 and interactions with co-regulators influenced both large-scale chromatin modeling and photobleaching recovery times. These data also showed that the ERalpha DNA-binding domain was insufficient for array targeting. Collectively, quantitative observations from this physiologically relevant biosensor suggest stochastic-based dynamics influence gene regulation at the promoter level. PMID:16968748

Sharp, Z Dave; Mancini, Maureen G; Hinojos, Cruz A; Dai, Fangyan; Berno, Valeria; Szafran, Adam T; Smith, Kelly P; Lele, Tanmay P; Lele, Tanmay T; Ingber, Donald E; Mancini, Michael A



Phosphorylated and dephosphorylated linker histone H1 reside in distinct chromatin domains in Tetrahymena macronuclei.  

PubMed Central

Phosphorylated and dephosphorylated isoforms of Tetrahymena macronuclear H1 were separated from each other by cation-exchange high performance liquid chromatography and used to generate a pairwise set of antisera that discriminate the phosphorylation state of this linker histone. Affinity-purified antibodies from each sera recognize appropriate H1 isoforms and stain macronuclei under appropriate physiological conditions. Immunogold localizations demonstrate that phosphorylated and dephosphorylated H1 localize nonrandomly in distinct subdomains of macronuclear chromatin. Dephosphorylated H1 is strongly enriched in the electron-dense chromatin bodies that punctuate macronuclear chromatin. In contrast, phosphorylated H1 isoforms, as well as an evolutionarily conserved H2A.F/Z-like variant (hv1) believed to function in the establishment of transcriptionally competent chromatin, are modestly enriched at the periphery of chromatin bodies and in the surrounding euchromatin. Using antibodies against TATA-binding protein, we show that transcriptionally active chromatin lies outside of the chromatin bodies in an area relatively devoid of H1. Antibodies against general core histones are more or less evenly distributed across these domains. Together, these data are consistent with a model in which phosphorylation of H1, perhaps in association with hv1, loosens the binding of H1 in chromatin leading to chromatin decondensation as part of a first-step mechanism in gene activation. In contrast, our data support the view that dephosphorylation of this linker histone facilitates or stabilizes condensed, transcriptionally silent chromatin. Images

Lu, M J; Mpoke, S S; Dadd, C A; Allis, C D



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



Chromatin Dynamics during DNA Repair Revealed by Pair Correlation Analysis of Molecular Flow in the Nucleus.  


Chromatin dynamics modulate DNA repair factor accessibility throughout the DNA damage response. The spatiotemporal scale upon which these dynamics occur render them invisible to live cell imaging. Here we present a believed novel assay to monitor the in vivo structural rearrangements of chromatin during DNA repair. By pair correlation analysis of EGFP molecular flow into chromatin before and after damage, this assay measures millisecond variations in chromatin compaction with submicron resolution. Combined with laser microirradiation we employ this assay to monitor the real-time accessibility of DNA at the damage site. We find from comparison of EGFP molecular flow with a molecule that has an affinity toward double-strand breaks (Ku-EGFP) that DNA damage induces a transient decrease in chromatin compaction at the damage site and an increase in compaction to adjacent regions, which together facilitate DNA repair factor recruitment to the lesion with high spatiotemporal control. PMID:24988341

Hinde, Elizabeth; Kong, Xiangduo; Yokomori, Kyoko; Gratton, Enrico



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.

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



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.

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



Transcription factors, chromatin and cancer  

Microsoft Academic Search

Transcription factors, chromatin and chromatin-modifying enzymes are key components in a complex network through which the genome interacts with its environment. For many transcription factors, binding motifs are found adjacent to the promoter regions of a large proportion of genes, requiring mechanisms that confer binding specificity in any given cell type. These include association of the factor with other proteins

James L. Thorne; Moray J. Campbell; Bryan M. Turner



Histone modifications influence mediator interactions with chromatin  

PubMed Central

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

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



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


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

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



Mesoscale Modeling of Chromatin Folding  

NASA Astrophysics Data System (ADS)

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

Schlick, Tamar



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.

Gilbert, Nick; Allan, James



Differential Chromatin Structure Encompassing Replication Origins in Transformed and Normal Cells  

PubMed Central

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

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



Biology of sperm chromatin structure and relationship to male fertility and embryonic survival.  


Embryonic mortality in mammals is typically thought to result from 'female factor' infertility. There is growing evidence, however, that the status of sperm chromatin (DNA) at the time of fertilisation can also influence embryonic survival. During the final stages of spermatogenesis (spermiogenesis) a number of unique biochemical, morphological and physiological processes take place that are associated with marked changes in the structure of sperm chromatin. In early stages of spermatogenesis, sperm DNA is associated with histone nucleoproteins and structured into classical nucleosome core particles similar to other somatic cells. As spermiogenesis proceeds, the histone nucleoproteins are replaced by transition proteins which are subsequently replaced by protamines. At the completion of spermiogenesis the chromatin of mature sperm has a toroidal structure that is tightly compacted and resistant to denaturation. The compaction is necessary to protect sperm chromatin during transit through the epididymis and female reproductive tract. Disruption to chromatin remodelling during spermiogenesis results in chromatin that is susceptible to denaturation. Inappropriate chromatin structure has been shown in a number of mammalian species to be related to male infertility, and specifically the failure of embryonic development. A range of techniques are available to assess chromatin status in sperm but arguably the most informative is the sperm chromatin structure assay (SCSA). The SCSA is a flow cytometric assay that uses the metachromatic properties of acridine orange to measure the susceptibility of sperm chromatin to acid-induced denaturation. A relationship has been demonstrated, primarily in men, between the SCSA outcome and the probability of continued embryonic development and the establishment of pregnancy after fertilisation. The contribution of sperm chromatin instability to reproductive wastage in both natural mating and assisted reproduction warrants further investigation as it may prove valuable as a means of decreasing the incidence of embryonic mortality. In this regard, it is possible that 'male factor' infertility may emerge as an even more important component in embryonic development. PMID:17303352

D'Occhio, M J; Hengstberger, K J; Johnston, S D



Atomic force microscopy of mammalian sperm chromatin  

Microsoft Academic Search

We have used the atomic force microscope (AFM) to image the surfaces of intact bull, mouse and rat sperm chromatin and partially decondensed mouse sperm chromatin attached to coverglass. High resolution AFM imaging was performed in air and saline using uncoated, unfixed and unstained chromatin. Images of the surfaces of intact chromatin from all three species and of an AFM-dissected

Michael J. Allen; Catherine Lee; Joseph Day Lee; Gilbert C. Pogany; Mehdi Balooch; Wigbert J. Siekhaus; Rod Balhorn



Chromatin remodeling by nuclear receptors  

Microsoft Academic Search

The eukaryotic genome is structurally organized into nucleosomes to form chromatin, which regulates gene expression, in part,\\u000a by controlling the accessibility of regulatory factors. When packaged as chromatin, many promoters are transcriptionally repressed,\\u000a thus reducing the access of transcription factors to their binding sites. However, nuclear receptors (NRs) are a group of\\u000a transcription factors that have the ability to access

Pratibha B. Hebbar; Trevor K. Archer



An overview of chromatin modifications.  


The last 15 years have witnessed tremendous progress in elucidating the roles of chromatin modifications in transcription regulation, DNA repair, replication, recombination, and other genomic processes. In this issue of Biopolymers, a series of reviews will summarize recent advances in our understanding of chromatin modifying enzymes and explore unresolved questions with respect to their regulation and functions in gene expression and other nuclear processes. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 95-97, 2013. PMID:23175384

Fick, Robert J; Trievel, Raymond C



ATP-dependent Chromatin Remodelling  

Microsoft Academic Search

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

Parul Choudhary; Patrick Varga-Weisz


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.

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



The insulation of genes from external enhancers and silencing chromatin  

PubMed Central

Insulators are DNA sequence elements that can serve in some cases as barriers to protect a gene against the encroachment of adjacent inactive condensed chromatin. Some insulators also can act as blocking elements to protect against the activating influence of distal enhancers associated with other genes. Although most of the insulators identified so far derive from Drosophila, they also are found in vertebrates. An insulator at the 5? end of the chicken ?-globin locus marks a boundary between an open chromatin domain and a region of constitutively condensed chromatin. Detailed analysis of this element shows that it possesses both enhancer blocking activity and the ability to screen reporter genes against position effects. Enhancer blocking is associated with binding of the protein CTCF; sites that bind CTCF are found at other critical points in the genome. Protection against position effects involves other properties that appear to be associated with control of histone acetylation and methylation. Insulators thus are complex elements that can help to preserve the independent function of genes embedded in a genome in which they are surrounded by regulatory signals they must ignore.

Burgess-Beusse, Bonnie; Farrell, Catherine; Gaszner, Miklos; Litt, Michael; Mutskov, Vesco; Recillas-Targa, Felix; Simpson, Melanie; West, Adam; Felsenfeld, Gary



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.

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



Sudemycin E influences alternative splicing and changes chromatin modifications.  


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; Webb, Thomas R; Stamm, Stefan



Globozoospermia is associated with chromatin structure abnormalities: case report.  


A recent study has shown normal sperm chromatin structure in a patient with globozoospermia. However, the poor success rate following intracytoplasmic sperm injection (ICSI) with the use of this sperm suggests that sperm nuclear abnormalities may be present. We report here a study of the sperm DNA integrity and chromosome aneuploidy and diploidy rates of a patient with 100% round-headed sperm. The sperm chromatin packaging quality was assessed by using flow cytometry after staining the DNA with propidium iodide. DNA fragmentation, possibly indicative of apoptosis, was evaluated by flow cytometry using the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labelling (TUNEL) assay. The sperm chromosome aneuploidy and diploidy rates were evaluated by fluorescence in-situ hybridization using alpha-centromeric probes for chromosomes 8, 12, 18, X and Y. The patient with globozoospermia had a significantly higher number of sperm with chromatin decondensation (35 +/- 1.1%) and positive for the TUNEL assay (37 +/- 1.7%) compared with that found in four normal controls (4.7 +/- 0.4 and 22.5 +/- 1.2% respectively). In contrast, the total sperm aneuploidy (0.16%) and diploidy (0.05%) rates for the chromosomes studied were within the range found in 14 normozoospermic men. To the best of our knowledge, this is the first case report of round-headed sperm that has shown an elevated number of sperm with abnormal chromatin structure and DNA strand breaks. PMID:12151448

Vicari, Enzo; Perdichizzi, Anna; De Palma, Adele; Burrello, Nunziatina; D'Agata, Rosario; Calogero, Aldo E



3,5,3'-triiodothyronine receptor-containing chromatin fragments: production by nuclease digestion.  


Nuclear thyroid hormone (T3) receptors are nonhistone proteins which are tightly bound to rat liver chromatin. The solubilization of the T3 receptors by micrococcal nuclease was studied using an assay which allows the delection of in vitro hormone binding and which is independent of the state of solubility of the chromatin. Nuclease digestion produces a receptor containing moiety which sediments at a rate of 5--6S. This form of the receptor is different than that released from chromatin at high ionic strength (3.8S) and potentially represents the stable association of the receptor which other elements of chromatin. Partial release of chromatin compaction by the use of dilute buffer solutions increases the rate of nuclease digestion, facilitates the release of the (5--6S) T3-receptor complex, and allows the isolation of sucrose gradient fractions which are enriched with receptor. PMID:6250802

Gruol, D J



The Spectrum of Anti-Chromatin/Nucleosome Autoantibodies: Independent and Interdependent Biomarkers of Disease  

PubMed Central

Autoantibodies directed to chromatin components date back to the discovery of the LE cell and the LE cell phenomenon circa 1950, and subsequent evidence that major components of that reaction were chromatin components and histones in particular. Over time, immunoassays ranging from ELISA and line immunoassays to more modern bead-based assays incorporated histone and DNA mixtures, purified histones, and purified nucleosomes leading to a more thorough understanding of the genesis and pathogenetic relationships of antibodies to chromatin components in systemic lupus erythematosus and other autoimmune conditions. More recently, interest has focussed on other components of chromatin such as high mobility group (HMG) proteins both as targets of B cell responses and pro-inflammatory mediators. This review will focus on immunoassays that utilize chromatin components, their clinical relationships, and newer evidence implicating HMG proteins and DNA neutrophil extracellular traps (NETs) as important players in systemic autoimmune rheumatic diseases.

Mehra, Sonal; Fritzler, Marvin J.



Condensation polyimides  

NASA Technical Reports Server (NTRS)

Polyimides belong to a class of polymers known as polyheterocyclics. Unlike most other high temperature polymers, polyimides can be prepared from a variety of inexpensive monomers by several synthetic routes. The glass transition and crystalline melt temperature, thermooxidative stability, toughness, dielectric constant, coefficient of thermal expansion, chemical stability, mechanical performance, etc. of polyimides can be controlled within certain boundaries. This versatility has permitted the development of various forms of polyimides. These include adhesives, composite matrices, coatings, films, moldings, fibers, foams and membranes. Polyimides are synthesized through both condensation (step-polymerization) and addition (chain growth polymerization) routes. The precursor materials used in addition polyimides or imide oligomers are prepared by condensation method. High molecular weight polyimide made via polycondensation or step-growth polymerization is studied. The various synthetic routes to condensation polyimides, structure/property relationships of condensation polyimides and composite properties of condensation polyimides are all studied. The focus is on the synthesis and chemical structure/property relationships of polyimides with particular emphasis on materials for composite application.

Hergenrother, P. M.



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

PubMed Central

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

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



Correlated spatio-temporal fluctuations in chromatin compaction states characterize stem cells.  


Stem cells integrate signals from the microenvironment to generate lineage-specific gene expression programs upon differentiation. Undifferentiated cell nuclei are easily deformable, with an active transcriptome, whereas differentiated cells have stiffer nuclei and condensed chromatin. Chromatin organization in the stem cell state is known to be highly dynamic but quantitative characterizations of its plasticity are lacking. Using fluorescence imaging, we study the spatio-temporal dynamics of nuclear architecture and chromatin compaction in mouse embryonic stem (ES) cells and differentiated states. Individual ES cells exhibit a relatively narrow variation in chromatin compaction, whereas primary mouse embryonic fibroblasts (PMEF) show broad distributions. However, spatial correlations in chromatin compaction exhibit an emergent length scale in PMEFs, although they are unstructured and longer ranged in ES cells. We provide evidence for correlated fluctuations with large amplitude and long intrinsic timescales, including an oscillatory component, in both chromatin compaction and nuclear area in ES cells. Such fluctuations are largely frozen in PMEF. The role of actin and Lamin A/C in modulating these fluctuations is described. A simple theoretical formulation reproduces the observed dynamics. Our results suggest that, in addition to nuclear plasticity, correlated spatio-temporal structural fluctuations of chromatin in undifferentiated cells characterize the stem cell state. PMID:23442906

Talwar, Shefali; Kumar, Abhishek; Rao, Madan; Menon, Gautam I; Shivashankar, G V



Snapshots: Chromatin Control of Viral Infection  

PubMed Central

Like their cellular host counterparts, many invading viral pathogens must contend with, modulate, and utilize the host cell’s chromatin machinery to promote efficient lytic infection or control persistent-latent states. While not intended to be comprehensive, this review represents a compilation of conceptual snapshots of the dynamic interplay of viruses with the chromatin environment. Contributions focus on chromatin dynamics during infection, viral circumvention of cellular chromatin repression, chromatin organization of large DNA viruses, tethering and persistence, viral interactions with cellular chromatin modulation machinery, and control of viral latency-reactivation cycles.

Knipe, David M.; Lieberman, Paul M.; Jung, Jae U.; McBride, Alison A.; Morris, Kevin V.; Ott, Melanie; Margolis, David; Nieto, Amelia; Nevels, Michael; Parks, Robin J.; Kristie, Thomas M.



Cyclin E Uses Cdc6 as a Chromatin-Associated Receptor Required for DNA Replication  

Microsoft Academic Search

Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specif- ically and saturably to chromatin in three phases. In the first phase, the origin recognition complex and Cdc6 pre- replication proteins, but not the minichromosome mainte- nance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin E-Cdk2 to DNA. We

Laura Furstenthal; Brett K. Kaiser; Craig Swanson; Peter K. Jackson



Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations  

Microsoft Academic Search

Chromatin immunoprecipitation (ChIP) defines the genomic distribution of proteins and their modifications but is limited by the cell numbers required (ideally >107). Here we describe a protocol that uses carrier chromatin and PCR, 'carrier' ChIP (CChIP), to permit analysis of as few as 100 cells. We assayed histone modifications at key regulator genes (such as Nanog, Pou5f1 (also known as

Laura P O'Neill; Matthew D VerMilyea; Bryan M Turner



Structural and functional genome analysis using extended chromatin  

SciTech Connect

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

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



Global Quantitative Modeling of Chromatin Factor Interactions  

PubMed Central

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.

Zhou, Jian; Troyanskaya, Olga G.



Large-scale chromatin de-compaction induced by low light is not accompanied by nucleosomal displacement  

PubMed Central

Arabidopsis thaliana is widely used as a model to study chromatin compaction dynamics during development and in response to the environment. Signals such as prolonged heat treatment, low light and pathogen infestation are known to induce large-scale de-condensation of nuclear chromatin. Here we demonstrate that the response to different environments varies at the nucleosomal level. Our results show that in contrast to previous reports on heat and biotic infestation, low light intensity signaling does not alter nucleosomal occupancy, despite the marked effects of low light on global chromatin compaction.

Peeters, Anton JM; Fransz, Paul



The DNA Binding and Catalytic Domains of Poly(ADP-Ribose) Polymerase 1 Cooperate in the Regulation of Chromatin Structure and Transcription?  

PubMed Central

We explored the mechanisms of chromatin compaction and transcriptional regulation by poly(ADP-ribose) polymerase 1 (PARP-1), a nucleosome-binding protein with an NAD+-dependent enzymatic activity. By using atomic force microscopy and a complementary set of biochemical assays with reconstituted chromatin, we showed that PARP-1 promotes the localized compaction of chromatin into supranucleosomal structures in a manner independent of the amino-terminal tails of core histones. In addition, we defined the domains of PARP-1 required for nucleosome binding, chromatin compaction, and transcriptional repression. Our results indicate that the DNA binding domain (DBD) of PARP-1 is necessary and sufficient for binding to nucleosomes, yet the DBD alone is unable to promote chromatin compaction and only partially represses RNA polymerase II-dependent transcription in an in vitro assay with chromatin templates (?50% of the repression observed with wild-type PARP-1). Furthermore, our results show that the catalytic domain of PARP-1, which does not bind nucleosomes on its own, cooperates with the DBD to promote chromatin compaction and efficient transcriptional repression in a manner independent of its enzymatic activity. Collectively, our results have revealed a novel function for the catalytic domain in chromatin compaction. In addition, they show that the DBD and catalytic domain cooperate to regulate chromatin structure and chromatin-dependent transcription, providing mechanistic insights into how these domains contribute to the chromatin-dependent functions of PARP-1.

Wacker, David A.; Ruhl, Donald D.; Balagamwala, Ehsan H.; Hope, Kristine M.; Zhang, Tong; Kraus, W. Lee



Plant chromatin: development and gene control.  


It is increasingly clear that chromatin is not just a device for packing DNA within the nucleus but also a dynamic material that changes as cellular environments alter. The precise control of chromatin modification in response to developmental and environmental cues determines the correct spatial and temporal expression of genes. Here, we review exciting discoveries that reveal chromatin participation in many facets of plant development. These include: chromatin modification from embryonic and meristematic development to flowering and seed formation, the involvement of DNA methylation and chromatin in controlling invasive DNA and in maintenance of epigenetic states, and the function of chromatin modifying and remodeling complexes such as SWI/SNF and histone acetylases and deacetylases in gene control. Given the role chromatin structure plays in every facet of plant development, chromatin research will undoubtedly be integral in both basic and applied plant biology. PMID:11891760

Li, Guofu; Hall, Timothy C; Holmes-Davis, Rachel



Mechanisms for ATP-dependent chromatin remodelling  

Microsoft Academic Search

During the past year, major advances have been made towards understanding the function of ATP-dependent chromatin-remodelling activities both in vitro and in vivo. These suggest that ATP-dependent chromatin-remodelling activities are capable of both altering the structure of individual nucleosomes and acting in concert with other forms of chromatin-modifying enzymes, to regulate the formation and decondensation of chromatin fibres.

Andrew Flaus; Tom Owen-Hughes



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 Chromatin Structure in Vivo  

Microsoft Academic Search

A number of important nuclear processes including replication, recombination, repair, and transcription involve the interaction of soluble nuclear proteins with DNA assembled as chromatin. Recent progress in a number of experimental systems has focused attention on the influence chromatin structure may exert on gene regulation in eukaryotes. With the advent of new technologies for the analysis of chromatin structurein vivo,studies

Joe S. Mymryk; Christy J. Fryer; Lee A. Jung; Trevor K. Archer



Polariton condensates  

SciTech Connect

Most students of physics know about the special properties of Bose-Einstein condensates (BECs) as demonstrated in the two best-known examples: superfluid helium-4, first reported in 1938, and condensates of trapped atomic gases, first observed in 1995. (See the article by Wolfgang Ketterle in PHYSICS TODAY, December 1999, page 30.) Many also know that superfluid {sup 3}He and superconducting metals contain BECs of fermion pairs. An underlying principle of all those condensed-matter systems, known as quantum fluids, is that an even number of fermions with half-integer spin can be combined to make a composite boson with integer spin. Such composite bosons, like all bosons, have the property that below some critical temperature--roughly the temperature at which the thermal de Broglie wavelength becomes comparable to the distance between the bosons--the total free energy is minimized by having a macroscopic number of bosons enter a single quantum state and form a macroscopic, coherent matter wave. Remarkably, the effect of interparticle repulsion is to lead to quantum mechanical exchange interactions that make that state robust, since the exchange interactions add coherently.

Snoke, David; Littlewood, Peter [University of Pittsburgh, Pennsylvania (United States); University of Cambridge (United Kingdom)



Localization of DNA in the condensed interphase chromosomes of Euglena  

Microsoft Academic Search

The localization of DNA in the condensed interphase chromosomes of Euglena was determined by immunoelectron microscopy. Deposits of gold particles that coincided with the localization of DNA followed threads that corresponded to the chromatin fibers. The threads were 55–80 nm in diameter and were assumed to be supersolenoids. The localization of gold deposits on chromosomes that had been sectioned in

K. Ueda; Y. Hayashi-Ishimaru



Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements)  

PubMed Central

The binding of sequence-specific regulatory factors and the recruitment of chromatin remodeling activities cause nucleosomes to be evicted from chromatin in eukaryotic cells. Traditionally, these active sites have been identified experimentally through their sensitivity to nucleases. Here we describe the details of a simple procedure for the genome-wide isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements). We also provide protocols for different methods of detecting FAIRE-enriched DNA, including use of PCR, DNA microarrays, and next-generation sequencing. FAIRE works on all eukaryotic chromatin tested to date. To perform FAIRE, chromatin is crosslinked with formaldehyde, sheared by sonication, and phenol-chloroform extracted. Most genomic DNA is crosslinked to nucleosomes and is sequestered to the interphase, whereas DNA recovered in the aqueous phase corresponds to nucleosome-depleted regions of the genome. The isolated regions are largely coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, enhancers, insulators, and active promoters. Given its speed and simplicity, FAIRE has utility in establishing chromatin profiles of diverse cell types in health and disease, isolating DNA regulatory elements en masse for further characterization, and as a screening assay for the effects of small molecules on chromatin organization.

Giresi, Paul G.; Lieb, Jason D.



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

PubMed Central

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

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



Chromatin Structure in Telomere Dynamics  

PubMed Central

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

Galati, Alessandra; Micheli, Emanuela; Cacchione, Stefano



Chromatin Organization and Transcriptional Regulation  

PubMed Central

Cell type specific transcriptional regulation must be adhered to in order to maintain cell identity throughout the lifetime of an organism, yet flexible enough to allow for responses to endogenous and exogenous stimuli. This regulation is mediated not only by molecular factors (e.g. cell type specific transcription factors, histone and DNA modifications), but also on the level of chromatin and genome organization. In this review we focus on recent findings that have contributed to our understanding of higher order chromatin structure and genome organization within the nucleus. We highlight new findings on the dynamic positioning of genes relative to each other, as well as to their chromosome territory and the nuclear lamina, and how the position of genes correlates with their transcriptional activity.

Hubner, Michael R.; Eckersley-Maslin, Melanie A.; Spector, David L.



Transposition of native chromatin for multimodal regulatory analysis and personal epigenomics  

PubMed Central

Several limitations of current epigenomic technology preclude their use in many experimental and clinical settings. Here we describe Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq)— based on direct in vitro transposition of sequencing adapters into native chromatin – as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple 2-step protocol from 500 to 50,000 cells, and reveals the interplay between genomic locations of open chromatin, DNA binding proteins, individual nucleosomes, and higher-order compaction at regulatory regions with nucleotide resolution. We discover classes of DNA binding factor that strictly avoid, can tolerate, or tend to overlap with nucleosomes. Using ATAC-seq, we measured and interpreted the serial daily epigenomes of resting human T cells from a proband via standard blood draws, demonstrating the feasibility of reading personal epigenomes in clinical timescales for monitoring health and disease.

Buenrostro, Jason D.; Giresi, Paul G.; Zaba, Lisa C.; Chang, Howard Y.; Greenleaf, William J.



Chromatin tandem affinity purification sequencing  

PubMed Central

Chromatin immunoprecipitation coupled with ultra-high-throughput sequencing (ChIP-seq) is a widely used method for mapping the interactions of proteins with DNA. However, the requirements for ChIP-grade antibodies impede wider application of this method, and variations in results can be high owing to differences in affinity and cross-reactivity of antibodies. Therefore, we developed chromatin tandem affinity purification (ChTAP) as an effective alternative to ChIP. Through the use of affinity tags and reagents that are identical for all proteins investigated, ChTAP enables one to directly compare the binding between different transcription factors and to directly assess the background in control experiments. Thus, ChTAP -seq can be used to rapidly map the genome-wide binding of multiple DNA -binding proteins in a wide range of cell types. ChTAP can be completed in 3–4 d, starting from cross-linking of chromatin to purification of ChIP DNA.

Soleimani, Vahab D; Palidwor, Gareth A; Ramachandran, Parameswaran; Perkins, Theodore J; Rudnicki, Michael A



Cyclin E uses Cdc6 as a chromatin-associated receptor required for DNA replication.  


Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specifically and saturably to chromatin in three phases. In the first phase, the origin recognition complex and Cdc6 prereplication proteins, but not the minichromosome maintenance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin E--Cdk2 to DNA. We find that cyclin E binds the NH(2)-terminal region of Cdc6 containing Cy--Arg-X-Leu (RXL) motifs. Cyclin E proteins with mutated substrate selection (Met-Arg-Ala-Ile-Leu; MRAIL) motifs fail to bind Cdc6, fail to compete with endogenous cyclin E--Cdk2 for chromatin binding, and fail to rescue replication in cyclin E--depleted extracts. Cdc6 proteins with mutations in the three consensus RXL motifs are quantitatively deficient for cyclin E binding and for rescuing replication in Cdc6-depleted extracts. Thus, the cyclin E--Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin E--Cdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin. Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro. These three phases of cyclin E association with chromatin may facilitate the diverse activities of cyclin E--Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis. PMID:11257126

Furstenthal, L; Kaiser, B K; Swanson, C; Jackson, P K



Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3?-OH Single-strand DNA Breaks*  

PubMed Central

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD?/? cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3?-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3?-OH ends in single-strand rather than double-strand DNA nicks/breaks.

Iglesias-Guimarais, Victoria; Gil-Guinon, Estel; Sanchez-Osuna, Maria; Casanelles, Elisenda; Garcia-Belinchon, Merce; Comella, Joan X.; Yuste, Victor J.



Understanding Condensation  

NSDL National Science Digital Library

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

Hartman, Monica



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



Noncoding RNA localisation mechanisms in chromatin regulation  

PubMed Central

An important challenge in biology has been to understand how cell-type-specific expression programs are orchestrated through regulated access to chromatin. Knowledge of the interaction between noncoding RNAs (ncRNAs) and chromatin regulators has the potential to help answer such questions, but how ncRNAs target chromatin regulators to specific sites in the genome is not well understood. Recently, Jeon and Lee proposed that DNA-binding proteins act as a bridge between ncRNAs and their target sites in chromatin. In this minireview, we examine their findings and place them in the wider context of how chromatin regulator-RNA complexes are targeted to specific sites in chromatin.



Genome-Wide Views of Chromatin Structure  

PubMed Central

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

Rando, Oliver J.; Chang, Howard Y.



HDACi--targets beyond chromatin.  


Histone deacetylases (HDACs) play an important role in gene regulation. Inhibitors of HDACs (HDACi) are novel anti-cancer drugs, which induce histone (hyper-) acetylation and counteract aberrant gene repression. On the other hand, HDACi treatment can also result in decreased gene expression, and targeting HDACs affects more than chromatin. Recently, HDACi were shown to evoke non-histone protein acetylation, which can alter signaling networks relevant for tumorgenesis. Furthermore, HDACi can promote the degradation of (proto-) oncoproteins. Here, we summarize these findings and discuss how these substances could be beneficial for the treatment and prevention of human ailments, such as cancer and unbalanced immune functions. PMID:19342155

Buchwald, Marc; Krämer, Oliver H; Heinzel, Thorsten



Sperm chromatin integrity of bucks transgenic for the WAP bGH gene.  


The aim of the study was to compare sperm chromatin structure of transgenic and non-transgenic rabbits. In addition, the effect of chromatin structure on semen fertility was determined. Twenty male rabbits transgenic (TG) for WAP bGH gene (Edison Biotechnology Institute Ohio University, USA) and nine non-transgenic (NTG) males were used. Both TG and NTG rabbits were 13-18 months old. Semen was collected at 1-week intervals and 3-7 ejaculates from each rabbit were examined in total. Sperm chromatin abnormalities were measured flow cytometrically according to the Sperm Chromatin Structure Assay method: after chromatin denaturation by low pH, sperm cells were stained with metachromatic fluorochrome acridine orange. Spermatozoa with abnormal chromatin structure and, subsequently, higher degree of denaturation, showed a shift in red fluorescence. Two different methods of semen fertility estimation were used: (1) for TG rabbits, AI of superovulated does and calculation of percentages of fertilised eggs and embryos developing in vitro to the blastocyst stage; (2) for NTG rabbits, AI of non-stimulated does and calculation of percentages of pregnant does and mean litter sizes. The mean value of COMPalpha(t) was 3.71 for TG rabbits and 2.89 for NTG rabbits (no significant difference, t-test). The mean values of S.D.alpha(t) for the TG and NTG rabbits were 10.94 and 10.40 (no significant difference, t-test), respectively. There were no significant correlations between sperm chromatin structure of TG males and the percentages of fertilised eggs or embryos developing to the blastocyst stage. A statistically significant correlation (-0.68, P<0.05) was found between S.D.alpha(t) of NTG males and percentages of pregnant does. The results showed chromatin stability was not different for sperm obtained from TG versus NTG bucks. The presence of WAP bGH gene construct in the genome of transgenic rabbits did not cause any spermatogenesis process disturbances leading to the production of spermatozoa with damaged chromatin structure. This suggests that the mere presence of the introduced gene construct does not lead to any abnormalities in DNA and chromatin proteins interaction. The possible chromatin damages in transgenic animals should be attributed to the activity of the introduced gene. The relationships between chromatin structure and fertility are only significant for sperm from NTG bucks. PMID:11078972

Gogol, P; Bochenek, M; Smorag, Z



Sperm chromatin structure correlates with spontaneous abortion and multiple pregnancy rates in assisted reproduction  

Microsoft Academic Search

The objective of this study was to determine if a relationship exists between sperm parameters, measured by sperm chromatin structure assay (SCSA), and spontaneous abortion and multiple births in couples undergoing assisted reproduction treatment. Retrospective analysis of infertility treatment outcomes and occurrence of spontaneous abortion and multiple births was conducted in 233 couples who underwent treatment by intracytoplasmic sperm injection

C. Kennedy; P. Ahlering; H. Rodriguez; S. Levy; P. Sutovsky



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

Microsoft Academic Search

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

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



Chromatin domain boundary element search tool for Drosophila  

PubMed Central

Chromatin domain boundary elements prevent inappropriate interaction between distant or closely spaced regulatory elements and restrict enhancers and silencers to correct target promoters. In spite of having such a general role and expected frequent occurrence genome wide, there is no DNA sequence analysis based tool to identify boundary elements. Here, we report chromatin domain Boundary Element Search Tool (cdBEST), to identify boundary elements. cdBEST uses known recognition sequences of boundary interacting proteins and looks for ‘motif clusters’. Using cdBEST, we identified boundary sequences across 12 Drosophila species. Of the 4576 boundary sequences identified in Drosophila melanogaster genome, >170 sequences are repetitive in nature and have sequence homology to transposable elements. Analysis of such sequences across 12 Drosophila genomes showed that the occurrence of repetitive sequences in the context of boundaries is a common feature of drosophilids. We use a variety of genome organization criteria and also experimental test on a subset of the cdBEST boundaries in an enhancer-blocking assay and show that 80% of them indeed function as boundaries in vivo. These observations highlight the role of cdBEST in better understanding of chromatin domain boundaries in Drosophila and setting the stage for comparative analysis of boundaries across closely related species.

Srinivasan, Arumugam; Mishra, Rakesh K.



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.

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



Chromatin structure analysis of the mouse Xist locus  

PubMed Central

The Xist gene is expressed exclusively from the inactive X chromosome and plays a central role in regulating X chromosome inactivation. Here we describe experiments aimed at defining the extent of the active chromatin domain of the expressed Xist allele. By using an allele-specific general DNaseI sensitivity assay we show that there is preferential digestion of the expressed allele at sites within the transcribed locus but not in flanking sites located up to 70 kb 5?. A putative proximal boundary for the Xist domain is located within 10 kb upstream of promoter P1. Chromatin in the expressed domain was found to be acetylated at H4 in XX somatic cells but also in XY cells, where Xist is never expressed. A single clear exception to this was the Xist promoter, which is acetylated only in XX cells. These observations concur with the view that H4 acetylation may not be a general marker of active chromatin domains and further support data implicating local promoter acetylation as being of primary functional significance in vivo.

McCabe, Veronica; Formstone, Emma J.; O'Neill, Laura P.; Turner, Bryan M.; Brockdorff, Neil



Tissue-Specific Regulation of Chromatin Insulator Function  

PubMed Central

Chromatin insulators organize the genome into distinct transcriptional domains and contribute to cell type–specific chromatin organization. However, factors regulating tissue-specific insulator function have not yet been discovered. Here we identify the RNA recognition motif-containing protein Shep as a direct interactor of two individual components of the gypsy insulator complex in Drosophila. Mutation of shep improves gypsy-dependent enhancer blocking, indicating a role as a negative regulator of insulator activity. Unlike ubiquitously expressed core gypsy insulator proteins, Shep is highly expressed in the central nervous system (CNS) with lower expression in other tissues. We developed a novel, quantitative tissue-specific barrier assay to demonstrate that Shep functions as a negative regulator of insulator activity in the CNS but not in muscle tissue. Additionally, mutation of shep alters insulator complex nuclear localization in the CNS but has no effect in other tissues. Consistent with negative regulatory activity, ChIP–seq analysis of Shep in a CNS-derived cell line indicates substantial genome-wide colocalization with a single gypsy insulator component but limited overlap with intact insulator complexes. Taken together, these data reveal a novel, tissue-specific mode of regulation of a chromatin insulator.

Matzat, Leah H.; Dale, Ryan K.; Moshkovich, Nellie; Lei, Elissa P.



A Conserved Chromatin Architecture Marks and Maintains the Restricted Germ Cell Lineage in Worms and Flies  

PubMed Central

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

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



Nuclease sensitivity of active chromatin.  


The active regions of chicken erythrocyte nuclei were labeled using the standard DNase I directed nick translation reaction. These nuclei were then used to study the characteristics and, in particular, the nuclease sensitivity of active genes. Although DNase I specifically attacks active genes, micrococcal nuclease solubilizes these regions to about the same degree as the total DNA. On the other hand micrococcal nuclease does selectively cut the internucleosomal regions of active genes resulting in the appearance of mononucleosomal fraction which is enriched in active gene DNA. A small percentage of the active chromatin is also released from the nucleus by low speed centrifugation following micrococcal nuclease treatment. The factors which make active genes sensitive to DNase I were shown to reside on individual nucleosomes from these regions. This was established by showing that isolated active mononucleosomes were preferentially sensitive to DNase I digestion. Although the high mobility group proteins are essential for the maintenance of DNase I sensitivity in active regions, these proteins are not necessary for the formation of the conformation which makes these genes preferentially accessible to micrococcal nuclease. The techniques employed in this paper enable one to study the chromatin structure of the entire population of actively expressed genes. Previous studies have elucidated the structure of a few special highly prevalent genes such as ovalbumin and hemoglobin. The results of this paper show that this special conformation is a general feature of all active genes irregardless of the extent of expression. PMID:6258137

Gazit, B; Cedar, H



A feel for the template: zinc finger protein transcription factors and chromatin.  


Transcription factors and chromatin collaborate in bringing the eukaryotic genome to life. An important, and poorly understood, aspect of this collaboration involves targeting the regulators to correct binding sites in vivo. An implicit and insufficiently tested assumption in the field has been that chromatin simply obstructs most sites and leaves only a few functionally relevant ones accessible. The major class of transcription factors in all metazoa, zinc finger proteins (ZFPs), can bind to chromatin in vitro (as clearly shown for Spl, GATA-1 and -4, and the nuclear hormone receptors, for example). Data on the accessibility of DNA within heterochromatin to nonhistone regulators (E.A. Sekinger and D.S. Gross. 2001. Mol. Cell 105: 403-414; C. Jolly et al. 2002. J. Cell. Biol. 156: 775-781) and the ability of the basal transcription machinery to reside within highly condensed chromatin (most recently, R. Christova and T. Oelgeschlaeger. 2002. Nat. Cell Biol. 4: 79-82) further weaken the argument that chromatin acts as an across-the-board deterrent to ZFP binding. These proteins, however, do not bind promiscuously in vivo, and recent data on human cells (C.E. Horak et al. 2002. Proc. Natl. Acad. Sci. U.S.A. 99: 2924-2929) confirm earlier data on budding yeast (B. Ren et al. 2000. Science (Washington, D.C.), 290: 2306-2309) that primary DNA sequence, i.e., density of binding sites per unit DNA length, is not the primary determinant of where a ZFP transcription factor will bind in vivo. This article reviews these data and uses ZFP transcription factors as a model system to compare in vitro binding to chromatin by transcription factors with their in vivo behavior in gene regulation. DNA binding domain structure, nonrandom nucleoprotein organization of chromatin at target promoters, and cooperativity of regulator action may all contribute to target site selection in vivo. PMID:12123285

Urnov, Fyodor D



Anti-chromatin and anti-histone antibodies in Egyptian patients with systemic lupus erythematosus.  


There has been a renewed interest in anti-chromatin and anti-histone antibodies in the last few years. To assess the prevalence of anti-chromatin and anti-histone antibodies in patients with systematic lupus erythematosus (SLE) and to correlate serum levels of these antibodies with clinical features of the disease, the presence of anti-chromatin and anti-histone antibodies in 38 patients with SLE was investigated by an enzyme-linked immunosorbent assay (ELISA). To determine the specificity of these antibodies, 15 patients with rheumatoid arthritis, 15 patients with systemic sclerosis, and 15 normal controls were also tested. Sensitivity of anti-chromatin antibodies in SLE patients was 89.5% and specificity was 80.0%, while sensitivity of anti-histone antibodies was 92.1% and specificity was 82.2%. Significant associations were found between the levels of anti-chromatin antibodies and arthritis, malar rash, oral ulcer, pulmonary affection (P < 0.05) also, lupus nephritis (P < 0.01), and disease activity score as measured by SLE disease activity index (SLEDAI; P < 0.001). Significant association was found between anti-histone antibodies and fatigue (P < 0.05). The incidence of positive anti-chromatin and anti-histone antibodies was significantly higher than that of anti-dsDNA antibodies in early stage of the disease. We conclude that anti-chromatin and anti-histone antibodies are both sensitive and specific for SLE and could be a useful addition to the laboratory tests that can help in the diagnosis of SLE. Anti-chromatin antibodies seem to be a promising marker useful in early diagnosis and assessment of disease activity in SLE patients especially in patients who are negative for anti-dsDNA antibodies. PMID:19291351

Shabana, Adel A; El-Ghawet, Atef E; Machaly, Shereen A; Abu Hashim, Ekbal M; El-Kady, Basma A; Shaat, Reham



Chromatin patterns associated with lung adenocarcinoma progression  

PubMed Central

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

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



Transcription of Walker 256 Carcinosarcoma Chromatin1  

Microsoft Academic Search

SUMMARY The in vitro transcriptions from chromatins of Walker256 carcinosarcoma, rat mammary gland, and rat liver were compared with respect to template activities and reciprocal double saturation hybridization of the transcribed RNA's. Walkertumor chromatin was found to have the least template activity in RNA synthesis in vitro, and the synthesized RNA, when annealed with DNA, also showed the least saturation

Nina C. Kostraba; T. Y. Wang



[Transcription-active portions of the chromatin].  


A review of data on transcriptionally active chromatin obtained during the last 5--7 years. The role of hypersensitive regions, of non-histone proteins, of DNA undermethylation and the participation of Hl histone in the organization of active chromatin are considered. The role of some of these processes in the mechanisms determining cell differentiation during ontogenesis is discussed. PMID:6194489

Karavanov, A A



Regulation of chromatin by histone modifications  

Microsoft Academic Search

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

Andrew J Bannister; Tony Kouzarides



An Oestrogen Receptor ?-bound Human Chromatin Interactome  

PubMed Central

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

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



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



TOPICAL REVIEW: The physics of chromatin  

NASA Astrophysics Data System (ADS)

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

Schiessel, Helmut



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

PubMed Central

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

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



Chromatin modifications associated with diabetes.  


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

Keating, Samuel T; El-Osta, Assam



The role of chromatin insulators in nuclear architecture and genome function.  


Eukaryotic genomes are intricately arranged into highly organized yet dynamic structures that underlie patterns of gene expression and cellular identity. The recent adaptation of novel genomic strategies for assaying nuclear architecture has significantly extended and accelerated our ability to query the nature of genome organization and the players involved. In particular, recent explorations of physical arrangements and chromatin landscapes in higher eukaryotes have demonstrated that chromatin insulators, which mediate functional interactions between regulatory elements, appear to play an important role in these processes. Here we reflect on current findings and our rapidly expanding understanding of insulators and their role in nuclear architecture and genome function. PMID:23298659

Van Bortle, Kevin; Corces, Victor G



The role of chromatin insulators in nuclear architecture and genome function  

PubMed Central

Eukaryotic genomes are intricately arranged into highly organized yet dynamic structures that underlie patterns of gene expression and cellular identity. The recent adaptation of novel genomic strategies for assaying nuclear architecture has significantly extended and accelerated our ability to query the nature of genome organization and the players involved. In particular, recent explorations of physical arrangements and chromatin landscapes in higher eukaryotes have demonstrated that chromatin insulators, which mediate functional interactions between regulatory elements, appear to play an important role in these processes. Here we reflect on current findings and our rapidly expanding understanding of insulators and their role in nuclear architecture and genome function.

Van Bortle, Kevin; Corces, Victor G.



Optimal use of tandem biotin and V5 tags in ChIP assays  

Microsoft Academic Search

BACKGROUND: Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition,

Katarzyna E Kolodziej; Farzin Pourfarzad; Ernie de Boer; Sanja Krpic; Frank Grosveld; John Strouboulis



The quinternary chromatin-DNA structure. Three-dimensional reconstruction and functional significance.  


Nuclear DNA-space images from Feulgen-stained HeLa cells synchronized at 1, 3, 5, 8, 12, 15, and 18 h following mitosis are digitized and their densitometric-geometric patterns are analyzed by means of a Quantimet 720-D image analyzer on line with a PDP11/40 computer. Frequency distributions of picture point optical densities for the phases and subphases as seen in nuclear images show that DNA packing changes are evident by means of ordinary optical microscopy. Radii of gyration of the images, and optical density profiles and distributions for several squashes of similar cells reveal that in particular instances chromatin DNA is distributed mostly towards the periphery, and usually with high circular isotropy. Cross power spectra of individual scan lines suggest that existence of higher order "quinternary" periodic structure for chromatin that modulates during the cell cycle. Three-dimensional reconstruction 2- micrometer sections of intact, Feulgen-stained mammalian tumor tissue show stainable material only toward the nuclear perimeter and not in the center (compatible with the evidence that initial thymidine incorporation in HeLa cells is generally at the nuclear border). Densitometric properties of reconstructed interphase chromatin-DNA bodies are highly coupled with similar properties of the whole nucleus, showing that a more condensed nucleus is always accompanied by a more condensed interphase chromatin DNA. The effect of micrococcal nuclease digestion on the digitized nuclear images is also presented. All the above data are then discussed in terms of a quinternary chromatin-DNA structure and its modulation during the cell cycle. PMID:6163547

Kendall, F M; Beltrame, F; Zietz, S; Belmont, A; Nicolini, C



Remodelling chromatin to shape development of plants.  


Establishment and dynamic regulation of a higher order chromatin structure is an essential component of development. Chromatin remodelling complexes such as the SWI2/SNF2 family of ATP-dependent chromatin remodellers can alter chromatin architecture by changing nucleosome positioning or substituting histones with histone variants. These remodellers often act in concert with chromatin modifiers such as the polycomb group proteins which confer repressive states through modification of histone tails. These mechanisms are highly conserved across the eukaryotic kingdom although in plants, owing to the maintenance of dedifferentiated cell states that allow for post-embyronic changes in development, strict control of chromatin remodelling is even more paramount. Recent and ongoing studies in the model plant Arabidopsis thaliana have found that while the major families of the SWI2/SNF2 ATPase chromatin remodellers are represented, a number of redundancies and divergent functions have emerged that show a break from the roles of their metazoan counterparts. This review focusses on the SNF2 and CHD families of ATP-dependent remodellers and their roles in plant development. PMID:24270012

Gentry, Matthew; Hennig, Lars



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



Chromatin remodelling initiation during human spermiogenesis.  


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

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



Chromatin remodelling initiation during human spermiogenesis  

PubMed Central

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

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



Histone variant H3.3 maintains a decondensed chromatin state essential for mouse preimplantation development.  


Histone variants can replace canonical histones in the nucleosome and modify chromatin structure and gene expression. The histone variant H3.3 preferentially associates with active chromatin and has been implicated in the regulation of a diverse range of developmental processes. However, the mechanisms by which H3.3 may regulate gene activity are unclear and gene duplication has hampered an analysis of H3.3 function in mouse. Here, we report that the specific knockdown of H3.3 in fertilized mouse zygotes leads to developmental arrest at the morula stage. This phenotype can be rescued by exogenous H3.3 but not by canonical H3.1 mRNA. Loss of H3.3 leads to over-condensation and mis-segregation of chromosomes as early as the two-cell stage, with corresponding high levels of aneuploidy, but does not appear to affect zygotic gene activation at the two-cell stage or lineage gene transcription at the morula stage. H3.3-deficient embryos have significantly reduced levels of markers of open chromatin, such as H3K36me2 and H4K16Ac. Importantly, a mutation in H3.3K36 that disrupts H3K36 methylation (H3.3K36R) does not rescue the H3.3 knockdown (KD) phenotype. In addition, H3.3 KD embryos have increased incorporation of linker H1. Knockdown of Mof (Kat8), an acetyltransferase specific for H4K16, similarly leads to excessive H1 incorporation. Remarkably, pan-H1 RNA interference (RNAi) partially rescues the chromosome condensation of H3.3 KD embryos and allows development to the blastocyst stage. These results reveal that H3.3 mediates a balance between open and condensed chromatin that is crucial for the fidelity of chromosome segregation during early mouse development. PMID:23903189

Lin, Chih-Jen; Conti, Marco; Ramalho-Santos, Miguel



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

PubMed Central

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

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



Different chromatin fractions of tomato (Solanum lycopersicum L.) and related species.  


Conventional chromosome staining has suggested that more than 75% of the tomato chromosomes are constituted by heterochromatin. In order to determine whether more deeply stained proximal regions are classic heterochromatin, the distributions of C-bands and chromomycin A(3) (CMA) bands, and the prophase condensation patterns, were analysed in tomato. In this and most other species of the tomato clade, the 5S and 45S rDNA sites were also localised. In tomato, CMA banding was similar to C-banding. After conventional staining, all species displayed large condensed heteropycnotic regions that did not correspond to C-bands or CMA bands. Analyses of the CMA banded karyotypes revealed a low heterochromatin content. Around 12-17% of the chromatin of tomato was CMA(+) and 1/4 to 1/5 of this heterochromatin corresponded to 45S rDNA. In other species, the CMA(+) heterochromatin showed extensive variation (8-35%), but was never near the values found in the literature for tomato. These data suggest the existence of three principal fractions of chromatin in tomato and related species: the late condensed euchromatin corresponding to the terminal regions of the chromosomes, the precocious condensed euchromatin that occupies the major part of the chromosomes and the constitutive heterochromatin that represents those regions revealed by C-bands. PMID:19646883

Brasileiro-Vidal, A C; Melo-Oliveira, M B; Carvalheira, G M G; Guerra, M



Chromatin: receiver and quarterback for cellular signals.  


Signal transduction pathways converge upon sequence-specific DNA binding factors to reprogram gene expression. Transcription factors, in turn, team up with chromatin modifying activities. However, chromatin is not simply an endpoint for signaling pathways. Histone modifications relay signals to other proteins to trigger more immediate responses than can be achieved through altered gene transcription, which might be especially important to time-urgent processes such as the execution of cell-cycle check points, chromosome segregation, or exit from mitosis. In addition, histone-modifying enzymes often have multiple nonhistone substrates, and coordination of activity toward different targets might direct signals both to and from chromatin. PMID:23375745

Johnson, David G; Dent, Sharon Y R



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.

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



Extensive Variation in Chromatin States Across Humans  

PubMed Central

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

Kasowski, Maya; Kyriazopoulou-Panagiotopoulou, Sofia; Grubert, Fabian; Zaugg, Judith B.; Kundaje, Anshul; Liu, Yuling; Boyle, Alan P.; Zhang, Qiangfeng Cliff; Zakharia, Fouad; Spacek, Damek V.; Li, Jingjing; Xie, Dan; Olarerin-George, Anthony; Steinmetz, Lars M.; Hogenesch, John B.; Kellis, Manolis; Batzoglou, Serafim; Snyder, Michael



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.

Papamichos-Chronakis, Manolis; Peterson, Craig L.



Chromatin Evolution and Molecular Drive in Speciation  

PubMed Central

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

Sawamura, Kyoichi



Nucleosome structure in chromatin from heated cells  

SciTech Connect

The effect of hyperthermia (40 to 80/sup 0/C) on the nucleosome structure of mammalian chromatin was determined using the enzyme micrococcal nuclease. At equivalent fractional DNA digestion it was found that neither the size of DNA nor the total fraction of cellular DNA associated with nucleosome structure is altered by heat exposure up to 48/sup 0/C for 30 min. It is proposed that this heat-induced reduction in the accessibility to nuclease attack of DNA in chromatin from heated cells is due to the increased protein mass associated with chromatin.

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



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.

Prinjha, Rab; Tarakhovsky, Alexander



Optimizing process vacuum condensers  

SciTech Connect

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

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



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

PubMed Central

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

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



The distribution of the DEK protein in mammalian chromatin  

Microsoft Academic Search

DEK is an abundant and ubiquitous chromatin protein. Here we investigate whether DEK is regularly distributed in the chromatin of human HeLa cells. We show that DEK appears to be excluded from the heterochromatic compartment. However, DEK seems to colocalize with a subfraction of chromatin bearing acetylated histone H4. We examined certain DNA sequences in specifically immunoprecipitated chromatin for four

Hong-gang Hu; Ingo Scholten; Claudia Gruss; Rolf Knippers



Targeting of the Yeast Ty5 Retrotransposon to Silent Chromatin Is Mediated by Interactions between Integrase and Sir4p†  

PubMed Central

The Ty5 retrotransposons of Saccharomyces cerevisiae integrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR and HML). We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP). The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed. As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin. This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity.

Xie, Weiwu; Gai, Xiaowu; Zhu, Yunxia; Zappulla, David C.; Sternglanz, Rolf; Voytas, Daniel F.



On the chromatin structure of eukaryotic telomeres  

PubMed Central

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

Vaquero-Sedas, Maria I



The Current State of Chromatin Immunoprecipitation  

Microsoft Academic Search

The biological significance of interactions of nuclear proteins with DNA in the context of gene expression, cell differentiation,\\u000a or disease has immensely been enhanced by the advent of chromatin immunoprecipitation (ChIP). ChIP is a technique whereby\\u000a a protein of interest is selectively immunoprecipitated from a chromatin preparation to determine the DNA sequences associated\\u000a with it. ChIP has been widely used

Philippe Collas



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


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


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



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.

Rudra, Soumya; Skibbens, Robert V.



Histone H1 Is a Specific Repressor of Core Histone Acetylation in Chromatin  

PubMed Central

Although a link between histone acetylation and transcription has been established, it is not clear how acetylases function in the nucleus of the cell and how they access their targets in a chromatin fiber containing H1 and folded into a highly condensed structure. Here we show that the histone acetyltransferase (HAT) p300/CBP-associated factor (PCAF), either alone or in a nuclear complex, can readily acetylate oligonucleosomal substrates. The linker histones, H1 and H5, specifically inhibit the acetylation of mono- and oligonucleosomes and not that of free histones or histone-DNA mixtures. We demonstrate that the inhibition is due mainly to steric hindrance of H3 by the tails of linker histones and not to condensation of the chromatin fiber. Cellular PCAF, which is complexed with accessory proteins in a multiprotein complex, can overcome the linker histone repression. We suggest that linker histones hinder access of PCAF, and perhaps other HATs, to their target acetylation sites and that perturbation of the linker histone organization in chromatin is a prerequisite for efficient acetylation of the histone tails in nucleosomes.

Herrera, Julio E.; West, Katherine L.; Schiltz, R. Louis; Nakatani, Yoshihiro; Bustin, Michael



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.

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.

Redi, Carlo Alberto; Zuccotti, Maurizio



Kinase-mediated changes in nucleosome conformation trigger chromatin decondensation via poly(ADP-ribosyl)ation.  


Dynamically controlled posttranslational modifications of nucleosomal histones alter chromatin condensation to regulate transcriptional activation. We report that a nuclear tandem kinase, JIL-1, controls gene expression by activating poly(ADP-ribose) polymerase-1 (PARP-1). JIL-1 phosphorylates the C terminus of the H2Av histone variant, which stimulates PARP-1 enzymatic activity in the surrounding chromatin, leading to further modification of histones and chromatin loosening. The H2Av nucleosome has a higher surface representation of PARP-1 binding patch, consisting of H3 and H4 epitopes. Phosphorylation of H2Av by JIL-1 restructures this surface patch, leading to activation of PARP-1. Exposure of Val61 and Leu23 of the H4 histone is critical for PARP-1 binding on nucleosome and PARP-1 activation following H2Av phosphorylation. We propose that chromatin loosening and associated initiation of gene expression is activated by phosphorylation of H2Av in a nucleosome positioned in promoter regions of PARP-1-dependent genes. PMID:24508391

Thomas, Colin J; Kotova, Elena; Andrake, Mark; Adolf-Bryfogle, Jared; Glaser, Robert; Regnard, Catherine; Tulin, Alexei V




Microsoft Academic Search

The air-cooled steam condenser and sub-cooler developed for use with the ; PL series of natural circulation boiling water nuclear power plants are designed ; to condense 13,000 lb\\/hr at 10% moisture at 15 in. Hg and to subcool the ; condensate to 100 deg F at an ambient temperature of 60 deg F. The design heat ; load at

V. R. Mardoc; R. G. Young



Efficient condensate removal  

Microsoft Academic Search

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




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



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

Microsoft Academic Search

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

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



Center for Barr body condensation on the proximal part of the human Xq: a hypothesis  

Microsoft Academic Search

The following hypothesis is put forward: X chromatin in man condenses around a center which is situated on Xq at a short distance from the centromere. The hypothesis is based on, and explains, two classes of observations. (1) Abnormal X chromosomes that have the assumed center in duplicate form bipartite Barr bodies in part of the cells. The frequency of

Eeva Therman; Gloria E. Sarto; Klaus Patau



Nucleosome acetylation sequencing to study the establishment of chromatin acetylation.  


The establishment of posttranslational chromatin modifications is a major mechanism for regulating how genomic DNA is utilized. However, current in vitro chromatin assays do not monitor histone modifications at individual nucleosomes. Here we describe a strategy, nucleosome acetylation sequencing, that allows us to read the amount of modification at each nucleosome. In this approach, a bead-bound trinucleosome substrate is enzymatically acetylated with radiolabeled acetyl CoA by the SAGA complex from Saccharomyces cerevisae. The product is digested by restriction enzymes that cut at unique sites between the nucleosomes and then counted to quantify the extent of acetylation at each nucleosomal site. We find that we can sensitively, specifically, and reproducibly follow enzyme-mediated nucleosome acetylation. Applying this strategy, when acetylation proceeds extensively, its distribution across nucleosomes is relatively uniform. However, when substrates are used that contain nucleosomes mutated at the major sites of SAGA-mediated acetylation, or that are studied under initial rate conditions, changes in the acetylation distribution can be observed. Nucleosome acetylation sequencing should be applicable to analyzing a wide range of modifications. Additionally, because our trinucleosomes synthesis strategy is highly modular and efficient, it can be used to generate nucleosomal systems in which nucleosome composition differs across the array. PMID:24769374

Mittal, Chitvan; Blacketer, Melissa J; Shogren-Knaak, Michael A



Sequential changes in macrostructural state and RNA-synthesizing activity of nucleolar and extranucleolar chromatin of rat liver cells during induction of DNA synthesis  

SciTech Connect

The dynamics of the structural changes in the RNA-synthesizing activity of rat liver cell chromatin was studied under conditions of sharp variations in the rate of protein synthesis. Inhibition of protein synthesis by the administration to animals of a single dose of cycloheximide (0.3 mg per 100 g weight) increased the total degree of condensation of the chromatin. Against this background the sequential activation of RNA polymerases I and II correlates with decondensation of the chromatin. In the 6th-12th h after the injection of cycloheximide a chromatin fraction enriched with RNA polymerase I and a fraction rich in RNA polymerase II were isolated from liver cell nuclei.

Shevchenko, N.A.; Boikov, P.Ya.; Todorov, I.N.



Effects of acrylamide on sperm parameters, chromatin quality, and the level of blood testosterone in mice  

PubMed Central

Background: Acrylamide (AA) is an important industrial chemical primarily. AA is also found in carbohydrate-rich foods that are prepared at high temperatures, such as French fries and potato chips. It is demonstrated that AA is a carcinogen and reproductive toxin and has ability to induce sperm damage. Objective: The aim of this study was to observe the effects of AA on sperm parameters and evaluation of sperm chromatin quality and testosterone hormone in mice. Materials and Methods: Totally, 16 adult male mice were divided into two groups. Mice of group A fed on basal diet; group B received basal diet and AA (10 mg/kg, water solution) for 35 days. The right cauda epididymis was incised and then placed in Ham’s F10 culture media at 37oC for 15 min. Released spermatozoa were used to analyze count, motility, morphology and viability. To determine the sperm DNA integrity and chromatin condensation, the cytochemical techniques including Aniline blue, Acridine orange and Chromomycin A3 staining were used. Results: AA-treated mice had poor parameters in comparison with control animals. In sperm chromatin assessments, except TB (p=0.16), significant differences were found in all of the tests between two groups. It was also seen a significant decrease in concentration of blood testosterone in AA-treated animals when compared to controls (p<0.001). Conclusion: According to our results, AA can affect sperm parameters as well as sperm chromatin condensation and DNA integrity in mice. These abnormalities may be related to the reduction in blood testosterone.

Pourentezari, Majid; Talebi, Alireza; Abbasi, Abulghasem; Khalili, Mohammad Ali; Mangoli, Esmat; Anvari, Morteza



Chromatin domains and regulation of transcription.  


Compartmentalization and compaction of DNA in the nucleus is the characteristic feature of eukaryotic cells. A fully extended DNA molecule has to be compacted 100,000 times to fit within the nucleus. At the same time it is critical that various DNA regions remain accessible for interaction with regulatory factors and transcription/replication factories. This puzzle is solved at the level of DNA packaging in chromatin that occurs in several steps: rolling of DNA onto nucleosomes, compaction of nucleosome fiber with formation of the so-called 30 nm fiber, and folding of the latter into the giant (50-200 kbp) loops, fixed onto the protein skeleton, the nuclear matrix. The general assumption is that DNA folding in the cell nucleus cannot be uniform. It has been known for a long time that a transcriptionally active chromatin fraction is more sensitive to nucleases; this was interpreted as evidence for the less tight compaction of this fraction. In this review we summarize the latest results on structure of transcriptionally active chromatin and the mechanisms of transcriptional regulation in the context of chromatin dynamics. In particular the significance of histone modifications and the mechanisms controlling dynamics of chromatin domains are discussed as well as the significance of spatial organization of the genome for functioning of distant regulatory elements. PMID:17466329

Razin, Sergey V; Iarovaia, Olga V; Sjakste, Nikolajs; Sjakste, Tatiana; Bagdoniene, Lida; Rynditch, Alla V; Eivazova, Elvira R; Lipinski, Marc; Vassetzky, Yegor S



PROTOCOLS: Chromatin Immunoprecipitation from Arabidopsis Tissues  

PubMed Central

The ability of proteins to associate with genomic DNA in the context of chromatin is critical for many nuclear processes including transcription, replication, recombination, and DNA repair. Chromatin immunoprecipication (ChIP) is a practical and useful technique for characterizing protein / DNA association in vivo. The procedure generally includes six steps: (1) crosslinking the protein to the DNA; (2) isolating the chromatin; (3) chromatin fragmentation; (4) imunoprecipitation with antibodies against the protein of interest; (5) DNA recovery; and (6) PCR identification of factor associated DNA sequences. In this protocol, we describe guidelines, experimental setup, and conditions for ChIP in intact Arabidopsis tissues. This protocol has been used to study association of histone modifications, of chromatin remodeling ATPases, as well as of sequence-specific transcription factors with the genomic DNA in various Arabidopsis thaliana tissues. The protocol described focuses on ChIP-qPCR, but can readily be adapted for use in ChIP-chip or ChIP-seq experiments. The entire procedure can be completed within 3 days.

Yamaguchi, Nobutoshi; Winter, Cara M.; Wu, Miin-Feng; Kwon, Chang Seob; William, Dilusha A.; Wagner, Doris



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



Quantitative Analysis of Chromosome Condensation in Fission Yeast  

PubMed Central

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

Petrova, Boryana; Dehler, Sascha; Kruitwagen, Tom; Heriche, Jean-Karim; Miura, Kota



Farnesyl diphosphate synthase assay.  


Farnesyl diphosphate synthase (FPS) catalyzes the sequential head-to-tail condensation of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) and geranyl diphosphate (GPP, C10) to produce farnesyl diphosphate (FPP, C15). This short-chain prenyl diphosphate constitutes a key branch-point of the isoprenoid biosynthetic pathway from which a variety of bioactive isoprenoids that are vital for normal plant growth and survival are produced. Here we describe a protocol to obtain highly purified preparations of recombinant FPS and a radiochemical assay method for measuring FPS activity in purified enzyme preparations and plant tissue extracts. PMID:24777789

Arró, Montserrat; Manzano, David; Ferrer, Albert



4D chromatin dynamics in cycling cells  

PubMed Central

This live cell study of chromatin dynamics in four dimensions (space and time) in cycling human cells provides direct evidence for three hypotheses first proposed by Theodor Boveri in seminal studies of fixed blastomeres from Parascaris equorum embryos: (I) Chromosome territory (CT) arrangements are stably maintained during interphase. (II) Chromosome proximity patterns change profoundly during prometaphase. (III) Similar CT proximity patterns in pairs of daughter nuclei reflect symmetrical chromosomal movements during anaphase and telophase, but differ substantially from the arrangement in mother cell nucleus. Hypothesis I could be confirmed for the majority of interphase cells. A minority, however, showed complex, rotational movements of CT assemblies with large-scale changes of CT proximity patterns, while radial nuclear arrangements were maintained. A new model of chromatin dynamics is proposed. It suggests that long-range DNA-DNA interactions in cell nuclei may depend on a combination of rotational CT movements and locally constrained chromatin movements.

Strickfaden, Hilmar; Zunhammer, Andreas; van Koningsbruggen, Silvana; Kohler, Daniela



Regulation of ISWI chromatin remodelling activity.  


The packaging of the eukaryotic genome into chromatin facilitates the storage of the genetic information within the nucleus, but prevents the access to the underlying DNA sequences. Structural changes in chromatin are mediated by several mechanisms. Among them, ATP-dependent remodelling complexes belonging to ISWI family provides one of the best examples that eukaryotic cells evolved to finely regulate these changes. ISWI-containing complexes use the energy derived from ATP hydrolysis to rearrange nucleosomes on chromatin in order to favour specific nuclear reactions. The combination of regulatory nuclear factors associated with the ATPase subunit as well as its modulation by specific histone modifications, specializes the nuclear function of each ISWI-containing complex. Here we review the different ways by which ISWI enzymatic activity can be modulated and regulated in the nucleus of eukaryotic cells. PMID:24414837

Toto, Maria; D'Angelo, Giulia; Corona, Davide F V



Doxorubicin, DNA torsion, and chromatin dynamics.  


Doxorubicin is one of the most important anti-cancer chemotherapeutic drugs, being widely used for the treatment of solid tumors and acute leukemias. The action of doxorubicin and other anthracycline drugs has been intensively investigated during the last several decades, but the mechanisms that have been proposed for cell killing remain disparate and controversial. In this review, we examine the proposed models for doxorubicin action from the perspective of the chromatin landscape, which is altered in many types of cancer due to recurrent mutations in chromatin modifiers. We highlight recent evidence for effects of anthracyclines on DNA torsion and chromatin dynamics that may underlie basic mechanisms of doxorubicin-mediated cell death and suggest new therapeutic strategies for cancer treatment. PMID:24361676

Yang, Fan; Teves, Sheila S; Kemp, Christopher J; Henikoff, Steven



Biotechnologies and therapeutics: chromatin as a target.  


As alterations in gene expression underlie a considerable proportion of human diseases, correcting such aberrant transcription in vivo is expected to provide therapeutic benefit to the patient. Attempts to control endogenous mammalian genes, however, face a significant obstacle in the form of chromatin. Aberrant gene repression can be alleviated by using small-molecule inhibitors that exert nucleus-wide effects on chromatin-based repressors. Genome-wide chromatin remodeling also occurs during cloning via nuclear transfer, and causes the deregulation of epigenetically controlled genes. Regulation of genes in vivo can be accomplished via the use of designed transcription factors - these result from a fusion of a designed DNA-binding domain based on the zinc finger protein motif to a functional domain of choice. PMID:11893498

Reik, Andreas; Gregory, Philip D; Urnov, Fyodor D



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.

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



Nuclease digestion studies of chromatin structure  

SciTech Connect

Micrococcal nuclease, which preferentially cleaves linker DNA in chromatin, was immobilized by covalent attachment to CNBr-activated agarose beads and used to study the accessibility of linker DNA in chromatin fibers prepared from chicken erythrocyte nuclei. This immobilized nuclease was able to cleave chromatin fibers into the typical pattern of fragments corresponding to multiples of mononucleosomes. Cleavage from only the ends of the fibers was ruled out by examining the products of cleavage of fibers end-labelled with /sup 35/P. Comparison of the rate of digestion by immobilized and soluble micrococcal nuclease indicated that the fiber structure does not significantly affect access to linker DNA. The absence of an effect of reducing temperatures on the rate of digestion of fibers, as compared to short oligonucleosomes, indicated that breathing motions to allow access to the fiber interior were not required for cleavage of linker DNA.

Deutsch, S.M.



Classifying leukemia types with chromatin conformation data  

PubMed Central

Background Although genetic or epigenetic alterations have been shown to affect the three-dimensional organization of genomes, the utility of chromatin conformation in the classification of human disease has never been addressed. Results Here, we explore whether chromatin conformation can be used to classify human leukemia. We map the conformation of the HOXA gene cluster in a panel of cell lines with 5C chromosome conformation capture technology, and use the data to train and test a support vector machine classifier named 3D-SP. We show that 3D-SP is able to accurately distinguish leukemias expressing MLL-fusion proteins from those expressing only wild-type MLL, and that it can also classify leukemia subtypes according to MLL fusion partner, based solely on 5C data. Conclusions Our study provides the first proof-of-principle demonstration that chromatin conformation contains the information value necessary for classification of leukemia subtypes.



Interplay between chromatin and RNA processing.  


The processing of pre-mRNAs, including the selection of polyadenylation sites, is influenced by the surrounding chromatin context. We review here recent studies in Arabidopsis thaliana highlighting the intricate and reciprocal interplay between chromatin state and RNA processing. The studies have revealed that transcription can be influenced by the presence, in gene introns, of combination of epigenetic marks typical of heterochromatin. New factors binding to these marks have been identified and shown to play key roles in controlling the use of polyadenylation sites and processing of functional mRNAs. Concomitantly, several proteins of both the splicing and the polyadenylation machineries are also emerging as regulators of DNA methylation patterns and chromatin silencing. PMID:24631845

Mathieu, Olivier; Bouché, Nicolas



Overcoming the chromatin barrier to end resection.  


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

Chen, Huan; Symington, Lorraine S



Dynamics of Histone Tails within Chromatin  

NASA Astrophysics Data System (ADS)

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

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



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

PubMed Central

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

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



Chromatin architecture: investigation of a subunit of chromatin by dark field electron microscopy.  

PubMed Central

Dark field scanning electron microscopy of unstained, unfixed samples of chromatin, histone-1-depleted chromatin, and nucleohistone has been used to identify an apparent subunit of chromatin, namely a disk-shaped structure we term the unit particle, which is probably about 135 A wide and 50 A thick in the hydrated state. The unit particles are found at rather uniform intervals along thin DNA-like fibers. Histone 1 depletion leads to a bimodal distribution of these spacings. Our observations suggest that the unit particle consists of a loop of nucleoprotein, perhaps around a histone core. Images

Langmore, J P; Wooley, J C



Condensed Matter Physics  

NSDL National Science Digital Library

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


Chromatin as an expansive canvas for chemical biology  

PubMed Central

Chromatin is extensively chemically modified and thereby acts as a dynamic signaling platform controlling gene function. Chromatin regulation is integral to cell differentiation, lineage commitment and organism development, whereas chromatin dysregulation can lead to age-related and neurodegenerative disorders as well as cancer. Investigating chromatin biology presents a unique challenge, as the issue spans many disciplines, including cell and systems biology, biochemistry and molecular biophysics. In recent years, the application of chemical biology methods for investigating chromatin processes has gained considerable traction. Indeed, chemical biologists now have at their disposal powerful chemical tools that allow chromatin biology to be scrutinized at the level of the cell all the way down to the single chromatin fiber. Here we present recent examples of how this rapidly expanding palette of chemical tools is being used to paint a detailed picture of chromatin function in organism development and disease.

Fierz, Beat; Muir, Tom W



Painting by Numbers: Increasing the Parts List for Chromatin Domains  

PubMed Central

In this issue of Molecular Cell, van Bemmel and colleagues (2013) report the genome-wide mapping of 42 novel chromatin factors, systematically identifying new components of the various chromatin domains present in fly cells.

Chen, Hsiuyi V.; Rando, Oliver J.



Chromatin as an expansive canvas for chemical biology.  


Chromatin is extensively chemically modified and thereby acts as a dynamic signaling platform controlling gene function. Chromatin regulation is integral to cell differentiation, lineage commitment and organism development, whereas chromatin dysregulation can lead to age-related and neurodegenerative disorders as well as cancer. Investigating chromatin biology presents a unique challenge, as the issue spans many disciplines, including cell and systems biology, biochemistry and molecular biophysics. In recent years, the application of chemical biology methods for investigating chromatin processes has gained considerable traction. Indeed, chemical biologists now have at their disposal powerful chemical tools that allow chromatin biology to be scrutinized at the level of the cell all the way down to the single chromatin fiber. Here we present recent examples of how this rapidly expanding palette of chemical tools is being used to paint a detailed picture of chromatin function in organism development and disease. PMID:22510649

Fierz, Beat; Muir, Tom W



Chromatin damage induced by fast neutrons or UV laser radiation.  


Chromatin samples from livers of Wistar rats were subjected to fast neutron irradiation in doses of 10-100 Gy or to a 248 nm excimer laser radiation, in doses of 0.5-3 MJ.m-2. The action of the radiation on chromatin was monitored by chromatin intrinsic fluorescence and fluorescence lifetimes (of bound ethidium bromide to chromatin) and by analysing fluorescence resonance energy transfer between dansyl chloride and acridine orange coupled to chromatin. For the mentioned doses of UV excimer laser radiation, the action on chromatin was more intense than in the case of fast neutrons. The same types of damage are produced by the two radiations: acidic and basic destruction of chromatin protein structure, DNA strand breaking and the increase of the distance between DNA and proteins in chromatin. PMID:12194271

Radu, L; Constantinescu, B; Gazdaru, D; Mihailescu, I



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.


DNA replication and chromatin structure of simian virus 40 insertion mutants.  

PubMed Central

Insertion of DNA segments into the nuclease-sensitive region of simian virus 40 alters both replication efficiency and chromatin structure. Mutants containing large insertions between the simian virus 40 origin of replication (ori site) and the 21-base-pair repeated sequences replicated poorly when assayed by transfection into COS-1 cells. Replication of mutants with shorter insertions was moderately reduced. This effect was cis-acting and independent of the nucleotide sequence of the insert. The nuclease-sensitive chromatin structure was retained in these mutants, but the pattern of cleavage sites was displaced in the late direction from the ori site. New cleavage sites appeared within the inserted sequences, suggesting that information specifying the nuclease-sensitive chromatin structure is located on the late side of the inserts. Accessibility to BglI (which cleaves within the ori site) was reduced in the larger insertion mutants. These results support the conclusion that efficient function of the viral origin of replication is correlated with its proximity to an altered chromatin structure. Images

Innis, J W; Scott, W A



Cell cycle regulation of chromatin at an origin of DNA replication  

PubMed Central

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein–Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h–HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.

Zhou, Jing; Chau, Charles M; Deng, Zhong; Shiekhattar, Ramin; Spindler, Mark-Peter; Schepers, Aloys; Lieberman, Paul M



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.

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



Models incorporating chromatin modification data identify functionally important p53 binding sites  

PubMed Central

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

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



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


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



Chromatin history: our view from the bridge.  


Thirty years ago, our conception of chromatin structure underwent a total metamorphosis as the nucleosome era began. In Kurosawa's classic movie 'Rashomon' (1951), each participant had a different perspective of the same pivotal event. This review outlines our perception of history. PMID:14570061

Olins, Donald E; Olins, Ada L



Regulating chromatin: On code and dynamic models  

NASA Astrophysics Data System (ADS)

In this short comment on the contributions by Arndt Benecke, Francois Fuks and Helmut Schiessel I refer to some recent experimental work which sheds light on the histone code hypothesis. Further, I make some remarks on the role theoretical approaches can play in decoding chromatin regulation.

Blossey, R.



Locus dependence in epigenetic chromatin silencing  

PubMed Central

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

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



Oncogene-mediated alterations in chromatin conformation  

PubMed Central

Emerging evidence suggests that chromatin adopts a nonrandom 3D topology and that the organization of genes into structural hubs and domains affects their transcriptional status. How chromatin conformation changes in diseases such as cancer is poorly understood. Moreover, how oncogenic transcription factors, which bind to thousands of sites across the genome, influence gene regulation by globally altering the topology of chromatin requires further investigation. To address these questions, we performed unbiased high-resolution mapping of intra- and interchromosome interactions upon overexpression of ERG, an oncogenic transcription factor frequently overexpressed in prostate cancer as a result of a gene fusion. By integrating data from genome-wide chromosome conformation capture (Hi-C), ERG binding, and gene expression, we demonstrate that oncogenic transcription factor overexpression is associated with global, reproducible, and functionally coherent changes in chromatin organization. The results presented here have broader implications, as genomic alterations in other cancer types frequently give rise to aberrant transcription factor expression, e.g., EWS-FLI1, c-Myc, n-Myc, and PML-RAR?.

Rickman, David S.; Soong, T. David; Moss, Benjamin; Mosquera, Juan Miguel; Dlabal, Jan; Terry, Stephane; MacDonald, Theresa Y.; Tripodi, Joseph; Bunting, Karen; Najfeld, Vesna; Demichelis, Francesca; Melnick, Ari M.; Elemento, Olivier; Rubin, Mark A.



Chromosomes without a 30-nm chromatin fiber  

PubMed Central

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

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



Epigenetic chromatin silencing: bistability and front propagation  

NASA Astrophysics Data System (ADS)

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

Sedighi, Mohammad; Sengupta, Anirvan M.



Centromeres: unique chromatin structures that drive chromosome segregation  

Microsoft Academic Search

Fidelity during chromosome segregation is essential to prevent aneuploidy. The proteins and chromatin at the centromere form a unique site for kinetochore attachment and allow the cell to sense and correct errors during chromosome segregation. Centromeric chromatin is characterized by distinct chromatin organization, epigenetics, centromere-associated proteins and histone variants. These include the histone H3 variant centromeric protein A (CENPA), the

Jolien S. Verdaasdonk; Kerry Bloom



Materials in Condensing Boilers.  

National Technical Information Service (NTIS)

This is a review of corrosion and materials problems in condensing boilers for oil and gas. In the condensing boiler the fluegas is cooled below the water dewpoint of 45-48sub(o)C with gasoil and 55-58sub(o)C with natural gas. Materials for the three zone...

P. Jansen



Incorporated evaporative condenser  

Microsoft Academic Search

The incorporated evaporative condenser developed in this work comprises of a system of fins, basin of water condensates, circuit pump and system of drop cloud via spraying. In the whole provision a system of drop collector is also included for the minimisation of water escapes now essential for the operation of system. Actually, the present work aims on the development

Michalis Gr. Vrachopoulos; Andronikos E. Filios; Georgios T. Kotsiovelos; Eleftherios D. Kravvaritis



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


The base excision repair (BER) pathway is a conserved DNA repair system required to maintain genomic integrity and prevent mutagenesis in all eukaryotic cells. Nevertheless, how BER operates in vivo (i.e. in the context of chromatin) is poorly understood. We have investigated the role of an essential ATP-dependent chromatin remodelling (ACR) complex RSC (Remodels the Structure of Chromatin) in BER of intact yeast cells. We show that depletion of STH1, the ATPase subunit of RSC, causes enhanced sensitivity to the DNA alkylating agent methyl methanesulfonate (MMS) and results in a substantial inhibition of BER, at the GAL1 locus and in the genome overall. Consistent with this observation, the DNA in chromatin is less accessible to micrococcal nuclease digestion in the absence of RSC. Quantitative PCR results indicate that repair deficiency in STH1 depleted cells is not due to changes in the expression of BER genes. Collectively, our data indicates the RSC complex promotes efficient BER in chromatin. These results provide, for the first time, a link between ATP-dependent chromatin remodelling and BER in living cells. PMID:24674626

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



Freeze-Tolerant Condensers  

NASA Technical Reports Server (NTRS)

Two condensers designed for use in dissipating heat carried by working fluids feature two-phase, self-adjusting configurations such that their working lengths automatically vary to suit their input power levels and/or heat-sink temperatures. A key advantage of these condensers is that they can function even if the temperatures of their heat sinks fall below the freezing temperatures of their working fluids and the fluids freeze. The condensers can even be restarted from the frozen condition. The top part of the figure depicts the layout of the first condenser. A two-phase (liquid and vapor) condenser/vapor tube is thermally connected to a heat sink typically, a radiatively or convectively cooled metal panel. A single-phase (liquid) condensate-return tube (return artery) is also thermally connected to the heat sink. At intervals along their lengths, the condenser/vapor tube and the return artery are interconnected through porous plugs. This condenser configuration affords tolerance of freezing, variable effective thermal conductance (such that the return temperature remains nearly constant, independently of the ultimate sink temperature), and overall pressure drop smaller than it would be without the porous interconnections. An additional benefit of this configuration is that the condenser can be made to recover from the completely frozen condition either without using heaters, or else with the help of heaters much smaller than would otherwise be needed. The second condenser affords the same advantages and is based on a similar principle, but it has a different configuration that affords improved flow of working fluid, simplified construction, reduced weight, and faster recovery from a frozen condition.

Crowley, Christopher J.; Elkouhk, Nabil



Mps1 phosphorylation of condensin II controls chromosome condensation at the onset of mitosis.  


During mitosis, genomic DNA is condensed into chromosomes to promote its equal segregation into daughter cells. Chromosome condensation occurs during cell cycle progression from G2 phase to mitosis. Failure of chromosome compaction at prophase leads to subsequent misregulation of chromosomes. However, the molecular mechanism that controls the early phase of mitotic chromosome condensation is largely unknown. Here, we show that Mps1 regulates initial chromosome condensation during mitosis. We identify condensin II as a novel Mps1-associated protein. Mps1 phosphorylates one of the condensin II subunits, CAP-H2, at Ser492 during mitosis, and this phosphorylation event is required for the proper loading of condensin II on chromatin. Depletion of Mps1 inhibits chromosomal targeting of condensin II and accurate chromosome condensation during prophase. These findings demonstrate that Mps1 governs chromosomal organization during the early stage of mitosis to facilitate proper chromosome segregation. PMID:24934155

Kagami, Yuya; Nihira, Keishi; Wada, Shota; Ono, Masaya; Honda, Mariko; Yoshida, Kiyotsugu



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

PubMed Central

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

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



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

PubMed Central

A comparative analysis of mitotic chromosomes of Theobroma cacao (cacao) and T. grandiflorum (cupuaçu) was performed aiming to identify cytological differences between the two most important species of this genus. Both species have symmetric karyotypes, with 2n = 20 metacentric chromosomes ranging in size from 2.00 to 1.19 ?m (cacao) and from 2.21 to 1.15 ?m (cupuaçu). The interphase nuclei of both species were of the arreticulate type, displaying up to 20 chromocentres, which were more regularly shaped in cacao than in cupuaçu. Prophase chromosomes of both species were more condensed in the proximal region, sometimes including the whole short arm. Both species exhibited only one pair of terminal heterochromatic bands, positively stained with chromomycin A 3 , which co-localized with the single 45S rDNA site. Each karyotype displayed a single 5S rDNA site in the proximal region of another chromosome pair. Heterochromatic bands were also observed on the centromeric/pericentromeric regions of all 20 chromosomes of cacao after C-banding followed by Giemsa or DAPI staining, whereas in cupuaçu they were never detected. These data suggest that the chromosomes of both species have been largely conserved and their pericentromeric chromatin is the only citologically differentiated region.



Epigenetic Regulation of Chromatin Structure and Gene Function by Biotin1,2  

PubMed Central

Covalent modifications of histones are a crucial component of epigenetic events that regulate chromatin structures and gene function. Evidence has been provided that distinct lysine residues in histones are modified by covalent attachment of the vitamin biotin, catalyzed by biotinidase and holocarboxylase synthetase. Biotinylation of histones appears to be conserved across species. The following biotinylation sites have been identified by using both mass spectrometry and enzymatic biotinylation of synthetic peptides: K9, K13, K125, K127, and K129 in histone H2A, K4, K9, and K18 in histone H3, and K8 and K12 in histone H4. Evidence has been provided that biotinylated histone H4 is enriched in pericentrometric heterochromatin, and that biotinylation of histone H4 participates in gene silencing, mitotic condensation of chromatin, and the cellular response to DNA damage. Biotinylation of histones is a reversible process and depends on exogenous biotin supply, but the identities of histone debiotinylases remain uncertain. We propose that some effects of biotin deficiency can be attributed to abnormal chromatin structures.

Hassan, Yousef I.; Zempleni, Janos



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.




Prp22 and Spliceosome Components Regulate Chromatin Dynamics in Germ-Line Polyploid Cells  

PubMed Central

During Drosophila oogenesis, the endopolyploid nuclei of germ-line nurse cells undergo a dramatic shift in morphology as oogenesis progresses; the easily-visible chromosomes are initially polytenic during the early stages of oogenesis before they transiently condense into a distinct ‘5-blob’ configuration, with subsequent dispersal into a diffuse state. Mutations in many genes, with diverse cellular functions, can affect the ability of nurse cells to fully decondense their chromatin, resulting in a ‘5-blob arrest’ phenotype that is maintained throughout the later stages of oogenesis. However, the mechanisms and significance of nurse-cell (NC) chromatin dispersal remain poorly understood. Here, we report that a screen for modifiers of the 5-blob phenotype in the germ line isolated the spliceosomal gene peanuts, the Drosophila Prp22. We demonstrate that reduction of spliceosomal activity through loss of peanuts promotes decondensation defects in NC nuclei during mid-oogenesis. We also show that the Prp38 spliceosomal protein accumulates in the nucleoplasm of nurse cells with impaired peanuts function, suggesting that spliceosomal recycling is impaired. Finally, we reveal that loss of additional spliceosomal proteins impairs the full decondensation of NC chromatin during later stages of oogenesis, suggesting that individual spliceosomal subcomplexes modulate expression of the distinct subset of genes that are required for correct morphology in endopolyploid nurse cells.

Klusza, Stephen; Novak, Amanda; Figueroa, Shirelle; Palmer, William; Deng, Wu-Min



Mapping Networks of Protein-mediated Physical Interactions between Chromatin Elements  

PubMed Central

Recent decade has witnessed an extensive advancement in our understanding of transcriptional regulation, in part due to a rapid progress in technologies which allow studying physical proximities between various chromatin regions at a resolution beyond that offered by conventional microscopy techniques. However, these methods do not specifically identify the protein component(s) that might mediate such interactions. Here we discusses the detailed protocol for Combined 3C-ChIP-Cloning (6C) technology which combines multiple techniques to simultaneously identify physical proximities between chromatin elements as well as the proteins that mediate these interactions. We further explore how the 6C assay can be incorporated with other techniques for a complete, cell-type-specific mapping of all inter and intrachromosomal interactions mediated by specific proteins. Thus, 6C assay provides an indispensable tool to address the role of specific proteins in nuclear organization and advances our understanding about the relation of chromatin higher order organization with transcriptional regulation to the next level.

Tiwari, Vijay K.; Baylin, Stephen B.



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



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



Formation of stress-specific p53 binding patterns is influenced by chromatin but not by modulation of p53 binding affinity to response elements†  

PubMed Central

The p53 protein is crucial for adapting programs of gene expression in response to stress. Recently, we revealed that this occurs partly through the formation of stress-specific p53 binding patterns. However, the mechanisms that generate these binding patterns remain largely unknown. It is not established whether the selective binding of p53 is achieved through modulation of its binding affinity to certain response elements (REs) or via a chromatin-dependent mechanism. To shed light on this issue, we used a microsphere assay for protein–DNA binding to measure p53 binding patterns on naked DNA. In parallel, we measured p53 binding patterns within chromatin using chromatin immunoprecipitation and DNase I coupled to ligation-mediated polymerase chain reaction footprinting. Through this experimental approach, we revealed that UVB and Nutlin-3 doses, which lead to different cellular outcomes, induce similar p53 binding patterns on naked DNA. Conversely, the same treatments lead to stress-specific p53 binding patterns on chromatin. We show further that altering chromatin remodeling using an histone acetyltransferase inhibitor reduces p53 binding to REs. Altogether, our results reveal that the formation of p53 binding patterns is not due to the modulation of sequence-specific p53 binding affinity. Rather, we propose that chromatin and chromatin remodeling are required in this process.

Millau, Jean-Francois; Bandele, Omari J.; Perron, Josiann; Bastien, Nathalie; Bouchard, Eric F.; Gaudreau, Luc; Bell, Douglas A.; Drouin, Regen



Formation of stress-specific p53 binding patterns is influenced by chromatin but not by modulation of p53 binding affinity to response elements.  


The p53 protein is crucial for adapting programs of gene expression in response to stress. Recently, we revealed that this occurs partly through the formation of stress-specific p53 binding patterns. However, the mechanisms that generate these binding patterns remain largely unknown. It is not established whether the selective binding of p53 is achieved through modulation of its binding affinity to certain response elements (REs) or via a chromatin-dependent mechanism. To shed light on this issue, we used a microsphere assay for protein-DNA binding to measure p53 binding patterns on naked DNA. In parallel, we measured p53 binding patterns within chromatin using chromatin immunoprecipitation and DNase I coupled to ligation-mediated polymerase chain reaction footprinting. Through this experimental approach, we revealed that UVB and Nutlin-3 doses, which lead to different cellular outcomes, induce similar p53 binding patterns on naked DNA. Conversely, the same treatments lead to stress-specific p53 binding patterns on chromatin. We show further that altering chromatin remodeling using an histone acetyltransferase inhibitor reduces p53 binding to REs. Altogether, our results reveal that the formation of p53 binding patterns is not due to the modulation of sequence-specific p53 binding affinity. Rather, we propose that chromatin and chromatin remodeling are required in this process. PMID:21177650

Millau, Jean-François; Bandele, Omari J; Perron, Josiann; Bastien, Nathalie; Bouchard, Eric F; Gaudreau, Luc; Bell, Douglas A; Drouin, Régen



Effect of antioxidant intake on sperm chromatin stability in healthy nonsmoking men.  


Oxidative stress is detrimental to sperm function and a significant factor in the etiology of male infertility. This report examines the association between dietary and supplementary intake of the antioxidants vitamin C, vitamin E, and beta-carotene and sperm chromatin integrity. Eighty-seven healthy male volunteers donated semen samples, completed food-frequency questionnaires, and provided information about their sociodemographic characteristics, medical and reproductive histories, and lifestyle habits. Sperm chromatin integrity was measured using the DNA fragmentation index (DFI) and related parameters, obtained from the sperm chromatin structure assay (SCSA). SCSA measures the susceptibility of sperm DNA to acid-induced denaturation in situ. After adjusting for age and duration of abstinence, there was no dose-response association between any DFI outcome and any antioxidant intake measure. Non-dose-related associations were found between beta-carotene intake and both the standard deviation of DFI (SD DFI) and the percent of immature sperm. Participants with moderate, but not high, beta-carotene intake had an increase in SD DFI compared with participants with low intake (adjusted means 206.7 and 180.5, respectively; P = .03), as well as an increase in the percentage of immature sperm (adjusted means 6.9% and 5.0%, respectively; P = .04). If antioxidant intake in the range studied is indeed beneficial for fertility in healthy men, it does not appear to be mediated through the integrity of sperm chromatin. The results of this study do not preclude possible beneficial effects of high antioxidant intake on sperm chromatin integrity for men with fertility problems. PMID:15955895

Silver, Elana W; Eskenazi, Brenda; Evenson, Donald P; Block, Gladys; Young, Suzanne; Wyrobek, Andrew J



Single-molecule and population probing of chromatin structure using DNA methyltransferases  

PubMed Central

Probing chromatin structure with DNA methyltransferases offers advantages over more commonly used nuclease-based and chromatin immunoprecipitation methods for detection of nucleosomes and non-histone protein-DNA interactions. Here we describe two related methods in which the readout of MTase accessibility is obtained by assaying 5-methylcytosine in DNA through the PCR-based technique of bisulfite genomic sequencing. The methyltransferase accessibility protocol (MAP) determines the relative frequency at which the enzyme accesses each of its target sites over an entire population of PCR amplified product. While MAP yields much quantitative information about relative accessibility of a region of chromatin, a complementary single-molecule view of methyltransferase accessibility, termed MAP for individual templates (MAP-IT), is provided by analysis of cloned PCR products. Absolute rather than relative methylation frequencies in a region are obtained by summing the methylation status at each site over a cohort of clones. Moreover, as the integrity of individual molecules is maintained in MAP-IT, unique information about the distribution of multiple footprints along continuous regions is gleaned. In principle, the population MAP and single-molecule MAP-IT strategies can be used to analyze chromatin structure in a variety of model systems. Here we describe the application of MAP in living S. cerevisiae cells and MAP-IT in the analysis of a mammalian tumor suppressor gene in nuclei. This application of MAP-IT provides the first means to simultaneously determine CpG methylation of mammalian genes and their overlying chromatin structure in the same single DNA molecule.

Kilgore, Jessica A.; Hoose, Scott A.; Gustafson, Tanya L.; Porter, Weston; Kladde, Michael P.



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

PubMed Central

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

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




PubMed Central

It has frequently been proposed that a variation in the relative content of lysine-rich, moderately lysine-rich, and arginine-rich histones might provide a mechanism by which specific portions of the genome may be genetically regulated. This possibility was investigated by comparing the electrophoretic pattern of these three fractions in cells differing markedly in their content of genetically active and genetically inactive chromatin. Three models were used: heterochromatin versus euchromatin; metaphase cells versus interphase cells, and mature lymphocytes versus phytohemagglutinin-stimulated lymphocytes. In no case was there a significant difference in the histone patterns of these contrasting models. It is concluded that, although histones may act as a generalized repressor and structural component of chromatin, factors other than a variation in histone pattern may be responsible for repression or derepression of specific segments of the genome.

Comings, David E.



Chromatin structure and phaseolin gene regulation.  


Chromatin structure, the organized packaging of DNA with histones in the nucleus, is now seen as a dynamic fabric that changes with development. Here, we use studies on the phaseolin (phas) gene that encodes a seed protein to show how chromatin structure interacts with the transcription machinery to accomplish rigorous spatial regulation of expression. In leaf and other vegetative tissues, a nucleosome is rotationally and translationally positioned over an ensemble of three phased TATA boxes, denying access to TBP. Current interest focuses on the mechanisms by which this architecture is remodeled during embryogenesis. The transcription factor PvALF is intrinsically involved, as are other non-histone proteins and abscisic acid. These concepts, and the possible modular nature of phas expression, are summarized together with speculations concerning the re-establishment of the nucleosome over the phas promoter during terminal stages of embryogenesis. PMID:11442053

Li, G; Chandrasekharan, M B; Wolffe, A P; Hall, T C



On the topology of chromatin fibres  

PubMed Central

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

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



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


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

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



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 remodeling during glucocoticoid receptor regulated transactivation  

PubMed Central

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

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



Chromatin maps, histone modifications and leukemia  

Microsoft Academic Search

Recent years have seen great advances in the understanding of epigenetic gene regulation. Many of the molecular players involved have recently been identified and are rapidly being characterized in detail. Genome scale studies, using chromatin immunoprecipitation followed by expression arrays (‘ChIP-Chip’) or next generation sequencing (‘ChIP-Seq’), have been applied to the study of transcription factor binding, DNA methylation, alternative histone

T Neff; S A Armstrong



SWI\\/SNF chromatin remodeling and cancer  

Microsoft Academic Search

The SWI\\/SNF complex contributes to the regulation of gene expression by altering the chromatin structure. Depending on the context, it can be involved in either transcriptional activation or repression. Growing genetic and molecular evidence indicate that subunits of the SWI\\/SNF complex act as tumor suppressors in human and mice. Results from biochemical and transfection studies suggest also that SWI\\/SNF participates

Agnès Klochendler-Yeivin; Christian Muchardt; Moshe Yaniv



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




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.




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.



Forecasting Aircraft Condensation Trails.  

National Technical Information Service (NTIS)

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



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)



Chromatin-associated protein phosphokinases of rat ventral prostate. Characteristics and effects of androgenic status.  


Protein phosphokinase activity endogenous to rat ventral prostate chromatin was assayed by using edphosphophosvitin as an exogenous substrate. For maximal activity of the kinase reaction, the presence of 200 mM NaCl, 5 mM MgCl2, and 1 mM dithiothreitol was essential. Two apparent pH optima were observed, a broad one between pH 7 and 7.4, and one at pH 7.89. At pH 7.4 the apparent Km for 31% dephosphophosvitin was 0.3 mg per ml. With respect to ATP, two apparent Km values (0.04 and 0.41 mM) were found. The kinase activity was minimal toward exogenous histones when used as substrates (3% for lysine-rich and 0.3% for arginine-rich (f3) histones, compared with dephosphophosvitin controls). The protein phosphokinases were not significantly stimulated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) when histones used as substrate. With dephosphophosvitin as substrate, cyclic AMP produced a small inhibition (5 to 15%). Orchiectomy of adult rats resulted in a rapid decline in the chromatin-associated protein phosphokinase activity assayed using optimal experimental condition described above. At 9 hours postorchiectomy, a 30% decline in the activity was observed; this was further reduced to about 50% of the control by 18 hours. This decrease in the kinase activity (e.g. at 9 hours postorchiectomy) appears to precede measurable changes in the protein and RNA complements of chromatin. Testosterone replacement following orchiectomy abolished this decline in the chromatin-associated activity. The chromatin-associated protein phosphokinase activity toward lysine-rich and arginine-rich histones was also sensitive to androgenic status of the animals and declined rapidly postorchiectomy. The results suggest the presence of multiple and androgen-sensitive protien phosphokinases associated with rat ventral prostate chromatin, which may modulate the phosphorylation of nuclear nonhistone phosphoproteins with changing gene action mediated by testosterone in this target tissue. PMID:234969

Ahmed, K; Wilson, M J



Higher order chromatin organization in cancer  

PubMed Central

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

Reddy, Karen L.; Feinberg, Andrew P.



Titration and hysteresis in epigenetic chromatin silencing.  


Epigenetic mechanisms of silencing via heritable chromatin modifications play a major role in gene regulation and cell fate specification. We consider a model of epigenetic chromatin silencing in budding yeast and study the bifurcation diagram and characterize the bistable and the monostable regimes. The main focus of this paper is to examine how the perturbations altering the activity of histone modifying enzymes affect the epigenetic states. We analyze the implications of having the total number of silencing proteins, given by the sum of proteins bound to the nucleosomes and the ones available in the ambient, to be constant. This constraint couples different regions of chromatin through the shared reservoir of ambient silencing proteins. We show that the response of the system to perturbations depends dramatically on the titration effect caused by the above constraint. In particular, for a certain range of overall abundance of silencing proteins, the hysteresis loop changes qualitatively with certain jump replaced by continuous merger of different states. In addition, we find a nonmonotonic dependence of gene expression on the rate of histone deacetylation activity of Sir2. We discuss how these qualitative predictions of our model could be compared with experimental studies of the yeast system under anti-silencing drugs. PMID:23588040

Dayarian, Adel; Sengupta, Anirvan M



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:

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



Chromatin remodeling by polyamines and polyamine analogs.  


Natural polyamines are involved in many molecular processes, including maintenance of DNA structure and RNA processing and translation. Our aim here is to present an overview of the literature concerning the significance of polyamines in the modulation of chromatin arrangement and the transcriptional regulation of gene expression. The pleiotropic picture emerging from the published data highlights that these polycations take part in apparently diverging effects, possibly depending on the heterogeneous experimental settings described, and on a methodological approach aimed at the evaluation of the global levels of the histone chemical modifications. Since the relevant changes observed appear to be rather local and gene specific, investigating histone modifications at the level of specific gene promoters of interest is thus to be recommended for future studies. Furthermore, decoding the multiple regulatory mechanisms by which polyamines exert their influence on chromatin-modifier enzymes will reasonably require focus on selected individual polyamine-regulated genes. The evaluation of the many known chromatin-remodeling enzymes for their individual susceptibility to polyamines or polyamine derivatives will also be helpful: determining how they discriminate between the different enzyme isoforms is expected to be a fruitful line of research for drug discovery, e.g., in cancer prevention and therapy. Indeed, polyamine derivatives acting as epigenetic modulators appear to be molecules with great potential as antitumor drugs. All these novel polyamine-based pharmacologically active molecules are thus promising tools, both as a stand-alone strategy and in combination with other anticancer compounds. PMID:23836422

Pasini, Alice; Caldarera, Claudio M; Giordano, Emanuele



Default assembly of early adenovirus chromatin  

SciTech Connect

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

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



Subfractionation of soluble macronuclear chromatin and enrichment of specific genes as chromatin from Euplotes eurystomus.  

PubMed Central

Euplotes eurystomus is a hypotrichous ciliated protozoan possessing within one cytoplasm a transcriptionally-inactive micronucleus with chromosomal-size DNA and a transcriptionally active macronucleus with "gene-size" DNA fragments. The chromatin in the macronucleus can be isolated in a soluble form without prior treatment by nucleases. In this study, macronuclear, soluble chromatin was subfractionated using isokinetic sucrose density gradient ultracentrifugation in a buffer consisting of 50 mM NaCl, 1 mM Na2 EDTA, 1 mM TEA HCl, pH 7.0, 0.1 mM TLCK and 0.1 mM PMSF. Fractions were collected and analyzed by DNA and protein gel electrophoresis, dot blot hybridization with specific gene probes, and modified Miller chromatin spreads. Analysis of the chromatin spreads showed that the sizes of the chromatin fragments in the various fractions correlate with the DNA size of the fragments. When dot blots of the fractions were hybridized with 5S rRNA, tubulin and rRNA gene probes we obtained about a 6 to 14-fold enrichment of hybridizable sequences in individual fractions. There appear to be differences in the non-histones present on each fraction as well as some overall similarities in histone and non-histone proteins. Images

Cadilla, C L; Roberson, A E; Harp, J; Olins, A L; Olins, D E



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.

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



Genetic factors underlying discordance in chromatin accessibility between monozygotic twins  

PubMed Central

Background Open chromatin is implicated in regulatory processes; thus, variations in chromatin structure may contribute to variations in gene expression and other phenotypes. In this work, we perform targeted deep sequencing for open chromatin, and array-based genotyping across the genomes of 72 monozygotic twins to identify genetic factors regulating co-twin discordance in chromatin accessibility. Results We show that somatic mutations cause chromatin discordance mainly via the disruption of transcription factor binding sites. Structural changes in DNA due to C:G to A:T transversions are under purifying selection due to a strong impact on chromatin accessibility. We show that CpGs whose methylation is specifically regulated during cellular differentiation appear to be protected from high mutation rates of 5?-methylcytosines, suggesting that the spectrum of CpG variations may be shaped fully at the developmental level but not through natural selection. Based on the association mapping of within-pair chromatin differences, we search for cases in which twin siblings with a particular genotype had chromatin discordance at the relevant locus. We identify 1,325 chromatin sites that are differentially accessible, depending on the genotype of a nearby locus, suggesting that epigenetic differences can control regulatory variations via interactions with genetic factors. Poised promoters present high levels of chromatin discordance in association with either somatic mutations or genetic-epigenetic interactions. Conclusion Our observations illustrate how somatic mutations and genetic polymorphisms may contribute to regulatory, and ultimately phenotypic, discordance.



Global Chromatin Domain Organization of the Drosophila Genome  

PubMed Central

In eukaryotes, neighboring genes can be packaged together in specific chromatin structures that ensure their coordinated expression. Examples of such multi-gene chromatin domains are well-documented, but a global view of the chromatin organization of eukaryotic genomes is lacking. To systematically identify multi-gene chromatin domains, we constructed a compendium of genome-scale binding maps for a broad panel of chromatin-associated proteins in Drosophila melanogaster. Next, we computationally analyzed this compendium for evidence of multi-gene chromatin domains using a novel statistical segmentation algorithm. We find that at least 50% of all fly genes are organized into chromatin domains, which often consist of dozens of genes. The domains are characterized by various known and novel combinations of chromatin proteins. The genes in many of the domains are coregulated during development and tend to have similar biological functions. Furthermore, during evolution fewer chromosomal rearrangements occur inside chromatin domains than outside domains. Our results indicate that a substantial portion of the Drosophila genome is packaged into functionally coherent, multi-gene chromatin domains. This has broad mechanistic implications for gene regulation and genome evolution.

de Wit, Elzo; Braunschweig, Ulrich; Greil, Frauke; Bussemaker, Harmen J.; van Steensel, Bas



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


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

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



Chromatin dynamics associated with HIV-1 Tat activated transcription  

PubMed Central

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

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



Diversity of operation in ATP-dependent chromatin remodelers.  


Chromatin is actively restructured by a group of proteins that belong to the family of ATP-dependent DNA translocases. These chromatin remodelers can assemble, relocate or remove nucleosomes, the fundamental building blocks of chromatin. The family of ATP-dependent chromatin remodelers has many properties in common, but there are also important differences that may account for their varying roles in the cell. Some of the important characteristics of these complexes have begun to be revealed such as their interactions with chromatin and their mechanism of operation. The different domains of chromatin remodelers are discussed in terms of their targets and functional roles in mobilizing nucleosomes. The techniques that have driven these findings are discussed and how these have helped develop the current models for how nucleosomes are remodeled. This article is part of a Special Issue entitled: Snf2/Swi2 ATPase structure and function. PMID:21616185

Hota, Swetansu K; Bartholomew, Blaine



Dissecting chromatin interactions in living cells from protein mobility maps  

Microsoft Academic Search

The genome of eukaryotes is organized into a dynamic nucleoprotein complex referred to as chromatin, which can adopt different\\u000a functional states. Both the DNA and the protein component of chromatin are subject to various post-translational modifications\\u000a that define the cell’s gene expression program. Their readout and establishment occurs in a spatio-temporally coordinated\\u000a manner that is controlled by numerous chromatin-interacting proteins.

Fabian Erdel; Katharina Müller-Ott; Michael Baum; Malte Wachsmuth; Karsten Rippe



Dosage compensation: an intertwined world of RNA and chromatin remodelling  

Microsoft Academic Search

Dosage compensation mechanisms in flies and mammals provide an exquisite example of chromatin associated RNAs in chromosome-wide transcription regulation. Recent progress shows that chromatin modifications are also closely linked to these processes. Concerted action of the RNA\\/chromatin-modifying enzymes may play a crucial role in determining transcriptional output. Furthermore, non-coding RNAs appear to play a dual role, being targeting modules as

Asifa Akhtar



Role of Polyamines in the Regulation of Chromatin Acetylation  

Microsoft Academic Search

Changes in chromatin structure can affect gene transcription, cell proliferation, and differentiation (1). The structural remodeling of chromatin associated with gene expression is mediated in part by the coordinated targeting\\u000a of various chromatin modifying enzymes to gene regulatory regions via recruitment by transcription factors and accessory proteins\\u000a (2). The dynamic interplay between various classes of enzymes that acetylate\\/deacetylate, phosphorylate\\/dephosphorylate, and

Cheryl A. Hobbs; Susan K. Gilmour


Chromatin remodeling and repair of DNA double-strand breaks  

Microsoft Academic Search

Eukaryotic cells have developed conserved mechanisms to efficiently sense and repair DNA damage that results from constant chromosomal lesions. DNA repair has to proceed in the context of chromatin, and both histone-modifiers and ATP-dependent chromatin remodelers have been implicated in this process. Here, we review the current understanding and new hypotheses on how different chromatin-modifying activities function in DNA repair

Lai-Yee Wong; Judith Recht; Brehon C. Laurent



Quantitative analysis of histone exchange for transcriptionally active chromatin  

Microsoft Academic Search

Background  Genome-wide studies use techniques, like chromatin immunoprecipitation, to purify small chromatin sections so that protein-protein\\u000a and protein-DNA interactions can be analyzed for their roles in modulating gene transcription. Histone post-translational\\u000a modifications (PTMs) are key regulators of gene transcription and are therefore prime targets for these types of studies.\\u000a Chromatin purification protocols vary in the amount of chemical cross-linking used to

Stephanie D Byrum; Sean D Taverna; Alan J Tackett




PubMed Central

Summary A major challenge in nuclear organization is the packaging of DNA into dynamic chromatin structures that can respond to changes in the transcriptional requirements of the cell. Posttranslational protein modifications, of histones and other chromatin-associated factors, are essential regulators of chromatin dynamics. In this review, we summarize studies demonstrating that posttranslational modification of proteins by small ubiquitin-related modifiers (SUMOs) regulates chromatin structure and function at multiple levels and through a variety of mechanisms to influence gene expression and maintain genome integrity.

Cubenas-Potts, Caelin; Matunis, Michael J.



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.

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



Long noncoding RNAs, chromatin, and development.  


The way in which the genome of a multicellular organism can orchestrate the differentiation of trillions of cells and many organs, all from a single fertilized egg, is the subject of intense study. Different cell types can be defined by the networks of genes they express. This differential expression is regulated at the epigenetic level by chromatin modifications, such as DNA and histone methylation, which interact with structural and enzymatic proteins, resulting in the activation or silencing of any given gene. While detailed mechanisms are emerging on the role of different chromatin modifications and how these functions are effected at the molecular level, it is still unclear how their deposition across the epigenomic landscape is regulated in different cells. A raft of recent evidence is accumulating that implicates long noncoding RNAs (lncRNAs) in these processes. Most genomes studied to date undergo widespread transcription, the majority of which is not translated into proteins. In this review, we will describe recent work suggesting that lncRNAs are more than transcriptional "noise", but instead play a functional role by acting as tethers and guides to bind proteins responsible for modifying chromatin and mediating their deposition at specific genomic locations. We suggest that lncRNAs are at the heart of developmental regulation, determining the epigenetic status and transcriptional network in any given cell type, and that they provide a means to integrate external differentiation cues with dynamic nuclear responses through the regulation of a metastable epigenome. Better characterization of the lncRNA-protein "interactome" may eventually lead to a new molecular toolkit, allowing researchers and clinicians to modulate the genome at the epigenetic level to treat conditions such as cancer. PMID:20062956

Caley, Daniel P; Pink, Ryan C; Trujillano, Daniel; Carter, David R F



Combinatorial probabilistic chromatin interactions produce transcriptional heterogeneity  

PubMed Central

Summary Gene regulation often appears deterministic in the average cell population, but transcription is a probabilistic process at the single-cell level. Although many mechanisms are invoked to account for this behavior, it is difficult to determine how cell-to-cell variation in the interactions of transcription factors with target chromatin impact transcriptional output. Here, we use cells that contain a 200-copy tandem array of promoter or reporter gene units to simultaneously visualize transient interaction, equilibrium or steady-state binding of fluorescent-protein-labeled glucocorticoid receptor with its DNA response elements, the recruitment of diverse coregulators, and transcriptional output at the single-cell level. These regulatory proteins associate with target chromatin via a probabilistic mechanism that produces cell-to-cell variability in binding. The multiple steps of this process are partially independent and differ between individual regulators. The association level of each regulator influences the transcriptional output in individual cells, but this does not account for all transcriptional heterogeneity. Additionally, specific combinatorial interactions of the glucocorticoid receptor and coregulators with response elements regulate transcription at the single-cell level. Like many endogenous genes, the average array transcriptional activity evolves over time. This apparently deterministic average temporal promoter progression involves changes in the probability that specific combinatorial glucocorticoid receptor and coregulator interactions will occur on the response elements in single cells. These data support the emerging `return-to-template' transcription model, which mechanistically unifies the observed extremely transient interactions between the transcription factor and response elements, cell-to-cell variability in steady-state association of factors with chromatin, and the resulting heterogeneous gene expression between individual cells.

Voss, Ty C.; Schiltz, R. Louis; Sung, Myong-Hee; Johnson, Thomas A.; John, Sam; Hager, Gordon L.



Role of chromatin states in transcriptional memory  

PubMed Central

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

Kundu, Sharmistha; Peterson, Craig L.



Chromatin regulation in drug addiction and depression  

PubMed Central

Alterations in gene expression are implicated in the pathogenesis of several neuropsychiatrie disorders, including drug addiction and depression, increasing evidence indicates that changes in gene expression in neurons, in the context of animal models of addiction and depression, are mediated in part by epigenetic mechanisms that alter chromatin structure on specific gene promoters. This review discusses recent findings from behavioral, molecular, and bioinformatic approaches that are being used to understand the complex epigenetic regulation of gene expression in brain by drugs of abuse and by stress. These advances promise to open up new avenues for improved treatments of these disorders.

Renthal, William; Nestler, Eric J.



Investigating physical chromatin associations across the Xenopus genome by chromatin immunoprecipitation.  


Chromatin immunoprecipitation (ChIP) combined with genomic analysis techniques provide a global snapshot of protein-DNA interactions in the context of chromatin, yielding insights into which genomic loci might be regulated by the DNA-associated protein under investigation. This protocol describes how to perform ChIP on intact or dissected Xenopus embryos. The ChIP-isolated DNA fragments are suitable for high-throughput sequencing (ChIP-Seq) or for quantitative PCR (ChIP-qPCR). In this protocol, embryonic tissue is harvested from Xenopus tropicalis or Xenopus laevis at the developmental stage of interest, and DNA-associated proteins are immobilized to their endogenous genomic binding sites with formaldehyde. Nuclei are extracted from embryos and subjected to sonication so as to shear the chromatin to a size that allows sufficient positional resolution of protein binding to genomic DNA. Chromatin fragments bound by the protein of interest are immunoprecipitated using antibody-coupled beads, washed under high-stringency conditions, and stripped from the beads with anionic detergents. The chemical cross-links are reversed, and the coimmunoprecipitated DNA is purified. The resulting DNA fragments can be analyzed by qPCR or used to create a ChIP-Seq library. General advice for qPCR and for making ChIP-Seq libraries is offered, and approaches for analyzing ChIP-Seq data are outlined. PMID:24786504

Gentsch, George E; Smith, James C



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



Keeping condensers clean  

SciTech Connect

The humble condenser is among the biggest contributors to a steam power plant's efficiency. But although a clean condenser can provide great economic benefit, a dirty one can raise plant heat rate, resulting in large losses of generation revenue and/or unnecessarily high fuel bills. Conventional methods for cleaning fouled tubes range form chemicals to scrapers to brushes and hydro-blasters. This article compares the available options and describes how one power station, Omaha Public Power District's 600 MW North Omaha coal-fired power station, cleaned up its act. The makeup and cooling water of all its five units comes from the Missouri River. 6 figs.

Wicker, K.



Vortices in condensate mixtures  

SciTech Connect

In a condensate made of two different atomic molecular species, Onsager's quantization condition implies that around a vortex, the velocity field cannot be the same for the two species. We explore some simple consequences of this observation. Thus, if the two condensates are in slow relative translation one over the other, the composite vortices are carried at a velocity that is a fraction of the single-species velocity. This property is valid for attractive interaction and below a critical velocity which corresponds to a saddle-node bifurcation.

Josserand, Christophe; Pomeau, Yves [Laboratoire de Modelisation en Mecanique, UPMC-CNRS UMR 7607, 4 Place Jussieu, 75252 Paris Cedex 05 (France); Laboratoire de Physique Statistique de l'Ecole Normale Superieure, 24 Rue Lhomond, 75231 Paris Cedex 05 (France); Departement of Mathematics, University of Arizona, Tucson, AZ 85720 (United States)



Chromatin immunoprecipitation reveals that the 180-bp satellite repeat is the key functional DNA element of Arabidopsis thaliana centromeres.  

PubMed Central

The centromeres of Arabidopsis thaliana chromosomes contain megabases of complex DNA consisting of numerous types of repetitive DNA elements. We developed a chromatin immunoprecipitation (ChIP) technique using an antibody against the centromeric H3 histone, HTR12, in Arabidopsis. ChIP assays showed that the 180-bp centromeric satellite repeat was precipitated with the antibody, suggesting that this repeat is the key component of the centromere/kinetochore complex in Arabidopsis.

Nagaki, Kiyotaka; Talbert, Paul B; Zhong, Cathy Xiaoyan; Dawe, R Kelly; Henikoff, Steven; Jiang, Jiming



Impaired methylation modifications of FZD3 alter chromatin accessibility and are involved in congenital hydrocephalus pathogenesis.  


Congenital hydrocephalus is heterogeneous in its etiology, and in addition to a genetic component, has been shown to be caused by environmental factors. Until now, however, no methylation alterations of target genes have been connected with congenital hydrocephalus in humans. Frizzled 3(FZD3) is a planar cell polarity (PCP) gene required for PCP signaling. Partial restoration of frizzled 3 activities in FZD3 mutant mice results in hydrocephalus. To analyze the possible roles of epigenetic modifications of the FZD3 gene in congenital hydrocephalus pathogenesis, DNA methylation in the promoter region of FZD3 was assayed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Gene expression and chromatin accessibility were also determined to assess the role of methylation alterations. Our study found methylation levels of the FZD3 gene were increased in congenital hydrocephalus, especially in males (10.57±3.90 vs. 7.08±0.94, p=0.001). Hypermethylation of FZD3 increased congenital hydrocephalus risk, with an odds ratio of 10.125 (p=0.003). Aberrant methylation modification of FZD3 altered both chromatin structure in this region and FZD3 expression levels. Totally, aberrant methylation modification of the FZD3 gene increases the risk of congenital hydrocephalus by altering chromatin structure and disturbing gene expression. PMID:24796881

Wang, Li; Shangguan, Shaofang; Chang, Shaoyan; Wang, Zhen; Lu, Xiaolin; Wu, Lihua; Li, Rui; Bao, Yihua; Qiu, Zhiyong; Niu, Bo; Zhang, Ting



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

PubMed Central

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

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



Hexosaminidase assays.  


beta-Hexosaminidases (EC are lysosomal enzymes that remove terminal beta-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited beta-hexosaminidase deficiency. More recently, aberrant hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors. PMID:18473163

Wendeler, Michaela; Sandhoff, Konrad



Effects of Myogenin on Expression of Late Muscle Genes through MyoD-Dependent Chromatin Remodeling Ability of Myogenin  

PubMed Central

MyoD and myogenin (Myog) recognize sets of distinct but overlapping target genes and play different roles in skeletal muscle differentiation. MyoD is sufficient for near-full expression of early targets, while Myog can only partially enhance expression of MyoD-initiated late muscle genes. However, the way in which Myog enhances the expression of MyoD-initiated late muscle genes remains unclear. Here, we examine the effects of Myog on chromatin remodeling at late muscle gene promoters and their activation within chromatin environment. Chromatin immunoprecipitation (ChIP) assay showed that Myog selectively bound to the regulatory sequences of late muscle genes. Overexpression of Myog was found to overcome sodium butyrate-inhibited chromatin at late muscle genes in differentiating C2C12 myoblasts, shifting the transcriptional activation of these genes to an earlier time period. Furthermore, overexpression of Myog led to increased hyperacetylation of core histone H4 in differentiating C2C12 myoblasts but not NIH3T3 fibroblasts, and hyperacetylated H4 was associated directly with the late muscle genes in differentiating C2C12, indicating that Myog can induce chromatin remodeling in the presence of MyoD. In addition, co-immunoprecipitation (CoIP) revealed that Myog was associated with the nuclear protein Brd4 in differentiating C2C12 myoblasts. Together, these results suggest that Myog enhances the expression of MyoD-initiated late muscle genes through MyoD-dependent ability of Myog to induce chromatin remodeling, in which Myog-Brd4 interaction may be involved.

Du, Chao; Jin, Ya-Qiong; Qi, Jun-Juan; Ji, Zhen-Xing; Li, Shu-Yan; An, Guo-Shun; Jia, Hong-Ti; Ni, Ju-Hua



Simple Simulations of DNA Condensation  

SciTech Connect

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




Global chromatin fibre compaction in response to DNA damage  

SciTech Connect

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

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



Chromatin dynamics at the hTERT promoter during transcriptional activation and repression by c-Myc and Mnt in Xenopus leavis oocytes.  


The transcription factors c-Myc and Mnt regulate gene expression through dimerization with Max and binding to E-boxes in target genes. While c-Myc activates gene expression via recruitment of histone modifying complexes, Mnt acts as a transcriptional repressor. Here, we used the Xenopus leavis oocyte system to address the effect of c-Myc and Mnt on transcription and chromatin remodeling over the E-box region in the human telomerase reverse transcriptase (hTERT) promoter. As expected we found elevated and decreased levels of hTERT transcription upon exogenously expressed c-Myc/Max and Mnt/Max, respectively. In addition, we confirmed binding of these heterodimers to both E-boxes already enriched with H3K9ac and H4K16ac. These chromatin marks were further enhanced upon c-Myc/Max binding followed by increased DNA accessibility in the E-box region. In contrast, Mnt/Max inhibited Myc-induced transcription and mediated repression through complete chromatin condensation and deacetylation of H3K9 and H4K16 across the E-box region. Importantly, Mnt was able to counteract c-Myc mediated activation even when expressed at low levels, suggesting Mnt to act as a strong repressor by closing the chromatin structure. Collectively our data demonstrate that the balance between c-Myc and Mnt activity determines the transcriptional outcome of the hTERT promoter by modulation of the chromatin architecture. PMID:23860446

Wahlström, Therese; Belikov, Sergey; Arsenian Henriksson, Marie



Municipal Landfill Gas Condensate,  

National Technical Information Service (NTIS)

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

J. Briggs



Cloud Condensation Nuclei  

Microsoft Academic Search

In this study the supersaturation spectra of Cloud Condensation Nuclei (CCN) and the size distribution spectra of aerosols were investigated. These studies were conducted because it is believed that atmospheric aerosols, especially CCN, can affect the climate of the Earth. First, the size distributions of aerosols and the number concentrations of CCN were measured at different times in different meteorological

Qiang Ji



Condensate removal device  


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

Maddox, James W. (Newport News, VA); Berger, David D. (Alexandria, VA)



Critical Temperature and Condensate Fraction of a Fermion Pair Condensate  

SciTech Connect

We report on measurements of the critical temperature and the temperature dependence of the condensate fraction for a fermion pair condensate of {sup 6}Li atoms. Bragg spectroscopy is employed to determine the critical temperature and the condensate fraction after a fast magnetic field ramp to the molecular side of the Feshbach resonance. Our measurements reveal evidence of level off of the critical temperature and limiting behavior of condensate fraction near the unitarity limit.

Inada, Yasuhisa; Kuwata-Gonokami, Makoto [ERATO Macroscopic Quantum Control Project, JST, 2-11-16 Yayoi, Bunkyo-Ku, Tokyo 113-8656 (Japan); Department of Applied Physics, University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-8656 (Japan); Horikoshi, Munekazu; Mukaiyama, Takashi [ERATO Macroscopic Quantum Control Project, JST, 2-11-16 Yayoi, Bunkyo-Ku, Tokyo 113-8656 (Japan); Nakajima, Shuta; Ueda, Masahito [ERATO Macroscopic Quantum Control Project, JST, 2-11-16 Yayoi, Bunkyo-Ku, Tokyo 113-8656 (Japan); Department of Physics, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)



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



Effects of airport screening X-irradiation on bovine sperm chromatin integrity and embryo development.  


Biological samples, including cryopreserved sperm, are routinely X-rayed during air shipment. The goal was to investigate the impact of X-irradiation used for checked and carry-on luggage on bovine sperm chromatin integrity and postfertilization in vitro embryonic development. Frozen domestic bull sperm (Bos taurus) (n=9 bulls) stored in a dry shipper (-160 degrees C) was screened by X-irradiation 0, 1, 2, and 3 times as either carry-on or checked luggage. Duplicate straws were thawed, and sperm were assessed for chromatin damage using the sperm chromatin structure assay (SCSA) and by postfertilization in vitro developmental competence of mature oocytes. Multiple exposure to X-rays did not significantly affect sperm chromatin integrity assessed by SCSA. There were lower proportions of oocytes cleaved (P=0.07; 21.6+/-3.1% vs. 29.4+/-3.1%, 24.9+/-3.1%, and 25.7+/-3.3% for 3 vs. 0, 1, and 2 times, respectively; least-squares means+/-SEM) and that developed to blastocysts (P=0.06; 9.0+/-1.7% vs. 13.8+/-1.7%, 11.5+/-1.7%, and 12.6+/-1.9%, respectively) when fertilization was performed with sperm X-rayed 3 times using checked luggage irradiation; developmental competence (percentage cleaved embryos becoming blastocysts) was unaffected. There were no deleterious effects of other X-irradiation treatments on embryo development. We inferred that screening by X-irradiation may reduce the ability of sperm to activate oocyte cleavage after multiple exposures at the checked luggage dose. However, there was no evidence that competence of embryos to become blastocysts was reduced by X-irradiation (45.4+/-5.7%, 40.4+/-5.7%, 46.4+/-6.1%, and 41.8+/-5.7% for 0, 1, 2, and 3 doses, respectively), but potential long-term epigenetic effects are unknown. PMID:19864012

Hendricks, K E M; Penfold, L M; Evenson, D P; Kaproth, M T; Hansen, P J



Biomarker reproducibility in exhaled breath condensate collected with different condensers  

Microsoft Academic Search

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

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



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



Establishing epigenetic domains via chromatin-bound histone modifiers.  


The eukaryotic nucleus harbors the DNA genome, which associates with histones and other chromosomal proteins into a complex referred to as chromatin. It provides an additional layer of so-called epigenetic information via histone modifications and DNA methylation on top of the DNA sequence that determines the cell's active gene expression program. The nucleus is devoid of internal organelles separated by membranes. Thus, free diffusive transport of proteins and RNA can occur throughout the space accessible for a given macromolecule. At the same time, chromatin is partitioned into different specialized structures such as nucleoli, chromosome territories, and heterochromatin domains that serve distinct functions. Here, we address the question of how the activity of chromatin-modifying enzymes is confined to chromatin subcompartments. We discuss mechanisms for establishing activity gradients of diffusive chromatin-modifying enzymes that could give rise to distinct chromatin domains within the cell nucleus. Interestingly, such gradients might directly result from immobilization of the enzymes on the flexible chromatin chain. Thus, locus-specific tethering of these enzymes to chromatin could have the potential to establish, maintain, or modulate epigenetic patterns of characteristic domain size. PMID:24033539

Erdel, Fabian; Müller-Ott, Katharina; Rippe, Karsten



Transcription and chromatin converge: lessons from yeast genetics.  


The control of transcription through the modification of chromatin has been a subject of intense study over the past year. The increasing use of genome-wide approaches to examine the role of chromatin and the complexes able to modify it is providing a global perspective that is profoundly altering our view of the transcription process. PMID:11250136

Gregory, P D




PubMed Central

Mouse two-celled embryos and blastulae were Feulgen stained and the DNA content of their nuclei was measured with an integrating microdensitometer. The cells considered on the basis of their nuclear DNA content to be in G1, S, and G2 phases of the cell cycle were selected and their total chromatin area and chromatin areas at different gray levels were measured by the image analyzing computer, Quantimet. The measurements were aimed at quantitation of several features of the chromatin morphology of cells in different functional states. The total area of chromatin was found to increase, and the mean density of chromatin to decrease, from the G1 to the G2 phase of the cell cycle in both two-celled embryos and blastulae. The area of chromatin decreased, and the mean density of chromatin increased, as embryos developed from two-celled to blastula stage. It was concluded that nuclear morphology in preimplantation mouse embryos depends on both the phase of the cell cycle and the stage of development. The method of image analysis described was found to be useful for quantitation of changes in chromatin morphology.

Sawicki, Wojciech; Rowinski, Jan; Abramczuk, Jan



Brd4 Shields Chromatin from ATM Kinase Signaling Storms  

PubMed Central

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

Choi, Serah; Bakkenist, Christopher J.



In vitro transcription of chromatin containing histones hyperacetylated in vivo.  

PubMed Central

The culture of cells in the presence of sodium n-butyrate causes an accumulation of histones that are highly acetylated. When chromatin containing these histones was transcribed with E. coli RNA polymerase, an increase in the template activity compared to control chromatin was observed. Titration of chromatin with polymerase under both reinitiating and non-reinitiating conditions showed there was no increase in the number of regions available for transcription. Comparison of the kinetics for single and multiple rounds of transcription indicated that the rate of elongation was increased and probably the rate of reinitiation as well. Comparison of the size of transcripts from control and acetylated chromatin showed a small increase in the average size of transcripts from acetylated chromatin. When transcription was compared using partially purified HeLa polymerase, an increase was also seen. Studies under various ionic conditions showed that control chromatin required a higher salt concentration for optimum activity than did acetylated chromatin. In addition, at the optimum salt concentration for each chromatin, there was very little difference in the transcriptional activity using exogenous HeLa RNA polymerase.

Dobson, M E; Ingram, V M



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



Multivalent engagement of chromatin modifications by linked binding modules  

Microsoft Academic Search

Various chemical modifications on histones and regions of associated DNA play crucial roles in genome management by binding specific factors that, in turn, serve to alter the structural properties of chromatin. These so-called effector proteins have typically been studied with the biochemist's paring knife — the capacity to recognize specific chromatin modifications has been mapped to an increasing number of

Haitao Li; Alexander J. Ruthenburg; Dinshaw J. Patel; C. David Allis



Comparative carcinogenicity of cigarette mainstream and sidestream smoke condensates on the mouse skin  

Microsoft Academic Search

Summary The direct carcinogenic effects of sidestream (SS) and mainstream (MS) smoke condensates of a filtered commercial brand of blond cigarettes were compared using a lifetime mouse skin tumorigenicity assay on female NMRI mice. Each cigarette was smoked by a smoking machine under the standard conditions, and the separately collected SS and MS smoke condensates were extracted with acetone\\/methanol as

Ed Mohtashamipur; Anke Mohtashamipur; Paul-Georg Germann; Heinrich Ernst; Klaus Norpoth; Ulrich Mohr



The AID-induced DNA damage response in chromatin.  


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

Daniel, Jeremy A; Nussenzweig, André



The chromatin regulatory code: Beyond a histone code  

NASA Astrophysics Data System (ADS)

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

Lesne, A.



Chromatin and epigenetic features of long-range gene regulation  

PubMed Central

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

Harmston, Nathan; Lenhard, Boris



Chromatin-linked determinants of zygotic genome activation.  


The merging of the maternal and paternal genomes into a single pronucleus after fertilization is accompanied by a remarkable reconfiguration of chromatin in the newly formed zygote. The first stages of embryonic chromatin remodeling take place in the absence of ongoing transcription, during a species-specific developmental time-frame. Once post-fertilization chromatin states are organized, zygotic genome activation (ZGA) is initiated, and embryonic transcripts gradually take control of development. We review here transitions in chromatin modifications associated with the onset of ZGA, and the role of transcription factors and DNA motifs in the regulation of ZGA. We propose a model of sequential chromatin remodeling events preceding ZGA, leading to the onset of embryonic transcription. PMID:22965566

Østrup, Olga; Andersen, Ingrid S; Collas, Philippe



Chromatin Dynamics during Nucleotide Excision Repair: Histones on the Move  

PubMed Central

It has been a long-standing question how DNA damage repair proceeds in a nuclear environment where DNA is packaged into chromatin. Several decades of analysis combining in vitro and in vivo studies in various model organisms ranging from yeast to human have markedly increased our understanding of the mechanisms underlying chromatin disorganization upon damage detection and re-assembly after repair. Here, we review the methods that have been developed over the years to delineate chromatin alterations in response to DNA damage by focusing on the well-characterized Nucleotide Excision Repair (NER) pathway. We also highlight how these methods have provided key mechanistic insight into histone dynamics coupled to repair in mammals, raising new issues about the maintenance of chromatin integrity. In particular, we discuss how NER factors and central players in chromatin dynamics such as histone modifiers, nucleosome remodeling factors, and histone chaperones function to mobilize histones during repair.

Adam, Salome; Polo, Sophie E.



Chromatin at the intersection of viral infection and DNA damage  

PubMed Central

During infection, viruses cause global disruption to nuclear architecture in their attempt to take over the cell. In turn, the host responds with various defenses, which include chromatin-mediated silencing of the viral genome and activation of DNA damage signaling pathways. Dynamic exchanges at chromatin, and specific post-translational modifications on histones have recently emerged as master controllers of DNA damage signaling and repair. Studying viral control of chromatin modifications is identifying histones as important players in the battle between host and virus for control of cell cycle and gene expression. These studies are revealing new complexities of the virus-host interaction, uncovering the potential of chromatin as an anti-viral defense mechanism, and also providing unique insights into the role of chromatin in DNA repair.

Lilley, Caroline E.; Chaurushiya, Mira S.; Weitzman, Matthew D.



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.

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



Effect of DNA Groove Binder Distamycin A upon Chromatin Structure  

PubMed Central

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

Majumder, Parijat; Dasgupta, Dipak



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



Chromatin structure along the ribosomal DNA of Dictyostelium. Regional differences and changes accompanying cell differentiation.  


The ribosomal genes of Dictyostelium discoideum are extrachromosomal palindromic DNA molecules situated in the nucleolus. Each molecule comprises ribosomal RNA coding regions and non-transcribed spacer regions. We used both biochemical and electron microscopic approaches to investigate the structure of transcribing and non-transcribing chromatin. Nucleoli from exponentially growing cells were digested with micrococcal nuclease, and the resulting DNA fragments were separated by gel electrophoresis and transferred to DBM paper. They were hybridized with cloned EcoRI fragments derived from different parts of the ribosomal gene. Probes of the coding region showed a smear, while probes of the non-transcribed regions gave pronounced banding patterns more complex than typical nucleosome repeats, but not due solely to sequence-specific cutting by micrococcal nuclease. The DNA of the coding region was digested more quickly than that of the non-transcribed ones. When nucleoli were digested with restriction enzymes, sites within the coding region were accessible and sites in the non-transcribed region were protected. The structure of ribosomal chromatin in differentiating cells, in which the rate of ribosomal RNA synthesis is reduced, was examined using essentially the same methods. The coding region, probed by hybridization to micrococcal digests, then showed a typical DNA repeat pattern indicating that this region had become condensed into nucleosomes, and its accessibility to restriction enzymes was very much reduced. On electron micrographs of lysed nucleoli from exponentially growing cells, two types of chromatin were observed, one with a beaded nucleosomal appearance, the other with putative RNA polymerase molecules attached to fibres indistinguishable from free DNA adsorbed to the same grid. The combined results suggest that whereas regions that are not transcribed are packaged with proteins that protect them from nuclease digestion, actively transcribing ribosomal genes are associated with few macromolecular constituents apart from those required for transcription and its regulation. PMID:6304325

Ness, P J; Labhart, P; Banz, E; Koller, T; Parish, R W



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



Designed transcription factors as structural, functional and therapeutic probes of chromatin in vivo. Fourth in review series on chromatin dynamics.  


Despite its central importance in gene regulation, chromatin in mammalian cells remains relatively poorly understood-a predicament due to the paucity of robust genetic tools in mammals, the complexity of the chromatin remodeling machinery, and the dynamic properties of chromatin in vivo. Here we review recent developments in understanding endogenous mammalian gene regulation via the use of designed transcription factors (TFs). These include mutated forms of naturally occurring TFs that exhibit dominant-negative activity, and designed proteins with novel, predetermined DNA-binding specificities. Systematic targeting of designed TFs to particular promoters is helping to illuminate the complex rules that chromatin imposes on TF access and action in vivo. We evaluate the potential applications of these proteins as probes of mammalian chromatin-based regulatory pathways and their potential for the therapy of human disease, highlighting leukemia in particular. PMID:12101091

Urnov, Fyodor D; Rebar, Edward J; Reik, Andreas; Pandolfi, Pier Paolo




PubMed Central

This unit describes a streamlined two-step protocol for the isolation of adult murine cardiomyocytes with subsequent Chromatin ImmunoPrecipitation (ChIP). Isolation and culturing of cardiomyocytes is a delicate process and the protocol presented here optimizes the combination of cardiomyocyte isolation with ChIP. ChIP is an invaluable method for analyzing molecular interactions occurring between a specific protein (or its post-translationally modified form) and a region of genomic DNA. ChIP has become a widely used technique in the last decade since several groundbreaking studies have focused attention on epigenetics and have identified many epigenetic regulatory mechanisms. However, epigenetics within cardiovascular biology is a new area of focus for many investigators, and we have optimized a method for performing ChIP in adult murine cardiomyocytes as we feel this will be an important aid to both the cardiovascular field and for the development of cell- and tissue-specific ChIP.

P, Bolli; C, Vardabasso; E, Bernstein; HW, Chaudhry



Identification of alternative topological domains in chromatin  

PubMed Central

Chromosome conformation capture experiments have led to the discovery of dense, contiguous, megabase-sized topological domains that are similar across cell types and conserved across species. These domains are strongly correlated with a number of chromatin markers and have since been included in a number of analyses. However, functionally-relevant domains may exist at multiple length scales. We introduce a new and efficient algorithm that is able to capture persistent domains across various resolutions by adjusting a single scale parameter. The ensemble of domains we identify allows us to quantify the degree to which the domain structure is hierarchical as opposed to overlapping, and our analysis reveals a pronounced hierarchical structure in which larger stable domains tend to completely contain smaller domains. The identified novel domains are substantially different from domains reported previously and are highly enriched for insulating factor CTCF binding and histone marks at the boundaries.



Chromatin dynamics in kidney development and function.  


Epigenetic mechanisms are fundamental key features of developing cells connecting developmental regulatory factors to chromatin modification. Changes in the environment during renal development can have long-lasting effects on the permanent tissue structure and the level of expression of important functional genes. These changes are believed to contribute to kidney disease occurrence and progression. Although the mechanisms of early patterning and cell fate have been well described for renal development, little is known about associated epigenetic modifications and their impact on how genes interact to specify the renal epithelial cells of nephrons and how this specification is relevant to maintaining normal renal function. A better understanding of the renal cell-specific epigenetic modifications and the interaction of different cell types to form this highly complex organ will not only help to better understand developmental defects and early loss of kidney function in children, but also help to understand and improve chronic disease progression, cell regeneration and renal aging. PMID:24817101

Bechtel-Walz, Wibke; Huber, Tobias B



Gravitational vacuum condensate stars  

PubMed Central

A new final state of gravitational collapse is proposed. By extending the concept of Bose–Einstein condensation to gravitational systems, a cold, dark, compact object with an interior de Sitter condensate pv = -?v and an exterior Schwarzschild geometry of arbitrary total mass M is constructed. These regions are separated by a shell with a small but finite proper thickness ? of fluid with equation of state p = +?, replacing both the Schwarzschild and de Sitter classical horizons. The new solution has no singularities, no event horizons, and a global time. Its entropy is maximized under small fluctuations and is given by the standard hydrodynamic entropy of the thin shell, which is of the order kB?Mc/, instead of the Bekenstein–Hawking entropy formula, SBH = 4?kBGM2/c. Hence, unlike black holes, the new solution is thermodynamically stable and has no information paradox.

Mazur, Pawel O.; Mottola, Emil



Transcriptional response to stress in the dynamic chromatin environment of cycling and mitotic cells  

PubMed Central

Heat shock factors (HSFs) are the master regulators of transcription under protein-damaging conditions, acting in an environment where the overall transcription is silenced. We determined the genomewide transcriptional program that is rapidly provoked by HSF1 and HSF2 under acute stress in human cells. Our results revealed the molecular mechanisms that maintain cellular homeostasis, including HSF1-driven induction of polyubiquitin genes, as well as HSF1- and HSF2-mediated expression patterns of cochaperones, transcriptional regulators, and signaling molecules. We characterized the genomewide transcriptional response to stress also in mitotic cells where the chromatin is tightly compacted. We found a radically limited binding and transactivating capacity of HSF1, leaving mitotic cells highly susceptible to proteotoxicity. In contrast, HSF2 occupied hundreds of loci in the mitotic cells and localized to the condensed chromatin also in meiosis. These results highlight the importance of the cell cycle phase in transcriptional responses and identify the specific mechanisms for HSF1 and HSF2 in transcriptional orchestration. Moreover, we propose that HSF2 is an epigenetic regulator directing transcription throughout cell cycle progression.

Vihervaara, Anniina; Sergelius, Christian; Vasara, Jenni; Blom, Malin A. H.; Elsing, Alexandra N.; Roos-Mattjus, Pia; Sistonen, Lea



The chromatin remodeler ACF acts as a dimeric motor to space nucleosomes  

PubMed Central

Evenly spaced nucleosomes directly correlate with condensed chromatin and gene silencing. The ATP-dependent chromatin assembly factor (ACF) forms such structures in vitro and is required for silencing in vivo. ACF generates and maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. But how the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish bidirectional movement is unknown. We show that nucleosome movement depends cooperatively on two ACF molecules, suggesting that ACF functions as a dimer of ATPases. Further, the nucleotide state determines whether the dimer closely engages one vs. both sides of the nucleosome. Three-dimensional reconstruction by single particle electron microscopy of the ATPase-nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases face each other. Our results suggest a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome, thereby allowing processive bidirectional movement. This novel dimeric motor mechanism differs from that of dimeric motors such as kinesin and dimeric helicases that processively translocate unidirectionally and reflects the unique challenges faced by motors that move nucleosomes.

Racki, Lisa R.; Yang, Janet G.; Naber, Nariman; Partensky, Peretz D.; Acevedo, Ashley; Purcell, Thomas J.; Cooke, Roger; Cheng, Yifan; Narlikar, Geeta J.



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.

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



Chromatin composition is changed by poly(ADP-ribosyl)ation during chromatin immunoprecipitation.  


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; Hüttner, Katharina; Bürkle, Alexander



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

Microsoft Academic Search

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

Sascha Beneke; Kirstin Meyer; Anja Holtz; Katharina Hüttner; Alexander Bürkle



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.

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



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


RNA polymerase III (Pol III) transcription is regulated by modifications of the chromatin. DNA methylation and post-translational modifications of histones, such as acetylation, phosphorylation and methylation have been linked to Pol III transcriptional activity. In addition to being regulated by modifications of DNA and histones, Pol III genes and its transcription factors have been implicated in the organization of nuclear chromatin in several organisms. In yeast, the ability of the Pol III transcription system to contribute to nuclear organization seems to be dependent on direct interactions of Pol III genes and/or its transcription factors TFIIIC and TFIIIB with the structural maintenance of chromatin (SMC) protein-containing complexes cohesin and condensin. In human cells, Pol III genes and transcription factors have also been shown to colocalize with cohesin and the transcription regulator and genome organizer CCCTC-binding factor (CTCF). Furthermore, chromosomal sites have been identified in yeast and humans that are bound by partial Pol III machineries (extra TFIIIC sites - ETC; chromosome organizing clamps - COC). These ETCs/COC as well as Pol III genes possess the ability to act as boundary elements that restrict spreading of heterochromatin. PMID:23150255

Pascali, Chiara; Teichmann, Martin



Simultaneously defining cell phenotypes, cell cycle, and chromatin modifications at single-cell resolution.  


Heterogeneity of cellular phenotypes in asynchronous cell populations placed in the same biochemical and biophysical environment may depend on cell cycle and chromatin modifications; however, no current method can measure these properties at single-cell resolution simultaneously and in situ. Here, we develop, test, and validate a new microscopy assay that rapidly quantifies global acetylation on histone H3 and measures a wide range of cell and nuclear properties, including cell and nuclear morphology descriptors, cell-cycle phase, and F-actin content of thousands of cells simultaneously, without cell detachment from their substrate, at single-cell resolution. These measurements show that isogenic, isotypic cells of identical DNA content and the same cell-cycle phase can still display large variations in H3 acetylation and that these variations predict specific phenotypic variations, in particular, nuclear size and actin cytoskeleton content, but not cell shape. The dependence of cell and nuclear properties on cell-cycle phase is assessed without artifact-prone cell synchronization. To further demonstrate its versatility, this assay is used to quantify the complex interplay among cell cycle, epigenetic modifications, and phenotypic variations following pharmacological treatments affecting DNA integrity, cell cycle, and inhibiting chromatin-modifying enzymes. PMID:23538711

Chambliss, Allison B; Wu, Pei-Hsun; Chen, Wei-Chiang; Sun, Sean X; Wirtz, Denis



Mutagenicity testing of condensates of smoke from titanium dioxide/hexachloroethane and zinc/hexachloroethane pyrotechnic mixtures.  


Condensates of smoke from titanium dioxide/hexachloroethane and zinc/hexachloroethane pyrotechnic mixtures were investigated for their potential to produce genetic damage in the tester strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium and in the mouse bone marrow micronucleus assay. Both smoke condensates contained several chlorinated hydrocarbons among which tetrachloroethylene, hexachloroethane, hexachlorobutadiene and hexachlorobenzene were identified by GC/MS. Condensate of smoke from titanium dioxide/hexachloroethane showed a dose-related positive response in the Salmonella assay with strains TA98 and TA100 in the absence of metabolic activation from rat liver S9 fraction. Both smoke condensates were negative in the micronucleus assay but produced a small but significant depression of erythropoietic activity. The results indicate that smoke condensate from titanium dioxide/hexachloroethane mixtures contains unidentified compound(s) that may be considered mutagenic in the Salmonella assay. PMID:2027339

Karlsson, N; Fängmark, I; Häggqvist, I; Karlsson, B; Rittfeldt, L; Marchner, H



High throughput automated chromatin immunoprecipitation as a platform for drug screening and antibody validation†,‡  

PubMed Central

Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein–DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large scale screening purposes. To address these challenges, we report HTChIP, an automated microfluidic-based platform for performing high-throughput ChIP screening measurements of 16 different targets simultaneously, with potential for further scale-up. From chromatin to analyzable PCR results only takes one day using HTChIP, as compared to several days up to one week for conventional protocols. HTChIP can also be used to test multiple antibodies and select the best performer for downstream ChIP applications, saving time and reagent costs of unsuccessful ChIP assays as a result of poor antibody quality. We performed a series of characterization assays to demonstrate that HTChIP can rapidly and accurately evaluate the epigenetic states of a cell, and that it is sensitive enough to detect the changes in the epigenetic state induced by a cytokine stimulant over a fine temporal resolution. With these results, we believe that HTChIP can introduce large improvements in routine ChIP, antibody screening, and drug screening efficiency, and further facilitate the use of ChIP as a valuable tool for research and discovery.

Wu, Angela R.; Kawahara, Tiara L.A.; Rapicavoli, Nicole A.; van Riggelen, Jan; Shroff, Emelyn H.; Xu, Liwen; Felsher, Dean W.; Chang, Howard Y.; Quake, Stephen R.



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.

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



Tracking the mechanical dynamics of human embryonic stem cell chromatin  

PubMed Central

Background A plastic chromatin structure has emerged as fundamental to the self-renewal and pluripotent capacity of embryonic stem (ES) cells. Direct measurement of chromatin dynamics in vivo is, however, challenging as high spatiotemporal resolution is required. Here, we present a new tracking-based method which can detect high frequency chromatin movement and quantify the mechanical dynamics of chromatin in live cells. Results We use this method to study how the mechanical properties of chromatin movement in human embryonic stem cells (hESCs) are modulated spatiotemporally during differentiation into cardiomyocytes (CM). Notably, we find that pluripotency is associated with a highly discrete, energy-dependent frequency of chromatin movement that we refer to as a ‘breathing’ state. We find that this ‘breathing’ state is strictly dependent on the metabolic state of the cell and is progressively silenced during differentiation. Conclusions We thus propose that the measured chromatin high frequency movements in hESCs may represent a hallmark of pluripotency and serve as a mechanism to maintain the genome in a transcriptionally accessible state. This is a result that could not have been observed without the high spatial and temporal resolution provided by this novel tracking method.



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.

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



Chromatin elimination in the hypotrichous ciliate Stylonychia mytilus.  


Chromatin elimination in the hypotrichous ciliate Stylonychia mytilus was studied by means of electron microscopy using a microspreading procedure. In the polytene chromosomes of the macronuclear anlagen three organization patterns are observed: Bands of various size composed of 300 A chromatin fibers, large blocks of 300 A nucleofilaments which probably represent the "heterochromatic" regions of the chromosome and axial 120 A filaments. Those DNA sequences which become eliminated belong to the 300 A fiber type. The eliminated chromatin occurs in the form of rings of variable size corresponding to a DNA content between 18 and 160 Kb while the axial 120 A filaments appear to be preserved. PMID:6768532

Meyer, G F; Lipps, H J



Out of register: How DNA determines the chromatin fiber geometry  

NASA Astrophysics Data System (ADS)

We present a model that predicts the geometry of chromatin fibers as a function of the DNA repeat length. Chromatin fibers are widely observed in vitro and are typically posited as the second level of the hierarchical organization of chromatin in the nuclei of cells. We postulate that the major driving force for fiber formation is the dense packing of the underlying DNA-protein spools, the nucleosomes, allowing for fibers with four possible diameters. We show that the diameters observed in experiments on reconstituted regular fibers correspond to the geometries that minimize the elastic energy of the DNA linking the nucleosomes.

Lanzani, G.; Schiessel, H.



Methods for the analysis of protein-chromatin interactions.  


The analysis of protein interactions with chromatin is vital for the understanding of DNA sequence recognition in vivo. Chromatin binding requires the interaction of proteins with DNA lying on the macromolecular protein surface of nucleosomes, a situation that can alter factor binding characteristics substantially when compared with naked DNA. It is therefore important to study these protein-DNA interactions in the context of a chromatin substrate, the more physiologically relevant binding situation. In this article we review techniques used in the investigation of protein interactions with defined nucleosomal templates. PMID:11876294

Brickwood, Sarah J; Myers, Fiona A; Chandler, Simon P



Histone Post-Translation Modifications Influence Chromatin Mechanical Stability  

NASA Astrophysics Data System (ADS)

Histone proteins organize the human genome into chromatin fibers while their post-translation modification (PTM) regulates genome replication, expression and repair. The mechanistic connections between histone PTMs and biological functions remain enigmatic. We find with a combination of magnetic tweezers mechanical measurements and biochemical studies that a number of histone PTMs influence the DNA mismatch repair process by mechanically destabilizing chromatin. The location of the PTM within the chromatin structure appears to determine the mechanism by which it alters the mechanical stability. These findings have direct implications for understanding the repair of the human genome.

Poirier, Michael




PubMed Central

Squashing salivary glands of Chironomus thummi larvae, Amblystoma tigrinum erythrocytes, or Spirostromum frequently results in stretched chromatin having highly oriented DNA as determined by polarized fluorescence microscopy of acridine orange-stained preparations. The examination of such material from C. thummi in the electron microscope indicates that the individual chromatin fibers have an average thickness of 80 A as is usually found in embedded and sectioned material. It is thus concluded that the DNA lies nearly parallel to the axis of these chromatin fibers. Detailed calculations of the polarization expected from various models of DNA packing are contained in an appendix.

Dusenbery, David B.; Uretz, Robert B.



The orientation of DNA within 80-Angstrom chromatin fibers.  


Squashing salivary glands of Chironomus thummi larvae, Amblystoma tigrinum erythrocytes, or Spirostromum frequently results in stretched chromatin having highly oriented DNA as determined by polarized fluorescence microscopy of acridine orange-stained preparations. The examination of such material from C. thummi in the electron microscope indicates that the individual chromatin fibers have an average thickness of 80 A as is usually found in embedded and sectioned material. It is thus concluded that the DNA lies nearly parallel to the axis of these chromatin fibers. Detailed calculations of the polarization expected from various models of DNA packing are contained in an appendix. PMID:4621586

Dusenbery, D B; Uretz, R B



HILS1 is a spermatid-specific linker histone H1-like protein implicated in chromatin remodeling during mammalian spermiogenesis  

PubMed Central

Chromatin remodeling is a major event that occurs during mammalian spermiogenesis, the process of spermatid maturation into spermatozoa. Nuclear condensation during spermiogenesis is accomplished by replacing somatic histones (linker histone H1 and core histones) and the testis-specific linker histone, H1t, with transition proteins and protamines. It has long been thought that H1t is the only testis-specific linker histone, and that all linker histones are replaced by transition proteins, and subsequently by protamines during spermiogenesis. Here, we report the identification and characterization of a spermatid-specific linker histone H1-like protein (termed HILS1) in the mouse and human. Both mouse and human HILS1 genes are located in intron 8 of the ?-sarcoglycan genes. HILS1 is highly expressed in nuclei of elongating and elongated spermatids (steps 9-15). HILS1 displays several biochemical properties that are similar to those of linker histones, including the abilities to bind reconstituted mononucleosomes, produce a chromatosome stop during micrococcal nuclease digestion, and aggregate chromatin. Because HILS1 is expressed in late spermatids that do not contain core histones, HILS1 may participate in spermatid nuclear condensation through a mechanism distinct from that of linker histones. Because HILS1 also belongs to the large winged helix/forkhead protein superfamily, HILS1 may also regulate gene transcription, DNA repair, and/or other chromosome processes during mammalian spermiogenesis.

Yan, Wei; Ma, Lang; Burns, Kathleen H.; Matzuk, Martin M.



Confinement Contains Condensates  

SciTech Connect

Dynamical chiral symmetry breaking and its connection to the generation of hadron masses has historically been viewed as a vacuum phenomenon. We argue that confinement makes such a position untenable. If quark-hadron duality is a reality in QCD, then condensates, those quantities that have commonly been viewed as constant empirical mass-scales that fill all spacetime, are instead wholly contained within hadrons; i.e., they are a property of hadrons themselves and expressed, e.g., in their Bethe-Salpeter or light-front wave functions. We explain that this paradigm is consistent with empirical evidence, and incidentally expose misconceptions in a recent Comment.

Brodsky, Stanley J.; Roberts, Craig D.; Shrock, Robert; Tandy, Peter C.



A Role for CARM1-Mediated Histone H3 Arginine Methylation in Protecting Histone Acetylation by Releasing Corepressors from Chromatin  

PubMed Central

Arginine methylation broadly occurs in histones and has been linked to transcriptional regulation, cell cycle regulation and DNA repair. While numerous proteins (histone code effectors) that specifically recognize or read the methylated lysine residues in core histones have been identified, little is known for effectors specific for methylated arginines in histones. In this study, we attempted to identify effector(s) recognizing asymmetrically methylated R17 and R26 in H3, which are catalyzed by CARM1/PRMT4, through an unbiased biochemical approach. Although we have yet to identify such effector using this approach, we find that these modifications function cooperatively with histone acetylation to inhibit the binding of the nucleosome remodeling and deacetylase complex (NuRD) and TIF1 family corepressors to H3 tail in vitro. In support of this finding, we show that overexpression of CARM1 in 293 T cells leads to reduced association of NuRD with chromatin, whereas knockdown of CARM1 in HeLa cells leads to increased association of NuRD with chromatin and decreased level of histone acetylation. Furthermore, in the Carm1?/? MEF cells there is an increased association of NuRD and TIF1? with chromatin and a global decrease in histone acetylation. By chromatin immunoprecipitation assay, we show that overexpression of CARM1 results in reduced association of NuRD complex and TIF1? with an episomal reporter and that CARM1 is required in MEF cells for LPS-induced dissociation of NuRD from a NF-?b target gene. Taking together, our study provides evidence for a role of CARM1-mediated arginine methylation in regulation of histone acetylation and transcription: facilitating transcription by discharging corepressors from chromatin.

Wang, Rui; Li, Jiwen; Wong, Jiemin



CYTOGENETIC ABNORMALITY IN MAN--Wider Implications of Theories of Sex Chromatin Origin  

PubMed Central

Female nuclei may be identified by means of sex chromatin. In general the number of sex chromatin bodies is one less than the number of X chromosomes. An exception to this rule is a case of sex chromatin-positive XO Turner's syndrome. This case suggests the possibility of sex chromatin-positive XY males, and it may be evidence for chromosomal differentiation.

Miles, Charles P.



Cohesin organizes chromatin loops at DNA replication factories  

PubMed Central

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

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



From neural development to cognition: unexpected roles for chromatin.  


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; Crabtree, Gerald R



Shaping the landscape: mechanistic consequences of ubiquitin modification of chromatin  

PubMed Central

The organization of eukaryotic chromosomes into transcriptionally active euchromatin and repressed heterochromatin requires mechanisms that establish, maintain and distinguish these canonical chromatin domains. Post-translational modifications are fundamental in these processes. Monoubiquitylation of histones was discovered more than three decades ago, but its precise function has been enigmatic until recently. It is now appreciated that the spectrum of chromatin ubiquitylation is not restricted to monoubiquitylation of histones, but includes degradatory ubiquitylation of histones, histone-modifying enzymes and non-histone chromatin factors. These occur in a spatially and temporally controlled manner. In this review, we summarize our understanding of these mechanisms with a particular emphasis on how ubiquitylation shapes the physical landscape of chromatin.

Braun, Sigurd; Madhani, Hiten D



Stimulation of transcription on chromatin by polar organic compounds.  

PubMed Central

Polar organic compounds, including DMSO, increase RNA synthesis on isolated chromatin by E. coli RNA polymerase and RNA polymerase II from calf thymus. Transcription is stimulated on chromatin from Friend-virus-infected erythroleukemia cells and from various other sources. Using procedures which inhibit specifically the formation of a stable initiation complex, it is shown that the stimulation does not result from an increase in initiation of both E. coli and the eukaryotic RNA polymerase. After separation of chromatin into template active and inactive fractions, DMSO increases RNA synthesis by a factor of about 1.5 using the template inactive fraction, while stimulation of transcription on the template active portion is lower (factor of 1.2). It is suggested that the effect on RNA synthesis is mediated by a weakening of the apolar interactions between histones in chromatin subunits, releasing transcription partially from the constraints imposed by histones.

Stratling, W H



The role of chromatin modifiers in normal and malignant hematopoiesis.  


Complex developmental processes such as hematopoiesis require a series of precise and coordinated changes in cellular identity to ensure blood homeostasis. Epigenetic mechanisms help drive changes in gene expression that accompany the transition from hematopoietic stem cells to terminally differentiated blood cells. Genome-wide profiling technologies now provide valuable glimpses of epigenetic changes that occur during normal hematopoiesis, and genetic mouse models developed to investigate the in vivo functions of chromatin-modifying enzymes clearly demonstrate significant roles for these enzymes during embryonic and adult hematopoiesis. Here, we will review the basic science aspects of chromatin modifications and the enzymes that add, remove, and interpret these epigenetic marks. This overview will provide a framework for understanding the roles that these molecules play during normal hematopoiesis. Moreover, many chromatin-modifying enzymes are involved in hematologic malignancies, underscoring the importance of establishing and maintaining appropriate chromatin modification patterns to normal hematology. PMID:23287864

Butler, Jill S; Dent, Sharon Y R



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



Human Histone Chaperone Nucleophosmin Enhances Acetylation-Dependent Chromatin Transcription†  

PubMed Central

Histone chaperones are a group of proteins that aid in the dynamic chromatin organization during different cellular processes. Here, we report that the human histone chaperone nucleophosmin interacts with the core histones H3, H2B, and H4 but that this histone interaction is not sufficient to confer the chaperone activity. Significantly, nucleophosmin enhances the acetylation-dependent chromatin transcription and it becomes acetylated both in vitro and in vivo. Acetylation of nucleophosmin and the core histones was found to be essential for the enhancement of chromatin transcription. The acetylated NPM1 not only shows an increased affinity toward acetylated histones but also shows enhanced histone transfer ability. Presumably, nucleophosmin disrupts the nucleosomal structure in an acetylation-dependent manner, resulting in the transcriptional activation. These results establish nucleophosmin (NPM1) as a human histone chaperone that becomes acetylated, resulting in the enhancement of chromatin transcription.

Swaminathan, V.; Kishore, A. Hari; Febitha, K. K.; Kundu, Tapas K.



Evidence for monomeric actin function in INO80 chromatin remodeling  

PubMed Central

Actin has well-established functions in the cytoplasm, but its roles in the nucleus remain poorly defined. Here, by studying the nuclear actin-containing yeast INO80 chromatin remodeling complex, we provide genetic and biochemical evidence for a role of monomeric actin in INO80 chromatin remodeling. In contrast to cytoplasmic actin, nuclear actin is present as a monomer in the INO80 complex and its barbed end is not accessible for polymerization. An actin mutation affecting in vivo nuclear functions is identified in subdomain 2, which reduces the chromatin remodeling activities of the INO80 complex in vitro. Importantly, the highly conserved subdomain 2 at the pointed end of actin contributes to INO80 interactions with chromatin. Our results establish an evolutionarily conserved function of nuclear actin in its monomeric form and suggest that nuclear actin can utilize a fundamentally distinct mechanism from cytoplasmic actin.

Kapoor, Prabodh; Chen, Mingming; Winkler, Duane David; Luger, Karolin; Shen, Xuetong



The Chromatin Landscape of Kaposi's Sarcoma-Associated Herpesvirus  

PubMed Central

Kaposi’s sarcoma-associated herpesvirus is an oncogenic ?-herpesvirus that causes latent infection in humans. In cells, the viral genome adopts a highly organized chromatin structure, which is controlled by a wide variety of cellular and viral chromatin regulatory factors. In the past few years, interrogation of the chromatinized KSHV genome by whole genome-analyzing tools revealed that the complex chromatin landscape spanning the viral genome in infected cells has important regulatory roles during the viral life cycle. This review summarizes the most recent findings regarding the role of histone modifications, histone modifying enzymes, DNA methylation, microRNAs, non-coding RNAs and the nuclear organization of the KSHV epigenome in the regulation of latent and lytic viral gene expression programs as well as their connection to KSHV-associated pathogenesis.

Toth, Zsolt; Brulois, Kevin; Jung, Jae U.



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.

Ronan, Jehnna L.; Wu, Wei



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.

Rosa, Stefanie; Shaw, Peter



Developmental regulation of chromatin conformation by Hox proteins in Drosophila  

PubMed Central

Summary We present a strategy to examine the chromatin conformation of individual loci in specific cell types during Drosophila embryogenesis. Regulatory DNA is tagged with binding sites (lacO) for LacI, which is used to immunopreciptiate the tagged chromatin from specific cell types. We applied this approach to Distalless (Dll), a gene required for limb development in Drosophila. We show that the local chromatin conformation at Dll depends on the cell type: in cells that express Dll, the 5’ regulatory region is in close proximity to the Dll promoter. In Dll nonexpressing cells this DNA is in a more extended configuration. In addition, transcriptional activators and repressors are bound to Dll regulatory DNA in a cell type specific manner. The pattern of binding by GAGA factor and the variant histone H2Av suggest that they play a role in the regulation of Dll chromatin conformation in expressing and non-expressing cell types, respectively.

Agelopoulos, Marios; McKay, Daniel J.; Mann, Richard S.



Superstructure and CD spectrum as probes of chromatin integrity.  

PubMed Central

Two types of chromatin were extracted from the same stock of rat liver nuclei by a short exposure to micrococcal nuclease and by shearing respectively. These two materials which are identical in their protein/DNA content and by the presence of the five histones, were compared by means of circular dichroism and electron microscopy. Under the electron microscope and in absence of any divalent cation a superstructure of the unfixed chromatin fiber can be viewed only with native material but is no more present in sheared one. The increase of CD signal at 280 nm (from 2000 to about 4000 cm2 deg.dmole-1) in the case of sheared chromatin is not related to the loss of superstructure but to the structural changes of DNA inside the nucleosomal core which are always produced by shearing. These two correlated observations offer new sensitive probes of the integrity of any native or reconstituted chromatin. Images

de Murcia, G; Das, G C; Erard, M; Daune, M



Absorption and emission spectroscopy in the analysis of laser radiation and fast neutron action on chromatin  

NASA Astrophysics Data System (ADS)

The actions of a 248 mm excimer laser radiation (0.5 - 3 MJ/m2) and of fast neutrons (d(13 MeV) + Be thick target, 5 - 100 Gy) on chromatin (the complex of DNA and proteins of eukaryotic cells) were analyzed. The parameters estimated were: the thermal transition, the fluorescence of chromatin-ethidium bromide complexes, the chromatin intrinsic fluorescence and the energy transfer efficiency between specific fluorescent ligands coupled at chromatin. The mechanism of UV excimer laser action is similar to the fast neutrons action on chromatin. The complex action includes the production of chromatin DNA strand breaks, the protein structure lesion and the chromatin conformation modification.

Radu, Liliana; Mihailescu, Ion N.; Constantinescu, B.; Gostian, O.



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.



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.

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



Strange Disoriented Chiral Condensates  

NASA Astrophysics Data System (ADS)

Enhancement of omega and anti-omega baryon production in Pb+Pb collisions at the CERN SPS can be explained by the formation of many small regions of disordered chiral condensate. This explanation implies that neutral and charged kaons as well as pions must exhibit novel isospin fluctuations. Fluctuations due to transient behavior of the Polyakov Loop condensate can produce similar phenomena. Kapusta and Gavin have computed the distribution of the fraction of neutral pions and kaons from such regions. We proposed robust statistical observables that can be used to extract the novel isospin fluctuations from background contributions in neutral/charged pion and K-short/K-charged correlation measurements at RHIC and LHC. The STAR experiment is currently examining K-short/K-charged correlations. Note that Pruneau, Voloshin and Gavin have proposed similar observables to study net-charge fluctuations. To obtain a baseline for comparison to RHIC and SPS experiments, Abdel-Aziz and Gavin compute these observables using numerical simulations using HIJING and URQMD event generators. We also obtain limits on the size and number of disordered regions by comparing to photon and charged-pion searches from WA98 and other SPS experiments. We will compare to the first results from STAR K-short/K-charged analysis.

Abdel-Aziz, Mohamed; Gavin, Sean



Chromatin insulators: linking genome organization to cellular function.  


A growing body of evidence suggests that insulators have a primary role in orchestrating the topological arrangement of higher-order chromatin architecture. Insulator-mediated long-range interactions can influence the epigenetic status of the genome and, in certain contexts, may have important effects on gene expression. Here we discuss higher-order chromatin organization as a unifying mechanism for diverse insulator actions across the genome. PMID:23706817

Phillips-Cremins, Jennifer E; Corces, Victor G



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.

Reeves, Raymond



Chromatin crosstalk in development and disease: lessons from REST  

Microsoft Academic Search

Protein complexes that contain chromatin-modifying enzymes have an important role in regulating gene expression. Recent studies have shown that a single transcription factor, the repressor element 1-silencing transcription factor (REST), can act as a hub for the recruitment of multiple chromatin-modifying enzymes, uncovering interdependencies among individual enzymes that affect gene regulation. Research into the function of REST and its corepressors

Lezanne Ooi; Ian C. Wood



Neutrophil extracellular traps: Is immunity the second function of chromatin?  

PubMed Central

Neutrophil extracellular traps (NETs) are made of processed chromatin bound to granular and selected cytoplasmic proteins. NETs are released by white blood cells called neutrophils, maybe as a last resort, to control microbial infections. This release of chromatin is the result of a unique form of cell death, dubbed “NETosis.” Here we review our understanding of how NETs are made, their function in infections and as danger signals, and their emerging importance in autoimmunity and coagulation.



The quantitative architecture of centromeric chromatin  

PubMed Central

The centromere, responsible for chromosome segregation during mitosis, is epigenetically defined by CENP-A containing chromatin. The amount of centromeric CENP-A has direct implications for both the architecture and epigenetic inheritance of centromeres. Using complementary strategies, we determined that typical human centromeres contain ?400 molecules of CENP-A, which is controlled by a mass-action mechanism. This number, despite representing only ?4% of all centromeric nucleosomes, forms a ?50-fold enrichment to the overall genome. In addition, although pre-assembled CENP-A is randomly segregated during cell division, this amount of CENP-A is sufficient to prevent stochastic loss of centromere function and identity. Finally, we produced a statistical map of CENP-A occupancy at a human neocentromere and identified nucleosome positions that feature CENP-A in a majority of cells. In summary, we present a quantitative view of the centromere that provides a mechanistic framework for both robust epigenetic inheritance of centromeres and the paucity of neocentromere formation. DOI:

Bodor, Dani L; Mata, Joao F; Sergeev, Mikhail; David, Ana Filipa; Salimian, Kevan J; Panchenko, Tanya; Cleveland, Don W; Black, Ben E; Shah, Jagesh V; Jansen, Lars ET



The quantitative architecture of centromeric chromatin.  


The centromere, responsible for chromosome segregation during mitosis, is epigenetically defined by CENP-A containing chromatin. The amount of centromeric CENP-A has direct implications for both the architecture and epigenetic inheritance of centromeres. Using complementary strategies, we determined that typical human centromeres contain ?400 molecules of CENP-A, which is controlled by a mass-action mechanism. This number, despite representing only ?4% of all centromeric nucleosomes, forms a ?50-fold enrichment to the overall genome. In addition, although pre-assembled CENP-A is randomly segregated during cell division, this amount of CENP-A is sufficient to prevent stochastic loss of centromere function and identity. Finally, we produced a statistical map of CENP-A occupancy at a human neocentromere and identified nucleosome positions that feature CENP-A in a majority of cells. In summary, we present a quantitative view of the centromere that provides a mechanistic framework for both robust epigenetic inheritance of centromeres and the paucity of neocentromere formation.DOI: PMID:25027692

Bodor, Dani L; Mata, João F; Sergeev, Mikhail; David, Ana Filipa; Salimian, Kevan J; Panchenko, Tanya; Cleveland, Don W; Black, Ben E; Shah, Jagesh V; Jansen, Lars Et



Evaluation of human sperm chromatin status after selection using a modified Diff-Quik stain indicates embryo quality and pregnancy outcomes following in vitro fertilization.  


Sperm chromatin/DNA damage can be measured by a variety of assays. However, it has been reported that these tests may lose prognostic value in Assisted Reproductive Technology (ART) cycles when assessed in post-prepared samples, possibly due to the normalizing effect promoted by sperm preparation procedures. We have recently implemented a modified version of the Diff-Quik staining assay that allows for the evaluation of human sperm chromatin status in native samples, together with standard sperm morphology assessment. However, the value of this parameter in terms of predicting in vitro fertilization (IVF) and Intracytoplasmic sperm injection (ICSI) outcomes after sperm selection is unknown. In this study, data from 138 couples undergoing in vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI) treatments showed that sperm chromatin integrity was significantly improved after density gradient centrifugation and swim up (p < 0.001), but no correlations were found with fertilization or embryo development rates (p > 0.05). However, sperm samples presenting lower percentages of damaged chromatin were associated with better quality (Grade I) embryos in both ART procedures (p < 0.05) and clinical pregnancy among IVF couples (p < 0.05). Furthermore, regression analysis confirmed the clinical value of Diff-Quik staining in predicting IVF (but not ICSI) clinical pregnancy (OR: 0.927, 95% CI: 0.871-0.985, p = 0.015), and a threshold value of 34.25% for this parameter was established. The proportion of IVF couples achieving a clinical pregnancy was reduced 1.9-fold when the percentage of abnormal dark staining was ?34.25% (p = 0.05). In conclusion, the Diff-Quik staining assay provides useful information regarding ART success, particularly in IVF cycles, where some degree of 'natural' sperm selection may occur; but not in ICSI, where sperm selection is operator dependent. This quick and low-cost assay is suggested as an alternative method to detect sperm chromatin status in minimal clinical settings, when no other well-established and robust assays (e.g. Sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling) are available. PMID:24124136

Tavares, R S; Silva, A F; Lourenço, B; Almeida-Santos, T; Sousa, A P; Ramalho-Santos, J



Developmentally Regulated Linker Histone H1c Promotes Heterochromatin Condensation and Mediates Structural Integrity of Rod Photoreceptors in Mouse Retina*  

PubMed Central

Mature rod photoreceptor cells contain very small nuclei with tightly condensed heterochromatin. We observed that during mouse rod maturation, the nucleosomal repeat length increases from 190 bp at postnatal day 1 to 206 bp in the adult retina. At the same time, the total level of linker histone H1 increased reaching the ratio of 1.3 molecules of total H1 per nucleosome, mostly via a dramatic increase in H1c. Genetic elimination of the histone H1c gene is functionally compensated by other histone variants. However, retinas in H1c/H1e/H10 triple knock-outs have photoreceptors with bigger nuclei, decreased heterochromatin area, and notable morphological changes suggesting that the process of chromatin condensation and rod cell structural integrity are partly impaired. In triple knock-outs, nuclear chromatin exposed several epigenetic histone modification marks masked in the wild type chromatin. Dramatic changes in exposure of a repressive chromatin mark, H3K9me2, indicate that during development linker histone plays a role in establishing the facultative heterochromatin territory and architecture in the nucleus. During retina development, the H1c gene and its promoter acquired epigenetic patterns typical of rod-specific genes. Our data suggest that histone H1c gene expression is developmentally up-regulated to promote facultative heterochromatin in mature rod photoreceptors.

Popova, Evgenya Y.; Grigoryev, Sergei A.; Fan, Yuhong; Skoultchi, Arthur I.; Zhang, Samuel S.; Barnstable, Colin J.



Higher chromatin mobility supports totipotency and precedes pluripotency in vivo.  


The fusion of the gametes upon fertilization results in the formation of a totipotent cell. Embryonic chromatin is expected to be able to support a large degree of plasticity. However, whether this plasticity relies on a particular conformation of the embryonic chromatin is unknown. Moreover, whether chromatin plasticity is functionally linked to cellular potency has not been addressed. Here, we adapted fluorescence recovery after photobleaching (FRAP) in the developing mouse embryo and show that mobility of the core histones H2A, H3.1, and H3.2 is unusually high in two-cell stage embryos and decreases as development proceeds. The transition toward pluripotency is accompanied by a decrease in histone mobility, and, upon lineage allocation, pluripotent cells retain higher mobility than the differentiated trophectoderm. Importantly, totipotent two-cell-like embryonic stem cells also display high core histone mobility, implying that reprogramming toward totipotency entails changes in chromatin mobility. Our data suggest that changes in chromatin dynamics underlie the transitions in cellular plasticity and that higher chromatin mobility is at the nuclear foundations of totipotency. PMID:24831699

Boškovi?, Ana; Eid, André; Pontabry, Julien; Ishiuchi, Takashi; Spiegelhalter, Coralie; Raghu Ram, Edupuganti V S; Meshorer, Eran; Torres-Padilla, Maria-Elena



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.

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



Predicting enhancer transcription and activity from chromatin modifications  

PubMed Central

Enhancers play a pivotal role in regulating the transcription of distal genes. Although certain chromatin features, such as the histone acetyltransferase P300 and the histone modification H3K4me1, indicate the presence of enhancers, only a fraction of enhancers are functionally active. Individual chromatin marks, such as H3K27ac and H3K27me3, have been identified to distinguish active from inactive enhancers. However, the systematic identification of the most informative single modification, or combination thereof, is still lacking. Furthermore, the discovery of enhancer RNAs (eRNAs) provides an alternative approach to directly predicting enhancer activity. However, it remains challenging to link chromatin modifications to eRNA transcription. Herein, we develop a logistic regression model to unravel the relationship between chromatin modifications and eRNA synthesis. We perform a systematic assessment of 24 chromatin modifications in fetal lung fibroblast and demonstrate that a combination of four modifications is sufficient to accurately predict eRNA transcription. Furthermore, we compare the ability of eRNAs and H3K27ac to discriminate enhancer activity. We demonstrate that eRNA is more indicative of enhancer activity. Finally, we apply our fibroblast trained model to six other cell-types and successfully predict eRNA synthesis. Thus, we demonstrate the learned relationships are general and independent of cell-type. We provided a powerful tool to identify active enhancers and reveal the relationship between chromatin modifications, eRNA production and enhancer activity.

Zhu, Yun; Sun, Lin; Chen, Zhao; Whitaker, John W.; Wang, Tao; Wang, Wei



Integrative annotation of chromatin elements from ENCODE data  

PubMed Central

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 regulatory elements, their interrelations contain much richer information for the systematic annotation of regulatory elements. To uncover these interrelations and to generate an interpretable summary of the massive datasets of the ENCODE Project, we apply unsupervised learning methodologies, converting dozens of chromatin datasets into discrete annotation maps of regulatory regions and other chromatin elements across the human genome. These methods rediscover and summarize diverse aspects of chromatin architecture, elucidate the interplay between chromatin activity and RNA transcription, and reveal that a large proportion of the genome lies in a quiescent state, even across multiple cell types. The resulting annotation of non-coding regulatory elements correlate strongly with mammalian evolutionary constraint, and provide an unbiased approach for evaluating metrics of evolutionary constraint in human. Lastly, we use the regulatory annotations to revisit previously uncharacterized disease-associated loci, resulting in focused, testable hypotheses through the lens of the chromatin landscape.

Hoffman, Michael M.; Ernst, Jason; Wilder, Steven P.; Kundaje, Anshul; Harris, Robert S.; Libbrecht, Max; Giardine, Belinda; Ellenbogen, Paul M.; Bilmes, Jeffrey A.; Birney, Ewan; Hardison, Ross C.; Dunham, Ian; Kellis, Manolis; Noble, William Stafford



Organization of chromatin and histone modifications at a transcription site  

PubMed Central

According to the transcription factory model, localized transcription sites composed of immobilized polymerase molecules transcribe chromatin by reeling it through the transcription site and extruding it to form a surrounding domain of recently transcribed decondensed chromatin. Although transcription sites have been identified in various cells, surrounding domains of recently transcribed decondensed chromatin have not. We report evidence that transcription sites associated with a tandem gene array in mouse cells are indeed surrounded by or adjacent to a domain of decondensed chromatin composed of sequences from the gene array. Formation of this decondensed domain requires transcription and topoisomerase II? activity. The decondensed domain is enriched for the trimethyl H3K36 mark that is associated with recently transcribed chromatin in yeast and several mammalian systems. Consistent with this, chromatin immunoprecipitation demonstrates a comparable enrichment of this mark in transcribed sequences at the tandem gene array. These results provide new support for the pol II factory model, in which an immobilized polymerase molecule extrudes decondensed, transcribed sequences into its surroundings.

Muller, Waltraud G.; Rieder, Dietmar; Karpova, Tatiana S.; John, Sam; Trajanoski, Zlatko; McNally, James G.



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.

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



On the physical and chemical dynamics of chromatin  

NASA Astrophysics Data System (ADS)

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

Apratim, Manjul


Evidence for two classes of chromatin-associated Epstein-Barr virus-determined nuclear antigen.  

PubMed Central

A new class of Epstein-Barr virus nuclear antigen (EBNA) was identified by the complement fixation assay. This new species of EBNA is more tightly bound to chromatin and was termed class II EBNA, as opposed to the more weakly associated species, class I EBNA. Preparations of this new antigen(s) specifically reduced absorption with the titer of anti-EBNA antibodies as determined by the anticomplement immunofluorescence assay. Therefore, the complement fixation antigens (class II EBNA) appear to be related to the classical EBNA (class I EBNA). The class I EBNA was found to focus at the same pH (4.6) as the soluble antigen found in the cytosol. The class II EBNA differed from the class I EBNA with regard to its overall charge, molecular size, antigenicity, and affinity for chromatin. The class II EBNA appeared to be a basic protein, based on its apparent pI of 9.2 and its binding to cation-exchange resins. It differed from histones with regard to its molecular size (molecular weight between 60,000 and 70,000) and its elution from hydroxylapatite chromatography. Steps were taken to prevent proteolysis and artifacts in the immunological assays and in the overall charge estimation of the new antigen by nonspecific basic histone protein-acidic protein interactions. Both class I and class II EBNA were identified by radioimmunoelectrophoresis on two-dimensional polyacrylamide gels with pI values of 5.0 and 8.5, respectively, and a molecular weight range of 60,000 to 70,000 for both. A lower-molecular-weight antigen identified by molecular sieve chromatography appeared to be due to interference by histones in the immunoassays since it was not observed by the two-dimensional gel electrophoresis. Further characterization of this class II EBNA is in progress. Images

Spelsberg, T C; Sculley, T B; Pikler, G M; Gilbert, J A; Pearson, G R



The rad9 gene of Coprinus cinereus encodes a proline-rich protein required for meiotic chromosome condensation and synapsis  

SciTech Connect

The rad9 gene of Coprinus cinereus is essential for the normal completion of meiosis. We examined surface-spread preparations of wild-type and rad9-1 nuclei from the meiotic stages of karyogamy through metaphase I, and we determined the primary sequence, structure, and meiotic expression of the rad9 gene. In wild-type C. cinereus, karyogamy is followed by condensation and alignment of homologous chromosomes. Condensation and axial core development largely precede synapsis, which often initiates at telomeres. A diffuse diplotene phase coincides with dissolution of the synaptonemal complex, and subsequently chromosomes further condense as the cells progress into metaphase I. In contrast, although karyogamy and nucleolar fusion are apparently normal in rad9-1 basidia, only short stretches of synaptonemal complex form. These correlate with stretches of condensed chromatin, mostly at apparent chromosome ends, and regions of presumptive triple synapsis are numerous. rad9-1 basidia enter the diffuse stages of early diplotene, and then 50% of these cells enter metaphase I by the criteria of nucleolar elimination and at least some chromatin condensation. rad9 gene expression is induced after gamma irradiation and during meiosis. The gene has 27 exons and encodes a predicted protein of 2157 amino acids, with a proline-rich amino terminus. 62 refs., 10 figs.

Seitz, L.C.; Tang, Keliang; Cummings, W.J.; Zolan, M.E. [Indiana Univ., Bloomington, IN (United States)



Sperm FISH and chromatin integrity in spermatozoa from a t(6;10;11) carrier.  


Complex chromosome rearrangements (CCRs) are structurally balanced or unbalanced aberrations involving more than two breakpoints on two or more chromosomes. CCRs can be a potential reason for genomic imbalance in gametes, which leads to a drastic reduction in fertility. In this study, the meiotic segregation pattern, aneuploidy of seven chromosomes uninvolved in the CCR and chromatin integrity were analysed in the ejaculated spermatozoa of a 46,XY,t(6;10;11)(q25.1;q24.3;q23.1)mat carrier with asthenozoospermia and a lack of conception. The frequency of genetically unbalanced spermatozoa was 78.8% with a prevalence of 4:2 segregants of 38.2%, while the prevalence of the adjacent 3:3 mode was 35.3%. Analysis of the aneuploidy of chromosomes 13, 15, 18, 21, 22, X and Y revealed an approximately fivefold increased level in comparison with that of the control group, indicating the presence of an interchromosomal effect. Sperm chromatin integrity status was evaluated using chromomycin A3 and aniline blue staining (deprotamination), acridine orange test and TUNEL assay (sperm DNA fragmentation). No differences were found when comparisons were made with a control group. We suggest that the accumulation of genetically unbalanced spermatozoa, significantly increased sperm aneuploidy level and decreased sperm motility (20%, progressive) were not responsible for the observed lack of reproductive success in the analysed infertile t(6;10;11) carrier. Interestingly, in the case described herein, a high level of sperm chromosomal imbalance appears not to be linked to sperm chromatin integrity status. PMID:24713394

Olszewska, Marta; Huleyuk, Nataliya; Fraczek, Monika; Zastavna, Danuta; Wiland, Ewa; Kurpisz, Maciej



Condensation Processes in Geothermal Systems  

NASA Astrophysics Data System (ADS)

We model condensation processes in geothermal systems to understand how this process changes fluid chemistry. We assume two processes operate in geothermal systems: 1) condensation of a vapor phase derived by boiling an aqueous geothermal fluid into a cool near surface water and 2) condensation of a magmatic vapor by a deep circulating meteoric thermal fluid. It is assumed that the condensation process has two stages. Initially the condensing fluid is under saturated in gaseous species. Condensation of the vapor phase continues until the pressure on the fluid equals the sum of the partial pressures of water and the dissolved gaseous species. At that time bubbles flux through the condensing fluid. In time the fluid and fluxing gas phase come to equilibrium. Calculation shows that during the second stage of the condensation process the liquid phase becomes enriched in more soluble gaseous species like CO2 and H2S, and depleted in less soluble species like CH4 and N2. Stage 2 condensation processes can therefore be monitored by ratios of more and less condensable species like CO2/N2. Condensation of vapor released by boiling geothermal fluids results in liquids with high concentrations of H2S and CO2 like is seen in geothermal system steam-heated waters. Condensation of a magmatic vapor into circulating meteoric water has been proposed, but not well demonstrated. We compare to our models the Cerro Prieto, Mexico gas analysis data set collected over twelve years time by USGS personnel. It was assumed for modeling that the Cerro Prieto geothermal fluids are circulating meteoritic fluids with N2/Ar ratios about 40 to which is added a magmatic vapor with N2/Ar ratio = 400. The Cerro Prieto analyses show a strong correlation between N2/Ar and CO2/N2 as predicted by calculation. Two dimensional image plots of well N2/Ar + CO2/N2 show a bull's-eye pattern on the geothermal field. Image plots of analyses collected over a year or less time show N2/Ar and CO2/N2 hot spots. Plotting data for individual wells show a hysteresis like loops on time vs. CO2/N2 diagrams. Our analysis demonstrates that condensation of magmatic vapor into convecting meteoric waters is a viable process. Condensation explains variations in Cerro Prieto geothermal system gas chemistry and is compatible with helium isotope data. Locally condensation appears to wax and wane over a time periods of about 10 years.

Norman, D. I.; Moore, J. N.



Microgravity condensing heat exchanger  

NASA Technical Reports Server (NTRS)

A heat exchanger having a plurality of heat exchanging aluminum fins with hydrophilic condensing surfaces which are stacked and clamped between two cold plates. The cold plates are aligned radially along a plane extending through the axis of a cylindrical duct and hold the stacked and clamped portions of the heat exchanging fins along the axis of the cylindrical duct. The fins extend outwardly from the clamped portions along approximately radial planes. The spacing between fins is symmetric about the cold plates, and are somewhat more closely spaced as the angle they make with the cold plates approaches Passageways extend through the fins between vertex spaces which provide capillary storage and communicate with passageways formed in the stacked and clamped portions of the fins, which communicate with water drains connected to a pump externally to the duct. Water with no entrained air is drawn from the capillary spaces.

Thomas, Christopher M. (Inventor); Ma, Yonghui (Inventor); North, Andrew (Inventor); Weislogel, Mark M. (Inventor)



Rejoining and misrejoining of radiation-induced chromatin breaks. IV. Charged particles  

NASA Technical Reports Server (NTRS)

We have recently reported the kinetics of chromosome rejoining and exchange formation in human lymphocytes exposed to gamma rays using the techniques of fluorescence in situ hybridization (FISH) and premature chromosome condensation (PCC). In this paper, we have extended previous measurements to cells exposed to charged particles. Our goal was to determine differences in chromatin break rejoining and misrejoining after exposure to low- and high-linear energy transfer (LET) radiation. Cells were irradiated with hydrogen, neon, carbon or iron ions in the LET range 0.3-140 keV/microm and were incubated at 37 degrees C for various times after exposure. Little difference was observed in the yield of early prematurely condensed chromosome breaks for the different ions. The kinetics of break rejoining was exponential for all ions and had similar time constants, but the residual level of unrejoined breaks after prolonged incubation was higher for high-LET radiation. The kinetics of exchange formation was also similar for the different ions, but the yield of chromosome interchanges measured soon after exposure was higher for high-LET particles, suggesting that a higher fraction of DNA breaks are misrejoined quickly. On the other hand, the rate of formation of complete exchanges was slightly lower for densely ionizing radiation. The ratios between the yields of different types of aberrations observed at 10 h postirradiation in prematurely condensed chromosome preparations were dependent on LET. We found significant differences between the yields of aberrations measured in interphase (after repair) and metaphase for densely ionizing radiation. This difference might be caused by prolonged mitotic delay and/or interphase death. Overall, the results point out significant differences between low- and high-LET radiation for the formation of chromosome aberrations.

Durante, M.; Furusawa, Y.; George, K.; Gialanella, G.; Greco, O.; Grossi, G.; Matsufuji, N.; Pugliese, M.; Yang, T. C.



Condensed Explosive Gas Dynamic Laser.  

National Technical Information Service (NTIS)

The condensed explosive of a gas dynamic laser is a condensed mixture of one or more nonhydrogenous organic explosive compounds, such as TNM, with a sufficient amount of aluminum or zirconium powder to supply energy to the products so that a temperature o...

J. Hershkowitz M. Y. D. Lanzerotti



Amine catalyzed condensation of tetraethylorthosilicate  

NASA Technical Reports Server (NTRS)

The catalysis of the condensation of hydrolyzed metal alkoxides by amines has been mentioned in the literature, but there has been no systematic study of their influence on the rate of the condensation reaction of the alkoxide and the microstructure of the resultant gel.

Jones, S.



Counterion condensation on ionic oligomers  

NASA Astrophysics Data System (ADS)

The Ramanathan-Woodbury formulas representing the charge density critical for the onset of counterion condensation on finite-length polymers are derived by three alternate methods, an extension of Debye-Huckel theory, a theory of end effects, and by density functional theory. For charged oligomers with length of the same order as the Debye length, the threshold for condensation is the same as for polymers of length much greater than the Debye lenght. However, the threshold depends both on length and salt concentration if the oligomer is shorter than the Debye length, in such a way as to recede to infinity as the ratio of oligomer length to Debye length tends to zero (i.e., condensation vanishes in this limit). The extended Debye-Huckel theory additionally provides a new result for the partition function of the condensed layer, showing that the free energy of the condensed counterions is different on an oligomer and a polymer, even when the fractional extent of condensation is the same. The end effect theory discloses a hitherto unnoticed connection between the number of counterions condensed at the ends of a long polymer and the number condensed on a short oligomer.

Manning, Gerald S.; Mohanty, Udayan



Interphase chromatin organisation in Arabidopsis nuclei: constraints versus randomness.  


The spatial chromatin organisation and molecular interactions within and between chromatin domains and chromosome territories (CTs) are essential for fundamental processes such as replication, transcription and DNA repair via homologous recombination. To analyse the distribution and interaction of whole CTs, centromeres, (sub)telomeres and ~100-kb interstitial chromatin segments in endopolyploid nuclei, specific FISH probes from Arabidopsis thaliana were applied to 2-64C differentiated leaf nuclei. Whereas CTs occupy a distinct and defined volume of the nucleus and do not obviously intermingle with each other in 2-64C nuclei, ~100-kb sister chromatin segments within these CTs become more non-cohesive with increasing endopolyploidy. Centromeres, preferentially located at the nuclear periphery, may show ring- or half-moon like shapes in 2C and 4C nuclei. Sister centromeres tend to associate up to the 8C level. From 16C nuclei on, they become progressively separated. The higher the polyploidy level gets, the more s