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Sample records for acetylation curtails nucleosome

  1. Acetylation curtails nucleosome binding, not stable nucleosome remodeling, by FoxO1

    SciTech Connect

    Hatta, M.; Liu, F.; Cirillo, L.A.

    2009-02-20

    Transcriptional activity of FoxO factors is controlled through the actions of multiple growth factors signaling through protein kinase B, whereby phosphorylation of FoxO factors inhibits FoxO-mediated transactivation by promoting nuclear export. Phosphorylation of FoxO factors is enhanced by p300-mediated acetylation, which decreases their affinity for DNA. The negative effect of acetylation on FoxO DNA binding, together with nuclear FoxO mobility, is eliminated by over-expression of the de-acetylase Sirt1, suggesting that acetylation mobilizes FoxO factors in chromatin for inducible gene expression. Here, we show that acetylation significantly curtails the affinity of FoxO1 for its binding sites in nucleosomal DNA but has no effect on either stable nucleosome binding or remodeling by this factor. We suggest that, while acetylation provides a first, essential step toward mobilizing FoxO factors for inducible gene repression, additional mechanisms exist for overcoming their inherent capacity to stably bind and remodel nuclear chromatin.

  2. Nucleosome acetylation sequencing to study the establishment of chromatin acetylation.

    PubMed

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

    2014-07-15

    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.

  3. Acetylated histone H3 increases nucleosome dissociation

    NASA Astrophysics Data System (ADS)

    Simon, Marek; Manohar, Mridula; Ottesen, Jennifer; Poirier, Michael

    2009-03-01

    Chromatin's basic unit structure is the nucleosome, i.e. genomic DNA wrapped around a particular class of proteins -- histones -- which due to their physical hindrance, block vital biological processes, such as DNA repair, DNA replication, and RNA transcription. Histone post-translational modifications, which are known to exist in vivo, are hypothesized to regulate these biological processes by directly altering DNA-histone interactions and thus nucleosome structure and stability. Using magnetic tweezers technique we studied the acetylation of histone H3 in the dyad region, i.e. at K115 and K122, on reconstituted arrays of nucleosomes under constant external force. Based on the measured increase in the probability of dissociation of modified nucleosomes, we infer that this double modification could facilitate histone chaperone mediated nucleosome disassembly in vivo.

  4. SWI/SNF Displaces SAGA-Acetylated Nucleosomes

    PubMed Central

    Chandy, Mark; Gutiérrez, José L.; Prochasson, Philippe; Workman, Jerry L.

    2006-01-01

    SWI/SNF is a well-characterized chromatin remodeling complex that remodels chromatin by sliding nucleosomes in cis and/or displacing nucleosomes in trans. The latter mechanism has the potential to remove promoter nucleosomes, allowing access to transcription factors and RNA polymerase. In vivo, histone acetylation often precedes apparent nucleosome loss; therefore, we sought to determine whether nucleosomes containing acetylated histones could be displaced by the SWI/SNF chromatin remodeling complex. We found that SAGA-acetylated histones were lost from an immobilized nucleosome array when treated with the SWI/SNF complex. When the nucleosome array was acetylated by SAGA in the presence of bound transcription activators, it generated a peak of acetylation surrounding the activator binding sites. Subsequent SWI/SNF treatment suppressed this acetylation peak. Immunoblots indicated that SWI/SNF preferentially displaced acetylated histones from the array relative to total histones. Moreover, the Swi2/Snf2 bromodomain, an acetyl-lysine binding domain, played a role in the displacement of acetylated histones. These data indicate that targeted histone acetylation by the SAGA complex predisposes promoter nucleosomes for displacement by the SWI/SNF complex. PMID:17030999

  5. Histone Acetylation near the Nucleosome Dyad Axis Enhances Nucleosome Disassembly by RSC and SWI/SNF

    PubMed Central

    Chatterjee, Nilanjana; North, Justin A.; Dechassa, Mekonnen Lemma; Manohar, Mridula; Prasad, Rashmi; Luger, Karolin; Ottesen, Jennifer J.; Poirier, Michael G.

    2015-01-01

    Signaling associated with transcription activation occurs through posttranslational modification of histones and is best exemplified by lysine acetylation. Lysines are acetylated in histone tails and the core domain/lateral surface of histone octamers. While acetylated lysines in histone tails are frequently recognized by other factors referred to as “readers,” which promote transcription, the mechanistic role of the modifications in the lateral surface of the histone octamer remains unclear. By using X-ray crystallography, we found that acetylated lysines 115 and 122 in histone H3 are solvent accessible, but in biochemical assays they appear not to interact with the bromodomains of SWI/SNF and RSC to enhance recruitment or nucleosome mobilization, as previously shown for acetylated lysines in H3 histone tails. Instead, we found that acetylation of lysines 115 and 122 increases the predisposition of nucleosomes for disassembly by SWI/SNF and RSC up to 7-fold, independent of bromodomains, and only in conjunction with contiguous nucleosomes. Thus, in combination with SWI/SNF and RSC, acetylation of lateral surface lysines in the histone octamer serves as a crucial regulator of nucleosomal dynamics distinct from the histone code readers and writers. PMID:26416878

  6. Histone acetylation dependent energy landscapes in tri-nucleosome revealed by residue-resolved molecular simulations

    PubMed Central

    Chang, Le; Takada, Shoji

    2016-01-01

    Histone tail acetylation is a key epigenetic marker that tends to open chromatin folding and activate transcription. Despite intensive studies, precise roles of individual lysine acetylation in chromatin folding have only been poorly understood. Here, we revealed structural dynamics of tri-nucleosomes with several histone tail acetylation states and analyzed histone tail interactions with DNA by performing molecular simulations at an unprecedentedly high resolution. We found versatile acetylation-dependent landscapes of tri-nucleosome. The H4 and H2A tail acetylation reduced the contact between the first and third nucleosomes mediated by the histone tails. The H3 tail acetylation reduced its interaction with neighboring linker DNAs resulting in increase of the distance between consecutive nucleosomes. Notably, two copies of the same histone in a single nucleosome have markedly asymmetric interactions with DNAs, suggesting specific pattern of nucleosome docking albeit high inherent flexibility. Estimated transcription factor accessibility was significantly high for the H4 tail acetylated structures. PMID:27698366

  7. Nucleosome competition reveals processive acetylation by the SAGA HAT module

    PubMed Central

    Ringel, Alison E.; Cieniewicz, Anne M.; Taverna, Sean D.; Wolberger, Cynthia

    2015-01-01

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex hyperacetylates histone tails in vivo in a manner that depends upon histone 3 lysine 4 trimethylation (H3K4me3), a histone mark enriched at promoters of actively transcribed genes. SAGA contains a separable subcomplex known as the histone acetyltransferase (HAT) module that contains the HAT, Gcn5, bound to Sgf29, Ada2, and Ada3. Sgf29 contains a tandem Tudor domain that recognizes H3K4me3-containing peptides and is required for histone hyperacetylation in vivo. However, the mechanism by which H3K4me3 recognition leads to lysine hyperacetylation is unknown, as in vitro studies show no effect of the H3K4me3 modification on histone peptide acetylation by Gcn5. To determine how H3K4me3 binding by Sgf29 leads to histone hyperacetylation by Gcn5, we used differential fluorescent labeling of histones to monitor acetylation of individual subpopulations of methylated and unmodified nucleosomes in a mixture. We find that the SAGA HAT module preferentially acetylates H3K4me3 nucleosomes in a mixture containing excess unmodified nucleosomes and that this effect requires the Tudor domain of Sgf29. The H3K4me3 mark promotes processive, multisite acetylation of histone H3 by Gcn5 that can account for the different acetylation patterns established by SAGA at promoters versus coding regions. Our results establish a model for Sgf29 function at gene promoters and define a mechanism governing crosstalk between histone modifications. PMID:26401015

  8. Linker histone H1 and H3K56 acetylation are antagonistic regulators of nucleosome dynamics

    PubMed Central

    Bernier, Morgan; Luo, Yi; Nwokelo, Kingsley C.; Goodwin, Michelle; Dreher, Sarah J.; Zhang, Pei; Parthun, Mark R.; Fondufe-Mittendorf, Yvonne; Ottesen, Jennifer J.; Poirier, Michael G.

    2015-01-01

    H1 linker histones are highly abundant proteins that compact nucleosomes and chromatin to regulate DNA accessibility and transcription. However, the mechanisms that target H1 regulation to specific regions of eukaryotic genomes are unknown. Here we report fluorescence measurements of human H1 regulation of nucleosome dynamics and transcription factor (TF) binding within nucleosomes. H1 does not block TF binding, instead it suppresses nucleosome unwrapping to reduce DNA accessibility within H1-bound nucleosomes. We then investigated H1 regulation by H3K56 and H3K122 acetylation, two transcriptional activating histone post translational modifications (PTMs). Only H3K56 acetylation, which increases nucleosome unwrapping, abolishes H1.0 reduction of TF binding. These findings show that nucleosomes remain dynamic, while H1 is bound and H1 dissociation is not required for TF binding within the nucleosome. Furthermore, our H3K56 acetylation measurements suggest that a single-histone PTM can define regions of the genome that are not regulated by H1. PMID:26648124

  9. Linker histone H1 and H3K56 acetylation are antagonistic regulators of nucleosome dynamics.

    PubMed

    Bernier, Morgan; Luo, Yi; Nwokelo, Kingsley C; Goodwin, Michelle; Dreher, Sarah J; Zhang, Pei; Parthun, Mark R; Fondufe-Mittendorf, Yvonne; Ottesen, Jennifer J; Poirier, Michael G

    2015-12-09

    H1 linker histones are highly abundant proteins that compact nucleosomes and chromatin to regulate DNA accessibility and transcription. However, the mechanisms that target H1 regulation to specific regions of eukaryotic genomes are unknown. Here we report fluorescence measurements of human H1 regulation of nucleosome dynamics and transcription factor (TF) binding within nucleosomes. H1 does not block TF binding, instead it suppresses nucleosome unwrapping to reduce DNA accessibility within H1-bound nucleosomes. We then investigated H1 regulation by H3K56 and H3K122 acetylation, two transcriptional activating histone post translational modifications (PTMs). Only H3K56 acetylation, which increases nucleosome unwrapping, abolishes H1.0 reduction of TF binding. These findings show that nucleosomes remain dynamic, while H1 is bound and H1 dissociation is not required for TF binding within the nucleosome. Furthermore, our H3K56 acetylation measurements suggest that a single-histone PTM can define regions of the genome that are not regulated by H1.

  10. Effects of histone acetylation by Piccolo NuA4 on the structure of a nucleosome and the interactions between two nucleosomes.

    PubMed

    Lee, Ju Yeon; Wei, Sijie; Lee, Tae-Hee

    2011-04-01

    We characterized the effect of histone acetylation on the structure of a nucleosome and the interactions between two nucleosomes. In this study, nucleosomes reconstituted with the Selex "Widom 601" sequence were acetylated with the Piccolo NuA4 complex, which acetylates mainly H4 N-terminal tail lysine residues and some H2A/H3 N-terminal tail lysine residues. Upon the acetylation, we observed directional unwrapping of nucleosomal DNA that accompanies topology change of the DNA. Interactions between two nucleosomes in solution were also monitored to discover multiple transient dinucleosomal states that can be categorized to short-lived and long-lived (∼1 s) states. The formation of dinucleosomes is strongly Mg(2+)-dependent, and unacetylated nucleosomes favor the formation of long-lived dinucleosomes 4-fold as much as the acetylated ones. These results suggest that the acetylation of histones by Piccolo NuA4 disturbs not only the structure of a nucleosome but also the interactions between two nucleosomes. Lastly, we suggest a structural model for a stable dinucleosomal state where the two nucleosomes are separated by ∼2 nm face-to-face and rotated by 34° with respect to each other.

  11. A bromodomain–DNA interaction facilitates acetylation-dependent bivalent nucleosome recognition by the BET protein BRDT

    PubMed Central

    Miller, Thomas C. R.; Simon, Bernd; Rybin, Vladimir; Grötsch, Helga; Curtet, Sandrine; Khochbin, Saadi; Carlomagno, Teresa; Müller, Christoph W.

    2016-01-01

    Bromodomains are critical components of many chromatin modifying/remodelling proteins and are emerging therapeutic targets, yet how they interact with nucleosomes, rather than acetylated peptides, remains unclear. Using BRDT as a model, we characterized how the BET family of bromodomains interacts with site-specifically acetylated nucleosomes. Here we report that BRDT interacts with nucleosomes through its first (BD1), but not second (BD2) bromodomain, and that acetylated histone recognition by BD1 is complemented by a bromodomain–DNA interaction. Simultaneous DNA and histone recognition enhances BRDT's nucleosome binding affinity and specificity, and its ability to localize to acetylated chromatin in cells. Conservation of DNA binding in bromodomains of BRD2, BRD3 and BRD4, indicates that bivalent nucleosome recognition is a key feature of these bromodomains and possibly others. Our results elucidate the molecular mechanism of BRDT association with nucleosomes and identify structural features of the BET bromodomains that may be targeted for therapeutic inhibition. PMID:27991587

  12. Acetylation of Histone H3 Lysine 56 Regulates Replication-Coupled Nucleosome Assembly

    PubMed Central

    Li, Qing; Zhou, Hui; Wurtele, Hugo; Davies, Brian; Horazdovsky, Bruce; Verreault, Alain; Zhang, Zhiguo

    2008-01-01

    SUMMARY Chromatin assembly factor 1 (CAF-1) and Rtt106 participate in the deposition of newly synthesized histones onto replicating DNA to form nucleosomes. This process is critical for the maintenance of genome stability and inheritance of functionally specialized chromatin structures in proliferating cells. However, the molecular functions of the acetylation of newly synthesized histones in this DNA replication-coupled nucleosome assembly pathway remain enigmatic. Here we show that histone H3 acetylated at lysine 56 (H3K56Ac) is incorporated onto replicating DNA and, by increasing the binding affinity of CAF-1 and Rtt106 for histone H3, H3K56Ac enhances the ability of these histone chaperones to assemble DNA into nucleosomes. Genetic analysis indicates that H3K56Ac acts in a nonredundant manner with the acetylation of the N-terminal residues of H3 and H4 in nucleosome assembly. These results reveal a mechanism by which H3K56Ac regulates replication-coupled nucleosome assembly mediated by CAF-1 and Rtt106. PMID:18662540

  13. Replication-independent nucleosome exchange is enhanced by local and specific acetylation of histone H4

    PubMed Central

    Elliott, Giles O.; Murphy, Kevin J.; Hayes, Jeffrey J.; Thiriet, Christophe

    2013-01-01

    We used a novel single-cell strategy to examine the fate of histones during G2-phase. Consistent with previous results, we find that in G2-phase, the majority of nuclear histones are assembled into chromatin, whereas a small fraction comprises an unassembled pool. Small increases in the amount of histones within the free pool affect the extent of exchange, suggesting that the free pool is in dynamic equilibrium with chromatin proteins. Unexpectedly, acetylated H4 is preferentially partitioned to the unassembled pool. Although an increase in global histone acetylation did not affect overall nucleosome dynamics, an H4 containing lysine to glutamine substitutions as mimics of acetylation significantly increased the rate of exchange, but did not affect the acetylation state of neighbouring nucleosomes. Interestingly, transcribed regions are particularly predisposed to exchange on incorporation of H4 acetylation mimics compared with surrounding regions. Our results support a model whereby histone acetylation on K8 and K16 specifically marks nucleosomes for eviction, with histones being rapidly deacetylated on reassembly. PMID:23303778

  14. The RSC Complex Exploits Histone Acetylation to Abrogate the Nucleosomal Barrier to RNA Polymerase II Elongation

    PubMed Central

    Carey, Michael; Li, Bing; Workman, Jerry L.

    2007-01-01

    Summary The coordinated action of histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling enzymes in promoter-dependent transcription initiation represents a paradigm for how epigenetic information regulates gene expression. However, little is known about how such enzymes function during transcription elongation. Here we investigated the role of RSC, a bromodomain-containing ATPase, in nucleosome transcription in vitro. Purified S. cerevisiae RNA polymerase II (pol II) arrests at two primary locations on a positioned mononucleosome. RSC stimulates passage of pol II through these sites. The function of RSC in elongation requires the energy of ATP hydrolysis. Moreover, the SAGA and NuA4 HATs strongly stimulated RSC’s effect on elongation. The stimulation correlates closely with Acetyl-CoA-dependent recruitment of RSC to nucleosomes. Thus, RSC can recognize acetylated nucleosomes and facilitate passage of pol II through them. These data support the view that histone modifications regulate accessibility of the coding region to pol II. PMID:17081996

  15. Opposing roles of H3- and H4-acetylation in the regulation of nucleosome structure––a FRET study.

    PubMed

    Gansen, Alexander; Tóth, Katalin; Schwarz, Nathalie; Langowski, Jörg

    2015-02-18

    Using FRET in bulk and on single molecules, we assessed the structural role of histone acetylation in nucleosomes reconstituted on the 170 bp long Widom 601 sequence. We followed salt-induced nucleosome disassembly, using donor–acceptor pairs on the ends or in the internal part of the nucleosomal DNA, and on H2B histone for measuring H2A/H2B dimer exchange. This allowed us to distinguish the influence of acetylation on salt-induced DNA unwrapping at the entry–exit site from its effect on nucleosome core dissociation. The effect of lysine acetylation is not simply cumulative, but showed distinct histone-specificity. Both H3- and H4-acetylation enhance DNA unwrapping above physiological ionic strength; however, while H3-acetylation renders the nucleosome core more sensitive to salt-induced dissociation and to dimer exchange, H4-acetylation counteracts these effects. Thus, our data suggest, that H3- and H4-acetylation have partially opposing roles in regulating nucleosome architecture and that distinct aspects of nucleosome dynamics might be independently controlled by individual histones.

  16. Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription

    PubMed Central

    Di Cerbo, Vincenzo; Mohn, Fabio; Ryan, Daniel P; Montellier, Emilie; Kacem, Salim; Tropberger, Philipp; Kallis, Eleni; Holzner, Monika; Hoerner, Leslie; Feldmann, Angelika; Richter, Florian Martin; Bannister, Andrew J; Mittler, Gerhard; Michaelis, Jens; Khochbin, Saadi; Feil, Robert; Schuebeler, Dirk; Owen-Hughes, Tom; Daujat, Sylvain; Schneider, Robert

    2014-01-01

    Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states. DOI: http://dx.doi.org/10.7554/eLife.01632.001 PMID:24668167

  17. Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription.

    PubMed

    Di Cerbo, Vincenzo; Mohn, Fabio; Ryan, Daniel P; Montellier, Emilie; Kacem, Salim; Tropberger, Philipp; Kallis, Eleni; Holzner, Monika; Hoerner, Leslie; Feldmann, Angelika; Richter, Florian Martin; Bannister, Andrew J; Mittler, Gerhard; Michaelis, Jens; Khochbin, Saadi; Feil, Robert; Schuebeler, Dirk; Owen-Hughes, Tom; Daujat, Sylvain; Schneider, Robert

    2014-03-25

    Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states. DOI: http://dx.doi.org/10.7554/eLife.01632.001.

  18. Intra- and inter-nucleosomal interactions of the histone H4 tail revealed with a human nucleosome core particle with genetically-incorporated H4 tetra-acetylation

    PubMed Central

    Wakamori, Masatoshi; Fujii, Yoshifumi; Suka, Noriyuki; Shirouzu, Mikako; Sakamoto, Kensaku; Umehara, Takashi; Yokoyama, Shigeyuki

    2015-01-01

    Post-translational modifications (PTMs) of histones, such as lysine acetylation of the N-terminal tails, play crucial roles in controlling gene expression. Due to the difficulty in reconstituting site-specifically acetylated nucleosomes with crystallization quality, structural analyses of histone acetylation are currently performed using synthesized tail peptides. Through engineering of the genetic code, translation termination, and cell-free protein synthesis, we reconstituted human H4-mono- to tetra-acetylated nucleosome core particles (NCPs), and solved the crystal structures of the H4-K5/K8/K12/K16-tetra-acetylated NCP and unmodified NCP at 2.4 Å and 2.2 Å resolutions, respectively. The structure of the H4-tetra-acetylated NCP resembled that of the unmodified NCP, and the DNA wrapped the histone octamer as precisely as in the unmodified NCP. However, the B-factors were significantly increased for the peripheral DNAs near the N-terminal tail of the intra- or inter-nucleosomal H4. In contrast, the B-factors were negligibly affected by the H4 tetra-acetylation in histone core residues, including those composing the acidic patch, and at H4-R23, which interacts with the acidic patch of the neighboring NCP. The present study revealed that the H4 tetra-acetylation impairs NCP self-association by changing the interactions of the H4 tail with DNA, and is the first demonstration of crystallization quality NCPs reconstituted with genuine PTMs. PMID:26607036

  19. Genomewide analysis of nucleosome density histone acetylation and HDAC function in fission yeast.

    PubMed

    Wirén, Marianna; Silverstein, Rebecca A; Sinha, Indranil; Walfridsson, Julian; Lee, Hang-Mao; Laurenson, Patricia; Pillus, Lorraine; Robyr, Daniel; Grunstein, Michael; Ekwall, Karl

    2005-08-17

    We have conducted a genomewide investigation into the enzymatic specificity, expression profiles, and binding locations of four histone deacetylases (HDACs), representing the three different phylogenetic classes in fission yeast (Schizosaccharomyces pombe). By directly comparing nucleosome density, histone acetylation patterns and HDAC binding in both intergenic and coding regions with gene expression profiles, we found that Sir2 (class III) and Hos2 (class I) have a role in preventing histone loss; Clr6 (class I) is the principal enzyme in promoter-localized repression. Hos2 has an unexpected role in promoting high expression of growth-related genes by deacetylating H4K16Ac in their open reading frames. Clr3 (class II) acts cooperatively with Sir2 throughout the genome, including the silent regions: rDNA, centromeres, mat2/3 and telomeres. The most significant acetylation sites are H3K14Ac for Clr3 and H3K9Ac for Sir2 at their genomic targets. Clr3 also affects subtelomeric regions which contain clustered stress- and meiosis-induced genes. Thus, this combined genomic approach has uncovered different roles for fission yeast HDACs at the silent regions in repression and activation of gene expression.

  20. Bivalent interaction of the PZP domain of BRPF1 with the nucleosome impacts chromatin dynamics and acetylation

    PubMed Central

    Klein, Brianna J.; Muthurajan, Uma M.; Lalonde, Marie-Eve; Gibson, Matthew D.; Andrews, Forest H.; Hepler, Maggie; Machida, Shinichi; Yan, Kezhi; Kurumizaka, Hitoshi; Poirier, Michael G.; Côté, Jacques; Luger, Karolin; Kutateladze, Tatiana G.

    2016-01-01

    BRPF1 (bromodomain PHD finger 1) is a core subunit of the MOZ histone acetyltransferase (HAT) complex, critical for normal developmental programs and implicated in acute leukemias. BRPF1 contains a unique assembly of zinc fingers, termed a PZP domain, the physiological role of which remains unclear. Here, we elucidate the structure-function relationship of this novel epigenetic reader and detail the biological and mechanistic consequences of its interaction with nucleosomes. PZP has a globular architecture and forms a 2:1 stoichiometry complex with the nucleosome, bivalently interacting with histone H3 and DNA. This binding impacts the nucleosome dynamics, shifting the DNA unwrapping/rewrapping equilibrium toward the unwrapped state and increasing DNA accessibility. We demonstrate that the DNA-binding function of the BRPF1 PZP domain is required for the MOZ-BRPF1-ING5-hEaf6 HAT complex to be recruited to chromatin and to acetylate nucleosomal histones. Our findings reveal a novel link between chromatin dynamics and MOZ-mediated acetylation. PMID:26626149

  1. SMARCAD1 is an ATP-dependent stimulator of nucleosomal H2A acetylation via CBP, resulting in transcriptional regulation

    PubMed Central

    Doiguchi, Masamichi; Nakagawa, Takeya; Imamura, Yuko; Yoneda, Mitsuhiro; Higashi, Miki; Kubota, Kazuishi; Yamashita, Satoshi; Asahara, Hiroshi; Iida, Midori; Fujii, Satoshi; Ikura, Tsuyoshi; Liu, Ziying; Nandu, Tulip; Kraus, W. Lee; Ueda, Hitoshi; Ito, Takashi

    2016-01-01

    Histone acetylation plays a pivotal role in transcriptional regulation, and ATP-dependent nucleosome remodeling activity is required for optimal transcription from chromatin. While these two activities have been well characterized, how they are coordinated remains to be determined. We discovered ATP-dependent histone H2A acetylation activity in Drosophila nuclear extracts. This activity was column purified and demonstrated to be composed of the enzymatic activities of CREB-binding protein (CBP) and SMARCAD1, which belongs to the Etl1 subfamily of the Snf2 family of helicase-related proteins. SMARCAD1 enhanced acetylation by CBP of H2A K5 and K8 in nucleosomes in an ATP-dependent fashion. Expression array analysis of S2 cells having ectopically expressed SMARCAD1 revealed up-regulated genes. Using native genome templates of these up-regulated genes, we found that SMARCAD1 activates their transcription in vitro. Knockdown analysis of SMARCAD1 and CBP indicated overlapping gene control, and ChIP-seq analysis of these commonly controlled genes showed that CBP is recruited to the promoter prior to SMARCAD1. Moreover, Drosophila genetic experiments demonstrated interaction between SMARCAD1/Etl1 and CBP/nej during development. The interplay between the remodeling activity of SMARCAD1 and histone acetylation by CBP sheds light on the function of chromatin and the genome-integrity network. PMID:26888216

  2. Histone H3 lysine 14 (H3K14) acetylation facilitates DNA repair in a positioned nucleosome by stabilizing the binding of the chromatin Remodeler RSC (Remodels Structure of Chromatin).

    PubMed

    Duan, Ming-Rui; Smerdon, Michael J

    2014-03-21

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

  3. Histone H4 K16Q mutation, an acetylation mimic, causes structural disorder of its N-terminal basic patch in the nucleosome.

    PubMed

    Zhou, Bing-Rui; Feng, Hanqiao; Ghirlando, Rodolfo; Kato, Hidenori; Gruschus, James; Bai, Yawen

    2012-08-03

    Histone tails and their posttranslational modifications play important roles in regulating the structure and dynamics of chromatin. For histone H4, the basic patch K(16)R(17)H(18)R(19) in the N-terminal tail modulates chromatin compaction and nucleosome sliding catalyzed by ATP-dependent ISWI chromatin remodeling enzymes while acetylation of H4 K16 affects both functions. The structural basis for the effects of this acetylation is unknown. Here, we investigated the conformation of histone tails in the nucleosome by solution NMR. We found that backbone amides of the N-terminal tails of histones H2A, H2B, and H3 are largely observable due to their conformational disorder. However, only residues 1-15 in H4 can be detected, indicating that residues 16-22 in the tails of both H4 histones fold onto the nucleosome core. Surprisingly, we found that K16Q mutation in H4, a mimic of K16 acetylation, leads to a structural disorder of the basic patch. Thus, our study suggests that the folded structure of the H4 basic patch in the nucleosome is important for chromatin compaction and nucleosome remodeling by ISWI enzymes while K16 acetylation affects both functions by causing structural disorder of the basic patch K(16)R(17)H(18)R(19). Published by Elsevier Ltd.

  4. Histone H3K56 Acetylation, CAF1, and Rtt106 Coordinate Nucleosome Assembly and Stability of Advancing Replication Forks

    PubMed Central

    Clemente-Ruiz, Marta; González-Prieto, Román; Prado, Félix

    2011-01-01

    Chromatin assembly mutants accumulate recombinogenic DNA damage and are sensitive to genotoxic agents. Here we have analyzed why impairment of the H3K56 acetylation-dependent CAF1 and Rtt106 chromatin assembly pathways, which have redundant roles in H3/H4 deposition during DNA replication, leads to genetic instability. We show that the absence of H3K56 acetylation or the simultaneous knock out of CAF1 and Rtt106 increases homologous recombination by affecting the integrity of advancing replication forks, while they have a minor effect on stalled replication fork stability in response to the replication inhibitor hydroxyurea. This defect in replication fork integrity is not due to defective checkpoints. In contrast, H3K56 acetylation protects against replicative DNA damaging agents by DNA repair/tolerance mechanisms that do not require CAF1/Rtt106 and are likely subsequent to the process of replication-coupled nucleosome deposition. We propose that the tight connection between DNA synthesis and histone deposition during DNA replication mediated by H3K56ac/CAF1/Rtt106 provides a mechanism for the stabilization of advancing replication forks and the maintenance of genome integrity, while H3K56 acetylation has an additional, CAF1/Rtt106-independent function in the response to replicative DNA damage. PMID:22102830

  5. Piccolo NuA4-catalyzed acetylation of nucleosomal histones: critical roles of an Esa1 Tudor/chromo barrel loop and an Epl1 enhancer of polycomb A (EPcA) basic region.

    PubMed

    Huang, Jiehuan; Tan, Song

    2013-01-01

    Piccolo NuA4 is an essential yeast histone acetyltransferase (HAT) complex that targets histones H4 and H2A in nucleosome substrates. While Piccolo NuA4's catalytic subunit Esa1 alone is unable to acetylate nucleosomal histones, its accessory subunits, Yng2 and Epl1, enable Esa1 to bind to and to act on nucleosomes. We previously determined that the Tudor domain of Esa1 and the EPcA homology domain of Epl1 play critical roles in Piccolo NuA4's ability to act on the nucleosome. In this work, we pinpoint a loop within the Esa1 Tudor domain and a short basic region at the N terminus of the Epl1 EPcA domain as necessary for this nucleosomal HAT activity. We also show that this Esa1 Tudor domain loop region is positioned close to nucleosomal DNA and that the Epl1 EPcA basic region is in proximity to the N-terminal histone H2A tail, the globular region of histone H4, and also to nucleosomal DNA when Piccolo NuA4 interacts with the nucleosome. Since neither region identified is required for Piccolo NuA4 to bind to nucleosomes and yet both are needed to acetylate nucleosomes, these regions may function after the enzyme binds nucleosomes to disengage substrate histone tails from nucleosomal DNA.

  6. A Statistical Thermodynamic Model Applied to Experimental AFM Population and Location Data Is Able to Quantify DNA-Histone Binding Strength and Internucleosomal Interaction Differences between Acetylated and Unacetylated Nucleosomal Arrays

    PubMed Central

    Solis, F. J.; Bash, R.; Yodh, J.; Lindsay, S. M.; Lohr, D.

    2004-01-01

    Imaging of nucleosomal arrays by atomic force microscopy allows a determination of the exact statistical distributions for the numbers of nucleosomes per array and the locations of nucleosomes on the arrays. This precision makes such data an excellent reference for testing models of nucleosome occupation on multisite DNA templates. The approach presented here uses a simple statistical thermodynamic model to calculate theoretical population and positional distributions and compares them to experimental distributions previously determined for 5S rDNA nucleosomal arrays (208-12,172-12). The model considers the possible locations of nucleosomes on the template, and takes as principal parameters an average free energy of interaction between histone octamers and DNA, and an average wrapping length of DNA around the octamers. Analysis of positional statistics shows that it is possible to consider interactions between nucleosomes and positioning effects as perturbations on a random positioning noninteracting model. Analysis of the population statistics is used to determine histone-DNA association constants and to test for differences in the free energies of nucleosome formation with different types of histone octamers, namely acetylated or unacetylated, and different DNA templates, namely 172-12 or 208-12 5S rDNA multisite templates. The results show that the two template DNAs bind histones with similar affinities but histone acetylation weakens the association of histones with both templates. Analysis of locational statistics is used to determine the strength of specific nucleosome positioning tendencies by the DNA templates, and the strength of the interactions between neighboring nucleosomes. The results show only weak positioning tendencies and that unacetylated nucleosomes interact much more strongly with one another than acetylated nucleosomes; in fact acetylation appears to induce a small anticooperative occupation effect between neighboring nucleosomes. PMID

  7. Nucleosome Histone Tail Conformation and Dynamics: Impacts of Lysine Acetylation and a Nearby Minor Groove Benzo[a]pyrene-Derived Lesion.

    PubMed

    Fu, Iwen; Cai, Yuqin; Geacintov, Nicholas E; Zhang, Yingkai; Broyde, Suse

    2017-04-11

    Histone tails in nucleosomes play critical roles in regulation of many biological processes, including chromatin compaction, transcription, and DNA repair. Moreover, post-translational modifications, notably lysine acetylation, are crucial to these functions. While the tails have been intensively studied, how the structures and dynamics of tails are impacted by the presence of a nearby bulky DNA lesion is a frontier research area, and how these properties are impacted by tail lysine acetylation remains unexplored. To obtain molecular insight, we have utilized all atom 3 μs molecular dynamics simulations of nucleosome core particles (NCPs) to determine the impact of a nearby DNA lesion, 10S (+)-trans-anti-B[a]P-N(2)-dG-the major adduct derived from the procarcinogen benzo[a]pyrene-on H2B tail behavior in unacetylated and acetylated states. We similarly studied lesion-free NCPs to investigate the normal properties of the H2B tail in both states. In the lesion-free NCPs, charge neutralization upon lysine acetylation causes release of the tail from the DNA. When the lesion is present, it stably engulfs part of the nearby tail, impairing the interactions between DNA and tail. With the tail in an acetylated state, the lesion still interacts with part of it, although unstably. The lesion's partial entrapment of the tail should hinder the tail from interacting with other nucleosomes, and other proteins such as acetylases, deacetylases, and acetyl-lysine binding proteins, and thus disrupt critical tail-governed processes. Hence, the lesion would impede tail functions modulated by acetylation or deacetylation, causing aberrant chromatin structures and impaired biological transactions such as transcription and DNA repair.

  8. Functional Organization of the Yeast SAGA Complex: Distinct Components Involved in Structural Integrity, Nucleosome Acetylation, and TATA-Binding Protein Interaction

    PubMed Central

    Sterner, David E.; Grant, Patrick A.; Roberts, Shannon M.; Duggan, Laura J.; Belotserkovskaya, Rimma; Pacella, Lisa A.; Winston, Fred; Workman, Jerry L.; Berger, Shelley L.

    1999-01-01

    SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription in vivo and possesses histone acetylation function. Here we report both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex. We demonstrate a correlation between the phenotypic severity of SAGA mutants and SAGA structural integrity. Specifically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtle structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complex and cause severe phenotypes. Interestingly, double mutants (gcn5Δ spt3Δ and gcn5Δ spt8Δ) causing loss of a member of each of the moderate classes have severe phenotypes, similar to spt7Δ, spt20Δ, or ada1Δ mutants. In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and find that first, normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromodomain. Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo. Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro. Overall, our data demonstrate that SAGA harbors multiple, distinct transcription-related functions, including direct TBP interaction and nucleosomal histone acetylation. Loss of either of these causes slight impairment in vivo, but loss of both is highly detrimental to growth and transcription. PMID:9858534

  9. Electricity demand curtailment planning

    SciTech Connect

    Allentuck, J; Carroll, O; Schnader, M

    1980-01-01

    The state of electricity demand curtailment planning for long term electricity supply disruptions is reviewed. Legal, institutional and technological problems associated with demand curtailment plans are examined, and the existence of well defined social objectives on the part of planners is questioned. A linear programming approach to electricity demand curtailment planning is presented.

  10. H3 Histone Tail Conformation within the Nucleosome and the Impact of K14 Acetylation Studied Using Enhanced Sampling Simulation

    PubMed Central

    Ikebe, Jinzen; Sakuraba, Shun; Kono, Hidetoshi

    2016-01-01

    Acetylation of lysine residues in histone tails is associated with gene transcription. Because histone tails are structurally flexible and intrinsically disordered, it is difficult to experimentally determine the tail conformations and the impact of acetylation. In this work, we performed simulations to sample H3 tail conformations with and without acetylation. The results show that irrespective of the presence or absence of the acetylation, the H3 tail remains in contact with the DNA and assumes an α-helix structure in some regions. Acetylation slightly weakened the interaction between the tail and DNA and enhanced α-helix formation, resulting in a more compact tail conformation. We inferred that this compaction induces unwrapping and exposure of the linker DNA, enabling DNA-binding proteins (e.g., transcription factors) to bind to their target sequences. In addition, our simulation also showed that acetylated lysine was more often exposed to the solvent, which is consistent with the fact that acetylation functions as a post-translational modification recognition site marker. PMID:26967163

  11. Acetylation of LYS-16 of H4 Histone Tail May Sequester the Tail and Inhibit its Interactions with Neighboring Nucleosomes

    NASA Astrophysics Data System (ADS)

    Potoyan, Davit; Papoian, Garegin

    2012-02-01

    Histone tails are highly flexible N terminal protrusions of histone proteins, which help to fold DNA into dense superstructures known as chromatin. On a molecular scale histone tails are poly-electrolites with high degree of conformational disorder, allowing them to function as bio-molecular ``switches,'' regulating various genetic regulatory processes via diverse types of covalent modifications. Because of being intrinsically disordered, the structural and dynamical aspects of histone tails are still poorly understood. Using multiple explicit solvent and coarse-grained MD simulations we have investigated the impact of the acetylation of LYS-16 residue on the conformational and DNA-binding propensities of H4 histone tail. The potential of mean force computed as a function of distance between a model DNA and histone tail center of mass showed a dramatic enhancement of binding affinity upon mono-acetylation of the H4 tail. The estimated binding free energy gain for the wild type is 2kT, while for the acetylated it reaches 4-5 kT. Additionally our structural analysis shows that acetylation is driving the chain into collapsed states, which get enriched in secondary structural elements upon binding to the DNA. We suggest a non-electrostatic mechanism that explains the enhanced binding affinity of the acetylated H4 tail. At last our findings lead us to propose a hypothesis that can potentially account for the celebrated chromatin ``fiber loosening effects'' observed in many experiments.

  12. Drought Water Right Curtailment

    NASA Astrophysics Data System (ADS)

    Walker, W.; Tweet, A.; Magnuson-Skeels, B.; Whittington, C.; Arnold, B.; Lund, J. R.

    2016-12-01

    California's water rights system allocates water based on priority, where lower priority, "junior" rights are curtailed first in a drought. The Drought Water Rights Allocation Tool (DWRAT) was developed to integrate water right allocation models with legal objectives to suggest water rights curtailments during drought. DWRAT incorporates water right use and priorities with a flow-forecasting model to mathematically represent water law and hydrology and suggest water allocations among water rights holders. DWRAT is compiled within an Excel workbook, with an interface and an open-source solver. By implementing California water rights law as an algorithm, DWRAT provides a precise and transparent framework for the complicated and often controversial technical aspects of curtailing water rights use during drought. DWRAT models have been developed for use in the Eel, Russian, and Sacramento river basins. In this study, an initial DWRAT model has been developed for the San Joaquin watershed, which incorporates all water rights holders in the basin and reference gage flows for major tributaries. The San Joaquin DWRAT can assess water allocation reliability by determining probability of rights holders' curtailment for a range of hydrologic conditions. Forecasted flow values can be input to the model to provide decision makers with the ability to make curtailment and water supply strategy decisions. Environmental flow allocations will be further integrated into the model to protect and improve ecosystem water reliability.

  13. Nucleosome Switches

    NASA Astrophysics Data System (ADS)

    Schwab, David J.; Bruinsma, Robijn F.; Rudnick, Joseph; Widom, Jonathan

    2008-06-01

    We present a statistical-mechanical model for the positioning of nucleosomes along genomic DNA molecules as a function of the strength of the binding potential and the chemical potential of the nucleosomes. We show that a significant section of the DNA is composed of two-level nucleosome switching regions where the nucleosome distribution undergoes a localized, first-order transition. The location of the nucleosome switches shows a strong correlation with the location of gene-regulation regions.

  14. Nucleosome switches.

    PubMed

    Schwab, David J; Bruinsma, Robijn F; Rudnick, Joseph; Widom, Jonathan

    2008-06-06

    We present a statistical-mechanical model for the positioning of nucleosomes along genomic DNA molecules as a function of the strength of the binding potential and the chemical potential of the nucleosomes. We show that a significant section of the DNA is composed of two-level nucleosome switching regions where the nucleosome distribution undergoes a localized, first-order transition. The location of the nucleosome switches shows a strong correlation with the location of gene-regulation regions.

  15. NuA4-dependent Acetylation of Nucleosomal Histones H4 and H2A Directly Stimulates Incorporation of H2A.Z by the SWR1 Complex*

    PubMed Central

    Altaf, Mohammed; Auger, Andréanne; Monnet-Saksouk, Julie; Brodeur, Joëlle; Piquet, Sandra; Cramet, Myriam; Bouchard, Nathalie; Lacoste, Nicolas; Utley, Rhea T.; Gaudreau, Luc; Côté, Jacques

    2010-01-01

    Structural and functional analyses of nucleosomes containing histone variant H2A.Z have drawn a lot of interest over the past few years. Important work in budding yeast has shown that H2A.Z (Htz1)-containing nucleosomes are specifically located on the promoter regions of genes, creating a specific chromatin structure that is poised for disassembly during transcription activation. The SWR1 complex is responsible for incorporation of Htz1 into nucleosomes through ATP-dependent exchange of canonical H2A-H2B dimers for Htz1-H2B dimers. Interestingly, the yeast SWR1 complex is functionally linked to the NuA4 acetyltransferase complex in vivo. NuA4 and SWR1 are physically associated in higher eukaryotes as they are homologous to the TIP60/p400 complex, which encompasses both histone acetyltransferase (Tip60) and histone exchange (p400/Domino) activities. Here we present work investigating the impact of NuA4-dependent acetylation on SWR1-driven incorporation of H2A.Z into chromatin. Using in vitro histone exchange assays with native chromatin, we demonstrate that prior chromatin acetylation by NuA4 greatly stimulates the exchange of H2A for H2A.Z. Interestingly, we find that acetylation of H2A or H4 N-terminal tails by NuA4 can independently stimulate SWR1 activity. Accordingly, we demonstrate that mutations of H4 or H2A N-terminal lysine residues have similar effects on H2A.Z incorporation in vivo, and cells carrying mutations in both tails are nonviable. Finally, depletion experiments indicate that the bromodomain-containing protein Bdf1 is important for NuA4-dependent stimulation of SWR1. These results provide important mechanistic insight into the functional cross-talk between chromatin acetylation and ATP-dependent exchange of histone H2A variants. PMID:20332092

  16. Regulation of the nucleosome unwrapping rate controls DNA accessibility

    PubMed Central

    North, Justin A.; Shimko, John C.; Javaid, Sarah; Mooney, Alex M.; Shoffner, Matthew A.; Rose, Sean D.; Bundschuh, Ralf; Fishel, Richard; Ottesen, Jennifer J.; Poirier, Michael G.

    2012-01-01

    Eukaryotic genomes are repetitively wrapped into nucleosomes that then regulate access of transcription and DNA repair complexes to DNA. The mechanisms that regulate extrinsic protein interactions within nucleosomes are unresolved. We demonstrate that modulation of the nucleosome unwrapping rate regulates protein binding within nucleosomes. Histone H3 acetyl-lysine 56 [H3(K56ac)] and DNA sequence within the nucleosome entry-exit region additively influence nucleosomal DNA accessibility by increasing the unwrapping rate without impacting rewrapping. These combined epigenetic and genetic factors influence transcription factor (TF) occupancy within the nucleosome by at least one order of magnitude and enhance nucleosome disassembly by the DNA mismatch repair complex, hMSH2–hMSH6. Our results combined with the observation that ∼30% of Saccharomyces cerevisiae TF-binding sites reside in the nucleosome entry–exit region suggest that modulation of nucleosome unwrapping is a mechanism for regulating transcription and DNA repair. PMID:22965129

  17. Curtailment and Stochastic Curtailment to Shorten the CES-D

    ERIC Educational Resources Information Center

    Finkelman, Matthew D.; Smits, Niels; Kim, Wonsuk; Riley, Barth

    2012-01-01

    The Center for Epidemiologic Studies-Depression (CES-D) scale is a well-known self-report instrument that is used to measure depressive symptomatology. Respondents who take the full-length version of the CES-D are administered a total of 20 items. This article investigates the use of curtailment and stochastic curtailment (SC), two sequential…

  18. Curtailment and Stochastic Curtailment to Shorten the CES-D

    ERIC Educational Resources Information Center

    Finkelman, Matthew D.; Smits, Niels; Kim, Wonsuk; Riley, Barth

    2012-01-01

    The Center for Epidemiologic Studies-Depression (CES-D) scale is a well-known self-report instrument that is used to measure depressive symptomatology. Respondents who take the full-length version of the CES-D are administered a total of 20 items. This article investigates the use of curtailment and stochastic curtailment (SC), two sequential…

  19. 10 CFR 580.03 - Curtailment priorities.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... DEPARTMENT OF ENERGY (CONTINUED) NATURAL GAS (ECONOMIC REGULATORY ADMINISTRATION) CURTAILMENT PRIORITIES FOR... than section 401(b) of the Natural Gas Policy Act of 1978, or any other rule, regulation, or order of... curtailment of deliveries of natural gas for any essential agricultural use, unless: (1) Such curtailment...

  20. 10 CFR 580.03 - Curtailment priorities.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 4 2011-01-01 2011-01-01 false Curtailment priorities. 580.03 Section 580.03 Energy DEPARTMENT OF ENERGY (CONTINUED) NATURAL GAS (ECONOMIC REGULATORY ADMINISTRATION) CURTAILMENT PRIORITIES FOR ESSENTIAL AGRICULTURAL USES § 580.03 Curtailment priorities. (a) Notwithstanding any provision of law...

  1. 10 CFR 580.03 - Curtailment priorities.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 4 2012-01-01 2012-01-01 false Curtailment priorities. 580.03 Section 580.03 Energy DEPARTMENT OF ENERGY (CONTINUED) NATURAL GAS (ECONOMIC REGULATORY ADMINISTRATION) CURTAILMENT PRIORITIES FOR ESSENTIAL AGRICULTURAL USES § 580.03 Curtailment priorities. (a) Notwithstanding any provision of law...

  2. RNF8-dependent histone modifications regulate nucleosome removal during spermatogenesis.

    PubMed

    Lu, Lin-Yu; Wu, Jiaxue; Ye, Lin; Gavrilina, Galina B; Saunders, Thomas L; Yu, Xiaochun

    2010-03-16

    During spermatogenesis, global nucleosome removal occurs where histones are initially replaced by transition proteins and subsequently by protamines. This chromatin reorganization is thought to facilitate the compaction of the paternal genome into the sperm head and to protect the DNA from damaging agents. Histone ubiquitination has been suggested to be important for sex chromosome inactivation during meiotic prophase and nucleosome removal at postmeiotic stages. However, the mechanisms regulating these ubiquitin-mediated processes are unknown. In this study, we investigate the role of the ubiquitin ligase RNF8 during spermatogenesis and find that RNF8-deficient mice are proficient in meiotic sex chromosome inactivation (MSCI) but deficient in global nucleosome removal. Moreover, we show that RNF8-dependent histone ubiquitination induces H4K16 acetylation, which may be an initial step in nucleosome removal. Thus, our results show that RNF8 plays an important role during spermatogenesis through histone ubiquitination, resulting in trans-histone acetylation and global nucleosome removal.

  3. Designing nucleosomal force sensors

    NASA Astrophysics Data System (ADS)

    Tompitak, M.; de Bruin, L.; Eslami-Mossallam, B.; Schiessel, H.

    2017-05-01

    About three quarters of our DNA is wrapped into nucleosomes: DNA spools with a protein core. It is well known that the affinity of a given DNA stretch to be incorporated into a nucleosome depends on the geometry and elasticity of the basepair sequence involved, causing the positioning of nucleosomes. Here we show that DNA elasticity can have a much deeper effect on nucleosomes than just their positioning: it affects their "identities". Employing a recently developed computational algorithm, the mutation Monte Carlo method, we design nucleosomes with surprising physical characteristics. Unlike any other nucleosomes studied so far, these nucleosomes are short-lived when put under mechanical tension whereas other physical properties are largely unaffected. This suggests that the nucleosome, the most abundant DNA-protein complex in our cells, might more properly be considered a class of complexes with a wide array of physical properties, and raises the possibility that evolution has shaped various nucleosome species according to their genomic context.

  4. The prenucleosome, a stable conformational isomer of the nucleosome.

    PubMed

    Fei, Jia; Torigoe, Sharon E; Brown, Christopher R; Khuong, Mai T; Kassavetis, George A; Boeger, Hinrich; Kadonaga, James T

    2015-12-15

    Chromatin comprises nucleosomes as well as nonnucleosomal histone-DNA particles. Prenucleosomes are rapidly formed histone-DNA particles that can be converted into canonical nucleosomes by a motor protein such as ACF. Here we show that the prenucleosome is a stable conformational isomer of the nucleosome. It consists of a histone octamer associated with ∼ 80 base pair (bp) of DNA, which is located at a position that corresponds to the central 80 bp of a nucleosome core particle. Monomeric prenucleosomes with free flanking DNA do not spontaneously fold into nucleosomes but can be converted into canonical nucleosomes by an ATP-driven motor protein such as ACF or Chd1. In addition, histone H3K56, which is located at the DNA entry and exit points of a canonical nucleosome, is specifically acetylated by p300 in prenucleosomes relative to nucleosomes. Prenucleosomes assembled in vitro exhibit properties that are strikingly similar to those of nonnucleosomal histone-DNA particles in the upstream region of active promoters in vivo. These findings suggest that the prenucleosome, the only known stable conformational isomer of the nucleosome, is related to nonnucleosomal histone-DNA species in the cell.

  5. 10 CFR 580.03 - Curtailment priorities.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... DEPARTMENT OF ENERGY (CONTINUED) NATURAL GAS (ECONOMIC REGULATORY ADMINISTRATION) CURTAILMENT PRIORITIES FOR ESSENTIAL AGRICULTURAL USES § 580.03 Curtailment priorities. (a) Notwithstanding any provision of law other than section 401(b) of the Natural Gas Policy Act of 1978, or any other rule, regulation, or order of...

  6. 10 CFR 580.03 - Curtailment priorities.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... DEPARTMENT OF ENERGY (CONTINUED) NATURAL GAS (ECONOMIC REGULATORY ADMINISTRATION) CURTAILMENT PRIORITIES FOR ESSENTIAL AGRICULTURAL USES § 580.03 Curtailment priorities. (a) Notwithstanding any provision of law other than section 401(b) of the Natural Gas Policy Act of 1978, or any other rule, regulation, or order of...

  7. Closing the gap between single molecule and bulk FRET analysis of nucleosomes.

    PubMed

    Gansen, Alexander; Hieb, Aaron R; Böhm, Vera; Tóth, Katalin; Langowski, Jörg

    2013-01-01

    Nucleosome structure and stability affect genetic accessibility by altering the local chromatin morphology. Recent FRET experiments on nucleosomes have given valuable insight into the structural transformations they can adopt. Yet, even if performed under seemingly identical conditions, experiments performed in bulk and at the single molecule level have given mixed answers due to the limitations of each technique. To compare such experiments, however, they must be performed under identical conditions. Here we develop an experimental framework that overcomes the conventional limitations of each method: single molecule FRET experiments are carried out at bulk concentrations by adding unlabeled nucleosomes, while bulk FRET experiments are performed in microplates at concentrations near those used for single molecule detection. Additionally, the microplate can probe many conditions simultaneously before expending valuable instrument time for single molecule experiments. We highlight this experimental strategy by exploring the role of selective acetylation of histone H3 on nucleosome structure and stability; in bulk, H3-acetylated nucleosomes were significantly less stable than non-acetylated nucleosomes. Single molecule FRET analysis further revealed that acetylation of histone H3 promoted the formation of an additional conformational state, which is suppressed at higher nucleosome concentrations and which could be an important structural intermediate in nucleosome regulation.

  8. Uncovering the forces between nucleosomes using DNA origami.

    PubMed

    Funke, Jonas J; Ketterer, Philip; Lieleg, Corinna; Schunter, Sarah; Korber, Philipp; Dietz, Hendrik

    2016-11-01

    Revealing the energy landscape for nucleosome association may contribute to the understanding of higher-order chromatin structures and their impact on genome regulation. We accomplish this in a direct measurement by integrating two nucleosomes into a DNA origami-based force spectrometer, which enabled subnanometer-resolution measurements of nucleosome-nucleosome distance frequencies via single-particle electron microscopy imaging. From the data, we derived the Boltzmann-weighted distance-dependent energy landscape for nucleosome pair interactions. We find a shallow but long-range (~6 nm) attractive nucleosome pair potential with a minimum of -1.6 kcal/mol close to direct contact distances. The relative nucleosome orientation had little influence, but histone H4 acetylation or removal of histone tails drastically decreased the interaction strength. Because of the weak and shallow pair potential, higher-order nucleosome assemblies will be compliant and experience dynamic shape fluctuations in the absence of additional cofactors. Our results contribute to a more accurate description of chromatin and our force spectrometer provides a powerful tool for the direct and high-resolution study of molecular interactions using imaging techniques.

  9. Uncovering the forces between nucleosomes using DNA origami

    PubMed Central

    Funke, Jonas J.; Ketterer, Philip; Lieleg, Corinna; Schunter, Sarah; Korber, Philipp; Dietz, Hendrik

    2016-01-01

    Revealing the energy landscape for nucleosome association may contribute to the understanding of higher-order chromatin structures and their impact on genome regulation. We accomplish this in a direct measurement by integrating two nucleosomes into a DNA origami–based force spectrometer, which enabled subnanometer-resolution measurements of nucleosome-nucleosome distance frequencies via single-particle electron microscopy imaging. From the data, we derived the Boltzmann-weighted distance-dependent energy landscape for nucleosome pair interactions. We find a shallow but long-range (~6 nm) attractive nucleosome pair potential with a minimum of −1.6 kcal/mol close to direct contact distances. The relative nucleosome orientation had little influence, but histone H4 acetylation or removal of histone tails drastically decreased the interaction strength. Because of the weak and shallow pair potential, higher-order nucleosome assemblies will be compliant and experience dynamic shape fluctuations in the absence of additional cofactors. Our results contribute to a more accurate description of chromatin and our force spectrometer provides a powerful tool for the direct and high-resolution study of molecular interactions using imaging techniques. PMID:28138524

  10. What controls nucleosome positions?

    PubMed

    Segal, Eran; Widom, Jonathan

    2009-08-01

    The DNA of eukaryotic genomes is wrapped in nucleosomes, which strongly distort and occlude the DNA from access to most DNA-binding proteins. An understanding of the mechanisms that control nucleosome positioning along the DNA is thus essential to understanding the binding and action of proteins that carry out essential genetic functions. New genome-wide data on in vivo and in vitro nucleosome positioning greatly advance our understanding of several factors that can influence nucleosome positioning, including DNA sequence preferences, DNA methylation, histone variants and post-translational modifications, higher order chromatin structure, and the actions of transcription factors, chromatin remodelers and other DNA-binding proteins. We discuss how these factors function and ways in which they might be integrated into a unified framework that accounts for both the preservation of nucleosome positioning and the dynamic nucleosome repositioning that occur across biological conditions, cell types, developmental processes and disease.

  11. Wind and solar energy curtailment: A review of international experience

    SciTech Connect

    Bird, Lori; Lew, Debra; Milligan, Michael; Carlini, E. Maria; Estanqueiro, Ana; Flynn, Damian; Gomez-Lazaro, Emilio; Holttinen, Hannele; Menemenlis, Nickie; Orths, Antje; Eriksen, Peter Børre; Smith, J. Charles; Soder, Lennart; Sorensen, Poul; Altiparmakis, Argyrios; Yasuda, Yoh; Miller, John

    2016-11-01

    Greater penetrations of variable renewable generation on some electric grids have resulted in increased levels of curtailment in recent years. Studies of renewable energy grid integration have found that curtailment levels may grow as the penetration of wind and solar energy generation increases. This paper reviews international experience with curtailment of wind and solar energy on bulk power systems in recent years, with a focus on eleven countries in Europe, North America, and Asia. It examines levels of curtailment, the causes of curtailment, curtailment methods and use of market-based dispatch, as well as operational, institutional, and other changes that are being made to reduce renewable energy curtailment.

  12. Nucleosome phasing - new insights

    NASA Astrophysics Data System (ADS)

    Chereji, Razvan

    2014-03-01

    Eukaryotic genomes are organized into arrays of nucleosomes, in which stretches of 147 base-pairs of DNA are wrapped around octameric histones. Recently, a new method of mapping nucleosome positions was developed, which gives a much higher accuracy than the typical MNase-seq method. I present a statistical mechanics model which is able to reproduce the high-resolution nucleosome positioning data. I show that the DNA sequence is not the main cause of the nucleosome phasing which is observed genome-wide, and I present the major nucleosome phasing elements. The statistical mechanics framework is general enough to be useful in explaining different experimental observations, and I present a few results of this model.

  13. Statistical mechanics of nucleosomes

    NASA Astrophysics Data System (ADS)

    Chereji, Razvan V.

    Eukaryotic cells contain long DNA molecules (about two meters for a human cell) which are tightly packed inside the micrometric nuclei. Nucleosomes are the basic packaging unit of the DNA which allows this millionfold compactification. A longstanding puzzle is to understand the principles which allow cells to both organize their genomes into chromatin fibers in the crowded space of their nuclei, and also to keep the DNA accessible to many factors and enzymes. With the nucleosomes covering about three quarters of the DNA, their positions are essential because these influence which genes can be regulated by the transcription factors and which cannot. We study physical models which predict the genome-wide organization of the nucleosomes and also the relevant energies which dictate this organization. In the last five years, the study of chromatin knew many important advances. In particular, in the field of nucleosome positioning, new techniques of identifying nucleosomes and the competing DNA-binding factors appeared, as chemical mapping with hydroxyl radicals, ChIP-exo, among others, the resolution of the nucleosome maps increased by using paired-end sequencing, and the price of sequencing an entire genome decreased. We present a rigorous statistical mechanics model which is able to explain the recent experimental results by taking into account nucleosome unwrapping, competition between different DNA-binding proteins, and both the interaction between histones and DNA, and between neighboring histones. We show a series of predictions of our new model, all in agreement with the experimental observations.

  14. Nucleosome Positioning and Epigenetics

    NASA Astrophysics Data System (ADS)

    Schwab, David; Bruinsma, Robijn

    2008-03-01

    The role of chromatin structure in gene regulation has recently taken center stage in the field of epigenetics, phenomena that change the phenotype without changing the DNA sequence. Recent work has also shown that nucleosomes, a complex of DNA wrapped around a histone octamer, experience a sequence dependent energy landscape due to the variation in DNA bend stiffness with sequence composition. In this talk, we consider the role nucleosome positioning might play in the formation of heterochromatin, a compact form of DNA generically responsible for gene silencing. In particular, we discuss how different patterns of nucleosome positions, periodic or random, could either facilitate or suppress heterochromatin stability and formation.

  15. Nucleosome Remodeling and Epigenetics

    PubMed Central

    Becker, Peter B.; Workman, Jerry L.

    2013-01-01

    Eukaryotic chromatin is kept flexible and dynamic to respond to environmental, metabolic, and developmental cues through the action of a family of so-called “nucleosome remodeling” ATPases. Consistent with their helicase ancestry, these enzymes experience conformation changes as they bind and hydrolyze ATP. At the same time they interact with DNA and histones, which alters histone–DNA interactions in target nucleosomes. Their action may lead to complete or partial disassembly of nucleosomes, the exchange of histones for variants, the assembly of nucleosomes, or the movement of histone octamers on DNA. “Remodeling” may render DNA sequences accessible to interacting proteins or, conversely, promote packing into tightly folded structures. Remodeling processes participate in every aspect of genome function. Remodeling activities are commonly integrated with other mechanisms such as histone modifications or RNA metabolism to assemble stable, epigenetic states. PMID:24003213

  16. Baculoviruses and nucleosome management

    SciTech Connect

    Volkman, Loy E.

    2015-02-15

    Negatively-supercoiled-ds DNA molecules, including the genomes of baculoviruses, spontaneously wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. Hence, nucleosome management should be essential for baculovirus genome replication and temporal regulation of transcription, but this has not been documented. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. SWI/SNF and INO80, two of the best-studied complexes, as well as chromatin modifier TIP60, all contain actin as a subunit. Retrospective analysis of results of AcMNPV time course experiments wherein actin polymerization was blocked by cytochalasin D drug treatment implicate actin-containing chromatin modifying complexes in decatenating baculovirus genomes, shutting down host transcription, and regulating late and very late phases of viral transcription. Moreover, virus-mediated nuclear localization of actin early during infection may contribute to nucleosome management. - Highlights: • Baculoviruses have negatively-supercoiled, circular ds DNA. • Negatively-supercoiled DNA spontaneously forms nucleosomes in the nucleus. • Nucleosomes must be mobilized for replication and transcription to proceed. • Actin-containing chromatin modifiers participate in baculovirus replication.

  17. Sustainable food consumption. Product choice or curtailment?

    PubMed

    Verain, Muriel C D; Dagevos, Hans; Antonides, Gerrit

    2015-08-01

    Food consumption is an important factor in shaping the sustainability of our food supply. The present paper empirically explores different types of sustainable food behaviors. A distinction between sustainable product choices and curtailment behavior has been investigated empirically and predictors of the two types of behavior have been identified. Respondents were classified into four segments based on their sustainable food behaviors: unsustainers, curtailers, product-oriented consumers, and sustainers. Significant differences between the segments were found with regard to food choice motives, personal and social norms, food involvement, subjective knowledge on sustainable food, ability to judge how sustainably a product has been produced and socio-demographics. It is concluded that distinguishing between behavioral strategies toward sustainable food consumption is important as consumer segments can be identified that differ both in their level of sustainable food consumption and in the type of behavior they employ.

  18. Altered nucleosomes of active nucleolar chromatin contain accessible histone H3 in its hyperacetylated forms

    SciTech Connect

    Johnson, E.M.; Sterner, R.; Allfrey, V.G.

    1987-05-25

    Chromatin of the organism Physarum polycephalum contains a class of conformationally altered nucleosomes previously localized to the transcribing regions of ribosomal genes in nucleoli. When nuclei are treated with 2-iodo(2-tritium)acetate, the histone H3 sulfhydryl group of the altered nucleosomes is derivatized while that of folded nucleosomes is not, and the labeled histones can then be identified by autoradiography of gels that separate H3 isoforms. The H3 derivatized is predominantly of tri- and tetraacetylated forms. In contrast, total free histone reacted with iodoacetate shows no preferential labeling of isoforms. Selective reaction of acetylated H3 is prevalent in both nucleolar and non-nucleolar chromatin. The results link specific patterns of H3 acetylation to changes in nucleosome conformation that occur during transcription.

  19. Nucleosome sliding: facts and fiction.

    PubMed

    Becker, Peter B

    2002-09-16

    Nucleosome sliding is a frequent result of energy-dependent nucleosome remodelling in vitro. This review discusses the possible roles for nucleosome sliding in the assembly and maintenance of dynamic chromatin and for the regulation of diverse functions in eukaryotic nuclei.

  20. Dynamics of Nucleosome Arrays

    NASA Astrophysics Data System (ADS)

    Poirier, Michael

    2007-03-01

    DNA sites wrapped into chromatin are sterically occluded from proteins that must bind for processes such as RNA transcription and DNA repair. However, the role of chromatin compaction in biological function is poorly understood. To understand the biological functions of chromatin compaction, we constructed nucleosome arrays that are built with a tandem repeat of high affinity nucleosome positioning sequences, which contain probes for DNA accessibility and chromatin structure. I will describe our results that use restriction enzyme digestion and fluorescence resonance energy transfer to determine the probability for DNA site exposure within compacted nucleosome arrays and the time scale for changes in chromatin compaction. I will then discuss how these results help explain how proteins gain access to DNA sites buried within chromatin.

  1. Epigenetic nucleosomes: Alu sequences and CG as nucleosome positioning element.

    PubMed

    Salih, F; Salih, B; Kogan, S; Trifonov, E N

    2008-08-01

    Alu sequences carry periodical pattern with CG dinucleotides (CpG) repeating every 31-32 bases. Similar distances are observed in distribution of DNA curvature in crystallized nucleosomes, at positions +/-1.5 and +/-4.5 periods of DNA from nucleosome DNA dyad. Since CG elements are also found to impart to nucleosomes higher stability when positioned at +/-1.5 sites, it suggests that CG dinucleotides may play a role in modulation of the nucleosome strength when the CG elements are methylated. Thus, Alu sequences may harbor special epigenetic nucleosomes with methylation-dependent regulatory functions. Nucleosome DNA sequence probe is suggested to detect locations of such regulatory nucleosomes in the sequences.

  2. Nucleosome Core Particle

    NASA Technical Reports Server (NTRS)

    1997-01-01

    Nucleosome Core Particle grown on STS-81. The fundamental structural unit of chromatin and is the basis for organization within the genome by compaction of DNA within the nucleus of the cell and by making selected regions of chromosomes available for transcription and replication. Principal Investigator's are Dr. Dan Carter and Dr. Gerard Bunick of New Century Pharmaceuticals.

  3. Nucleosome Core Particle

    NASA Technical Reports Server (NTRS)

    1997-01-01

    Nucleosome Core Particle grown on STS-81. The fundamental structural unit of chromatin and is the basis for organization within the genome by compaction of DNA within the nucleus of the cell and by making selected regions of chromosomes available for transcription and replication. Principal Investigator's are Dr. Dan Carter and Dr. Gerard Bunick of New Century Pharmaceuticals.

  4. Sequence-dependent nucleosome positioning.

    PubMed

    Chung, Ho-Ryun; Vingron, Martin

    2009-03-13

    Eukaryotic DNA is organized into a macromolecular structure called chromatin. The basic repeating unit of chromatin is the nucleosome, which consists of two copies of each of the four core histones and DNA. The nucleosomal organization and the positions of nucleosomes have profound effects on all DNA-dependent processes. Understanding the factors that influence nucleosome positioning is therefore of general interest. Among the many determinants of nucleosome positioning, the DNA sequence has been proposed to have a major role. Here, we analyzed more than 860,000 nucleosomal DNA sequences to identify sequence features that guide the formation of nucleosomes in vivo. We found that both a periodic enrichment of AT base pairs and an out-of-phase oscillating enrichment of GC base pairs as well as the overall preference for GC base pairs are determinants of nucleosome positioning. The preference for GC pairs can be related to a lower energetic cost required for deformation of the DNA to wrap around the histones. In line with this idea, we found that only incorporation of both signal components into a sequence model for nucleosome formation results in maximal predictive performance on a genome-wide scale. In this manner, one achieves greater predictive power than published approaches. Our results confirm the hypothesis that the DNA sequence has a major role in nucleosome positioning in vivo.

  5. New insights into nucleosome unwrapping

    NASA Astrophysics Data System (ADS)

    Chereji, Razvan; Morozov, Alexandre

    2013-03-01

    Eukaryotic genomes are organized into arrays of nucleosomes, in which stretches of 147 base-pairs (bp) of DNA are wrapped around octameric histones. Recently, a new approach for direct mapping of nucleosome centers at bp resolution was developed [Brogaard et al., Nature 486, 496-501 (2012)] and some intriguing results appeared. About 40% of the inter-dyad distances are smaller than 147 bp, which imply massive nucleosome unwrapping, genome-wide, in vivo. The histogram of the inter-dyad distances presents small oscillations which indicate a step-wise unwrapping of the nucleosomal DNA from the histone. We present a statistical mechanics model for the nucleosome unwrapping, which is able to take into account sequence-dependent binding energies, sequence-independent potential barriers and wells, effective two-body interactions between the nucleosomes, competition between different species, cooperative-binding, and other important factors which dictate the nucleosome distribution along the DNA. We are able to reproduce the distribution of the inter-dyad distances, which cannot be obtained if there is no nucleosome unwrapping. The nucleosome unwrapping model can explain also the variable DNA accessibility and the nucleosome-induced cooperativity, which were observed experimentally.

  6. Properties of active nucleosomes as revealed by HMG 14 and 17 chromatography.

    PubMed Central

    Weisbrod, S T

    1982-01-01

    Nucleosomes from actively transcribed genes (active nucleosomes) contain nonhistone proteins HMG 14 and 17 and are preferentially sensitive to digestion by DNAse I. Active nucleosomes isolated by chromatography on an HMG 14 and 17 glass bead affinity column were analyzed with respect to overall structure, accessory nonhistone components and modifications to the DNA and histones. The experiments lead to the following conclusions: the DNA in the active nucleosome is undermethylated compared to bulk DNA; topoisomerase I is a non-stoichiometric component of the active nucleosome fraction; the level of histone acetylation is enriched in active nucleosomes, but the extent of enrichment cannot account for HMG binding; and the two histone H3 molecules in the active nucleosome can dimerize more readily and are, therefore, probably closer together than those in the bulk of the nucleosomes. Additionally it is shown that HMG 14 and 17 prefer to bind to single- vs. double-stranded nucleic acids. The role of HMG 14 and 17 in producing a highly DNAse I sensitive structure and correspondingly helping to facilitate transcription is discussed in terms of these properties. Images PMID:6210882

  7. Nucleosome positioning patterns derived from human apoptotic nucleosomes.

    PubMed

    Frenkel, Zakharia M; Trifonov, Edward N; Volkovich, Zeev; Bettecken, Thomas

    2011-12-01

    This communication reports on the nucleosome positioning patterns (bendability matrices) for the human genome, derived from over 8_million nucleosome DNA sequences obtained from apoptotically digested lymphocytes. This digestion procedure is used here for the first time for the purpose of extraction and sequencing of the nucleosome DNA fragments. The dominant motifs suggested by the matrices of DNA bendability calculated for light and heavy isochores are significantly different. Both, however, are in full agreement with the linear description YRRRRRYYYYYR, and with earlier derivations by N-gram extensions. Thus, the choice of the nucleosome positioning patterns crucially depends on the G + C composition of the analyzed sequences.

  8. Electrophoresis of Positioned Nucleosomes

    PubMed Central

    Castelnovo, Martin; Grauwin, Sébastian

    2007-01-01

    We present in this article an original approach to compute the electrophoretic mobility of rigid nucleo-protein complexes like nucleosomes. This model allows us to address theoretically the influence of complex position along DNA, as well as wrapped length of DNA on the electrophoretic mobility of the complex. The predictions of the model are in qualitative agreement with experimental results on mononucleosomes assembled on short DNA fragments (<400 bp). Influences of additional experimental parameters like gel concentration, ionic strength, and effective charges are also discussed in the framework of the model, and are found to be qualitatively consistent with experiments when available. Based on the present model, we propose a simple semi-empirical formula describing positioning of nucleosomes as seen through electrophoresis. PMID:17277181

  9. Efficacy of curtailment announcements as a predictor of lumber supply

    Treesearch

    Henry. Spelter

    2001-01-01

    A practical method for tracking the effect of curtailment announcements on lumber supply is described and tested. Combining announcements of closures and curtailments with mill capacities enables the creation of accurate forward-looking assessments of lumber supply 1 to 2 months into the future. For three American and Canadian lumber- producing regions, the method...

  10. Charge State of the Globular Histone Core Controls Stability of the Nucleosome

    PubMed Central

    Fenley, Andrew T.; Adams, David A.; Onufriev, Alexey V.

    2010-01-01

    Presented here is a quantitative model of the wrapping and unwrapping of the DNA around the histone core of the nucleosome that suggests a mechanism by which this transition can be controlled: alteration of the charge state of the globular histone core. The mechanism is relevant to several classes of posttranslational modifications such as histone acetylation and phosphorylation; several specific scenarios consistent with recent in vivo experiments are considered. The model integrates a description based on an idealized geometry with one based on the atomistic structure of the nucleosome, and the model consistently accounts for both the electrostatic and nonelectrostatic contributions to the nucleosome free energy. Under physiological conditions, isolated nucleosomes are predicted to be very stable (38 ± 7 kcal/mol). However, a decrease in the charge of the globular histone core by one unit charge, for example due to acetylation of a single lysine residue, can lead to a significant decrease in the strength of association with its DNA. In contrast to the globular histone core, comparable changes in the charge state of the histone tail regions have relatively little effect on the nucleosome's stability. The combination of high stability and sensitivity explains how the nucleosome is able to satisfy the seemingly contradictory requirements for thermodynamic stability while allowing quick access to its DNA informational content when needed by specific cellular processes such as transcription. PMID:20816070

  11. Charge state of the globular histone core controls stability of the nucleosome.

    PubMed

    Fenley, Andrew T; Adams, David A; Onufriev, Alexey V

    2010-09-08

    Presented here is a quantitative model of the wrapping and unwrapping of the DNA around the histone core of the nucleosome that suggests a mechanism by which this transition can be controlled: alteration of the charge state of the globular histone core. The mechanism is relevant to several classes of posttranslational modifications such as histone acetylation and phosphorylation; several specific scenarios consistent with recent in vivo experiments are considered. The model integrates a description based on an idealized geometry with one based on the atomistic structure of the nucleosome, and the model consistently accounts for both the electrostatic and nonelectrostatic contributions to the nucleosome free energy. Under physiological conditions, isolated nucleosomes are predicted to be very stable (38 +/- 7 kcal/mol). However, a decrease in the charge of the globular histone core by one unit charge, for example due to acetylation of a single lysine residue, can lead to a significant decrease in the strength of association with its DNA. In contrast to the globular histone core, comparable changes in the charge state of the histone tail regions have relatively little effect on the nucleosome's stability. The combination of high stability and sensitivity explains how the nucleosome is able to satisfy the seemingly contradictory requirements for thermodynamic stability while allowing quick access to its DNA informational content when needed by specific cellular processes such as transcription. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  12. A split personality for nucleosomes.

    PubMed

    McKay, Daniel J; Lieb, Jason D

    2014-12-04

    A high-resolution look at where histones touch DNA reveals a surprisingly intricate, dynamic, and modular nucleosome. Three advances in the study by Rhee et al. include unexpected interactions between the H3 tail and linker DNA, new evidence for existence of subnucleosomal particles, and asymmetric patterns of histone modification within a single nucleosome that correspond to the direction of transcription.

  13. Nucleosome signalling; an evolving concept.

    PubMed

    Turner, Bryan M

    2014-08-01

    The nucleosome core particle is the first stage of DNA packaging in virtually all eukaryotes. It both organises nuclear DNA and protects it from adventitious binding of transcription factors and the consequent deregulation of gene expression. Both properties are essential to allow the genome expansion characteristic of complex eukaryotes. The nucleosome is a flexible structure in vivo, allowing selective relaxation of its intrinsically inhibitory effects in response to external signals. Structural changes are brought about by dedicated remodelling enzymes and by posttranslational modifications of the core histones. Histone modifications occasionally alter nucleosome structure directly, but their more usual roles are to act as receptors on the nucleosome surface that are recognised by specific protein domains. The bound proteins, in turn, affect nucleosome structure and function. This strategy enormously expands the signalling capacity of the nucleosome and its ability to influence both the initiation and elongation stages of transcription. The enzymes responsible for placing and removing histone modifications, and the modification-binding proteins themselves, are ubiquitous, numerous and conserved amongst eukaryotes. Like the nucleosome, they date back to the earliest eukaryotes and may have played integral and essential roles in eukaryotic evolution. The present properties and epigenetic functions of the nucleosome reflect its evolutionary past and the selective pressures to which it has responded and can be better understood in this context. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.

  14. Thermodynamics of Intragenic Nucleosome Ordering

    NASA Astrophysics Data System (ADS)

    Chevereau, G.; Palmeira, L.; Thermes, C.; Arneodo, A.; Vaillant, C.

    2009-10-01

    The nucleosome ordering observed in vivo along yeast genes is described by a thermodynamical model of nonuniform fluid of 1D hard rods confined by two excluding energy barriers at gene extremities. For interbarrier distances L≲1.5kbp, nucleosomes equilibrate into a crystal-like configuration with a nucleosome repeat length (NRL) L/ñ165bp, where n is the number of regularly positioned nucleosomes. We also observe “bistable” genes with a fuzzy chromatin resulting from a statistical mixing of two crystal states, one with an expanded chromatin (NRL ˜L/n) and the other with a compact one (NRL ˜L/(n+1)). By means of single nucleosome switching, bistable genes may drastically alter their expression level as suggested by their higher transcriptional plasticity. These results enlighten the role of the intragenic chromatin on gene expression regulation.

  15. Nucleosome Positioning in Saccharomyces cerevisiae

    PubMed Central

    Jansen, An; Verstrepen, Kevin J.

    2011-01-01

    Summary: The DNA of eukaryotic cells is spooled around large histone protein complexes, forming nucleosomes that make up the basis for a high-order packaging structure called chromatin. Compared to naked DNA, nucleosomal DNA is less accessible to regulatory proteins and regulatory processes. The exact positions of nucleosomes therefore influence several cellular processes, including gene expression, chromosome segregation, recombination, replication, and DNA repair. Here, we review recent technological advances enabling the genome-wide mapping of nucleosome positions in the model eukaryote Saccharomyces cerevisiae. We discuss the various parameters that determine nucleosome positioning in vivo, including cis factors like AT content, variable tandem repeats, and poly(dA:dT) tracts that function as chromatin barriers and trans factors such as chromatin remodeling complexes, transcription factors, histone-modifying enzymes, and RNA polymerases. In the last section, we review the biological role of chromatin in gene transcription, the evolution of gene regulation, and epigenetic phenomena. PMID:21646431

  16. DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila.

    PubMed

    Liu, Jun; Zimmer, Kurt; Rusch, Douglas B; Paranjape, Neha; Podicheti, Ram; Tang, Haixu; Calvi, Brian R

    2015-10-15

    Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. We have been examining the activation of origins responsible for developmental gene amplification in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. Here, we examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. We propose that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC.

  17. DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila

    PubMed Central

    Liu, Jun; Zimmer, Kurt; Rusch, Douglas B.; Paranjape, Neha; Podicheti, Ram; Tang, Haixu; Calvi, Brian R.

    2015-01-01

    Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. We have been examining the activation of origins responsible for developmental gene amplification in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. Here, we examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. We propose that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC. PMID:26227968

  18. Fluid curtailment during childhood diarrhea: a countdown analysis.

    PubMed

    Perin, Jamie; Carvajal-Velez, Liliana; Carter, Emily; Bryce, Jennifer; Newby, Holly

    2015-06-26

    The foundation of recommended diarrhea management in young children is increased fluids and continued feeding. This increase in fluids is necessary to replace those lost during diarrhea and ultimately prevent dehydration. There may be an opportunity to prevent deaths in children under five by discouraging the practice of reducing or curtailing fluids during diarrhea episodes across different settings worldwide. We quantify and describe the extent of fluid curtailment in children with diarrhea in a selection of countries (Burkina Faso, Democratic Republic of Congo, Ethiopia, Nigeria, Tanzania, and Uganda) with high burden of diarrhea-related mortality with national cross sectional survey data. We examine the practice of fluid curtailment in these countries and its relationship to child and household traits and to characteristics of diarrhea management. The prevalence of fluid curtailment among children under five with diarrhea is strikingly high in these countries: 55 % in Nigeria, 49 % in Ethiopia, 44 % in Uganda, 37 % in Tanzania, 36 % in DR Congo and 32 % in Burkina Faso. Fluid curtailment is associated with giving less food, potentially worsening the impact of this harmful practice. Children who were reported to have had fluids curtailed during diarrhea episodes were also 3.51 (95 % confidence, 2.66 - 4.64) times more likely to be reported to have food withheld (α = 0.05; p < 0.001). Children who received care from non-governmental providers, and those who were breastfed were more likely to have their fluids curtailed, as were children with an unimproved water source. Children of poorer or less educated mothers and those living in rural areas are more likely to have curtailed fluids, compared to children of less poor or more educated mothers, or those living in urban areas. The harmful practice of curtailing fluids for a child with diarrhea is highly prevalent, representing an increased risk of dehydration and complications due to diarrhea, including death

  19. Stimulation of V(D)J recombination by histone acetylation.

    PubMed

    McBlane, F; Boyes, J

    2000-04-20

    V(D)J recombination assembles functional immunoglobulin and T cell receptor genes from individual gene segments [1]. A common recombination mechanism, initiated by the proteins RAG1 and RAG2 at conserved recombination signal sequences (RSSs), operates at all rearranging loci [2] [3]. It has been proposed that the key regulator of the reaction is 'accessibility' of the RSS within chromatin [4]. Recently, the packaging of RSSs into nucleosomes was shown to inhibit initiation of V(D)J recombination [5] [6]. Nevertheless, the tight tissue specificity of regulation cannot be explained by nucleosome-mediated repression alone because a significant fraction of RSSs would be predicted to lie in linker regions between nucleosomes. Therefore, some aspect of the regulation of the recombination reaction must rely on the disruption of higher-order chromatin structure. Here, we report that histone acetylation directly stimulates the recombination reaction in vivo in the correct cell- and stage-specific manner. Neither expression of RAG genes nor activity of RAG proteins was increased by acetylation. Furthermore, histone acetylation failed to overcome nucleosome-mediated repression of RSS recognition and cleavage in vitro. Our data suggest a role for histone acetylation in stimulating recombination in vivo through disruption of higher-order chromatin structures.

  20. E APPROACH, FACING W SHOWING PARAPET CURTAILS Stanley Brook ...

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

    E APPROACH, FACING W SHOWING PARAPET CURTAILS - Stanley Brook Bridge, Spanning Stanley Brook, Stanley Brook Motor Road, & Seaside Trail on Barr Hill-Day Mountain Carriage Road, Seal Harbor, Hancock County, ME

  1. Tail-induced attraction between nucleosome core particles.

    PubMed

    Mühlbacher, F; Schiessel, H; Holm, C

    2006-09-01

    We study a possible electrostatic mechanism underlying the compaction of DNA inside the nuclei of eucaryotes: the tail-bridging effect between nucleosomes, the fundamental DNA packaging units of the chromatin complex. As a simple model of the nucleosome we introduce the eight-tail colloid, a charged sphere with eight oppositely charged, flexible, grafted chains that represent the terminal histone tails. We show that our complexes attract each other via the formation of chain bridges and contrast this to the effect of attraction via charge patches. We demonstrate that the attraction between eight-tail colloids can be tuned by changing the fraction of charged monomers on the tails. This suggests a physical mechanism of chromatin compaction where the degree of DNA condensation is controlled via biochemical means, namely the acetylation and deacetylation of lysines in the histone tails.

  2. Circulating Nucleosomes and Nucleosome Modifications as Biomarkers in Cancer

    PubMed Central

    McAnena, Peter; Brown, James A. L.; Kerin, Michael J.

    2017-01-01

    Traditionally the stratification of many cancers involves combining tumour and clinicopathological features (e.g., patient age; tumour size, grade, receptor status and location) to inform treatment options and predict recurrence risk and survival. However, current biomarkers often require invasive excision of the tumour for profiling, do not allow monitoring of the response to treatment and stratify patients into broad heterogeneous groups leading to inconsistent treatment responses. Here we explore and describe the benefits of using circulating biomarkers (nucleosomes and/or modifications to nucleosomes) as a non-invasive method for detecting cancer and monitoring response to treatment. Nucleosomes (DNA wound around eight core histone proteins) are responsible for compacting our genome and their composition and post-translational modifications are responsible for regulating gene expression. Here, we focus on breast and colorectal cancer as examples where utilizing circulating nucleosomes as biomarkers hold real potential as liquid biopsies. Utilizing circulating nucleosomes as biomarkers is an exciting new area of research that promises to allow both the early detection of cancer and monitoring of treatment response. Nucleosome-based biomarkers combine with current biomarkers, increasing both specificity and sensitivity of current tests and have the potential to provide individualised precision-medicine based treatments for patients. PMID:28075351

  3. A distinct switch in interactions of the histone H4 tail domain upon salt-dependent folding of nucleosome arrays.

    PubMed

    Pepenella, Sharon; Murphy, Kevin J; Hayes, Jeffrey J

    2014-09-26

    The core histone tail domains mediate inter-nucleosomal interactions that direct folding and condensation of nucleosome arrays into higher-order chromatin structures. The histone H4 tail domain facilitates inter-array interactions by contacting both the H2A/H2B acidic patch and DNA of neighboring nucleosomes. Likewise, H4 tail-H2A contacts stabilize array folding. However, whether the H4 tail domains stabilize array folding via inter-nucleosomal interactions with the DNA of neighboring nucleosomes remains unclear. We utilized defined oligonucleosome arrays containing a single specialized nucleosome with a photo-inducible cross-linker in the N terminus of the H4 tail to characterize these interactions. We observed that the H4 tail participates exclusively in intra-array interactions with DNA in unfolded arrays. These interactions are diminished during array folding, yet no inter-nucleosome, intra-array H4 tail-DNA contacts are observed in condensed chromatin. However, we document contacts between the N terminus of the H4 tail and H2A. Installation of acetylation mimics known to disrupt H4-H2A surface interactions did not increase observance of H4-DNA inter-nucleosomal interactions. These results suggest the multiple functions of the H4 tail require targeted distinct interactions within condensed chromatin.

  4. DNA looping mediates nucleosome transfer

    PubMed Central

    Brennan, Lucy D.; Forties, Robert A.; Patel, Smita S.; Wang, Michelle D.

    2016-01-01

    Proper cell function requires preservation of the spatial organization of chromatin modifications. Maintenance of this epigenetic landscape necessitates the transfer of parental nucleosomes to newly replicated DNA, a process that is stringently regulated and intrinsically linked to replication fork dynamics. This creates a formidable setting from which to isolate the central mechanism of transfer. Here we utilized a minimal experimental system to track the fate of a single nucleosome following its displacement, and examined whether DNA mechanics itself, in the absence of any chaperones or assembly factors, may serve as a platform for the transfer process. We found that the nucleosome is passively transferred to available dsDNA as predicted by a simple physical model of DNA loop formation. These results demonstrate a fundamental role for DNA mechanics in mediating nucleosome transfer and preserving epigenetic integrity during replication. PMID:27808093

  5. Fuzziness and noise in nucleosomal architecture

    PubMed Central

    Flores, Oscar; Deniz, Özgen; Soler-López, Montserrat; Orozco, Modesto

    2014-01-01

    Nucleosome organization plays a key role in the regulation of gene expression. However, despite the striking advances in the accuracy of nucleosome maps, there are still severe discrepancies on individual nucleosome positioning and how this influences gene regulation. The variability among nucleosome maps, which precludes the fine analysis of nucleosome positioning, might emerge from diverse sources. We have carefully inspected the extrinsic factors that may induce diversity by the comparison of microccocal nuclease (MNase)-Seq derived nucleosome maps generated under distinct conditions. Furthermore, we have also explored the variation originated from intrinsic nucleosome dynamics by generating additional maps derived from cell cycle synchronized and asynchronous yeast cultures. Taken together, our study has enabled us to measure the effect of noise in nucleosome occupancy and positioning and provides insights into the underlying determinants. Furthermore, we present a systematic approach that may guide the standardization of MNase-Seq experiments in order to generate reproducible genome-wide nucleosome patterns. PMID:24586063

  6. Nucleosome dynamics during chromatin remodeling in vivo.

    PubMed

    Ramachandran, Srinivas; Henikoff, Steven

    2016-01-01

    Precise positioning of nucleosomes around regulatory sites is achieved by the action of chromatin remodelers, which use the energy of ATP to slide, evict or change the composition of nucleosomes. Chromatin remodelers act to bind nucleosomes, disrupt histone-DNA interactions and translocate the DNA around the histone core to reposition nucleosomes. Hence, remodeling is expected to involve nucleosomal intermediates with a structural organization that is distinct from intact nucleosomes. We describe the identification of a partially unwrapped nucleosome structure using methods that map histone-DNA contacts genome-wide. This alternative nucleosome structure is likely formed as an intermediate or by-product during nucleosome remodeling by the RSC complex. Identification of the loss of histone-DNA contacts during chromatin remodeling by RSC in vivo has implications for the regulation of transcriptional initiation.

  7. Nucleosome structure and conformational changes

    SciTech Connect

    McGhee, J.D.; Felsenfeld, G.; Eisenberg, H.

    1980-10-01

    We have used a variety of chemical probes to measure the accessibility of DNA on the surface of the nucleosome. We review these results, and describe new experiments which show that T4 phage DNA can form complexes with the core histones, possessing the properties of normal nucleosomes. Since T4 DNA is largely occupied by glucose residues in the major groove, this suggests that the major groove is not filled with histone amino acid side chains. We also report results of recent measurements which appear to show that only a few strong charge interactions are involved in the attachment of the terminal 20 nucleotide pairs at each end of nucleosome core DNA. We speculate on the possible functional significance of the accessibility of DNA revealed by all of these experiments. We have also examined conformational changes induced in nucleosomes at high ionic strength (0.5 to 0.7M NaCl). The frictional coefficient is found to undergo a small increase in this region, not consistent with models in which the nucleosome is completely unfolded, but possibly reflecting the dissociation of terminal DNA from the nucleosome surface.

  8. ISWI Remodelling of Physiological Chromatin Fibres Acetylated at Lysine 16 of Histone H4

    PubMed Central

    Klinker, Henrike; Mueller-Planitz, Felix; Yang, Renliang; Forné, Ignasi; Liu, Chuan-Fa; Nordenskiöld, Lars; Becker, Peter B.

    2014-01-01

    ISWI is the catalytic subunit of several ATP-dependent chromatin remodelling factors that catalyse the sliding of nucleosomes along DNA and thereby endow chromatin with structural flexibility. Full activity of ISWI requires residues of a basic patch of amino acids in the N-terminal ‘tail’ of histone H4. Previous studies employing oligopeptides and mononucleosomes suggested that acetylation of the H4 tail at lysine 16 (H4K16) within the basic patch may inhibit the activity of ISWI. On the other hand, the acetylation of H4K16 is known to decompact chromatin fibres. Conceivably, decompaction may enhance the accessibility of nucleosomal DNA and the H4 tail for ISWI interactions. Such an effect can only be evaluated at the level of nucleosome arrays. We probed the influence of H4K16 acetylation on the ATPase and nucleosome sliding activity of Drosophila ISWI in the context of defined, in vitro reconstituted chromatin fibres with physiological nucleosome spacing and linker histone content. Contrary to widespread expectations, the acetylation did not inhibit ISWI activity, but rather stimulated ISWI remodelling under certain conditions. Therefore, the effect of H4K16 acetylation on ISWI remodelling depends on the precise nature of the substrate. PMID:24516652

  9. Acetylation mimics within individual core histone tail domains indicate distinct roles in regulating the stability of higher-order chromatin structure.

    PubMed

    Wang, Xiaodong; Hayes, Jeffrey J

    2008-01-01

    Nucleosome arrays undergo salt-dependent self-association into large oligomers in a process thought to recapitulate essential aspects of higher-order tertiary chromatin structure formation. Lysine acetylation within the core histone tail domains inhibits self-association, an effect likely related to its role in facilitating transcription. As acetylation of specific tail domains may encode distinct functions, we investigated biochemical and self-association properties of model nucleosome arrays containing combinations of native and mutant core histones with lysine-to-glutamine substitutions to mimic acetylation. Acetylation mimics within the tail domains of H2B and H4 caused the largest inhibition of array self-association, while modification of the H3 tail uniquely affected the stability of DNA wrapping within individual nucleosomes. In addition, the effect of acetylation mimics on array self-association is inconsistent with a simple charge neutralization mechanism. For example, acetylation mimics within the H2A tail can have either a positive or negative effect on self-association, dependent upon the acetylation state of the other tails and nucleosomal repeat length. Finally, we demonstrate that glutamine substitutions and lysine acetylation within the H4 tail domain have identical effects on nucleosome array self-association. Our results indicate that acetylation of specific tail domains plays distinct roles in the regulation of chromatin structure.

  10. H4K44 Acetylation Facilitates Chromatin Accessibility during Meiosis.

    PubMed

    Hu, Jialei; Donahue, Greg; Dorsey, Jean; Govin, Jérôme; Yuan, Zuofei; Garcia, Benjamin A; Shah, Parisha P; Berger, Shelley L

    2015-12-01

    Meiotic recombination hotspots are associated with histone post-translational modifications and open chromatin. However, it remains unclear how histone modifications and chromatin structure regulate meiotic recombination. Here, we identify acetylation of histone H4 at Lys44 (H4K44ac) occurring on the nucleosomal lateral surface. We show that H4K44 is acetylated at pre-meiosis and meiosis and displays genome-wide enrichment at recombination hotspots in meiosis. Acetylation at H4K44 is required for normal meiotic recombination, normal levels of double-strand breaks (DSBs) during meiosis, and optimal sporulation. Non-modifiable H4K44R results in increased nucleosomal occupancy around DSB hotspots. Our results indicate that H4K44ac functions to facilitate chromatin accessibility favorable for normal DSB formation and meiotic recombination. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Conditions for positioning of nucleosomes on DNA

    NASA Astrophysics Data System (ADS)

    Sheinman, Michael; Chung, Ho-Ryun

    2015-08-01

    Positioning of nucleosomes along a eukaryotic genome plays an important role in its organization and regulation. There are many different factors affecting the location of nucleosomes. Some can be viewed as preferential binding of a single nucleosome to different locations along the DNA and some as interactions between neighboring nucleosomes. In this study, we analyze positioning of nucleosomes and derive conditions for their good positioning. Using analytic and numerical approaches we find that, if the binding preferences are very weak, an interplay between the interactions and the binding preferences is essential for a good positioning of nucleosomes, especially on correlated energy landscapes. Analyzing the empirical energy landscape, we conclude that good positioning of nucleosomes in vivo is possible only if they strongly interact. In this case, our model, predicting long-length-scale fluctuations of nucleosomes' occupancy along the DNA, accounts well for the empirical observations.

  12. Conditions for positioning of nucleosomes on DNA.

    PubMed

    Sheinman, Michael; Chung, Ho-Ryun

    2015-08-01

    Positioning of nucleosomes along a eukaryotic genome plays an important role in its organization and regulation. There are many different factors affecting the location of nucleosomes. Some can be viewed as preferential binding of a single nucleosome to different locations along the DNA and some as interactions between neighboring nucleosomes. In this study, we analyze positioning of nucleosomes and derive conditions for their good positioning. Using analytic and numerical approaches we find that, if the binding preferences are very weak, an interplay between the interactions and the binding preferences is essential for a good positioning of nucleosomes, especially on correlated energy landscapes. Analyzing the empirical energy landscape, we conclude that good positioning of nucleosomes in vivo is possible only if they strongly interact. In this case, our model, predicting long-length-scale fluctuations of nucleosomes' occupancy along the DNA, accounts well for the empirical observations.

  13. Acetyl chloride

    Integrated Risk Information System (IRIS)

    Acetyl chloride ; CASRN 75 - 36 - 5 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  14. Wind and Solar Energy Curtailment: Experience and Practices in the United States

    SciTech Connect

    Bird, L.; Cochran, J.; Wang, X.

    2014-03-01

    This report examines U.S. curtailment practices, with a particular emphasis on utilities in the Western states. The information presented here is based on a series of interviews conducted with utilities, system operators, wind energy developers, and non-governmental organizations. The report provides case studies of curtailment experience and examines the reasons for curtailment, curtailment procedures, compensation, and practices that can minimize curtailment.

  15. Folding of Nucleosome Arrays

    NASA Astrophysics Data System (ADS)

    Howell, Steven; Jimenez-Useche, Isabel; Andresen, Kurt; Yuan, Chongli; Qiu, Xiangyun

    2014-03-01

    Chromatin conformation and dynamics is central to gene functions including packaging, regulation, and repair. At the molecular level, the basic building block of chromatin is a nucleosome core particle (NCP) made of 147 base pairs (bp) of dsDNA wrapped around an octamer of histone proteins. These NCPs are connected by short 10-90 bps of linker DNA as beads on a string. Key factors determining the packaging of NCP arrays to form chromatin include ionic condition, linker DNA length, and epigenetic modifications, especially of the histone tails. We have investigated how the conformations of model tetra-NCP arrays are modulated by these factors using small angle x-ray scattering (SAXS). Here we present recent studies of the effects of ion (KCl and MgCl2), linker length, and histone modification (tail deletions) on NCP arrays. Our SAXS measurement makes it possible to learn about both the global compaction of NCP arrays and local inter-NCP spatial correlations within the same array.

  16. Nucleosome Organization in Human Embryonic Stem Cells.

    PubMed

    Yazdi, Puya G; Pedersen, Brian A; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Chen, Yumay; Jacobsen, Steven E; Wang, Ping H

    2015-01-01

    The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a "ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this "ground state" by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states) are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational analysis of nucleosome

  17. Distribution of acetylated histones resulting from Gal4-VP16 recruitment of SAGA and NuA4 complexes

    PubMed Central

    Vignali, Marissa; Steger, David J.; Neely, Kristen E.; Workman, Jerry L.

    2000-01-01

    We analyzed the targeting of histone acetyltransferase (HAT) complexes by DNA-binding activators during transcriptional activation and the resulting distribution of acetylated histones. An in vitro competition assay was developed to acetylate and transcribe a nucleosomal array template in the presence of excess non-specific chromatin, which mimics in vivo conditions. Stimulation of transcription from the nucleosomal array template under competitive conditions by the SAGA and NuA4 HAT complexes depended on the presence of the Gal4-VP16 activator, which recognizes sites in the promoter and directly interacts with these HATs. Importantly, the stimulation of transcription by SAGA and NuA4 depended on the presence of Gal4-VP16 during histone acetylation, and Gal4-VP16-bound nucleosomal templates were acetylated preferentially by SAGA and NuA4 relative to the competitor chromatin. While targeting of the SAGA complex led to H3 acetylation of promoter-proximal nucleosomes, targeting of the NuA4 complex led to a broader domain of H4 acetylation of >3 kbp. Thus, either promoter-proximal H3 acetylation by SAGA or broadly distributed acetylation of H4 by NuA4 activated transcription from chromatin templates. PMID:10835360

  18. Nucleosome repositioning underlies dynamic gene expression.

    PubMed

    Nocetti, Nicolas; Whitehouse, Iestyn

    2016-03-15

    Nucleosome repositioning at gene promoters is a fundamental aspect of the regulation of gene expression. However, the extent to which nucleosome repositioning is used within eukaryotic genomes is poorly understood. Here we report a comprehensive analysis of nucleosome positions as budding yeast transit through an ultradian cycle in which expression of >50% of all genes is highly synchronized. We present evidence of extensive nucleosome repositioning at thousands of gene promoters as genes are activated and repressed. During activation, nucleosomes are relocated to allow sites of general transcription factor binding and transcription initiation to become accessible. The extent of nucleosome shifting is closely related to the dynamic range of gene transcription and generally related to DNA sequence properties and use of the coactivators TFIID or SAGA. However, dynamic gene expression is not limited to SAGA-regulated promoters and is an inherent feature of most genes. While nucleosome repositioning occurs pervasively, we found that a class of genes required for growth experience acute nucleosome shifting as cells enter the cell cycle. Significantly, our data identify that the ATP-dependent chromatin-remodeling enzyme Snf2 plays a fundamental role in nucleosome repositioning and the expression of growth genes. We also reveal that nucleosome organization changes extensively in concert with phases of the cell cycle, with large, regularly spaced nucleosome arrays being established in mitosis. Collectively, our data and analysis provide a framework for understanding nucleosome dynamics in relation to fundamental DNA-dependent transactions.

  19. Visible periodicity of strong nucleosome DNA sequences.

    PubMed

    Salih, Bilal; Tripathi, Vijay; Trifonov, Edward N

    2015-01-01

    Fifteen years ago, Lowary and Widom assembled nucleosomes on synthetic random sequence DNA molecules, selected the strongest nucleosomes and discovered that the TA dinucleotides in these strong nucleosome sequences often appear at 10-11 bases from one another or at distances which are multiples of this period. We repeated this experiment computationally, on large ensembles of natural genomic sequences, by selecting the strongest nucleosomes--i.e. those with such distances between like-named dinucleotides, multiples of 10.4 bases, the structural and sequence period of nucleosome DNA. The analysis confirmed the periodicity of TA dinucleotides in the strong nucleosomes, and revealed as well other periodic sequence elements, notably classical AA and TT dinucleotides. The matrices of DNA bendability and their simple linear forms--nucleosome positioning motifs--are calculated from the strong nucleosome DNA sequences. The motifs are in full accord with nucleosome positioning sequences derived earlier, thus confirming that the new technique, indeed, detects strong nucleosomes. Species- and isochore-specific variations of the matrices and of the positioning motifs are demonstrated. The strong nucleosome DNA sequences manifest the highest hitherto nucleosome positioning sequence signals, showing the dinucleotide periodicities in directly observable rather than in hidden form.

  20. Brownian dynamics simulation of the effect of histone modification on nucleosome structure

    NASA Astrophysics Data System (ADS)

    Li, Wei; Dou, Shuo-Xing; Xie, Ping; Wang, Peng-Ye

    2007-05-01

    Using Brownian dynamics we simulate the effect of histone modification, such as phosphorylation, acetylation, and methylation, on nucleosome structure by varying the interaction force between DNA and the histone octamer. The simulation shows that the structural stability of nucleosome is very sensitive to the interaction force, and the DNA unwrapping from the modified histone octamer usually occurs turn by turn. Furthermore, the effects of temperature and DNA break as well as the competition between modified and normal histone octamers are investigated, with the simulation results being in agreement with the experimental observation that phosphorylated nucleosomes near DNA breaks are more easily depleted. Though the simulation study may only give a coarse grained view of the DNA unwrapping process for the modified histone octamer, it may provide insight into the mechanism of DNA repair.

  1. Crystal structure of the nucleosome containing histone H3 with crotonylated lysine 122.

    PubMed

    Suzuki, Yuya; Horikoshi, Naoki; Kato, Daiki; Kurumizaka, Hitoshi

    2016-01-15

    The crotonylation of histones is an important post-translational modification, and epigenetically functions in the regulation of genomic DNA activity. The histone modifications in the structured "histone-fold" domains are considered to have an especially important impact on the nucleosome structure and dynamics. In the present study, we reconstituted the human nucleosome containing histone H3.2 crotonylated at the Lys122 residue, and determined its crystal structure at 2.56 Å resolution. We found that the crotonylation of the H3 Lys122 residue does not affect the overall nucleosome structure, but locally impedes the formation of the water-mediated hydrogen bond with the DNA backbone. Consistently, thermal stability assays revealed that the H3 Lys122 crotonylation, as well as the H3 Lys122 acetylation, clearly reduced the histone-DNA association. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Variations on Stochastic Curtailment in Sequential Mastery Testing

    ERIC Educational Resources Information Center

    Finkelman, Matthew David

    2010-01-01

    In sequential mastery testing (SMT), assessment via computer is used to classify examinees into one of two mutually exclusive categories. Unlike paper-and-pencil tests, SMT has the capability to use variable-length stopping rules. One approach to shortening variable-length tests is stochastic curtailment, which halts examination if the probability…

  3. 27 CFR 40.114 - Extension or curtailment of factory.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... of factory. 40.114 Section 40.114 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND... Products Changes in Location of Factory § 40.114 Extension or curtailment of factory. Where a tobacco products factory is to be changed to an extent which will make inaccurate the description of the factory as...

  4. Variations on Stochastic Curtailment in Sequential Mastery Testing

    ERIC Educational Resources Information Center

    Finkelman, Matthew David

    2010-01-01

    In sequential mastery testing (SMT), assessment via computer is used to classify examinees into one of two mutually exclusive categories. Unlike paper-and-pencil tests, SMT has the capability to use variable-length stopping rules. One approach to shortening variable-length tests is stochastic curtailment, which halts examination if the probability…

  5. Review fifteen years of search for strong nucleosomes.

    PubMed

    Trifonov, Edward N; Nibhani, Reshma

    2015-08-01

    Don Crothers, Mikael Kubista, Jon Widom, and their teams have been first to look for strong nucleosomes, in a bid to reveal the nucleosome positioning pattern(s) carried by the nucleosome DNA sequences. They were first to demonstrate that the nucleosome stability correlates with 10-11 base sequence periodicity, and that the strong nucleosomes localize preferentially in centromeres. This review describes these findings and their connection to recent discovery of the strong nucleosomes (SNs) with visibly periodic nucleosome DNA sequences.

  6. Conserved nucleosome positioning defines replication origins

    PubMed Central

    Eaton, Matthew L.; Galani, Kyriaki; Kang, Sukhyun; Bell, Stephen P.; MacAlpine, David M.

    2010-01-01

    The origin recognition complex (ORC) specifies replication origin location. The Saccharomyces cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function. PMID:20351051

  7. Functional roles of nucleosome stability and dynamics.

    PubMed

    Chereji, Răzvan V; Morozov, Alexandre V

    2015-01-01

    Nucleosome is a histone-DNA complex known as the fundamental repeating unit of chromatin. Up to 90% of eukaryotic DNA is wrapped around consecutive octamers made of the core histones H2A, H2B, H3 and H4. Nucleosome positioning affects numerous cellular processes that require robust and timely access to genomic DNA, which is packaged into the tight confines of the cell nucleus. In living cells, nucleosome positions are determined by intrinsic histone-DNA sequence preferences, competition between histones and other DNA-binding proteins for genomic sequence, and ATP-dependent chromatin remodelers. We discuss the major energetic contributions to nucleosome formation and remodeling, focusing especially on partial DNA unwrapping off the histone octamer surface. DNA unwrapping enables efficient access to nucleosome-buried binding sites and mediates rapid nucleosome removal through concerted action of two or more DNA-binding factors. High-resolution, genome-scale maps of distances between neighboring nucleosomes have shown that DNA unwrapping and nucleosome crowding (mutual invasion of nucleosome territories) are much more common than previously thought. Ultimately, constraints imposed by nucleosome energetics on the rates of ATP-dependent and spontaneous chromatin remodeling determine nucleosome occupancy genome-wide, and shape pathways of cellular response to environmental stresses.

  8. Relationship between nucleosome positioning and DNA methylation

    PubMed Central

    Chodavarapu, Ramakrishna K.; Feng, Suhua; Bernatavichute, Yana V.; Chen, Pao-Yang; Stroud, Hume; Yu, Yanchun; Hetzel, Jonathan; Kuo, Frank; Kim, Jin; Cokus, Shawn J.; Casero, David; Bernal, Maria; Huijser, Peter; Clark, Amander T.; Krämer, Ute; Merchant, Sabeeha S.; Zhang, Xiaoyu; Jacobsen, Steven E.; Pellegrini, Matteo

    2010-01-01

    Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer1, 2. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana utilizing massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified ten base periodicities in the DNA methylation status of nucleosome-bound DNA and found that nucleosomal DNA was more highly methylated than flanking DNA. These results suggest that nucleosome positioning strongly influences DNA methylation patterning throughout the genome and that DNA methyltransferases preferentially target nucleosome-bound DNA. We also observed similar trends in human nucleosomal DNA suggesting that the relationships between nucleosomes and DNA methyltransferases are conserved. Finally, as has been observed in animals, nucleosomes were highly enriched on exons, and preferentially positioned at intron-exon and exon-intron boundaries. RNA Pol II was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition. PMID:20512117

  9. spFRET reveals changes in nucleosome breathing by neighboring nucleosomes.

    PubMed

    Buning, Ruth; Kropff, Wietske; Martens, Kirsten; van Noort, John

    2015-02-18

    Chromatin, the structure in which DNA is compacted in eukaryotic cells, plays a key role in regulating DNA accessibility. FRET experiments on single nucleosomes, the basic units in chromatin, have revealed a dynamic nucleosome where spontaneous DNA unwrapping from the ends provides access to the nucleosomal DNA. Here we investigated how this DNA breathing is affected by extension of the linker DNA and by the presence of a neighboring nucleosome. We found that both electrostatic interactions between the entering and exiting linker DNA and nucleosome-nucleosome interactions increase unwrapping. Interactions between neighboring nucleosomes are more likely in dinucleosomes spaced by 55 bp of linker DNA than in dinucleosomes spaced by 50 bp of linker DNA. Such increased unwrapping may not only increase the accessibility of nucleosomal DNA in chromatin fibers, it may also be key to folding of nucleosomes into higher order structures.

  10. spFRET reveals changes in nucleosome breathing by neighboring nucleosomes

    NASA Astrophysics Data System (ADS)

    Buning, Ruth; Kropff, Wietske; Martens, Kirsten; van Noort, John

    2015-02-01

    Chromatin, the structure in which DNA is compacted in eukaryotic cells, plays a key role in regulating DNA accessibility. FRET experiments on single nucleosomes, the basic units in chromatin, have revealed a dynamic nucleosome where spontaneous DNA unwrapping from the ends provides access to the nucleosomal DNA. Here we investigated how this DNA breathing is affected by extension of the linker DNA and by the presence of a neighboring nucleosome. We found that both electrostatic interactions between the entering and exiting linker DNA and nucleosome-nucleosome interactions increase unwrapping. Interactions between neighboring nucleosomes are more likely in dinucleosomes spaced by 55 bp of linker DNA than in dinucleosomes spaced by 50 bp of linker DNA. Such increased unwrapping may not only increase the accessibility of nucleosomal DNA in chromatin fibers, it may also be key to folding of nucleosomes into higher order structures.

  11. Structure of nucleosome-HMG complexes

    SciTech Connect

    Paton, A.E.

    1982-12-01

    This dissertation concentrates on the structure of HMG-nucleosome complexes, and how they differ from nucleosomes alone. The first chapter provides an introduction to chromatin and an overview of the field. The second and third chapters describe what kinds of nucleosome-HMG protein complexes form in solution, and where the HMG proteins may bind on the nucleosome. A model is proposed that locates the HMG binding sites on the nucleosome core particle. The fourth chapter describes the biophysical characterization of the complex. The methods include thermal denaturation, circular dichroism and sedimentation velocity, all done under variety of solvent conditions. These methods reveal a great deal of information on the stability and interactions of the complex. The fifth chapter describes conformational probes of the complex. These results reveal the structural transitions that occur when HMG protein binds to the nucleosome as well as the parts of the nucleosome essential for the binding reaction.

  12. Histone hypoacetylation-activated genes are repressed by acetyl-CoA- and chromatin-mediated mechanism

    PubMed Central

    Mehrotra, Swati; Galdieri, Luciano; Zhang, Tiantian; Zhang, Man; Pemberton, Lucy F.; Vancura, Ales

    2014-01-01

    Transcriptional activation is typically associated with increased acetylation of promoter histones. However, this paradigm does not apply to transcriptional activation of all genes. In this study we have characterized a group of genes that are repressed by histone acetylation. These histone hypoacetylation-activated genes (HHAAG) are normally repressed during exponential growth, when the cellular level of acetyl-CoA is high and global histone acetylation is also high. The HHAAG are induced during diauxic shift, when the levels of acetyl-CoA and global histone acetylation decrease. The histone hypoacetylation-induced activation of HHAAG is independent of Msn2/Msn4. The repression of HSP12, one of the HHAAG, is associated with well-defined nucleosomal structure in the promoter region, while histone hypoacetylation-induced activation correlates with delocalization of positioned nucleosomes or with reduced nucleosome occupancy. Correspondingly, unlike the majority of yeast genes, HHAAG are transcriptionally upregulated when expression of histone genes is reduced. Taken together, these results suggest a model in which histone acetylation is required for proper positioning of promoter nucleosomes and repression of HHAAG. PMID:24907648

  13. Using Atomic Force Microscopy To Study Chromatin Structure and Nucleosome Remodeling

    PubMed Central

    Lohr, D.; Bash, R.; Wang, H.; Yodh, J.; Lindsay, S.

    2007-01-01

    Atomic Force Microscopy (AFM) is a technique that can directly image single molecules in solution and it therefore provides a powerful tool for obtaining unique insights into the basic properties of biological materials and the functional processes in which they are involved. We have used AFM to analyze basic features of nucleosomes in arrays, such as DNA-histone binding strength, cooperativity in template occupation, nucleosome stabilities, nucleosome locations and the effects of acetylation, to compare these features in different types of arrays and to track the response of array nucleosomes to the action of the human Swi-Snf ATP-dependent nucleosome remodeling complex. These experiments required several specific adaptations of basic AFM methods, such as repetitive imaging of the same fields of molecules in liquid, the ability to change the environmental conditions of the sample being imaged and detection of specific types of molecules within compositionally complex samples. Here we describe the techniques that allowed such analyses to be carried out. PMID:17309844

  14. Nucleosome architecture throughout the cell cycle.

    PubMed

    Deniz, Özgen; Flores, Oscar; Aldea, Martí; Soler-López, Montserrat; Orozco, Modesto

    2016-01-28

    Nucleosomes provide additional regulatory mechanisms to transcription and DNA replication by mediating the access of proteins to DNA. During the cell cycle chromatin undergoes several conformational changes, however the functional significance of these changes to cellular processes are largely unexplored. Here, we present the first comprehensive genome-wide study of nucleosome plasticity at single base-pair resolution along the cell cycle in Saccharomyces cerevisiae. We determined nucleosome organization with a specific focus on two regulatory regions: transcription start sites (TSSs) and replication origins (ORIs). During the cell cycle, nucleosomes around TSSs display rearrangements in a cyclic manner. In contrast to gap (G1 and G2) phases, nucleosomes have a fuzzier organization during S and M phases, Moreover, the choreography of nucleosome rearrangements correlate with changes in gene expression during the cell cycle, indicating a strong association between nucleosomes and cell cycle-dependent gene functionality. On the other hand, nucleosomes are more dynamic around ORIs along the cell cycle, albeit with tighter regulation in early firing origins, implying the functional role of nucleosomes on replication origins. Our study provides a dynamic picture of nucleosome organization throughout the cell cycle and highlights the subsequent impact on transcription and replication activity.

  15. Nucleosome architecture throughout the cell cycle

    PubMed Central

    Deniz, Özgen; Flores, Oscar; Aldea, Martí; Soler-López, Montserrat; Orozco, Modesto

    2016-01-01

    Nucleosomes provide additional regulatory mechanisms to transcription and DNA replication by mediating the access of proteins to DNA. During the cell cycle chromatin undergoes several conformational changes, however the functional significance of these changes to cellular processes are largely unexplored. Here, we present the first comprehensive genome-wide study of nucleosome plasticity at single base-pair resolution along the cell cycle in Saccharomyces cerevisiae. We determined nucleosome organization with a specific focus on two regulatory regions: transcription start sites (TSSs) and replication origins (ORIs). During the cell cycle, nucleosomes around TSSs display rearrangements in a cyclic manner. In contrast to gap (G1 and G2) phases, nucleosomes have a fuzzier organization during S and M phases, Moreover, the choreography of nucleosome rearrangements correlate with changes in gene expression during the cell cycle, indicating a strong association between nucleosomes and cell cycle-dependent gene functionality. On the other hand, nucleosomes are more dynamic around ORIs along the cell cycle, albeit with tighter regulation in early firing origins, implying the functional role of nucleosomes on replication origins. Our study provides a dynamic picture of nucleosome organization throughout the cell cycle and highlights the subsequent impact on transcription and replication activity. PMID:26818620

  16. Changing chromatin fiber conformation by nucleosome repositioning.

    PubMed

    Müller, Oliver; Kepper, Nick; Schöpflin, Robert; Ettig, Ramona; Rippe, Karsten; Wedemann, Gero

    2014-11-04

    Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ?10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.

  17. Changing Chromatin Fiber Conformation by Nucleosome Repositioning

    PubMed Central

    Müller, Oliver; Kepper, Nick; Schöpflin, Robert; Ettig, Ramona; Rippe, Karsten; Wedemann, Gero

    2014-01-01

    Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell. PMID:25418099

  18. A cooperative activation loop among SWI/SNF, gamma-H2AX and H3 acetylation for DNA double-strand break repair.

    PubMed

    Lee, Han-Sae; Park, Ji-Hye; Kim, So-Jung; Kwon, Su-Jung; Kwon, Jongbum

    2010-04-21

    Although recent studies highlight the importance of histone modifications and ATP-dependent chromatin remodelling in DNA double-strand break (DSB) repair, how these mechanisms cooperate has remained largely unexplored. Here, we show that the SWI/SNF chromatin remodelling complex, earlier known to facilitate the phosphorylation of histone H2AX at Ser-139 (S139ph) after DNA damage, binds to gamma-H2AX (the phosphorylated form of H2AX)-containing nucleosomes in S139ph-dependent manner. Unexpectedly, BRG1, the catalytic subunit of SWI/SNF, binds to gamma-H2AX nucleosomes by interacting with acetylated H3, not with S139ph itself, through its bromodomain. Blocking the BRG1 interaction with gamma-H2AX nucleosomes either by deletion or overexpression of the BRG1 bromodomain leads to defect of S139ph and DSB repair. H3 acetylation is required for the binding of BRG1 to gamma-H2AX nucleosomes. S139ph stimulates the H3 acetylation on gamma-H2AX nucleosomes, and the histone acetyltransferase Gcn5 is responsible for this novel crosstalk. The H3 acetylation on gamma-H2AX nucleosomes is induced by DNA damage. These results collectively suggest that SWI/SNF, gamma-H2AX and H3 acetylation cooperatively act in a feedback activation loop to facilitate DSB repair.

  19. SWI/SNF has intrinsic nucleosome disassembly activity that is dependent on adjacent nucleosomes.

    PubMed

    Dechassa, Mekonnen Lemma; Sabri, Abdellah; Pondugula, Santhi; Kassabov, Stefan R; Chatterjee, Nilanjana; Kladde, Michael P; Bartholomew, Blaine

    2010-05-28

    The ATP-dependent chromatin remodeling complex SWI/SNF regulates transcription and has been implicated in promoter nucleosome eviction. Efficient nucleosome disassembly by SWI/SNF alone in biochemical assays, however, has not been directly observed. Employing a model system of dinucleosomes rather than mononucleosomes, we demonstrate that remodeling leads to ordered and efficient disassembly of one of the two nucleosomes. An H2A/H2B dimer is first rapidly displaced, and then, in a slower reaction, an entire histone octamer is lost. Nucleosome disassembly by SWI/SNF did not require additional factors such as chaperones or acceptors of histones. Observations in single molecules as well as bulk measurement suggest that a key intermediate in this process is one in which a nucleosome is moved toward the adjacent nucleosome. SWI/SNF recruited by the transcriptional activator Gal4-VP16 preferentially mobilizes the proximal nucleosome and destabilizes the adjacent nucleosome.

  20. Transcription of nucleosomes from human chromatin.

    PubMed Central

    Shaw, P A; Sahasrabuddhe, C G; Hodo, H G; Saunders, G F

    1978-01-01

    Nucleosomes (chromatin subunits) prepared by micrococcal nuclease digestion of human nuclei are similar in histone content but substantially reduced in non-histone proteins as compared to undigested chromatin. Chromatin transcription experiments indicate that the DNA in the nucleosomes is accessible to DNA-dependent RNA polymerase in vitro. The template capacities of chromatin and nucleosomes are 1.5 and 10%, respectively, relative to high molecular weight DNA, with intermediate values for oligonucleosomes. Three distinct sizes of transcripts, 150, 120 and 95 nucleotides in length, are obtained when nucleosomes are used as templates. However, when nucleosomal DNA is used as a template, the predominant size of transcripts is 150 nucleotides. When oligonucleosomes are used as templates longer transcripts are obtained. This indicates that RNA polymerase can transcribe the DNA contained in the nucleosomes. PMID:693325

  1. The N-terminal domains of histones H3 and H4 are not necessary for chromatin assembly factor-1- mediated nucleosome assembly onto replicated DNA in vitro

    PubMed Central

    Shibahara, Kei-ichi; Verreault, Alain; Stillman, Bruce

    2000-01-01

    An in vitro reconstitution system for the analysis of replication-coupled nucleosome assembly is described. In this “two-step system,” nucleosome assembly is performed in a separate reaction from DNA replication, wherein purified newly replicated DNA remains noncovalently marked for subsequent chromatin assembly factor-1 (CAF-1)-dependent nucleosome assembly. Because the nucleosome assembly is performed separately from the DNA replication step, this system is more versatile and biochemically tractable when compared with nucleosome assembly during simian virus 40 (SV40) DNA replication. The N-terminal domains of histones H3 and H4 play an important but redundant function in nucleosome assembly in the budding yeast, Saccharomyces cerevisiae. It had been proposed that at least one tail of histone H3 or H4 is required for replication-coupled nucleosome assembly. However, we demonstrate that the N-terminal domains of both histone H3 and H4 are dispensable for CAF-1-mediated formation of nucleosome cores onto newly replicated DNA in vitro. CAF-1 and each of its individual subunits stably bound to recombinant (H3.H4)2 tetramers lacking the N-terminal domains of both H3 and H4. Therefore, the N-terminal tails of histone H3 and H4 that contain the specific acetylation sites are not necessary for CAF-1-dependent nucleosome assembly onto replicated DNA. We suggest that the histone acetylation may be required for a CAF-1 independent pathway or function after deposition, by marking of newly replicated chromatin. PMID:10884407

  2. Recognition Imaging of Acetylated Chromatin Using a DNA Aptamer

    PubMed Central

    Lin, Liyun; Fu, Qiang; Williams, Berea A.R.; Azzaz, Abdelhamid M.; Shogren-Knaak, Michael A.; Chaput, John C.; Lindsay, Stuart

    2009-01-01

    Histone acetylation plays an important role in the regulation of gene expression. A DNA aptamer generated by in vitro selection to be highly specific for histone H4 protein acetylated at lysine 16 was used as a recognition element for atomic force microscopy-based recognition imaging of synthetic nucleosomal arrays with precisely controlled acetylation. The aptamer proved to be reasonably specific at recognizing acetylated histones, with recognition efficiencies of 60% on-target and 12% off-target. Though this selectivity is much poorer than the >2000:1 equilibrium specificity of the aptamer, it is a large improvement on the performance of a ChIP-quality antibody, which is not selective at all in this application, and it should permit high-fidelity recognition with repeated imaging. The ability to image the precise location of posttranslational modifications may permit nanometer-scale investigation of their effect on chromatin structure. PMID:19751687

  3. Mapping nucleosome positions using DNase-seq.

    PubMed

    Zhong, Jianling; Luo, Kaixuan; Winter, Peter S; Crawford, Gregory E; Iversen, Edwin S; Hartemink, Alexander J

    2016-03-01

    Although deoxyribonuclease I (DNase I) was used to probe the structure of the nucleosome in the 1960s and 1970s, in the current high-throughput sequencing era, DNase I has mainly been used to study genomic regions devoid of nucleosomes. Here, we reveal for the first time that DNase I can be used to precisely map the (translational) positions of in vivo nucleosomes genome-wide. Specifically, exploiting a distinctive DNase I cleavage profile within nucleosome-associated DNA--including a signature 10.3 base pair oscillation that corresponds to accessibility of the minor groove as DNA winds around the nucleosome--we develop a Bayes-factor-based method that can be used to map nucleosome positions along the genome. Compared to methods that require genetically modified histones, our DNase-based approach is easily applied in any organism, which we demonstrate by producing maps in yeast and human. Compared to micrococcal nuclease (MNase)-based methods that map nucleosomes based on cuts in linker regions, we utilize DNase I cuts both outside and within nucleosomal DNA; the oscillatory nature of the DNase I cleavage profile within nucleosomal DNA enables us to identify translational positioning details not apparent in MNase digestion of linker DNA. Because the oscillatory pattern corresponds to nucleosome rotational positioning, it also reveals the rotational context of transcription factor (TF) binding sites. We show that potential binding sites within nucleosome-associated DNA are often centered preferentially on an exposed major or minor groove. This preferential localization may modulate TF interaction with nucleosome-associated DNA as TFs search for binding sites.

  4. Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density

    PubMed Central

    Lieleg, Corinna; Ketterer, Philip; Nuebler, Johannes; Ludwigsen, Johanna; Gerland, Ulrich; Dietz, Hendrik

    2015-01-01

    Arrays of regularly spaced nucleosomes are a hallmark of chromatin, but it remains unclear how they are generated. Recent genome-wide studies, in vitro and in vivo, showed constant nucleosome spacing even if the histone concentration was experimentally reduced. This counters the long-held assumption that nucleosome density determines spacing and calls for factors keeping spacing constant regardless of nucleosome density. We call this a clamping activity. Here, we show in a purified system that ISWI- and CHD1-type nucleosome remodelers have a clamping activity such that they not only generate regularly spaced nucleosome arrays but also generate constant spacing regardless of nucleosome density. This points to a functionally attractive nucleosome interaction that could be mediated either directly by nucleosome-nucleosome contacts or indirectly through the remodelers. Mutant Drosophila melanogaster ISWI without the HAND-SANT-SLIDE (HSS) domain had no detectable spacing activity even though it is known to remodel and slide nucleosomes. This suggests that the role of ISWI remodelers in generating constant spacing is not just to mediate nucleosome sliding; they actively contribute to the attractive interaction. Additional factors are necessary to set physiological spacing in absolute terms. PMID:25733687

  5. Nucleosome spacing generated by ISWI and CHD1 remodelers is constant regardless of nucleosome density.

    PubMed

    Lieleg, Corinna; Ketterer, Philip; Nuebler, Johannes; Ludwigsen, Johanna; Gerland, Ulrich; Dietz, Hendrik; Mueller-Planitz, Felix; Korber, Philipp

    2015-05-01

    Arrays of regularly spaced nucleosomes are a hallmark of chromatin, but it remains unclear how they are generated. Recent genome-wide studies, in vitro and in vivo, showed constant nucleosome spacing even if the histone concentration was experimentally reduced. This counters the long-held assumption that nucleosome density determines spacing and calls for factors keeping spacing constant regardless of nucleosome density. We call this a clamping activity. Here, we show in a purified system that ISWI- and CHD1-type nucleosome remodelers have a clamping activity such that they not only generate regularly spaced nucleosome arrays but also generate constant spacing regardless of nucleosome density. This points to a functionally attractive nucleosome interaction that could be mediated either directly by nucleosome-nucleosome contacts or indirectly through the remodelers. Mutant Drosophila melanogaster ISWI without the Hand-Sant-Slide (HSS) domain had no detectable spacing activity even though it is known to remodel and slide nucleosomes. This suggests that the role of ISWI remodelers in generating constant spacing is not just to mediate nucleosome sliding; they actively contribute to the attractive interaction. Additional factors are necessary to set physiological spacing in absolute terms.

  6. ISWI remodels nucleosomes through a random walk.

    PubMed

    Al-Ani, Gada; Malik, Shuja Shafi; Eastlund, Allen; Briggs, Koan; Fischer, Christopher J

    2014-07-15

    The chromatin remodeler ISWI is capable of repositioning clusters of nucleosomes to create well-ordered arrays or moving single nucleosomes from the center of DNA fragments toward the ends without disrupting their integrity. Using standard electrophoresis assays, we have monitored the ISWI-catalyzed repositioning of different nucleosome samples each containing a different length of DNA symmetrically flanking the initially centrally positioned histone octamer. We find that ISWI moves the histone octamer between distinct and thermodynamically stable positions on the DNA according to a random walk mechanism. Through the application of a spectrophotometric assay for nucleosome repositioning, we further characterized the repositioning activity of ISWI using short nucleosome substrates and were able to determine the macroscopic rate of nucleosome repositioning by ISWI. Additionally, quantitative analysis of repositioning experiments performed at various ISWI concentrations revealed that a monomeric ISWI is sufficient to obtain the observed repositioning activity as the presence of a second ISWI bound had no effect on the rate of nucleosome repositioning. We also found that ATP hydrolysis is poorly coupled to nucleosome repositioning, suggesting that DNA translocation by ISWI is not energetically rate-limiting for the repositioning reaction. This is the first calculation of a microscopic ATPase coupling efficiency for nucleosome repositioning and also further supports our conclusion that a second bound ISWI does not contribute to the repositioning reaction.

  7. ISWI Remodels Nucleosomes through a Random Walk

    PubMed Central

    2015-01-01

    The chromatin remodeler ISWI is capable of repositioning clusters of nucleosomes to create well-ordered arrays or moving single nucleosomes from the center of DNA fragments toward the ends without disrupting their integrity. Using standard electrophoresis assays, we have monitored the ISWI-catalyzed repositioning of different nucleosome samples each containing a different length of DNA symmetrically flanking the initially centrally positioned histone octamer. We find that ISWI moves the histone octamer between distinct and thermodynamically stable positions on the DNA according to a random walk mechanism. Through the application of a spectrophotometric assay for nucleosome repositioning, we further characterized the repositioning activity of ISWI using short nucleosome substrates and were able to determine the macroscopic rate of nucleosome repositioning by ISWI. Additionally, quantitative analysis of repositioning experiments performed at various ISWI concentrations revealed that a monomeric ISWI is sufficient to obtain the observed repositioning activity as the presence of a second ISWI bound had no effect on the rate of nucleosome repositioning. We also found that ATP hydrolysis is poorly coupled to nucleosome repositioning, suggesting that DNA translocation by ISWI is not energetically rate-limiting for the repositioning reaction. This is the first calculation of a microscopic ATPase coupling efficiency for nucleosome repositioning and also further supports our conclusion that a second bound ISWI does not contribute to the repositioning reaction. PMID:24898619

  8. The Genomic Code for Nucleosome Positioning

    NASA Astrophysics Data System (ADS)

    Widom, Jonathan

    2008-03-01

    Eukaryotic genomes encode an additional layer of genetic information, superimposed on top of the regulatory and coding information, that controls the organization of the genomic DNA into arrays of nucleosomes. We have developed a partial ability to read this nucleosome positioning code and predict the in vivo locations of nucleosomes. Our results suggest that genomes utilize the nucleosome positioning code to facilitate specific chromosome functions including to delineate functional versus nonfunctional binding sites for key gene regulatory proteins, and to define the next higher level of chromosome structure itself.

  9. Reading sequence-directed computational nucleosome maps.

    PubMed

    Nibhani, Reshma; Trifonov, Edward N

    2015-01-01

    Recently developed latest version of the sequence-directed single-base resolution nucleosome mapping reveals existence of strong nucleosomes and chromatin columnar structures (columns). Broad application of this simple technique for further studies of chromatin and chromosome structure requires some basic understanding as to how it works and what information it affords. The paper provides such an introduction to the method. The oscillating maps of singular nucleosomes, of short and long oligonucleosome columns, are explained, as well as maps of chromatin on satellite DNA and occurrences of counter-phase (antiparallel) nucleosome neighbors.

  10. Effects of DNA methylation on nucleosome stability.

    PubMed

    Collings, Clayton K; Waddell, Peter J; Anderson, John N

    2013-03-01

    Methylation of DNA at CpG dinucleotides represents one of the most important epigenetic mechanisms involved in the control of gene expression in vertebrate cells. In this report, we conducted nucleosome reconstitution experiments in conjunction with high-throughput sequencing on 572 KB of human DNA and 668 KB of mouse DNA that was unmethylated or methylated in order to investigate the effects of this epigenetic modification on the positioning and stability of nucleosomes. The results demonstrated that a subset of nucleosomes positioned by nucleotide sequence was sensitive to methylation where the modification increased the affinity of these sequences for the histone octamer. The features that distinguished these nucleosomes from the bulk of the methylation-insensitive nucleosomes were an increase in the frequency of CpG dinucleotides and a unique rotational orientation of CpGs such that their minor grooves tended to face toward the histones in the nucleosome rather than away. These methylation-sensitive nucleosomes were preferentially associated with exons as compared to introns while unmethylated CpG islands near transcription start sites became enriched in nucleosomes upon methylation. The results of this study suggest that the effects of DNA methylation on nucleosome stability in vitro can recapitulate what has been observed in the cell and provide a direct link between DNA methylation and the structure and function of chromatin.

  11. Ubiquitous nucleosome crowding in the yeast genome.

    PubMed

    Chereji, Răzvan V; Morozov, Alexandre V

    2014-04-08

    Nucleosomes may undergo a conformational change in which a stretch of DNA peels off the histone octamer surface as a result of thermal fluctuations or interactions with chromatin remodelers. Thus, neighboring nucleosomes may invade each other's territories by DNA unwrapping and translocation, or through initial assembly in partially wrapped states. A recent high-resolution map of distances between dyads of neighboring nucleosomes in Saccharomyces cerevisiae reveals that nucleosomes frequently overlap DNA territories of their neighbors. This conclusion is supported by lower-resolution maps of S. cerevisiae nucleosome lengths based on micrococcal nuclease digestion and paired-end sequencing. The average length of wrapped DNA follows a stereotypical pattern in genes and promoters, correlated with the well-known distribution of nucleosome occupancy: nucleosomal DNA tends to be shorter in promoters and longer in coding regions. To explain these observations, we have developed a biophysical model that uses a 10-11-bp periodic histone-DNA binding energy profile. The profile is based on the pattern of histone-DNA contacts in nucleosome crystal structures, as well as the idea of linker length discretization caused by higher-order chromatin structure. Our model is in agreement with the observed genome-wide distributions of interdyad distances, wrapped DNA lengths, and nucleosome occupancies. Furthermore, our approach explains in vitro measurements of the accessibility of nucleosome-covered target sites and nucleosome-induced cooperativity between DNA-binding factors. We rule out several alternative scenarios of histone-DNA interactions as inconsistent with the genomic data.

  12. Chromatin remodelers clear nucleosomes from intrinsically unfavorable sites to establish nucleosome-depleted regions at promoters.

    PubMed

    Tolkunov, Denis; Zawadzki, Karl A; Singer, Cara; Elfving, Nils; Morozov, Alexandre V; Broach, James R

    2011-06-15

    Most promoters in yeast contain a nucleosome-depleted region (NDR), but the mechanisms by which NDRs are established and maintained in vivo are currently unclear. We have examined how genome-wide nucleosome placement is altered in the absence of two distinct types of nucleosome remodeling activity. In mutants of both SNF2, which encodes the ATPase component of the Swi/Snf remodeling complex, and ASF1, which encodes a histone chaperone, distinct sets of gene promoters carry excess nucleosomes in their NDRs relative to wild-type. In snf2 mutants, excess promoter nucleosomes correlate with reduced gene expression. In both mutants, the excess nucleosomes occupy DNA sequences that are energetically less favorable for nucleosome formation, indicating that intrinsic histone-DNA interactions are not sufficient for nucleosome positioning in vivo, and that Snf2 and Asf1 promote thermodynamic equilibration of nucleosomal arrays. Cells lacking SNF2 or ASF1 still accomplish the changes in promoter nucleosome structure associated with large-scale transcriptional reprogramming. However, chromatin reorganization in the mutants is reduced in extent compared to wild-type cells, even though transcriptional changes proceed normally. In summary, active remodeling is required for distributing nucleosomes to energetically favorable positions in vivo and for reorganizing chromatin in response to changes in transcriptional activity.

  13. Canonical nucleosome organization at promoters forms during genome activation.

    PubMed

    Zhang, Yong; Vastenhouw, Nadine L; Feng, Jianxing; Fu, Kai; Wang, Chenfei; Ge, Ying; Pauli, Andrea; van Hummelen, Paul; Schier, Alexander F; Liu, X Shirley

    2014-02-01

    The organization of nucleosomes influences transcriptional activity by controlling accessibility of DNA binding proteins to the genome. Genome-wide nucleosome binding profiles have identified a canonical nucleosome organization at gene promoters, where arrays of well-positioned nucleosomes emanate from nucleosome-depleted regions. The mechanisms of formation and the function of canonical promoter nucleosome organization remain unclear. Here we analyze the genome-wide location of nucleosomes during zebrafish embryogenesis and show that well-positioned nucleosome arrays appear on thousands of promoters during the activation of the zygotic genome. The formation of canonical promoter nucleosome organization is independent of DNA sequence preference, transcriptional elongation, and robust RNA polymerase II (Pol II) binding. Instead, canonical promoter nucleosome organization correlates with the presence of histone H3 lysine 4 trimethylation (H3K4me3) and affects future transcriptional activation. These findings reveal that genome activation is central to the organization of nucleosome arrays during early embryogenesis.

  14. Nucleosome dynamics: HMGB1 relaxes canonical nucleosome structure to facilitate estrogen receptor binding.

    PubMed

    Joshi, Sachindra R; Sarpong, Yaw C; Peterson, Ronald C; Scovell, William M

    2012-11-01

    High mobility group protein 1 (HMGB1) interacts with DNA and chromatin to influence the regulation of transcription, DNA repair and recombination. We show that HMGB1 alters the structure and stability of the canonical nucleosome (N) in a nonenzymatic, ATP-independent manner. Although estrogen receptor (ER) does not bind to its consensus estrogen response element within a nucleosome, HMGB1 restructures the nucleosome to facilitate strong ER binding. The isolated HMGB1-restructured nucleosomes (N' and N″) remain stable and exhibit characteristics distinctly different from the canonical nucleosome. These findings complement previous studies that showed (i) HMGB1 stimulates in vivo transcriptional activation at estrogen response elements and (ii) knock down of HMGB1 expression by siRNA precipitously reduced transcriptional activation. The findings indicate that one aspect of the mechanism of HMGB1 action involves a restructuring of the nucleosome that appears to relax structural constraints within the nucleosome.

  15. Sequence-based prediction of single nucleosome positioning and genome-wide nucleosome occupancy.

    PubMed

    van der Heijden, Thijn; van Vugt, Joke J F A; Logie, Colin; van Noort, John

    2012-09-18

    Nucleosome positioning dictates eukaryotic DNA compaction and access. To predict nucleosome positions in a statistical mechanics model, we exploited the knowledge that nucleosomes favor DNA sequences with specific periodically occurring dinucleotides. Our model is the first to capture both dyad position within a few base pairs, and free binding energy within 2 k(B)T, for all the known nucleosome positioning sequences. By applying Percus's equation to the derived energy landscape, we isolate sequence effects on genome-wide nucleosome occupancy from other factors that may influence nucleosome positioning. For both in vitro and in vivo systems, three parameters suffice to predict nucleosome occupancy with correlation coefficients of respectively 0.74 and 0.66. As predicted, we find the largest deviations in vivo around transcription start sites. This relatively simple algorithm can be used to guide future studies on the influence of DNA sequence on chromatin organization.

  16. Nucleosomes influence multiple steps during replication initiation

    PubMed Central

    Azmi, Ishara F; Watanabe, Shinya; Maloney, Michael F; Kang, Sukhyun; Belsky, Jason A; MacAlpine, David M; Peterson, Craig L; Bell, Stephen P

    2017-01-01

    Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin. DOI: http://dx.doi.org/10.7554/eLife.22512.001 PMID:28322723

  17. A physical analysis of nucleosome positioning

    NASA Astrophysics Data System (ADS)

    Gerland, Ulrich

    2015-03-01

    The first level of genome packaging in eukaryotic cells involves the formation of dense nucleosome arrays, with DNA coverage near 90% in yeasts. A high nucleosome coverage is essential for cells, e.g. to prevent cryptic transcription, and the local positions of specific nucleosomes can play an important role in gene regulation. It is known that in vivo nucleosome positions are affected by a complex mix of passive and active mechanisms, including sequence-specific histone-DNA binding, nucleosome-nucleosome interactions, ATP-dependent remodeling enzymes, transcription, and DNA replication. Yet, the statistical distribution of nucleosome positions is extremely well described by simple physical models that treat the chromatin fiber as an interacting one-dimensional gas. I will discuss how can we interpret this surprising observation from a mechanistic perspective. I will also discuss the kinetics of the interacting gas model, which is pertinent to the question of how cells achieve the high nucleosome coverage within a short time, e.g. after DNA replication.

  18. Exploring Nucleosome Unwrapping Using DNA Origami.

    PubMed

    Funke, Jonas J; Ketterer, Philip; Lieleg, Corinna; Korber, Philipp; Dietz, Hendrik

    2016-12-14

    We establish a DNA origami based tool for quantifying conformational equilibria of biomolecular assemblies as a function of environmental conditions. As first application, we employed the tool to study the salt-induced disassembly of nucleosome core particles. To extract binding constants and energetic penalties, we integrated nucleosomes in the spectrometer such that unwrapping of the nucleosomal template DNA, leading from bent to more extended states was directly coupled to the conformation of the spectrometer. Nucleosome unwrapping was induced by increasing the ionic strength. The corresponding shifts in conformation equilibrium of the spectrometer were followed by direct conformation imaging using negative staining TEM and by FRET read out after gel electrophoretic separation of conformations. We find nucleosome dissociation constants in the picomolar range at low ionic strength (11 mM MgCl2), in the nanomolar range at intermediate ionic strength (11 mM MgCl2 with 0.5-1 M NaCl) and in the micromolar range at larger ionic strength (11 mM MgCl2 with ≥1.5 M NaCl). Integration of up to four nucleosomes stacked side-by-side, as it might occur within chromatin fibers, did not appear to affect the salt-induced unwrapping of nucleosomes. Presumably, such stacking interactions are already effectively screened at the nucleosome unwrapping conditions. Our spectrometer provides a modular platform with a direct read out to study conformational equilibria for targets from small biomolecules up to large macromolecular assemblies.

  19. The organization of nucleosomes around splice sites

    PubMed Central

    Chen, Wei; Luo, Liaofu; Zhang, Lirong

    2010-01-01

    The occupancy of nucleosomes along chromosome is a key factor for gene regulation. However, except promoter regions, genome-wide properties and functions of nucleosome organization remain unclear in mammalian genomes. Using the computational model of Increment of Diversity with Quadratic Discriminant (IDQD) trained from the microarray data, the nucleosome occupancy score (NOScore) was defined and applied to splice junction regions of constitutive, cassette exon, alternative 3′ and 5′ splicing events in the human genome. We found an interesting relation between NOScore and RNA splicing: exon regions have higher NOScores compared with their flanking intron sequences in both constitutive and alternative splicing events, indicating the stronger nucleosome occupation potential of exon regions. In addition, NOScore valleys present at ∼25 bp upstream of the acceptor site in all splicing events. By defining folding diversity-to-energy ratio to describe RNA structural flexibility, we demonstrated that primary RNA transcripts from nucleosome occupancy regions are relatively rigid and those from nucleosome depleted regions are relatively flexible. The negative correlation between nucleosome occupation/depletion of DNA sequence and structural flexibility/rigidity of its primary transcript around splice junctions may provide clues to the deeper understanding of the unexpected role for nucleosome organization in the regulation of RNA splicing. PMID:20097656

  20. Structural analysis of nucleosomal barrier to transcription

    PubMed Central

    Gaykalova, Daria A.; Kulaeva, Olga I.; Volokh, Olesya; Shaytan, Alexey K.; Hsieh, Fu-Kai; Kirpichnikov, Mikhail P.; Sokolova, Olga S.; Studitsky, Vasily M.

    2015-01-01

    Thousands of human and Drosophila genes are regulated at the level of transcript elongation and nucleosomes are likely targets for this regulation. However, the molecular mechanisms of formation of the nucleosomal barrier to transcribing RNA polymerase II (Pol II) and nucleosome survival during/after transcription remain unknown. Here we show that both DNA–histone interactions and Pol II backtracking contribute to formation of the barrier and that nucleosome survival during transcription likely occurs through allosterically stabilized histone–histone interactions. Structural analysis indicates that after Pol II encounters the barrier, the enzyme backtracks and nucleosomal DNA recoils on the octamer, locking Pol II in the arrested state. DNA is displaced from one of the H2A/H2B dimers that remains associated with the octamer. The data reveal the importance of intranucleosomal DNA–protein and protein–protein interactions during conformational changes in the nucleosome structure on transcription. Mechanisms of nucleosomal barrier formation and nucleosome survival during transcription are proposed. PMID:26460019

  1. A Brief Review of Nucleosome Structure

    PubMed Central

    Cutter, Amber; Hayes, Jeffrey J.

    2015-01-01

    The nucleosomal subunit organization of chromatin provides a multitude of functions. Nucleosomes elicit an initial ~7-fold linear compaction of genomic DNA. They provide a critical mechanism for stable repression of genes and other DNA-dependent activities by restricting binding of trans-acting factors to cognate DNA sequences. Conversely they are engineered to be nearly meta-stable and disassembled (and reassembled) in a facile manner to allow rapid access to the underlying DNA during processes such as transcription, replication and DNA repair. Nucleosomes protect the genome from DNA damaging agents and provide a lattice onto which a myriad of epigenetic signals are deposited. Moreover, vast strings of nucleosomes provide a framework for assembly of the chromatin fiber and higher-order chromatin structures. Thus, in order to provide a foundation for understanding these functions, we present a review of the basic elements of nucleosome structure and stability, including the association of linker histones. PMID:25980611

  2. Genome-wide nucleosome mapping of Plasmodium falciparum reveals histone-rich coding and histone-poor intergenic regions and chromatin remodeling of core and subtelomeric genes.

    PubMed

    Westenberger, Scott J; Cui, Long; Dharia, Neekesh; Winzeler, Elizabeth; Cui, Liwang

    2009-12-16

    start sites or transcription factor binding sites. Using the nucleosomal occupancy data as the baseline, we further mapped the genome-wide enrichment of H3K9 acetylation and detected general enrichment of this mark in intergenic regions. These data on nucleosome enrichment changes add to our understanding of the influence of chromatin structure on the regulation of gene expression. Histones are generally enriched in coding regions, and relatively poor in intergenic regions. Histone enrichment patterns allow for identification of new putative gene-coding regions. Most genes do not show correlation between chromatin structure and steady-state mRNA levels, indicating the dominant roles of other regulatory mechanisms. We present a genome-wide nucleosomal occupancy map, which can be used as a reference for future experiments of histone modification mapping.

  3. A barrier nucleosome model for statistical positioning of nucleosomes throughout the yeast genome.

    PubMed

    Mavrich, Travis N; Ioshikhes, Ilya P; Venters, Bryan J; Jiang, Cizhong; Tomsho, Lynn P; Qi, Ji; Schuster, Stephan C; Albert, Istvan; Pugh, B Franklin

    2008-07-01

    Most nucleosomes are well-organized at the 5' ends of S. cerevisiae genes where "-1" and "+1" nucleosomes bracket a nucleosome-free promoter region (NFR). How nucleosomal organization is specified by the genome is less clear. Here we establish and inter-relate rules governing genomic nucleosome organization by sequencing DNA from more than one million immunopurified S. cerevisiae nucleosomes (displayed at http://atlas.bx.psu.edu/). Evidence is presented that the organization of nucleosomes throughout genes is largely a consequence of statistical packing principles. The genomic sequence specifies the location of the -1 and +1 nucleosomes. The +1 nucleosome forms a barrier against which nucleosomes are packed, resulting in uniform positioning, which decays at farther distances from the barrier. We present evidence for a novel 3' NFR that is present at >95% of all genes. 3' NFRs may be important for transcription termination and anti-sense initiation. We present a high-resolution genome-wide map of TFIIB locations that implicates 3' NFRs in gene looping.

  4. Replication-guided nucleosome packing and nucleosome breathing expedite the formation of dense arrays.

    PubMed

    Osberg, Brendan; Nuebler, Johannes; Korber, Philipp; Gerland, Ulrich

    2014-12-16

    The first level of genome packaging in eukaryotic cells involves the formation of dense nucleosome arrays, with DNA coverage near 90% in yeasts. How cells achieve such high coverage within a short time, e.g. after DNA replication, remains poorly understood. It is known that random sequential adsorption of impenetrable particles on a line reaches high density extremely slowly, due to a jamming phenomenon. The nucleosome-shifting action of remodeling enzymes has been proposed as a mechanism to resolve such jams. Here, we suggest two biophysical mechanisms which assist rapid filling of DNA with nucleosomes, and we quantitatively characterize these mechanisms within mathematical models. First, we show that the 'softness' of nucleosomes, due to nucleosome breathing and stepwise nucleosome assembly, significantly alters the filling behavior, speeding up the process relative to 'hard' particles with fixed, mutually exclusive DNA footprints. Second, we explore model scenarios in which the progression of the replication fork could eliminate nucleosome jamming, either by rapid filling in its wake or via memory of the parental nucleosome positions. Taken together, our results suggest that biophysical effects promote rapid nucleosome filling, making the reassembly of densely packed nucleosomes after DNA replication a simpler task for cells than was previously thought.

  5. Causal role of histone acetylations in enhancer function

    PubMed Central

    Pradeepa, Madapura M.

    2017-01-01

    ABSTRACT Enhancers control development and cellular function by spatiotemporal regulation of gene expression. Co-occurrence of acetylation of histone H3 at lysine 27 (H3K27ac) and mono methylation of histone H3 at lysine 4 (H3K4me1) has been widely used for identification of active enhancers. However, increasing evidence suggests that using this combination of marks alone for enhancer identification gives an incomplete picture of the active enhancer repertoire. We have shown that the H3 globular domain acetylations, H3K64ac and H3K122ac, and an H4 tail acetylation, H4K16ac, are enriched at active enhancers together with H3K27ac, and also at a large number of enhancers without detectable H3K27ac. We propose that acetylations at these lysine residues of histones H3 and H4 might function by directly affecting chromatin structure, nucleosome–nucleosome interactions, nucleosome stability, and transcription factor accessibility. PMID:27792455

  6. Stability of nucleosome placement in newly repaired regions of DNA

    SciTech Connect

    Nissen, K.A.; Lan, S.Y.; Smerdon, M.J.

    1986-07-05

    Rearrangements of chromatin structure during excision repair of UV-damaged DNA appear to involve unfolding of nucleosomal DNA while repair is taking place, followed by refolding of this DNA into a native nucleosome structure. Recently, we found that repair patches are not distributed uniformly along the DNA in nucleosome core particles immediately following their refolding into nucleosomes. Therefore, the distribution of repair patches in nucleosome core DNA was used to monitor the stability of nucleosome placement in these regions. Our results indicate that in nondividing human cells undergoing excision repair there is a slow change in the positioning of nucleosomes in newly repaired regions of chromatin, resulting in the eventual randomization of repair patches in nucleosome core DNA. Furthermore, the nonrandom placement of nucleosomes observed just after the refolding event is not re-established during DNA replication. Possible mechanisms for this change in nucleosome placement along the DNA are discussed.

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

    PubMed

    Salih, Bilal; Trifonov, Edward N

    2015-01-01

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

  8. Overcoming a nucleosomal barrier to replication

    PubMed Central

    Chang, Han-Wen; Pandey, Manjula; Kulaeva, Olga I.; Patel, Smita S.; Studitsky, Vasily M.

    2016-01-01

    Efficient overcoming and accurate maintenance of chromatin structure and associated histone marks during DNA replication are essential for normal functioning of the daughter cells. However, the molecular mechanisms of replication through chromatin are unknown. We have studied traversal of uniquely positioned mononucleosomes by T7 replisome in vitro. Nucleosomes present a strong, sequence-dependent barrier for replication, with particularly strong pausing of DNA polymerase at the +(31–40) and +(41–65) regions of the nucleosomal DNA. The exonuclease activity of T7 DNA polymerase increases the overall rate of progression of the replisome through a nucleosome, likely by resolving nonproductive complexes. The presence of nucleosome-free DNA upstream of the replication fork facilitates the progression of DNA polymerase through the nucleosome. After replication, at least 50% of the nucleosomes assume an alternative conformation, maintaining their original positions on the DNA. Our data suggest a previously unpublished mechanism for nucleosome maintenance during replication, likely involving transient formation of an intranucleosomal DNA loop. PMID:27847876

  9. Structural basis for retroviral integration into nucleosomes.

    PubMed

    Maskell, Daniel P; Renault, Ludovic; Serrao, Erik; Lesbats, Paul; Matadeen, Rishi; Hare, Stephen; Lindemann, Dirk; Engelman, Alan N; Costa, Alessandro; Cherepanov, Peter

    2015-07-16

    Retroviral integration is catalysed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration.

  10. Stepwise nucleosome translocation by RSC remodeling complexes.

    PubMed

    Harada, Bryan T; Hwang, William L; Deindl, Sebastian; Chatterjee, Nilanjana; Bartholomew, Blaine; Zhuang, Xiaowei

    2016-02-19

    The SWI/SNF-family remodelers regulate chromatin structure by coupling the free energy from ATP hydrolysis to the repositioning and restructuring of nucleosomes, but how the ATPase activity of these enzymes drives the motion of DNA across the nucleosome remains unclear. Here, we used single-molecule FRET to monitor the remodeling of mononucleosomes by the yeast SWI/SNF remodeler, RSC. We observed that RSC primarily translocates DNA around the nucleosome without substantial displacement of the H2A-H2B dimer. At the sites where DNA enters and exits the nucleosome, the DNA moves largely along or near its canonical wrapping path. The translocation of DNA occurs in a stepwise manner, and at both sites where DNA enters and exits the nucleosome, the step size distributions exhibit a peak at approximately 1-2 bp. These results suggest that the movement of DNA across the nucleosome is likely coupled directly to DNA translocation by the ATPase at its binding site inside the nucleosome.

  11. Histone chaperone-mediated nucleosome assembly process.

    PubMed

    Fan, Hsiu-Fang; Liu, Zi-Ning; Chow, Sih-Yao; Lu, Yi-Han; Li, Hsin

    2015-01-01

    A huge amount of information is stored in genomic DNA and this stored information resides inside the nucleus with the aid of chromosomal condensation factors. It has been reported that the repeat nucleosome core particle (NCP) consists of 147-bp of DNA and two copies of H2A, H2B, H3 and H4. Regulation of chromosomal structure is important to many processes inside the cell. In vivo, a group of histone chaperones facilitate and regulate nucleosome assembly. How NCPs are constructed with the aid of histone chaperones remains unclear. In this study, the histone chaperone-mediated nucleosome assembly process was investigated using single-molecule tethered particle motion (TPM) experiments. It was found that Asf1 is able to exert more influence than Nap1 and poly glutamate acid (PGA) on the nucleosome formation process, which highlights Asf1's specific role in tetrasome formation. Thermodynamic parameters supported a model whereby energetically favored nucleosomal complexes compete with non-nucleosomal complexes. In addition, our kinetic findings propose the model that histone chaperones mediate nucleosome assembly along a path that leads to enthalpy-favored products with free histones as reaction substrates.

  12. Assembly of nucleosomal arrays from recombinant core histones and nucleosome positioning DNA.

    PubMed

    Rogge, Ryan A; Kalashnikova, Anna A; Muthurajan, Uma M; Porter-Goff, Mary E; Luger, Karolin; Hansen, Jeffrey C

    2013-09-10

    Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.

  13. Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases

    PubMed Central

    Dahlin, Jayme L; Chen, Xiaoyue; Walters, Michael A.; Zhang, Zhiguo

    2015-01-01

    During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies. PMID:25365782

  14. Improved nucleosome-positioning algorithm iNPS for accurate nucleosome positioning from sequencing data.

    PubMed

    Chen, Weizhong; Liu, Yi; Zhu, Shanshan; Green, Christopher D; Wei, Gang; Han, Jing-Dong Jackie

    2014-09-18

    Accurate determination of genome-wide nucleosome positioning can provide important insights into global gene regulation. Here, we describe the development of an improved nucleosome-positioning algorithm-iNPS-which achieves significantly better performance than the widely used NPS package. By determining nucleosome boundaries more precisely and merging or separating shoulder peaks based on local MNase-seq signals, iNPS can unambiguously detect 60% more nucleosomes. The detected nucleosomes display better nucleosome 'widths' and neighbouring centre-centre distance distributions, giving rise to sharper patterns and better phasing of average nucleosome profiles and higher consistency between independent data subsets. In addition to its unique advantage in classifying nucleosomes by shape to reveal their different biological properties, iNPS also achieves higher significance and lower false positive rates than previously published methods. The application of iNPS to T-cell activation data demonstrates a greater ability to facilitate detection of nucleosome repositioning, uncovering additional biological features underlying the activation process.

  15. Nucleosomal signatures impose nucleosome positioning in coding and noncoding sequences in the genome

    PubMed Central

    González, Sara; García, Alicia; Vázquez, Enrique; Serrano, Rebeca; Sánchez, Mar; Quintales, Luis; Antequera, Francisco

    2016-01-01

    In the yeast genome, a large proportion of nucleosomes occupy well-defined and stable positions. While the contribution of chromatin remodelers and DNA binding proteins to maintain this organization is well established, the relevance of the DNA sequence to nucleosome positioning in the genome remains controversial. Through quantitative analysis of nucleosome positioning, we show that sequence changes distort the nucleosomal pattern at the level of individual nucleosomes in three species of Schizosaccharomyces and in Saccharomyces cerevisiae. This effect is equally detected in transcribed and nontranscribed regions, suggesting the existence of sequence elements that contribute to positioning. To identify such elements, we incorporated information from nucleosomal signatures into artificial synthetic DNA molecules and found that they generated regular nucleosomal arrays indistinguishable from those of endogenous sequences. Strikingly, this information is species-specific and can be combined with coding information through the use of synonymous codons such that genes from one species can be engineered to adopt the nucleosomal organization of another. These findings open the possibility of designing coding and noncoding DNA molecules capable of directing their own nucleosomal organization. PMID:27662899

  16. A Novel H2A/H4 Nucleosomal Histone Acetyltransferase in Tetrahymena thermophila

    PubMed Central

    Ohba, Reiko; Steger, David J.; Brownell, James E.; Mizzen, Craig A.; Cook, Richard G.; Côté, Jacques; Workman, Jerry L.; Allis, C. David

    1999-01-01

    Recently, we reported the identification of a 55-kDa polypeptide (p55) from Tetrahymena macronuclei as a catalytic subunit of a transcription-associated histone acetyltransferase (HAT A). Extensive homology between p55 and Gcn5p, a component of the SAGA and ADA transcriptional coactivator complexes in budding yeast, suggests an immediate link between the regulation of chromatin structure and transcriptional output. Here we report the characterization of a second transcription-associated HAT activity from Tetrahymena macronuclei. This novel activity is distinct from complexes containing p55 and putative ciliate SAGA and ADA components and shares several characteristics with NuA4 (for nucleosomal H2A/H4), a 1.8-MDa, Gcn5p-independent HAT complex recently described in yeast. A key feature of both the NuA4 and Tetrahymena activities is their acetylation site specificity for lysines 5, 8, 12, and 16 of H4 and lysines 5 and 9 of H2A in nucleosomal substrates, patterns that are distinct from those of known Gcn5p family members. Moreover, like NuA4, the Tetrahymena activity is capable of activating transcription from nucleosomal templates in vitro in an acetyl coenzyme A-dependent fashion. Unlike NuA4, however, sucrose gradient analyses of the ciliate enzyme, following sequential denaturation and renaturation, estimate the molecular size of the catalytically active subunit to be ∼80 kDa, consistent with the notion that a single polypeptide or a stable subcomplex is sufficient for this H2A/H4 nucleosomal HAT activity. Together, these data document the importance of this novel HAT activity for transcriptional activation from chromatin templates and suggest that a second catalytic HAT subunit, in addition to p55/Gcn5p, is conserved between yeast and Tetrahymena. PMID:10022893

  17. Nucleosome

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The fundamental structural unit of chromatin and is the basis for organization within the genome by compaction of DNA within the nucleus of the cell and by making selected regions of chromosomes available for transcription and replication.

  18. Nucleosome

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The fundamental structural unit of chromatin and is the basis for organization within the genome by compaction of DNA within the nucleus of the cell and by making selected regions of chromosomes available for transcription and replication.

  19. Dynamics of nucleosome invasion by DNA binding proteins.

    PubMed

    Tims, Hannah S; Gurunathan, Kaushik; Levitus, Marcia; Widom, Jonathan

    2011-08-12

    Nucleosomes sterically occlude their wrapped DNA from interacting with many large protein complexes. How proteins gain access to nucleosomal DNA target sites in vivo is not known. Outer stretches of nucleosomal DNA spontaneously unwrap and rewrap with high frequency, providing rapid and efficient access to regulatory DNA target sites located there; however, rates for access to the nucleosome interior have not been measured. Here we show that for a selected high-affinity nucleosome positioning sequence, the spontaneous DNA unwrapping rate decreases dramatically with distance inside the nucleosome. The rewrapping rate also decreases, but only slightly. Our results explain the previously known strong position dependence on the equilibrium accessibility of nucleosomal DNA, which is characteristic of both selected and natural sequences. Our results point to slow nucleosome conformational fluctuations as a potential source of cell-cell variability in gene activation dynamics, and they reveal the dominant kinetic path by which multiple DNA binding proteins cooperatively invade a nucleosome.

  20. Nucleosome repeat lengths and columnar chromatin structure.

    PubMed

    Trifonov, Edward N

    2016-06-01

    Thorough quantitative study of nucleosome repeat length (NRL) distributions, conducted in 1992 by J. Widom, resulted in a striking observation that the linker lengths between the nucleosomes are quantized. Comparison of the NRL average values with the MNase cut distances predicted from the hypothetical columnar structure of chromatin (this work) shows a close correspondence between the two. This strongly suggests that the NRL distribution, actually, reflects the dominant role of columnar chromatin structure common for all eukaryotes.

  1. Toxoplasma histone acetylation remodelers as novel drug targets

    PubMed Central

    Vanagas, Laura; Jeffers, Victoria; Bogado, Silvina S; Dalmasso, Maria C; Sullivan, William J; Angel, Sergio O

    2013-01-01

    Toxoplasma gondii is a leading cause of neurological birth defects and a serious opportunistic pathogen. The authors and others have found that Toxoplasma uses a unique nucleosome composition supporting a fine gene regulation together with other factors. Post-translational modifications in histones facilitate the establishment of a global chromatin environment and orchestrate DNA-related biological processes. Histone acetylation is one of the most prominent post-translational modifications influencing gene expression. Histone acetyltransferases and histone deacetylases have been intensively studied as potential drug targets. In particular, histone deacetylase inhibitors have activity against apicomplexan parasites, underscoring their potential as a new class of antiparasitic compounds. In this review, we summarize what is known about Toxoplasma histone acetyltransferases and histone deacetylases, and discuss the inhibitors studied to date. Finally, the authors discuss the distinct possibility that the unique nucleosome composition of Toxoplasma, which harbors a nonconserved H2Bv variant histone, might be targeted in novel therapeutics directed against this parasite. PMID:23199404

  2. Genomic Nucleosome Organization Reconstituted with Pure Proteins.

    PubMed

    Krietenstein, Nils; Wal, Megha; Watanabe, Shinya; Park, Bongsoo; Peterson, Craig L; Pugh, B Franklin; Korber, Philipp

    2016-10-20

    Chromatin remodelers regulate genes by organizing nucleosomes around promoters, but their individual contributions are obfuscated by the complex in vivo milieu of factor redundancy and indirect effects. Genome-wide reconstitution of promoter nucleosome organization with purified proteins resolves this problem and is therefore a critical goal. Here, we reconstitute four stages of nucleosome architecture using purified components: yeast genomic DNA, histones, sequence-specific Abf1/Reb1, and remodelers RSC, ISW2, INO80, and ISW1a. We identify direct, specific, and sufficient contributions that in vivo observations validate. First, RSC clears promoters by translating poly(dA:dT) into directional nucleosome removal. Second, partial redundancy is recapitulated where INO80 alone, or ISW2 at Abf1/Reb1sites, positions +1 nucleosomes. Third, INO80 and ISW2 each align downstream nucleosomal arrays. Fourth, ISW1a tightens the spacing to canonical repeat lengths. Such a minimal set of rules and proteins establishes core mechanisms by which promoter chromatin architecture arises through a blend of redundancy and specialization. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Regulatory analysis for review and establishment of natural gas curtailment priorities. Volume 1

    SciTech Connect

    1980-05-01

    This report discusses important differences among alternatives for establishing natural gas curtailment priorities to deal with long-run supply shortages and short-run capacity shortages. Results from surveys and simulation of shortage costs for each alternative curtailment plan produced and major findings that are directly relevant for considering possible changes in the present curtailment system are discussed. This volume should be used to identify basic alternatives, to review findings which can guide review of any proposed policy change, and to gain a basic understanding of how curtailment alternatives were evaluated. This volume is sufficient for gaining an overall understanding of the effect that natural gas curtailment can have on both users and suppliers.

  4. SWI/SNF- and RSC-catalyzed nucleosome mobilization requires internal DNA loop translocation within nucleosomes.

    PubMed

    Liu, Ning; Peterson, Craig L; Hayes, Jeffrey J

    2011-10-01

    The multisubunit SWI/SNF and RSC complexes utilize energy derived from ATP hydrolysis to mobilize nucleosomes and render the DNA accessible for various nuclear processes. Here we test the idea that remodeling involves intermediates with mobile DNA bulges or loops within the nucleosome by cross-linking the H2A N- or C-terminal tails together to generate protein "loops" that constrict separation of the DNA from the histone surface. Analyses indicate that this intranucleosomal cross-linking causes little or no change in remodeling-dependent exposure of DNA sequences within the nucleosome to restriction enzymes. However, cross-linking inhibits nucleosome mobilization and blocks complete movement of nucleosomes to extreme end positions on the DNA fragments. These results are consistent with evidence that nucleosome remodeling involves intermediates with DNA loops on the nucleosome surface but indicate that such loops do not freely diffuse about the surface of the histone octamer. We propose a threading model for movement of DNA loops around the perimeter of the nucleosome core.

  5. Two arginine residues suppress the flexibility of nucleosomal DNA in the canonical nucleosome core.

    PubMed

    Kono, Hidetoshi; Shirayama, Kazuyoshi; Arimura, Yasuhiro; Tachiwana, Hiroaki; Kurumizaka, Hitoshi

    2015-01-01

    The dynamics of nucleosomes containing either canonical H3 or its centromere-specific variant CENP-A were investigated using molecular dynamics simulations. The simulations showed that the histone cores were structurally stable during simulation periods of 100 ns and 50 ns, while DNA was highly flexible at the entry and exit regions and partially dissociated from the histone core. In particular, approximately 20-25 bp of DNA at the entry and exit regions of the CENP-A nucleosome exhibited larger fluctuations than DNA at the entry and exit regions of the H3 nucleosome. Our detailed analysis clarified that this difference in dynamics was attributable to a difference in two basic amino acids in the αN helix; two arginine (Arg) residues in H3 were substituted by lysine (Lys) residues at the corresponding sites in CENP-A. The difference in the ability to form hydrogen bonds with DNA of these two residues regulated the flexibility of nucleosomal DNA at the entry and exit regions. Our exonuclease III assay consistently revealed that replacement of these two Arg residues in the H3 nucleosome by Lys enhanced endonuclease susceptibility, suggesting that the DNA ends of the CENP-A nucleosome are more flexible than those of the H3 nucleosome. This difference in the dynamics between the two types of nucleosomes may be important for forming higher order structures in different phases.

  6. Interacting epidemics? Sleep curtailment, insulin resistance, and obesity

    PubMed Central

    Lucassen, Eliane A; Rother, Kristina I; Cizza, Giovanni

    2012-01-01

    In the last 50 years, the average self-reported sleep duration in the United States has decreased by 1.5–2 hours in parallel with an increasing prevalence of obesity and diabetes. Epidemiological studies and meta-analyses report a strong relationship between short or disturbed sleep, obesity, and abnormalities in glucose metabolism. This relationship is likely to be bidirectional and causal in nature, but many aspects remain to be elucidated. Sleep and the internal circadian clock influence a host of endocrine parameters. Sleep curtailment in humans alters multiple metabolic pathways, leading to more insulin resistance, possibly decreased energy expenditure, increased appetite, and immunological changes. On the other hand, psychological, endocrine, and anatomical abnormalities in individuals with obesity and/or diabetes can interfere with sleep duration and quality, thus creating a vicious cycle. In this review, we address mechanisms linking sleep with metabolism, highlight the need for studies conducted in real-life settings, and explore therapeutic interventions to improve sleep, with a potential beneficial effect on obesity and its comorbidities. PMID:22827862

  7. Changes in taste preference and steps taken after sleep curtailment.

    PubMed

    Smith, Shannon L; Ludy, Mary-Jon; Tucker, Robin M

    2016-09-01

    A substantial proportion of the population does not achieve the recommended amount of sleep. Previous work demonstrates that sleep alterations perturb energy balance by disrupting appetite hormones, increasing energy intake, and decreasing physical activity. This study explored the influence of sleep duration on taste perception as well as effects on dietary intake and physical activity. Participants (n=24 habitual short sleepers and n=27 habitual long sleepers, 82.4% female, 88.2% white, 25.2±7.7years) completed two randomized taste visits; one following short sleep duration (≤7h) and one following long sleep duration (>7h). Taste perception measures included sweet and salt detection thresholds (ascending 3-alternative, forced-choice method), as well as sweet preference (Monell 2-series, forced-choice, paired-comparison, tracking method). Steps and sleep were tracked via FitBit, an activity monitoring device. Dietary intake was assessed using 24-hour recalls and analyzed using Nutritionist Pro. Habitual long-sleepers had a higher sweet taste preference (p=0.042) and took fewer steps (p=0.036) following sleep curtailment compared to the night where they slept >7h but did not experience changes in dietary intake or detection thresholds. Habitual short-sleepers did not experience changes in taste perception, activity, or dietary intake following sleep alteration. Habitual long-sleepers may be at greater risk of gaining weight when typical sleep patterns are disrupted. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Evaluating the effectiveness of localized control strategies to curtail chikungunya

    PubMed Central

    Ndeffo-Mbah, Martial L.; Durham, David P.; Skrip, Laura A.; Nsoesie, Elaine O.; Brownstein, John S.; Fish, Durland; Galvani, Alison P.

    2016-01-01

    Chikungunya, a re-emerging arbovirus transmitted to humans by Aedes aegypti and Ae. albopictus mosquitoes, causes debilitating disease characterized by an acute febrile phase and chronic joint pain. Chikungunya has recently spread to the island of St. Martin and subsequently throughout the Americas. The disease is now affecting 42 countries and territories throughout the Americas. While chikungunya is mainly a tropical disease, the recent introduction and subsequent spread of Ae. albopictus into temperate regions has increased the threat of chikungunya outbreaks beyond the tropics. Given that there are currently no vaccines or treatments for chikungunya, vector control remains the primary measure to curtail transmission. To investigate the effectiveness of a containment strategy that combines disease surveillance, localized vector control and transmission reduction measures, we developed a model of chikungunya transmission dynamics within a large residential neighborhood, explicitly accounting for human and mosquito movement. Our findings indicate that prompt targeted vector control efforts combined with measures to reduce transmission from symptomatic cases to mosquitoes may be highly effective approaches for controlling outbreaks of chikungunya, provided that sufficient detection of chikungunya cases can be achieved. PMID:27045523

  9. Interacting epidemics? Sleep curtailment, insulin resistance, and obesity.

    PubMed

    Lucassen, Eliane A; Rother, Kristina I; Cizza, Giovanni

    2012-08-01

    In the last 50 years, the average self-reported sleep duration in the United States has decreased by 1.5-2 hours in parallel with an increasing prevalence of obesity and diabetes. Epidemiological studies and meta-analyses report a strong relationship between short or disturbed sleep, obesity, and abnormalities in glucose metabolism. This relationship is likely to be bidirectional and causal in nature, but many aspects remain to be elucidated. Sleep and the internal circadian clock influence a host of endocrine parameters. Sleep curtailment in humans alters multiple metabolic pathways, leading to more insulin resistance, possibly decreased energy expenditure, increased appetite, and immunological changes. On the other hand, psychological, endocrine, and anatomical abnormalities in individuals with obesity and/or diabetes can interfere with sleep duration and quality, thus creating a vicious cycle. In this review, we address mechanisms linking sleep with metabolism, highlight the need for studies conducted in real-life settings, and explore therapeutic interventions to improve sleep, with a potential beneficial effect on obesity and its comorbidities.

  10. A role for Snf2 related nucleosome spacing enzymes in genome-wide nucleosome organization

    PubMed Central

    Gkikopoulos, Triantaffyllos; Schofield, Pieta; Singh, Vijender; Pinskaya, Marina; Mellor, Jane; Smolle, Michaela; Workman, Jerry L.; Barton, Geoffrey; Owen-Hughes, Tom

    2012-01-01

    The positioning of nucleosomes within the coding regions of eukaryotic genes is aligned with respect to transcriptional start sites. This organization is likely to influence many genetic processes, requiring access to the underlying DNA. Here we show that the combined action of Isw1 and Chd1 nucleosome spacing enzymes is required to maintain this organization. In the absence of these enzymes regular positioning of the majority of nucleosomes is lost. Exceptions include the region upstream of the promoter, the +1 nucleosome and a subset of locations distributed throughout coding regions where other factors are likely to be involved. These observations indicated that ATP-dependent remodeling enzymes are responsible for directing the positioning of the majority of nucleosomes within the Saccharomyces cerevisiae genome. PMID:21940898

  11. Assembly of RNA polymerase II preinitiation complexes before assembly of nucleosomes allows efficient initiation of transcription on nucleosomal templates

    SciTech Connect

    Knezetic, J.A.; Jacob, G.A.; Luse, D.S.

    1988-08-01

    The authors have previously shown that assembly of nucleosomes on the DNA template blocks transcription initiation by RNA polymerase II in vitro. In the studies reported here, they demonstrate that assembly of a complete RNA polymerase II preinitiation complex before nucleosome assembly results in nucleosomal templates which support initiation in vitro as efficiently as naked DNA. Control experiments prove that the observations are not the result of slow displacemnt of nucleosomes by the transcription machinery during chromatin assembly, nor are they an artifact of inefficient nucleosome deposition on templates already bearing an RNA polymerase. Thus, the RNA polymerase II preinitiation complex appears to be resistant to disruption by subsequent nucleosome assembly.

  12. Histone H4 hyperacetylation and rapid turnover of its acetyl groups in transcriptionally inactive rooster testis spermatids.

    PubMed Central

    Oliva, R; Mezquita, C

    1982-01-01

    In order to study the relationship between acetylation of histones, chromatin structure and gene activity, the distribution and turnover of acetyl groups among nucleosomal core histones and the extent of histone H4 acetylation were examined in rooster testis cell nuclei at different stages of spermatogenesis. Histone H4 was the predominant acetylated histone in mature testes. Hyperacetylation of H4 and rapid turnover of its acetyl groups are not univocally correlated with transcriptional activity since they were detected in both genetically active testicular cells and genetically inactive elongated spermatids. During the transition from nucleohistone to nucleoprotamine in elongated spermatids the chromatin undergoes dramatic structural changes with exposition of binding sites on DNA (1). Hyperacetylation of H4 and rapid turnover of its acetyl groups could be correlated with the particular conformation of chromatin in elongated spermatids and might represent a necessary condition for binding of chromosomal proteins to DNA. Images PMID:7162988

  13. Dynamic regulation of transcription factors by nucleosome remodeling.

    PubMed

    Li, Ming; Hada, Arjan; Sen, Payel; Olufemi, Lola; Hall, Michael A; Smith, Benjamin Y; Forth, Scott; McKnight, Jeffrey N; Patel, Ashok; Bowman, Gregory D; Bartholomew, Blaine; Wang, Michelle D

    2015-06-05

    The chromatin landscape and promoter architecture are dominated by the interplay of nucleosome and transcription factor (TF) binding to crucial DNA sequence elements. However, it remains unclear whether nucleosomes mobilized by chromatin remodelers can influence TFs that are already present on the DNA template. In this study, we investigated the interplay between nucleosome remodeling, by either yeast ISW1a or SWI/SNF, and a bound TF. We found that a TF serves as a major barrier to ISW1a remodeling, and acts as a boundary for nucleosome repositioning. In contrast, SWI/SNF was able to slide a nucleosome past a TF, with concurrent eviction of the TF from the DNA, and the TF did not significantly impact the nucleosome positioning. Our results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes.

  14. Visualization and analysis of unfolded nucleosomes associated with transcribing chromatin.

    PubMed Central

    Bazett-Jones, D P; Mendez, E; Czarnota, G J; Ottensmeyer, F P; Allfrey, V G

    1996-01-01

    We have characterized the structure of transcriptionally active nucleosome subunits using electron spectroscopic imaging. Individual nucleosomes were analyzed in terms of total mass, DNA and protein content, while the ensemble of images of active nucleosomes was used to calculate a three-dimensional reconstruction. Transcriptionally active nucleosomes were separated from inactive nucleosomes by mercury-affinity chromatography thus making it possible to compare their structures. The chromatographic results combined with electron spectroscopic imaging confirm that active nucleosomes unfold to form extended U-shaped particles. Phosphorus mapping indicated that the nucleosomal DNA also underwent a conformational change consistent with particle unfolding. The three-dimensional structure of the Hg-affinity purified nucleosomes determined using quaternion-assisted angular reconstitution methods unites and resolves the different electron microscopic views of the particle and is concordant with a sulphydryl-exposing disruption of the H3-H4 tetramer. PMID:8628657

  15. Featuring the nucleosome surface as a therapeutic target.

    PubMed

    da Silva, Isabel Torres Gomes; de Oliveira, Paulo Sergio Lopes; Santos, Guilherme Martins

    2015-05-01

    Chromatin is the major regulator of gene expression and genome maintenance. Proteins that bind the nucleosome, the repetitive unit of chromatin, and the histone H4 tail are critical to establishing chromatin architecture and phenotypic outcomes. Intriguingly, nucleosome-binding proteins (NBPs) and the H4 tail peptide compete for the same binding site at an acidic region on the nucleosome surface. Although the essential facts about the nucleosome were revealed 17 years ago, new insights into its atomic structure and molecular mechanisms are still emerging. Several complex nucleosome:NBP structures were recently revealed, characterizing the NBP-binding sites on the nucleosome surface. Here we discuss the potential of the nucleosome surface as a therapeutic target and the impact and development of exogenous nucleosome-binding molecules (eNBMs).

  16. Nucleosome positioning in yeasts: methods, maps, and mechanisms.

    PubMed

    Lieleg, Corinna; Krietenstein, Nils; Walker, Maria; Korber, Philipp

    2015-06-01

    Eukaryotic nuclear DNA is packaged into nucleosomes. During the past decade, genome-wide nucleosome mapping across species revealed the high degree of order in nucleosome positioning. There is a conserved stereotypical nucleosome organization around transcription start sites (TSSs) with a nucleosome-depleted region (NDR) upstream of the TSS and a TSS-aligned regular array of evenly spaced nucleosomes downstream over the gene body. As nucleosomes largely impede access to DNA and thereby provide an important level of genome regulation, it is of general interest to understand the mechanisms generating nucleosome positioning and especially the stereotypical NDR-array pattern. We focus here on the most advanced models, unicellular yeasts, and review the progress in mapping nucleosomes and which nucleosome positioning mechanisms are discussed. There are four mechanistic aspects: How are NDRs generated? How are individual nucleosomes positioned, especially those flanking the NDRs? How are nucleosomes evenly spaced leading to regular arrays? How are regular arrays aligned at TSSs? The main candidates for nucleosome positioning determinants are intrinsic DNA binding preferences of the histone octamer, specific DNA binding factors, nucleosome remodeling enzymes, transcription, and statistical positioning. We summarize the state of the art in an integrative model where nucleosomes are positioned by a combination of all these candidate determinants. We highlight the predominance of active mechanisms involving nucleosome remodeling enzymes which may be recruited by DNA binding factors and the transcription machinery. While this mechanistic framework emerged clearly during recent years, the involved factors and their mechanisms are still poorly understood and require future efforts combining in vivo and in vitro approaches.

  17. Predicting Nucleosome Positioning Using Multiple Evidence Tracks

    NASA Astrophysics Data System (ADS)

    Reynolds, Sheila M.; Weng, Zhiping; Bilmes, Jeff A.; Noble, William Stafford

    We describe a probabilistic model, implemented as a dynamic Bayesian network, that can be used to predict nucleosome positioning along a chromosome based on one or more genomic input tracks containing position-specific information (evidence). Previous models have either made predictions based on primary DNA sequence alone, or have been used to infer nucleosome positions from experimental data. Our framework permits the combination of these two distinct types of information. We show how this flexible framework can be used to make predictions based on either sequence-model scores or experimental data alone, or by using the two in combination to interpret the experimental data and fill in gaps. The model output represents the posterior probability, at each position along the chromosome, that a nucleosome core overlaps that position, given the evidence. This posterior probability is computed by integrating the information contained in the input evidence tracks along the entire input sequence, and fitting the evidence to a simple grammar of alternating nucleosome cores and linkers. In addition to providing a novel mechanism for the prediction of nucleosome positioning from arbitrary heterogeneous data sources, this framework is also applicable to other genomic segmentation tasks in which local scores are available from models or from data that can be interpreted as defining a probability assignment over labels at that position. The ability to combine sequence-based predictions and data from experimental assays is a significant and novel contribution to the ongoing research regarding the primary structure of chromatin and its effects upon gene regulation.

  18. A brief review of nucleosome structure.

    PubMed

    Cutter, Amber R; Hayes, Jeffrey J

    2015-10-07

    The nucleosomal subunit organization of chromatin provides a multitude of functions. Nucleosomes elicit an initial ∼7-fold linear compaction of genomic DNA. They provide a critical mechanism for stable repression of genes and other DNA-dependent activities by restricting binding of trans-acting factors to cognate DNA sequences. Conversely they are engineered to be nearly meta-stable and disassembled (and reassembled) in a facile manner to allow rapid access to the underlying DNA during processes such as transcription, replication and DNA repair. Nucleosomes protect the genome from DNA damaging agents and provide a lattice onto which a myriad of epigenetic signals are deposited. Moreover, vast strings of nucleosomes provide a framework for assembly of the chromatin fiber and higher-order chromatin structures. Thus, in order to provide a foundation for understanding these functions, we present a review of the basic elements of nucleosome structure and stability, including the association of linker histones. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. Nucleosome–nucleosome interactions via histone tails and linker DNA regulate nuclear rigidity

    PubMed Central

    Shimamoto, Yuta; Tamura, Sachiko; Masumoto, Hiroshi; Maeshima, Kazuhiro

    2017-01-01

    Cells, as well as the nuclei inside them, experience significant mechanical stress in diverse biological processes, including contraction, migration, and adhesion. The structural stability of nuclei must therefore be maintained in order to protect genome integrity. Despite extensive knowledge on nuclear architecture and components, however, the underlying physical and molecular mechanisms remain largely unknown. We address this by subjecting isolated human cell nuclei to microneedle-based quantitative micromanipulation with a series of biochemical perturbations of the chromatin. We find that the mechanical rigidity of nuclei depends on the continuity of the nucleosomal fiber and interactions between nucleosomes. Disrupting these chromatin features by varying cation concentration, acetylating histone tails, or digesting linker DNA results in loss of nuclear rigidity. In contrast, the levels of key chromatin assembly factors, including cohesin, condensin II, and CTCF, and a major nuclear envelope protein, lamin, are unaffected. Together with in situ evidence using living cells and a simple mechanical model, our findings reveal a chromatin-based regulation of the nuclear mechanical response and provide insight into the significance of local and global chromatin structures, such as those associated with interdigitated or melted nucleosomal fibers. PMID:28428255

  20. Triple helix DNA alters nucleosomal histone-DNA interactions and acts as a nucleosome barrier.

    PubMed

    Westin, L; Blomquist, P; Milligan, J F; Wrange, O

    1995-06-25

    Oligonucleotides which form triple helical complexes on double-stranded DNA have been previously reported to selectively inhibit transcription both in vitro and in vivo by physically blocking RNA polymerase or transcription factor access to the DNA template. Here we show that a 16mer oligonucleotide, which forms triple helix DNA by binding to a 16 bp homopurine segment, alters the formation of histone-DNA contacts during in vitro nucleosome reconstitution. This effect was DNA sequence-specific and required the oligonucleotide to be present during in vitro nucleosome reconstitution. Binding of the triple helix oligonucleotide on a 199 bp mouse mammary tumour virus promoter DNA fragment with a centrally located triplex DNA resulted in interruption of histone-DNA contacts flanking the triplex DNA segment. When nucleosome reconstitution is carried out on a longer, 279 bp DNA fragment with an asymmetrically located triplex site, nucleosome formation occurred at the border of the triple helical DNA. In this case the triplex DNA functioned as a nucleosome barrier. We conclude that triplex DNA cannot be accommodated within a nucleosome context and thus may be used to site-specifically manipulate nucleosome organization.

  1. Triple helix DNA alters nucleosomal histone-DNA interactions and acts as a nucleosome barrier.

    PubMed Central

    Westin, L; Blomquist, P; Milligan, J F; Wrange, O

    1995-01-01

    Oligonucleotides which form triple helical complexes on double-stranded DNA have been previously reported to selectively inhibit transcription both in vitro and in vivo by physically blocking RNA polymerase or transcription factor access to the DNA template. Here we show that a 16mer oligonucleotide, which forms triple helix DNA by binding to a 16 bp homopurine segment, alters the formation of histone-DNA contacts during in vitro nucleosome reconstitution. This effect was DNA sequence-specific and required the oligonucleotide to be present during in vitro nucleosome reconstitution. Binding of the triple helix oligonucleotide on a 199 bp mouse mammary tumour virus promoter DNA fragment with a centrally located triplex DNA resulted in interruption of histone-DNA contacts flanking the triplex DNA segment. When nucleosome reconstitution is carried out on a longer, 279 bp DNA fragment with an asymmetrically located triplex site, nucleosome formation occurred at the border of the triple helical DNA. In this case the triplex DNA functioned as a nucleosome barrier. We conclude that triplex DNA cannot be accommodated within a nucleosome context and thus may be used to site-specifically manipulate nucleosome organization. Images PMID:7610046

  2. Distal chromatin structure influences local nucleosome positions and gene expression.

    PubMed

    Jansen, An; van der Zande, Elisa; Meert, Wim; Fink, Gerald R; Verstrepen, Kevin J

    2012-05-01

    The positions of nucleosomes across the genome influence several cellular processes, including gene transcription. However, our understanding of the factors dictating where nucleosomes are located and how this affects gene regulation is still limited. Here, we perform an extensive in vivo study to investigate the influence of the neighboring chromatin structure on local nucleosome positioning and gene expression. Using truncated versions of the Saccharomyces cerevisiae URA3 gene, we show that nucleosome positions in the URA3 promoter are at least partly determined by the local DNA sequence, with so-called 'anti-nucleosomal elements' like poly(dA:dT) tracts being key determinants of nucleosome positions. In addition, we show that changes in the nucleosome positions in the URA3 promoter strongly affect the promoter activity. Most interestingly, in addition to demonstrating the effect of the local DNA sequence, our study provides novel in vivo evidence that nucleosome positions are also affected by the position of neighboring nucleosomes. Nucleosome structure may therefore be an important selective force for conservation of gene order on a chromosome, because relocating a gene to another genomic position (where the positions of neighboring nucleosomes are different from the original locus) can have dramatic consequences for the gene's nucleosome structure and thus its expression.

  3. Global nucleosome distribution and the regulation of transcription in yeast

    PubMed Central

    Ercan, Sevinc; Carrozza, Michael J; Workman, Jerry L

    2004-01-01

    Recent studies show that active regulatory regions of the yeast genome have a lower density of nucleosomes than other regions, and that there is an inverse correlation between nucleosome density and the transcription rate of a gene. This may be the result of transcription factors displacing nucleosomes. PMID:15461807

  4. A Simple Model of Nucleosome Localization

    NASA Astrophysics Data System (ADS)

    Schwab, David; Bruinsma, Robijn

    2007-03-01

    It has recently been shown that nucleosomes localize to preferred locations along DNA. This localization is a result of the sequence dependent bending stiffness of dsDNA, which must be wrapped around a histone protein to form a nucleosome. As a simple model of nucleosome localization, we study a one-dimensional hard-core gas in a random potential. We numerically solve for the density profile and other thermodynamic quantities using as input both randomly generated potential profiles and experimental energy landscapes. We compare with the annealed average, inspired by the Random Energy Model, and find that the quenched and annealed averages differ significantly above the localization temperature, implying sequence induced structural organization long before the system has frozen. Although information about the ground state is preserved at higher temperatures, there exist massive structural reorganizations at fixed temperature when the chemical potential is lowered. This offers another perspective on why different cells, with different chemical potentials, have different gene expression.

  5. Dynamics and function of compact nucleosome arrays.

    PubMed

    Poirier, Michael G; Oh, Eugene; Tims, Hannah S; Widom, Jonathan

    2009-09-01

    The packaging of eukaryotic DNA into chromatin sterically occludes polymerases, recombinases and repair enzymes. How chromatin structure changes to allow their actions is unknown. We constructed defined fluorescently labeled trinucleosome arrays, allowing analysis of chromatin conformational dynamics via fluorescence resonance energy transfer (FRET). The arrays undergo reversible Mg2+-dependent folding similar to that of longer arrays studied previously. We define two intermediate conformational states in the reversible folding of the nucleosome arrays and characterize the microscopic rate constants. Nucleosome arrays are highly dynamic even when compact, undergoing conformational fluctuations on timescales in the second to microsecond range. Compact states of the arrays allow binding to DNA within the central nucleosome via site exposure. Protein binding can also drive decompaction of the arrays. Thus, our results reveal multiple modes by which spontaneous chromatin fiber dynamics allow for the invasion and action of DNA-processing protein complexes.

  6. Nucleosomal TATA-switch: competing orientations of TATA on the nucleosome.

    PubMed

    Hapala, Jan; Trifonov, Edward N

    2013-09-15

    Transcription is known to be affected by the rotational setting of the transcription response elements within nucleosomes. We studied the rotational positioning of the TATA box, the most universal promoter motif. We applied a bioinformatic high-resolution nucleosome mapping technique to eukaryotic promoters. Our results show that the nucleosome DNA sequence harboring the TATA box encodes alternative rotational positions for the same piece of DNA. This may serve for switching the gene activity on and off. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Nucleosome structure in chromatin from heated cells

    SciTech Connect

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

    1980-12-01

    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.

  8. Patching Broken DNA: Nucleosome Dynamics and the Repair of DNA Breaks.

    PubMed

    Gursoy-Yuzugullu, Ozge; House, Nealia; Price, Brendan D

    2016-05-08

    The ability of cells to detect and repair DNA double-strand breaks (DSBs) is dependent on reorganization of the surrounding chromatin structure by chromatin remodeling complexes. These complexes promote access to the site of DNA damage, facilitate processing of the damaged DNA and, importantly, are essential to repackage the repaired DNA. Here, we will review the chromatin remodeling steps that occur immediately after DSB production and that prepare the damaged chromatin template for processing by the DSB repair machinery. DSBs promote rapid accumulation of repressive complexes, including HP1, the NuRD complex, H2A.Z and histone methyltransferases at the DSB. This shift to a repressive chromatin organization may be important to inhibit local transcription and limit mobility of the break and to maintain the DNA ends in close contact. Subsequently, the repressive chromatin is rapidly dismantled through a mechanism involving dynamic exchange of the histone variant H2A.Z. H2A.Z removal at DSBs alters the acidic patch on the nucleosome surface, promoting acetylation of the H4 tail (by the NuA4-Tip60 complex) and shifting the chromatin to a more open structure. Further, H2A.Z removal promotes chromatin ubiquitination and recruitment of additional DSB repair proteins to the break. Modulation of the nucleosome surface and nucleosome function during DSB repair therefore plays a vital role in processing of DNA breaks. Further, the nucleosome surface may function as a central hub during DSB repair, directing specific patterns of histone modification, recruiting DNA repair proteins and modulating chromatin packing during processing of the damaged DNA template.

  9. Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro.

    PubMed

    Hinz, John M; Laughery, Marian F; Wyrick, John J

    2015-12-08

    During Cas9 genome editing in eukaryotic cells, the bacterial Cas9 enzyme cleaves DNA targets within chromatin. To understand how chromatin affects Cas9 targeting, we characterized Cas9 activity on nucleosome substrates in vitro. We find that Cas9 endonuclease activity is strongly inhibited when its target site is located within the nucleosome core. In contrast, the nucleosome structure does not affect Cas9 activity at a target site within the adjacent linker DNA. Analysis of target sites that partially overlap with the nucleosome edge indicates that the accessibility of the protospacer-adjacent motif (PAM) is the critical determinant of Cas9 activity on a nucleosome.

  10. Quantitative test of the barrier nucleosome model for statistical positioning of nucleosomes up- and downstream of transcription start sites.

    PubMed

    Möbius, Wolfram; Gerland, Ulrich

    2010-08-19

    The positions of nucleosomes in eukaryotic genomes determine which parts of the DNA sequence are readily accessible for regulatory proteins and which are not. Genome-wide maps of nucleosome positions have revealed a salient pattern around transcription start sites, involving a nucleosome-free region (NFR) flanked by a pronounced periodic pattern in the average nucleosome density. While the periodic pattern clearly reflects well-positioned nucleosomes, the positioning mechanism is less clear. A recent experimental study by Mavrich et al. argued that the pattern observed in Saccharomyces cerevisiae is qualitatively consistent with a "barrier nucleosome model," in which the oscillatory pattern is created by the statistical positioning mechanism of Kornberg and Stryer. On the other hand, there is clear evidence for intrinsic sequence preferences of nucleosomes, and it is unclear to what extent these sequence preferences affect the observed pattern. To test the barrier nucleosome model, we quantitatively analyze yeast nucleosome positioning data both up- and downstream from NFRs. Our analysis is based on the Tonks model of statistical physics which quantifies the interplay between the excluded-volume interaction of nucleosomes and their positional entropy. We find that although the typical patterns on the two sides of the NFR are different, they are both quantitatively described by the same physical model with the same parameters, but different boundary conditions. The inferred boundary conditions suggest that the first nucleosome downstream from the NFR (the +1 nucleosome) is typically directly positioned while the first nucleosome upstream is statistically positioned via a nucleosome-repelling DNA region. These boundary conditions, which can be locally encoded into the genome sequence, significantly shape the statistical distribution of nucleosomes over a range of up to approximately 1,000 bp to each side.

  11. Probing Nucleosome Stability with a DNA Origami Nanocaliper.

    PubMed

    Le, Jenny V; Luo, Yi; Darcy, Michael A; Lucas, Christopher R; Goodwin, Michelle F; Poirier, Michael G; Castro, Carlos E

    2016-07-26

    The organization of eukaryotic DNA into nucleosomes and chromatin undergoes dynamic structural changes to regulate genome processing, including transcription and DNA repair. Critical chromatin rearrangements occur over a wide range of distances, including the mesoscopic length scale of tens of nanometers. However, there is a lack of methodologies that probe changes over this mesoscopic length scale within chromatin. We have designed, constructed, and implemented a DNA-based nanocaliper that probes this mesoscopic length scale. We developed an approach of integrating nucleosomes into our nanocaliper at two attachment points with over 50% efficiency. Here, we focused on attaching the two DNA ends of the nucleosome to the ends of the two nanocaliper arms, so the hinge angle is a readout of the nucleosome end-to-end distance. We demonstrate that nucleosomes integrated with 6, 26, and 51 bp linker DNA are partially unwrapped by the nanocaliper by an amount consistent with previously observed structural transitions. In contrast, the nucleosomes integrated with the longer 75 bp linker DNA remain fully wrapped. We found that the nanocaliper angle is a sensitive measure of nucleosome disassembly and can read out transcription factor (TF) binding to its target site within the nucleosome. Interestingly, the nanocaliper not only detects TF binding but also significantly increases the probability of TF occupancy at its site by partially unwrapping the nucleosome. These studies demonstrate the feasibility of using DNA nanotechnology to both detect and manipulate nucleosome structure, which provides a foundation of future mesoscale studies of nucleosome and chromatin structural dynamics.

  12. Structure and function of human histone H3.Y nucleosome

    PubMed Central

    Kujirai, Tomoya; Horikoshi, Naoki; Sato, Koichi; Maehara, Kazumitsu; Machida, Shinichi; Osakabe, Akihisa; Kimura, Hiroshi; Ohkawa, Yasuyuki; Kurumizaka, Hitoshi

    2016-01-01

    Histone H3.Y is a primate-specific, distant H3 variant. It is evolutionarily derived from H3.3, and may function in transcription regulation. However, the mechanism by which H3.Y regulates transcription has not been elucidated. In the present study, we determined the crystal structure of the H3.Y nucleosome, and found that many H3.Y-specific residues are located on the entry/exit sites of the nucleosome. Biochemical analyses revealed that the DNA ends of the H3.Y nucleosome were more flexible than those of the H3.3 nucleosome, although the H3.Y nucleosome was stable in vitro and in vivo. Interestingly, the linker histone H1, which compacts nucleosomal DNA, appears to bind to the H3.Y nucleosome less efficiently, as compared to the H3.3 nucleosome. These characteristics of the H3.Y nucleosome are also conserved in the H3.Y/H3.3 heterotypic nucleosome, which may be the predominant form in cells. In human cells, H3.Y preferentially accumulated around transcription start sites (TSSs). Taken together, H3.Y-containing nucleosomes around transcription start sites may form relaxed chromatin that allows transcription factor access, to regulate the transcription status of specific genes. PMID:27016736

  13. The effect of micrococcal nuclease digestion on nucleosome positioning data.

    PubMed

    Chung, Ho-Ryun; Dunkel, Ilona; Heise, Franziska; Linke, Christian; Krobitsch, Sylvia; Ehrenhofer-Murray, Ann E; Sperling, Silke R; Vingron, Martin

    2010-12-29

    Eukaryotic genomes are packed into chromatin, whose basic repeating unit is the nucleosome. Nucleosome positioning is a widely researched area. A common experimental procedure to determine nucleosome positions involves the use of micrococcal nuclease (MNase). Here, we show that the cutting preference of MNase in combination with size selection generates a sequence-dependent bias in the resulting fragments. This strongly affects nucleosome positioning data and especially sequence-dependent models for nucleosome positioning. As a consequence we see a need to re-evaluate whether the DNA sequence is a major determinant of nucleosome positioning in vivo. More generally, our results show that data generated after MNase digestion of chromatin requires a matched control experiment in order to determine nucleosome positions.

  14. Nucleosome structure(s) and stability: variations on a theme.

    PubMed

    Andrews, Andrew J; Luger, Karolin

    2011-01-01

    Chromatin is a highly regulated, modular nucleoprotein complex that is central to many processes in eukaryotes. The organization of DNA into nucleosomes and higher-order structures has profound implications for DNA accessibility. Alternative structural states of the nucleosome, and the thermodynamic parameters governing its assembly and disassembly, need to be considered in order to understand how access to nucleosomal DNA is regulated. In this review, we provide a brief historical account of how the overriding perception regarding aspects of nucleosome structure has changed over the past thirty years. We discuss recent technical advances regarding nucleosome structure and its physical characterization and review the evidence for alternative nucleosome conformations and their implications for nucleosome and chromatin dynamics.

  15. The convergent chemical synthesis of histone H3 protein for site-specific acetylation at Lys56 and ubiquitination at Lys122.

    PubMed

    Qi, Yun-Kun; He, Qiao-Qiao; Ai, Hua-Song; Guo, Jing; Li, Jia-Bin

    2017-03-29

    Deposition of (H3-H4)2 tetramers is believed to be the critical step in nucleosome assembly. Site-specific acetylation and ubiquitination of histone H3 have been speculated to synergistically facilitate the formation and deposition of (H3-H4)2 tetramers. Here we report our endeavors toward the first chemical synthesis of homogenous histone H3, which bears Lys56 acetylation and Lys122 ubiquitination, for in vitro biochemical and biophysical studies.

  16. Plasmodium falciparum Nucleosomes Exhibit Reduced Stability and Lost Sequence Dependent Nucleosome Positioning

    PubMed Central

    Silberhorn, Elisabeth; Schwartz, Uwe; Symelka, Anne; de Koning-Ward, Tania; Längst, Gernot

    2016-01-01

    The packaging and organization of genomic DNA into chromatin represents an additional regulatory layer of gene expression, with specific nucleosome positions that restrict the accessibility of regulatory DNA elements. The mechanisms that position nucleosomes in vivo are thought to depend on the biophysical properties of the histones, sequence patterns, like phased di-nucleotide repeats and the architecture of the histone octamer that folds DNA in 1.65 tight turns. Comparative studies of human and P. falciparum histones reveal that the latter have a strongly reduced ability to recognize internal sequence dependent nucleosome positioning signals. In contrast, the nucleosomes are positioned by AT-repeat sequences flanking nucleosomes in vivo and in vitro. Further, the strong sequence variations in the plasmodium histones, compared to other mammalian histones, do not present adaptations to its AT-rich genome. Human and parasite histones bind with higher affinity to GC-rich DNA and with lower affinity to AT-rich DNA. However, the plasmodium nucleosomes are overall less stable, with increased temperature induced mobility, decreased salt stability of the histones H2A and H2B and considerable reduced binding affinity to GC-rich DNA, as compared with the human nucleosomes. In addition, we show that plasmodium histone octamers form the shortest known nucleosome repeat length (155bp) in vitro and in vivo. Our data suggest that the biochemical properties of the parasite histones are distinct from the typical characteristics of other eukaryotic histones and these properties reflect the increased accessibility of the P. falciparum genome. PMID:28033404

  17. Plasmodium falciparum Nucleosomes Exhibit Reduced Stability and Lost Sequence Dependent Nucleosome Positioning.

    PubMed

    Silberhorn, Elisabeth; Schwartz, Uwe; Löffler, Patrick; Schmitz, Samuel; Symelka, Anne; de Koning-Ward, Tania; Merkl, Rainer; Längst, Gernot

    2016-12-01

    The packaging and organization of genomic DNA into chromatin represents an additional regulatory layer of gene expression, with specific nucleosome positions that restrict the accessibility of regulatory DNA elements. The mechanisms that position nucleosomes in vivo are thought to depend on the biophysical properties of the histones, sequence patterns, like phased di-nucleotide repeats and the architecture of the histone octamer that folds DNA in 1.65 tight turns. Comparative studies of human and P. falciparum histones reveal that the latter have a strongly reduced ability to recognize internal sequence dependent nucleosome positioning signals. In contrast, the nucleosomes are positioned by AT-repeat sequences flanking nucleosomes in vivo and in vitro. Further, the strong sequence variations in the plasmodium histones, compared to other mammalian histones, do not present adaptations to its AT-rich genome. Human and parasite histones bind with higher affinity to GC-rich DNA and with lower affinity to AT-rich DNA. However, the plasmodium nucleosomes are overall less stable, with increased temperature induced mobility, decreased salt stability of the histones H2A and H2B and considerable reduced binding affinity to GC-rich DNA, as compared with the human nucleosomes. In addition, we show that plasmodium histone octamers form the shortest known nucleosome repeat length (155bp) in vitro and in vivo. Our data suggest that the biochemical properties of the parasite histones are distinct from the typical characteristics of other eukaryotic histones and these properties reflect the increased accessibility of the P. falciparum genome.

  18. Global remodeling of nucleosome positions in C. elegans

    PubMed Central

    2013-01-01

    Background Eukaryotic chromatin architecture is affected by intrinsic histone-DNA sequence preferences, steric exclusion between nucleosome particles, formation of higher-order structures, and in vivo activity of chromatin remodeling enzymes. Results To disentangle sequence-dependent nucleosome positioning from the other factors, we have created two high-throughput maps of nucleosomes assembled in vitro on genomic DNA from the nematode worm Caenorhabditis elegans. A comparison of in vitro nucleosome positions with those observed in a mixed-stage, mixed-tissue population of C. elegans cells reveals that in vivo sequence preferences are modified on the genomic scale. Indeed, G/C dinucleotides are predicted to be most favorable for nucleosome formation in vitro but not in vivo. Nucleosome sequence read coverage in vivo is distinctly lower in chromosome arms than in central regions; the observed changes in apparent nucleosome sequence specificity, likely due to genome-wide chromatin remodeler activity, contribute to the formation of these megabase-scale chromatin domains. We also observe that the majority of well-positioned in vivo nucleosomes do not occupy thermodynamically favorable sequences observed in vitro. Finally, we find that exons are intrinsically more amenable to nucleosome formation compared to introns. Nucleosome occupancy of introns and exons consistently increases with G/C content in vitro but not in vivo, in agreement with our observation that G/C dinucleotide enrichment does not strongly promote in vivo nucleosome formation. Conclusions Our findings highlight the importance of both sequence specificity and active nucleosome repositioning in creating large-scale chromatin domains, and the antagonistic roles of intrinsic sequence preferences and chromatin remodelers in C. elegans. Sequence read data has been deposited into Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra; accession number SRA050182). Additional data, software and computational

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

    PubMed

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

    2015-02-18

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

  20. Dynamic nucleosome organization at hox promoters during zebrafish embryogenesis.

    PubMed

    Weicksel, Steven E; Xu, Jia; Sagerström, Charles G

    2013-01-01

    Nucleosome organization at promoter regions plays an important role in regulating gene activity. Genome-wide studies in yeast, flies, worms, mammalian embryonic stem cells and transformed cell lines have found well-positioned nucleosomes flanking a nucleosome depleted region (NDR) at transcription start sites. This nucleosome arrangement depends on DNA sequence (cis-elements) as well as DNA binding factors and ATP-dependent chromatin modifiers (trans-factors). However, little is understood about how the nascent embryonic genome positions nucleosomes during development. This is particularly intriguing since the embryonic genome must undergo a broad reprogramming event upon fusion of sperm and oocyte. Using four stages of early embryonic zebrafish development, we map nucleosome positions at the promoter region of 37 zebrafish hox genes. We find that nucleosome arrangement at the hox promoters is a progressive process that takes place over several stages. At stages immediately after fertilization, nucleosomes appear to be largely disordered at hox promoter regions. At stages after activation of the embryonic genome, nucleosomes are detectable at hox promoters, with positions becoming more uniform and more highly occupied. Since the genomic sequence is invariant during embryogenesis, this progressive change in nucleosome arrangement suggests that trans-factors play an important role in organizing nucleosomes during embryogenesis. Separating hox genes into expressed and non-expressed groups shows that expressed promoters have better positioned and occupied nucleosomes, as well as distinct NDRs, than non-expressed promoters. Finally, by blocking the retinoic acid-signaling pathway, we disrupt early hox gene transcription, but observe no effect on nucleosome positions, suggesting that active hox transcription is not a driving force behind the arrangement of nucleosomes at the promoters of hox genes during early development.

  1. Chromatin remodeling: nucleosomes bulging at the seams.

    PubMed

    Peterson, Craig L

    2002-04-02

    ATP-dependent chromatin remodeling enzymes, such as SWI/SNF, hydrolyze thousands of ATPs to regulate gene expression on chromatin fibers. Recent mechanistic studies suggest that these enzymes generate localized changes in DNA topology that drive formation of multiple, remodeled nucleosomal states.

  2. Stabilization of the promoter nucleosomes in nucleosome-free regions by the yeast Cyc8-Tup1 corepressor.

    PubMed

    Chen, Kaifu; Wilson, Marenda A; Hirsch, Calley; Watson, Anjanette; Liang, Shoudan; Lu, Yue; Li, Wei; Dent, Sharon Y R

    2013-02-01

    The yeast Cyc8 (also known as Ssn6)-Tup1 complex regulates gene expression through a variety of mechanisms, including positioning of nucleosomes over promoters of some target genes to limit accessibility to the transcription machinery. To further define the functions of Cyc8-Tup1 in gene regulation and chromatin remodeling, we performed genome-wide profiling of changes in nucleosome organization and gene expression that occur upon loss of CYC8 or TUP1 and observed extensive nucleosome alterations in both promoters and gene bodies of derepressed genes. Our improved nucleosome profiling and analysis approaches revealed low-occupancy promoter nucleosomes (P nucleosomes) at locations previously defined as nucleosome-free regions. In the absence of CYC8 or TUP1, this P nucleosome is frequently lost, whereas nucleosomes are gained at -1 and +1 positions, accompanying up-regulation of downstream genes. Our analysis of public ChIP-seq data revealed that Cyc8 and Tup1 preferentially bind TATA-containing promoters, which are also enriched in genes derepressed upon loss of CYC8 or TUP1. These results suggest that stabilization of the P nucleosome on TATA-containing promoters may be a central feature of the repressive chromatin architecture created by the Cyc8-Tup1 corepressor, and that releasing the P nucleosome contributes to gene activation.

  3. Prediction of nucleosome DNA formation potential and nucleosome positioning using increment of diversity combined with quadratic discriminant analysis.

    PubMed

    Zhao, Xiujuan; Pei, Zhiyong; Liu, Jia; Qin, Sheng; Cai, Lu

    2010-11-01

    In this work, a novel method was developed to distinguish nucleosome DNA and linker DNA based on increment of diversity combined with quadratic discriminant analysis (IDQD), using k-mer frequency of nucleotides in genome. When used to predict DNA potential for forming nucleosomes, the model achieved a high accuracy of 94.94%, 77.60%, and 86.81%, respectively, for Saccharomyces cerevisiae, Homo sapiens, and Drosophila melanogaster. The area under the receiver operator characteristics curve of our classifier was 0.982 for S. cerevisiae. Our results indicate that DNA sequence preference is critical for nucleosome formation potential and is likely conserved across eukaryotes. The model successfully identified nucleosome-enriched or nucleosome-depleted regions in S. cerevisiae genome, suggesting nucleosome positioning depends on DNA sequence preference. Thus, IDQD classifier is useful for predicting nucleosome positioning.

  4. RAPID SEMISYNTHESIS OF ACETYLATED AND SUMOYLATED HISTONE ANALOGS

    PubMed Central

    Dhall, Abhinav; Weller, Caroline E.

    2016-01-01

    The density and diversity of post-translational modifications (PTMs) observed in histone proteins typically limits their purification to homogeneity from biological sources. Access to quantities of uniformly modified histones is, however, critical for investigating the downstream effects of histone PTMs on chromatin-templated processes. Therefore, a number of semisynthetic methodologies have been developed to generate histones bearing precisely defined PTMs or close analogs thereof. In this chapter, we present two optimized and rapid strategies for generating functional analogs of site-specifically acetylated and sumoylated histones. First, we describe a convergent strategy to site-specifically attach the small ubiquitin-like modifier-3 (SUMO-3) protein to the site of Lys12 in histone H4 by means of a disulfide linkage. We then describe the generation of thialysine analogs of histone H3 acetylated at Lys 14 or Lys 56, using thiol-ene coupling chemistry. Both strategies afford multi-milligram quantities of uniformly modified histones that are easily incorporated into mononucleosomes and nucleosome arrays for biophysical and biochemical investigations. These methods are readily extendable to any desired sites in the four core nucleosomal histones and their variant forms. PMID:27423861

  5. THE EXCHANGE REACTION OF ACETYL FLUORIDE AND ACETYL HEXAFLUOROARSENATE,

    DTIC Science & Technology

    From the temperature dependence of the exchange rate of the methyl protons between acetyl fluoride and acetyl hexafluoroarsenate an Arrhenius...the reaction was found to be one-half order in acetyl hexafluoroarsenate and zero order in acetyl fluoride. (Author)

  6. Chronic sleep curtailment impairs the flexible implementation of task goals in new parents.

    PubMed

    Plessow, Franziska; Kiesel, Andrea; Petzold, Antje; Kirschbaum, Clemens

    2011-06-01

    Chronic sleep curtailment is a major concern for health in Western societies. Yet, research on potential consequences of long-term sleep curtailment on cognitive functions is still scarce. The present study investigated the link between chronic sleep limitation and executive functions that enable adaptation to changing environmental demands, i.e. the ability to flexibly implement task goals. To address the effects of chronic sleep restriction under real-life conditions, we considered a sample of adults who often suffer from reduced sleep durations over many months. One-hundred and six new parents (infant's age: 6-18months) were assigned to a sleep-curtailed group (<7h of nighttime sleep) and a non-sleep-curtailed group (≥7h of nighttime sleep), respectively, based on their self-reported average nighttime sleep duration over the preceding 6months. The ability to implement task goals was addressed applying a task-switching paradigm in which participants randomly switched between two tasks. While the two groups did not differ with regard to overall performance level, number of nighttime awakenings, naps during the day, daytime sleepiness, mood, chronic stress level and subjectively perceived cognitive capability, sleep-curtailed new parents showed higher costs for switching between tasks compared with repeating a task than non-sleep-curtailed new parents. This finding on the group level was further substantiated by a negative correlation between nighttime sleep duration and switch costs. With this study, we provide the first evidence for an impairment of the ability to flexibly implement task goals in chronically sleep-deprived new parents and, thus, for a link between chronic sleep curtailment and executive functions.

  7. NuA4 links methylation of histone H3 lysines 4 and 36 to acetylation of histones H4 and H3.

    PubMed

    Ginsburg, Daniel S; Anlembom, Timi Elvuchio; Wang, Jianing; Patel, Sanket R; Li, Bing; Hinnebusch, Alan G

    2014-11-21

    Cotranscriptional methylation of histone H3 lysines 4 and 36 by Set1 and Set2, respectively, stimulates interaction between nucleosomes and histone deacetylase complexes to block cryptic transcription in budding yeast. We previously showed that loss of all H3K4 and H3K36 methylation in a set1Δset2Δ mutant reduces interaction between native nucleosomes and the NuA4 lysine acetyltransferase (KAT) complex. We now provide evidence that NuA4 preferentially binds H3 tails mono- and dimethylated on H3K4 and di- and trimethylated on H3K36, an H3 methylation pattern distinct from that recognized by the RPD3C(S) and Hos2/Set3 histone deacetylase complexes (HDACs). Loss of H3K4 or H3K36 methylation in set1Δ or set2Δ mutants reduces NuA4 interaction with bulk nucleosomes in vitro and in vivo, and reduces NuA4 occupancy of transcribed coding sequences at particular genes. We also provide evidence that NuA4 acetylation of lysine residues in the histone H4 tail stimulates SAGA interaction with nucleosomes and its recruitment to coding sequences and attendant acetylation of histone H3 in vivo. Thus, H3 methylation exerts opposing effects of enhancing nucleosome acetylation by both NuA4 and SAGA as well as stimulating nucleosome deacetylation by multiple HDACs to maintain the proper level of histone acetylation in transcribed coding sequences.

  8. Nanoscale Nucleosome Dynamics Assessed with Time-lapse AFM

    PubMed Central

    Lyubchenko, Yuri L.

    2013-01-01

    A fundamental challenge associated with chromosomal gene regulation is accessibility of DNA within nucleosomes. Recent studies performed by various techniques, including single-molecule approaches, led to the realization that nucleosomes are dynamic structures rather than static systems, as it was once believed. Direct data is required in order to understand the dynamics of nucleosomes more clearly and answer fundamental questions, including: What is the range of nucleosome dynamics? Does a non-ATP dependent unwrapping process of nucleosomes exist? What are the factors facilitating the large scale opening and unwrapping of nucleosomes? This review summarizes the results of nucleosome dynamics obtained with time-lapse AFM, including a high-speed version (HS-AFM) capable of visualizing molecular dynamics on the millisecond time scale. With HS-AFM, the dynamics of nucleosomes at a sub-second time scale was observed allowing one to visualize various pathways of nucleosome dynamics, such as sliding and unwrapping, including complete dissociation. Overall, these findings reveal new insights into the dynamics of nucleosomes and the novel mechanisms controlling spontaneous chromatin dynamics. PMID:24839467

  9. Asymmetric nucleosomes flank promoters in the budding yeast genome.

    PubMed

    Ramachandran, Srinivas; Zentner, Gabriel E; Henikoff, Steven

    2015-03-01

    Nucleosomes in active chromatin are dynamic, but whether they have distinct structural conformations is unknown. To identify nucleosomes with alternative structures genome-wide, we used H4S47C-anchored cleavage mapping, which revealed that 5% of budding yeast (Saccharomyces cerevisiae) nucleosome positions have asymmetric histone-DNA interactions. These asymmetric interactions are enriched at nucleosome positions that flank promoters. Micrococcal nuclease (MNase) sequence-based profiles of asymmetric nucleosome positions revealed a corresponding asymmetry in MNase protection near the dyad axis, suggesting that the loss of DNA contacts around H4S47 is accompanied by protection of the DNA from MNase. Chromatin immunoprecipitation mapping of selected nucleosome remodelers indicated that asymmetric nucleosomes are bound by the RSC chromatin remodeling complex, which is required for maintaining nucleosomes at asymmetric positions. These results imply that the asymmetric nucleosome-RSC complex is a metastable intermediate representing partial unwrapping and protection of nucleosomal DNA on one side of the dyad axis during chromatin remodeling.

  10. Quantifying the role of steric constraints in nucleosome positioning.

    PubMed

    Rube, H Tomas; Song, Jun S

    2014-02-01

    Statistical positioning, the localization of nucleosomes packed against a fixed barrier, is conjectured to explain the array of well-positioned nucleosomes at the 5' end of genes, but the extent and precise implications of statistical positioning in vivo are unclear. We examine this hypothesis quantitatively and generalize the idea to include moving barriers as well as nucleosomes actively packed against a barrier. Early experiments noted a similarity between the nucleosome profile aligned and averaged across genes and that predicted by statistical positioning; however, we demonstrate that aligning random nucleosomes also generates the same profile, calling the previous interpretation into question. New rigorous results reformulate statistical positioning as predictions on the variance structure of nucleosome locations in individual genes. In particular, a quantity termed the variance gradient, describing the change in variance between adjacent nucleosomes, is tested against recent high-throughput nucleosome sequencing data. Constant variance gradients provide support for generalized statistical positioning in ∼ 50% of long genes. Genes that deviate from predictions have high nucleosome turnover and cell-to-cell gene expression variability. The observed variance gradient suggests an effective nucleosome size of 158 bp, instead of the commonly perceived 147 bp. Our analyses thus clarify the role of statistical positioning in vivo.

  11. Incorporating nucleosomes into thermodynamic models of transcription regulation

    PubMed Central

    Raveh-Sadka, Tali; Levo, Michal; Segal, Eran

    2009-01-01

    Transcriptional control is central to many cellular processes, and, consequently, much effort has been devoted to understanding its underlying mechanisms. The organization of nucleosomes along promoter regions is important for this process, since most transcription factors cannot bind nucleosomal sequences and thus compete with nucleosomes for DNA access. This competition is governed by the relative concentrations of nucleosomes and transcription factors and by their respective sequence binding preferences. However, despite its importance, a mechanistic understanding of the quantitative effects that the competition between nucleosomes and factors has on transcription is still missing. Here we use a thermodynamic framework based on fundamental principles of statistical mechanics to explore theoretically the effect that different nucleosome organizations along promoters have on the activation dynamics of promoters in response to varying concentrations of the regulating factors. We show that even simple landscapes of nucleosome organization reproduce experimental results regarding the effect of nucleosomes as general repressors and as generators of obligate binding cooperativity between factors. Our modeling framework also allows us to characterize the effects that various sequence elements of promoters have on the induction threshold and on the shape of the promoter activation curves. Finally, we show that using only sequence preferences for nucleosomes and transcription factors, our model can also predict expression behavior of real promoter sequences, thereby underscoring the importance of the interplay between nucleosomes and factors in determining expression kinetics. PMID:19451592

  12. Incorporating nucleosomes into thermodynamic models of transcription regulation.

    PubMed

    Raveh-Sadka, Tali; Levo, Michal; Segal, Eran

    2009-08-01

    Transcriptional control is central to many cellular processes, and, consequently, much effort has been devoted to understanding its underlying mechanisms. The organization of nucleosomes along promoter regions is important for this process, since most transcription factors cannot bind nucleosomal sequences and thus compete with nucleosomes for DNA access. This competition is governed by the relative concentrations of nucleosomes and transcription factors and by their respective sequence binding preferences. However, despite its importance, a mechanistic understanding of the quantitative effects that the competition between nucleosomes and factors has on transcription is still missing. Here we use a thermodynamic framework based on fundamental principles of statistical mechanics to explore theoretically the effect that different nucleosome organizations along promoters have on the activation dynamics of promoters in response to varying concentrations of the regulating factors. We show that even simple landscapes of nucleosome organization reproduce experimental results regarding the effect of nucleosomes as general repressors and as generators of obligate binding cooperativity between factors. Our modeling framework also allows us to characterize the effects that various sequence elements of promoters have on the induction threshold and on the shape of the promoter activation curves. Finally, we show that using only sequence preferences for nucleosomes and transcription factors, our model can also predict expression behavior of real promoter sequences, thereby underscoring the importance of the interplay between nucleosomes and factors in determining expression kinetics.

  13. DANPOS: Dynamic analysis of nucleosome position and occupancy by sequencing

    PubMed Central

    Chen, Kaifu; Xi, Yuanxin; Pan, Xuewen; Li, Zhaoyu; Kaestner, Klaus; Tyler, Jessica; Dent, Sharon; He, Xiangwei; Li, Wei

    2013-01-01

    Recent developments in next-generation sequencing have enabled whole-genome profiling of nucleosome organizations. Although several algorithms for inferring nucleosome position from a single experimental condition have been available, it remains a challenge to accurately define dynamic nucleosomes associated with environmental changes. Here, we report a comprehensive bioinformatics pipeline, DANPOS, explicitly designed for dynamic nucleosome analysis at single-nucleotide resolution. Using both simulated and real nucleosome data, we demonstrated that bias correction in preliminary data processing and optimal statistical testing significantly enhances the functional interpretation of dynamic nucleosomes. The single-nucleotide resolution analysis of DANPOS allows us to detect all three categories of nucleosome dynamics, such as position shift, fuzziness change, and occupancy change, using a uniform statistical framework. Pathway analysis indicates that each category is involved in distinct biological functions. We also analyzed the influence of sequencing depth and suggest that even 200-fold coverage is probably not enough to identify all the dynamic nucleosomes. Finally, based on nucleosome data from the human hematopoietic stem cells (HSCs) and mouse embryonic stem cells (ESCs), we demonstrated that DANPOS is also robust in defining functional dynamic nucleosomes, not only in promoters, but also in distal regulatory regions in the mammalian genome. PMID:23193179

  14. Nucleosome positioning and composition modulate in silico chromatin flexibility

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  15. Overlapping Functions between SWR1 Deletion and H3K56 Acetylation in Candida albicans

    PubMed Central

    Guan, Zhiyun

    2015-01-01

    Nucleosome destabilization by histone variants and modifications has been implicated in the epigenetic regulation of gene expression, with the histone variant H2A.Z and acetylation of H3K56 (H3K56ac) being two examples. Here we find that deletion of SWR1, the major subunit of the SWR1 complex depositing H2A.Z into chromatin in exchange for H2A, promotes epigenetic white-opaque switching in Candida albicans. We demonstrate through nucleosome mapping that SWR1 is required for proper nucleosome positioning on the promoter of WOR1, the master regulator of switching, and that its effects differ in white and opaque cells. Furthermore, we find that H2A.Z is enriched adjacent to nucleosome-free regions at the WOR1 promoter in white cells, suggesting a role in the stabilization of a repressive chromatin state. Deletion of YNG2, a subunit of the NuA4 H4 histone acetyltransferase (HAT) that targets SWR1 activity through histone acetylation, produces a switching phenotype similar to that of swr1, and both may act downstream of the GlcNAc signaling pathway. We further uncovered a genetic interaction between swr1 and elevated H3K56ac with the discovery that the swr1 deletion mutant is highly sensitive to nicotinamide. Our results suggest that the interaction of H2A.Z and H3K56ac regulates epigenetic switching at the nucleosome level, as well as having global effects. PMID:25862154

  16. Racial/Ethnic and Socio-Contextual Correlates of Chronic Sleep Curtailment in Childhood

    PubMed Central

    Peña, Michelle-Marie; Rifas-Shiman, Sheryl L.; Gillman, Matthew W.; Redline, Susan; Taveras, Elsie M.

    2016-01-01

    Study Objectives: To examine the association between race/ethnicity and sleep curtailment from infancy to mid-childhood, and to determine the extent to which socioeconomic and contextual factors both explain racial/ethnic differences and are independently associated with sleep curtailment. Methods: We studied 1,288 children longitudinally in Project Viva, a pre-birth cohort study, from 6 months to 7 years of age. The main exposure was the child's race/ethnicity. The main outcome was a sleep curtailment score from 6 months to 7 years. The score ranged from 0–13, where 0 indicated maximal sleep curtailment and 13 indicated never having curtailed sleep. Results: The mean (standard deviation) sleep curtailment score was 10.2 (2.7) points. In adjusted models (β [95% CI]), black (−1.92, [−2.39, −1.45] points), Hispanic (−1.58, [−2.43, −0.72] points), and Asian (−1.71, [−2.55, −0.86] points) children had lower sleep scores than white children. Adjustment for sociodemographic covariates attenuated racial/ethnic differences in sleep scores for black (by 24%) and Hispanic children (by 32%) but strengthened the differences for Asian children by 14%. Further adjustment for environmental and behavioral variables did not substantially change these differences. Independently, low maternal education, living in households with incomes < $70,000, viewing more TV, and having a TV in the child's bedroom were associated with lower sleep scores. Conclusions: Chronic sleep curtailment from infancy to mid-childhood was more prevalent among black, Hispanic, and Asian children. These differences were partially but not entirely explained by socio-contextual variables. Independently, children from lower socioeconomic status and those with greater exposures to TV also had greater sleep curtailment. Citation: Peña MM, Rifas-Shiman SL, Gillman MW, Redline S, Taveras EM. Racial/ethnic and socio-contextual correlates of chronic sleep curtailment in childhood. SLEEP 2016

  17. The energetic implications of curtailing versus storing wind- and solar-generated electricity

    NASA Astrophysics Data System (ADS)

    Barnhart, C. J.; Dale, M.; Brandt, A. R.; Benson, S. M.

    2013-12-01

    Rapid deployment of power generation technologies harnessing wind and solar resources continues to reduce the carbon intensity of the power grid. But as these technologies comprise a larger fraction of power supply, their variable, weather-dependent nature poses challenges to power grid operation. Today, during times of power oversupply or unfavorable market conditions, power grid operators curtail these resources. Rates of curtailment are expected to increase with increased renewable electricity production. That is unless technologies are implemented that can provide grid flexibility to balance power supply with power demand. Curtailment is an obvious forfeiture of energy and it decreases the profitability of electricity from curtailed generators. What are less obvious are the energetic costs for technologies that provide grid flexibility. We present a theoretical framework to calculate how storage affects the energy return on energy investment (EROI) ratios of wind and solar resources. Our methods identify conditions under which it is more energetically favorable to store energy than it is to simply curtail electricity production. Electrochemically based storage technologies result in much smaller EROI ratios than large-scale geologically based storage technologies like compressed air energy storage (CAES) and pumped hydroelectric storage (PHS). All storage technologies paired with solar photovoltaic (PV) generation yield EROI ratios that are greater than curtailment. Due to their low energy stored on electrical energy invested (ESOIe) ratios, conventional battery technologies reduce the EROI ratios of wind generation below curtailment EROI ratios. To yield a greater net energy return than curtailment, battery storage technologies paired with wind generation need an ESOIe>80. We identify improvements in cycle life as the most feasible way to increase battery ESOIe. Depending upon the battery's embodied energy requirement, an increase of cycle life to 10

  18. Substantial Histone Reduction Modulates Genomewide Nucleosomal Occupancy and Global Transcriptional Output

    PubMed Central

    Celona, Barbara; Weiner, Assaf; Di Felice, Francesca; Mancuso, Francesco M.; Cesarini, Elisa; Rossi, Riccardo L.; Gregory, Lorna; Baban, Dilair; Rossetti, Grazisa; Grianti, Paolo; Pagani, Massimiliano; Bonaldi, Tiziana; Ragoussis, Jiannis; Friedman, Nir; Camilloni, Giorgio; Bianchi, Marco E.; Agresti, Alessandra

    2011-01-01

    The basic unit of genome packaging is the nucleosome, and nucleosomes have long been proposed to restrict DNA accessibility both to damage and to transcription. Nucleosome number in cells was considered fixed, but recently aging yeast and mammalian cells were shown to contain fewer nucleosomes. We show here that mammalian cells lacking High Mobility Group Box 1 protein (HMGB1) contain a reduced amount of core, linker, and variant histones, and a correspondingly reduced number of nucleosomes, possibly because HMGB1 facilitates nucleosome assembly. Yeast nhp6 mutants lacking Nhp6a and -b proteins, which are related to HMGB1, also have a reduced amount of histones and fewer nucleosomes. Nucleosome limitation in both mammalian and yeast cells increases the sensitivity of DNA to damage, increases transcription globally, and affects the relative expression of about 10% of genes. In yeast nhp6 cells the loss of more than one nucleosome in four does not affect the location of nucleosomes and their spacing, but nucleosomal occupancy. The decrease in nucleosomal occupancy is non-uniform and can be modelled assuming that different nucleosomal sites compete for available histones. Sites with a high propensity to occupation are almost always packaged into nucleosomes both in wild type and nucleosome-depleted cells; nucleosomes on sites with low propensity to occupation are disproportionately lost in nucleosome-depleted cells. We suggest that variation in nucleosome number, by affecting nucleosomal occupancy both genomewide and gene-specifically, constitutes a novel layer of epigenetic regulation. PMID:21738444

  19. SWR-C and INO80 chromatin remodelers recognize nucleosome-free regions near +1 nucleosomes.

    PubMed

    Yen, Kuangyu; Vinayachandran, Vinesh; Pugh, B Franklin

    2013-09-12

    SWR-C/SWR1 and INO80 are multisubunit complexes that catalyze the deposition and removal, respectively, of histone variant H2A.Z from the first nucleosome at the start of genes. How they target and engage these +1 nucleosomes is unclear. Using ChIP-exo, we identified the subnucleosomal placement of 20 of their subunits across the yeast genome. The Swc2 subunit of SWR-C bound a narrowly defined region in the adjacent nucleosome-free region (NFR), where it positioned the Swr1 subunit over one of two sites of H2A.Z deposition at +1. The genomic binding maps suggest that many subunits have a rather plastic organization that allows subunits to exchange between the two complexes. One outcome of promoting H2A/H2A.Z exchange was an enhanced turnover of entire nucleosomes, thereby creating dynamic chromatin at the start of genes. Our findings provide unifying concepts on how these two opposing chromatin remodeling complexes function selectively at the +1 nucleosome of nearly all genes.

  20. Scanning force microscopy reveals ellipsoid shape of chicken erythrocyte nucleosomes.

    PubMed Central

    Fritzsche, W; Henderson, E

    1996-01-01

    Scanning force microscopy was used to investigate the conformation of hypotonic spread chicken erythrocyte nucleosomes. Nucleosomal chains were prepared in low-salt conditions and fixed before centrifugation onto glass coverslips and air drying. The images of single nucleosomes were isolated by image processing, and the height and geometry of the resulting three-dimensional structures were investigated. An average nucleosome height of 4.2 +/- 1.1 nm was determined. A virtual cross section at half-maximum height of the nucleosome structure was used for a characterization of the nucleosome geometry. The shape of this cross section was best described by an ellipse with an aspect ratio (major/minor axis) of approximately 1.30. Images FIGURE 1 FIGURE 2 FIGURE 3 PMID:8889198

  1. Nucleosomes suppress spontaneous mutations base-specifically in eukaryotes.

    PubMed

    Chen, Xiaoshu; Chen, Zhidong; Chen, Han; Su, Zhijian; Yang, Jianfeng; Lin, Fangqin; Shi, Suhua; He, Xionglei

    2012-03-09

    It is unknown how the composition and structure of DNA within the cell affect spontaneous mutations. Theory suggests that in eukaryotic genomes, nucleosomal DNA undergoes fewer C→T mutations because of suppressed cytosine hydrolytic deamination relative to nucleosome-depleted DNA. Comparative genomic analyses and a mutation accumulation experiment showed that nucleosome occupancy nearly eliminated cytosine deamination, resulting in an ~50% decrease of the C→T mutation rate in nucleosomal DNA. Furthermore, the rates of G→T and A→T mutations were also about twofold suppressed by nucleosomes. On the basis of these results, we conclude that nucleosome-dependent mutation spectra affect eukaryotic genome structure and evolution and may have implications for understanding the origin of mutations in cancers and in induced pluripotent stem cells.

  2. DNA bending potentials for loop-mediated nucleosome repositioning

    SciTech Connect

    Langowski, Jorg

    2012-01-01

    Nucleosome repositioning is a fundamental process in gene function. DNA elasticity is a key element of loop-mediated nucleosome repositioning. Two analytical models for DNA elasticity have been proposed: the linear sub-elastic chain (SEC), which allows DNA kinking, and the worm-like chain (WLC), with a harmonic bending potential. In vitro studies have shown that nucleosomes reposition in a discontiguous manner on a segment of DNA and this has also been found in ground-state calculations with the WLC analytical model. Here we study using Monte Carlo simulation the dynamics of DNA loop-mediated nucleosome repositioning at physiological temperatures using the SEC and WLC potentials. At thermal energies both models predict nearest-neighbor repositioning of nucleosomes on DNA, in contrast to the repositioning in jumps observed in experiments. This suggests a crucial role of DNA sequence in nucleosome repositioning.

  3. Universal full-length nucleosome mapping sequence probe.

    PubMed

    Tripathi, Vijay; Salih, Bilal; Trifonov, Edward N

    2015-01-01

    For the computational sequence-directed mapping of the nucleosomes, the knowledge of the nucleosome positioning motifs - 10-11 base long sequences - and respective matrices of bendability, is not sufficient, since there is no justified way to fuse these motifs in one continuous nucleosome DNA sequence. Discovery of the strong nucleosome (SN) DNA sequences, with visible sequence periodicity allows derivation of the full-length nucleosome DNA bendability pattern as matrix or consensus sequence. The SN sequences of three species (A. thaliana, C. elegans, and H. sapiens) are aligned (512 sequences for each species), and long (115 dinucleotides) matrices of bendability derived for the species. The matrices have strong common property - alternation of runs of purine-purine (RR) and pyrimidine-pyrimidine (YY) dinucleotides, with average period 10.4 bases. On this basis the universal [R,Y] consensus of the nucleosome DNA sequence is derived, with exactly defined positions of respective penta- and hexamers RRRRR, RRRRRR, YYYYY, and YYYYYY.

  4. MNase titration reveals differences between nucleosome occupancy and chromatin accessibility

    PubMed Central

    Mieczkowski, Jakub; Cook, April; Bowman, Sarah K.; Mueller, Britta; Alver, Burak H.; Kundu, Sharmistha; Deaton, Aimee M.; Urban, Jennifer A.; Larschan, Erica; Park, Peter J.; Kingston, Robert E.; Tolstorukov, Michael Y.

    2016-01-01

    Chromatin accessibility plays a fundamental role in gene regulation. Nucleosome placement, usually measured by quantifying protection of DNA from enzymatic digestion, can regulate accessibility. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC (MNase accessibility), quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both nucleosome location and accessibility in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares histone and non-histone protection. We find that changes in accessibility at enhancers, promoters and other regulatory regions do not correlate with changes in nucleosome occupancy. Moreover, high nucleosome occupancy does not necessarily preclude high accessibility, which reveals novel principles of chromatin regulation. PMID:27151365

  5. Histone acetylation: from code to web and router via intrinsically disordered regions.

    PubMed

    Horikoshi, Masami

    2013-01-01

    Structural changes of chromatin, which consists of nucleosomes and nucleosome-associated factors, lead to functional changes that are important determinants of eukaryotic gene regulation. These structural changes are regulated by modifications of histones and DNA, both of which are components of nucleosomes, as well as by replacement of histone variants and the actions of noncoding RNAs. In studies of chromatin modifications, a great deal of attention has been paid to histone acetylation. Progress in understanding this subject has been extensive, including i) elucidation of the relationship of histone acetylation and gene activity; ii) the first isolation of a histonemodifying enzyme; iii) the first identification of a factor that recognizes a modified site; iv) elucidation of the mechanism by which histone modification leads to structural changes in nucleosomes; and v) elucidation of the mechanism of border formation between euchromatin and heterochromatin. Histone acetylation is considered to be fundamental in several fields, including studies of a) the role of chromatin and epigenetics in higher-order biochemical systems such as transcription, DNA replication, and repair; b) biological phenomena such as cell proliferation and differentiation; and c) cancer and aging, potentially leading to clinical applications. In this review, I will discuss the histone code hypothesis, at one time believed to represent a unified theory regarding the functions of histone modification. In addition, I will describe the "modification web theory, " by which the problems in the histone code hypothesis can be overcome, as well as the "signal router theory, " which explains the mechanisms of formation, development, and evolution of the modification web from a structural viewpoint. Lastly, I will illustrate how these novel theories partially explain the robustness of biological systems against various perturbations, and elucidate the strategy that a cell employs to avoid fatal

  6. "Anticipated" nucleosome positioning pattern in prokaryotes.

    PubMed

    Rapoport, Alexandra E; Trifonov, Edward N

    2011-11-15

    Linguistic (word count) analysis of prokaryotic genome sequences, by Shannon N-gram extension, reveals that the dominant hidden motifs in A+T rich genomes are T(A)(T)A and G(A)(T)C with uncertain number of repeating A and T. Since prokaryotic sequences are largely protein-coding, the motifs would correspond to amphipathic alpha-helices with alternating lysine and phenylalanine as preferential polar and non-polar residues. The motifs are also known in eukaryotes, as nucleosome positioning patterns. Their existence in prokaryotes as well may serve for binding of histone-like proteins to DNA. In this case the above patterns in prokaryotes may be considered as "anticipated" nucleosome positioning patterns which, quite likely, existed in prokaryotic genomes before the evolutionary separation between eukaryotes and prokaryotes.

  7. Interaction of influenza virus proteins with nucleosomes

    SciTech Connect

    Garcia-Robles, Inmaculada; Akarsu, Hatice; Mueller, Christoph W.; Ruigrok, Rob W.H.; Baudin, Florence . E-mail: baudin@embl-grenoble.fr

    2005-02-05

    During influenza virus infection, transcription and replication of the viral RNA take place in the cell nucleus. Directly after entry in the nucleus the viral ribonucleoproteins (RNPs, the viral subunits containing vRNA, nucleoprotein and the viral polymerase) are tightly associated with the nuclear matrix. Here, we have analysed the binding of RNPs, M1 and NS2/NEP proteins to purified nucleosomes, reconstituted histone octamers and purified single histones. RNPs and M1 both bind to the chromatin components but at two different sites, RNP to the histone tails and M1 to the globular domain of the histone octamer. NS2/NEP did not bind to nucleosomes at all. The possible consequences of these findings for nuclear release of newly made RNPs and for other processes during the infection cycle are discussed.

  8. Elastic Correlations in Nucleosomal DNA Structure

    NASA Astrophysics Data System (ADS)

    Mohammad-Rafiee, Farshid; Golestanian, Ramin

    2005-06-01

    The structure of DNA in the nucleosome core particle is studied using an elastic model that incorporates anisotropy in the bending energetics and twist-bend coupling. Using the experimentally determined structure of nucleosomal DNA [T. J. Richmond and C. A. Davey, Nature (London), NATUAS, 0028-0836 423, 145 (2003), 10.1038/nature01595], it is shown that elastic correlations exist between twist, roll, tilt, and stretching of DNA, as well as the distance between phosphate groups. The twist-bend coupling term is shown to be able to capture these correlations to a large extent, and a fit to the experimental data yields a new estimate of G=25 nm for the value of the twist-bend coupling constant.

  9. Imputed outage costs under a proposed curtailable rate program in Taiwan

    SciTech Connect

    Hsu, G.J.Y. ); Chang, P.L.; Chen, T.Y. )

    1991-01-01

    The implementation of a curtailable rate program through an appropriately designed menu, mainly determined by the customer's outage costs, is one feasibly solution to a power shortage problem. In Taiwan, this issue is particularly important because currently Taiwan is facing a shortage of power generation supply in the summer peak period. In this paper, the authors conducted a survey to examine the market acceptance for the proposed curtailable rate menu and investigated customers' imputed outage costs in relation to their attributes. The survey results show that the imputed outage costs range from $2.25/KW to $3.14/KW and a potentiality of 5.6% to 18.1% of high-tension power peak load supply of the surveyed customers could be curtailed. The economic implications of the research results are presented and further research is recommended.

  10. Unwrapping of Nucleosomal DNA Ends: A Multiscale Molecular Dynamics Study

    PubMed Central

    Voltz, Karine; Trylska, Joanna; Calimet, Nicolas; Smith, Jeremy C.; Langowski, Jörg

    2012-01-01

    To permit access to DNA-binding proteins involved in the control and expression of the genome, the nucleosome undergoes structural remodeling including unwrapping of nucleosomal DNA segments from the nucleosome core. Here we examine the mechanism of DNA dissociation from the nucleosome using microsecond timescale coarse-grained molecular dynamics simulations. The simulations exhibit short-lived, reversible DNA detachments from the nucleosome and long-lived DNA detachments not reversible on the timescale of the simulation. During the short-lived DNA detachments, 9 bp dissociate at one extremity of the nucleosome core and the H3 tail occupies the space freed by the detached DNA. The long-lived DNA detachments are characterized by structural rearrangements of the H3 tail including the formation of a turn-like structure at the base of the tail that sterically impedes the rewrapping of DNA on the nucleosome surface. Removal of the H3 tails causes the long-lived detachments to disappear. The physical consistency of the CG long-lived open state was verified by mapping a CG structure representative of this state back to atomic resolution and performing molecular dynamics as well as by comparing conformation-dependent free energies. Our results suggest that the H3 tail may stabilize the nucleosome in the open state during the initial stages of the nucleosome remodeling process. PMID:22385856

  11. The effect of DNA supercoiling on nucleosome structure and stability

    NASA Astrophysics Data System (ADS)

    Elbel, Tabea; Langowski, Jörg

    2015-02-01

    Nucleosomes have to open to allow access to DNA in transcription, replication, and DNA damage repair. Changes in DNA torsional strain (e.g. during transcription elongation) influence the accessibility of nucleosomal DNA. Here we investigated the effect of DNA supercoiling-induced torsional strain on nucleosome structure and stability by scanning force microscopy (SFM) and fluorescence correlation spectroscopy (FCS). Nucleosomes were reconstituted onto 2.7 kb DNA plasmids with varying superhelical densities. The SFM results show a clear dependence of the amount of DNA wrapped around the nucleosome core on the strength and type of supercoiling. Negative supercoiling led to smaller nucleosome opening angles as compared to relaxed or positively supercoiled DNA. FCS experiments show that nucleosomes reconstituted on negatively superhelical DNA are more resistant to salt-induced destabilization, as seen by reduced H2A-H2B dimer eviction from the nucleosome. Our results show that changes in DNA topology, e.g. during transcription elongation, affect the accessibility of nucleosomal DNA.

  12. Genome-wide nucleosome specificity and directionality of chromatin remodelers

    PubMed Central

    Yen, Kuangyu; Vinayachandran, Vinesh; Batta, Kiran; Koerber, R. Thomas; Pugh, B. Franklin

    2012-01-01

    How chromatin remodelers cooperate to organize nucleosomes around the start and end of genes is not known. We determined the genome-wide binding of remodeler complexes SWI/SNF, RSC, ISW1a, ISW1b, ISW2, and INO80 to individual nucleosomes in Saccharomyces, and determined their functional contributions to nucleosome positioning through deletion analysis. We applied ultra-high resolution ChIP-exo mapping to Isw2 to determine its sub-nucleosomal orientation and organization on a genomic scale. Remodelers interacted with selected nucleosome positions relative to the start and end of genes, and produced net directionality in moving nucleosomes either away or towards nucleosome-free regions at the 5′ and 3′ ends of genes. Isw2 possessed a sub-nucleosomal organization in accord with biochemical and crystallographic-based models that place its linker binding region within promoters and abutted against Reb1-bound locations. Together these findings reveal a coordinated position-specific approach taken by remodelers to organize genic nucleosomes into arrays. PMID:22726434

  13. A positioned +1 nucleosome enhances promoter-proximal pausing.

    PubMed

    Jimeno-González, Silvia; Ceballos-Chávez, María; Reyes, José C

    2015-03-31

    Chromatin distribution is not uniform along the human genome. In most genes there is a promoter-associated nucleosome free region (NFR) followed by an array of nucleosomes towards the gene body in which the first (+1) nucleosome is strongly positioned. The function of this characteristic chromatin distribution in transcription is not fully understood. Here we show in vivo that the +1 nucleosome plays a role in modulating RNA polymerase II (RNAPII) promoter-proximal pausing. When a +1 nucleosome is strongly positioned, elongating RNAPII has a tendency to stall at the promoter-proximal region, recruits more negative elongation factor (NELF) and produces less mRNA. The nucleosome-induced pause favors pre-mRNA quality control by promoting the addition of the cap to the nascent RNA. Moreover, the uncapped RNAs produced in the absence of a positioned nucleosome are degraded by the 5'-3' exonuclease XRN2. Interestingly, reducing the levels of the chromatin remodeler ISWI factor SNF2H decreases +1 nucleosome positioning and increases RNAPII pause release. This work demonstrates a function for +1 nucleosome in regulation of transcription elongation, pre-mRNA processing and gene expression.

  14. The effect of DNA supercoiling on nucleosome structure and stability.

    PubMed

    Elbel, Tabea; Langowski, Jörg

    2015-02-18

    Nucleosomes have to open to allow access to DNA in transcription, replication, and DNA damage repair. Changes in DNA torsional strain (e.g. during transcription elongation) influence the accessibility of nucleosomal DNA. Here we investigated the effect of DNA supercoiling-induced torsional strain on nucleosome structure and stability by scanning force microscopy (SFM) and fluorescence correlation spectroscopy (FCS). Nucleosomes were reconstituted onto 2.7 kb DNA plasmids with varying superhelical densities. The SFM results show a clear dependence of the amount of DNA wrapped around the nucleosome core on the strength and type of supercoiling. Negative supercoiling led to smaller nucleosome opening angles as compared to relaxed or positively supercoiled DNA. FCS experiments show that nucleosomes reconstituted on negatively superhelical DNA are more resistant to salt-induced destabilization, as seen by reduced H2A-H2B dimer eviction from the nucleosome. Our results show that changes in DNA topology, e.g. during transcription elongation, affect the accessibility of nucleosomal DNA.

  15. A positioned +1 nucleosome enhances promoter-proximal pausing

    PubMed Central

    Jimeno-González, Silvia; Ceballos-Chávez, María; Reyes, José C.

    2015-01-01

    Chromatin distribution is not uniform along the human genome. In most genes there is a promoter-associated nucleosome free region (NFR) followed by an array of nucleosomes towards the gene body in which the first (+1) nucleosome is strongly positioned. The function of this characteristic chromatin distribution in transcription is not fully understood. Here we show in vivo that the +1 nucleosome plays a role in modulating RNA polymerase II (RNAPII) promoter-proximal pausing. When a +1 nucleosome is strongly positioned, elongating RNAPII has a tendency to stall at the promoter-proximal region, recruits more negative elongation factor (NELF) and produces less mRNA. The nucleosome-induced pause favors pre-mRNA quality control by promoting the addition of the cap to the nascent RNA. Moreover, the uncapped RNAs produced in the absence of a positioned nucleosome are degraded by the 5′-3′ exonuclease XRN2. Interestingly, reducing the levels of the chromatin remodeler ISWI factor SNF2H decreases +1 nucleosome positioning and increases RNAPII pause release. This work demonstrates a function for +1 nucleosome in regulation of transcription elongation, pre-mRNA processing and gene expression. PMID:25735750

  16. Extranucleosomal DNA Binding Directs Nucleosome Sliding by Chd1 ▿

    PubMed Central

    McKnight, Jeffrey N.; Jenkins, Katherine R.; Nodelman, Ilana M.; Escobar, Thelma; Bowman, Gregory D.

    2011-01-01

    Chd1- and ISWI-type chromatin remodelers can sense extranucleosomal DNA and preferentially shift nucleosomes toward longer stretches of available DNA. The DNA-binding domains of these chromatin remodelers are believed to be responsible for sensing extranucleosomal DNA and are needed for robust sliding, but it is unclear how these domains contribute to directional movement of nucleosomes. Here, we show that the DNA-binding domain of Chd1 is not essential for nucleosome sliding but is critical for centering mononucleosomes on short DNA fragments. Remarkably, nucleosome centering was achieved by replacing the native DNA-binding domain of Chd1 with foreign DNA-binding domains of Escherichia coli AraC or Drosophila melanogaster engrailed. Introducing target DNA sequences recognized by the foreign domains enabled the remodelers to rapidly shift nucleosomes toward these binding sites, demonstrating that these foreign DNA-binding domains dictated the direction of sliding. Sequence-directed sliding occluded the target DNA sequences on the nucleosome enough to promote release of the remodeler. Target DNA sequences were highly stimulatory at multiple positions flanking the nucleosome and had the strongest influence when separated from the nucleosome by 23 or fewer base pairs. These results suggest that the DNA-binding domain's affinity for extranucleosomal DNA is the key determinant for the direction that Chd1 shifts the nucleosome. PMID:21969605

  17. Nucleosomes Selectively Inhibit Cas9 Off-target Activity at a Site Located at the Nucleosome Edge.

    PubMed

    Hinz, John M; Laughery, Marian F; Wyrick, John J

    2016-11-25

    Nucleosomes affect Cas9 binding and activity at on-target sites, but their impact at off-target sites is unknown. To investigate how nucleosomes affect Cas9 cleavage at off-target sites in vitro, we used a single guide RNA (sgRNA) that has been previously shown to efficiently direct Cas9 cleavage at the edge of the strongly positioned 601 nucleosome. Our data indicate that single mismatches between the sgRNA and DNA target have relatively little effect on Cas9 cleavage of naked DNA substrates, but strongly inhibit cleavage of nucleosome substrates, particularly when the mismatch is in the sgRNA "seed" region. These findings indicate that nucleosomes may enhance Cas9 specificity by inhibiting cleavage of off-target sites at the nucleosome edge.

  18. Control of nucleosome movement: to space or not to space nucleosomes?

    PubMed

    Prasad, Punit; Bartholomew, Blaine

    2010-05-16

    A key feature of ATP-dependent chromatin remodeling complexes is how they control the ability of the complex to translocate along DNA within the context of a nucleosome. Although these complexes generally initiate DNA translocation near the dyad axis of the nucleosome, the progression and eventual termination is regulated in quite distinct ways. The best studied examples of these are the ISWI type which has strong extranucleosomal DNA dependent activity or the SWI/SNF type which has no linker DNA requirement. Recent data provide insights into the mechanism of regulation of DNA translocation by the ISWI type complexes and how the structure of the ISWI-nucleosome complex changes during chromatin remodeling.

  19. A Study on the Installation Promotion Program of Distributed Generation with Load Curtailment Contract

    NASA Astrophysics Data System (ADS)

    Sasaki, Yutaka; Hara, Ryoichi; Kita, Hiroyuki; Hasegawa, Jun

    Distribution generation using new energy resources have been expected in the future power system. However, the interconnection cost and equipment cost of distributed generation (DG) are still expensive and stunt the growth of installed DG capacity. From the economic and environmental points of view, promotion of DG installation is indispensable in the future. This paper proposes a DG installation promotion program based on the load curtailment contract. In the proposed program, the load curtailment contract saves investments required for future power system reinforcement and induces the customers to install DG. Economical and environmental feasibility of the proposed program are discussed through numerical studies.

  20. Nucleosomes protect DNA from DNA methylation in vivo and in vitro

    PubMed Central

    Felle, Max; Hoffmeister, Helen; Rothammer, Julia; Fuchs, Andreas; Exler, Josef H.; Längst, Gernot

    2011-01-01

    Positioned nucleosomes limit the access of proteins to DNA. However, the impact of nucleosomes on DNA methylation in vitro and in vivo is poorly understood. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the de novo methyltransferases. We show that compared to linker DNA, nucleosomal DNA is largely devoid of CpG methylation. ATP-dependent chromatin remodelling frees nucleosomal CpG dinucleotides and renders the remodelled nucleosome a 2-fold better substrate for Dnmt3a methyltransferase compared to free DNA. These results reflect the situation in vivo, as quantification of nucleosomal DNA methylation levels in HeLa cells shows a 2-fold decrease of nucleosomal DNA methylation levels compared to linker DNA. Our findings suggest that nucleosomal positions are stably maintained in vivo and nucleosomal occupancy is a major determinant of global DNA methylation patterns in vivo. PMID:21622955

  1. On Using Stochastic Curtailment to Shorten the SPRT in Sequential Mastery Testing

    ERIC Educational Resources Information Center

    Finkelman, Matthew

    2008-01-01

    Sequential mastery testing (SMT) has been researched as an efficient alternative to paper-and-pencil testing for pass/fail examinations. One popular method for determining when to cease examination in SMT is the truncated sequential probability ratio test (TSPRT). This article introduces the application of stochastic curtailment in SMT to shorten…

  2. Aristotelian-Inspired Model for Curtailing Academic Dishonesty in the United States

    ERIC Educational Resources Information Center

    Sanders, Maria A.

    2012-01-01

    This dissertation explores the growing epidemic of academic dishonesty in the United States in order to propose an Aristotelian-inspired model for developing moral character to curtail this epidemic. The task is laid out in four parts. Chapter one responds to the problem of "akrasia," adopting a modified version of Devin Henry's…

  3. Reducing Wind Curtailment through Transmission Expansion in a Wind Vision Future

    SciTech Connect

    Jorgensen, Jennie; Mai, Trieu; Brinkman, Greg

    2017-01-01

    The Department of Energy's 2015 Wind Vision study, which analyzed an ambitious scenario where wind power served 35% of U.S. electricity consumption in 2050, showed the potential for wind energy to provide substantial health, environmental, and economic benefits. Using a commercial unit commitment and economic dispatch model, we build on this research by assessing the hourly operational feasibility of a similar high wind future in the Western United States. Our detailed simulations found no hours of unmet load or reserve violations with more than 35% potential wind (and 12% potential solar) available on the system, which highlights the technical possibility of integrating large amounts of wind energy. However, absent significant changes to the western grid, we find that substantial wind curtailment could be an issue, as it could degrade the potential for wind power to reduce fuel costs and lowering the emission benefits. To assess the value of transmission to mitigate wind curtailment, we model a suite of transmission expansion scenarios. We find that wind curtailment could be reduced by approximately half under a scenario where new transmission is based only on proposed projects. This avoided wind curtailment could lower annual production costs and reduce carbon dioxide emissions substantially. Greater transmission expansion was found to yield further benefits, although the marginal benefits of these new lines were found to decline. Overall, these results suggest that power systems operation can be realized with more than 35% wind penetration, but that transmission expansion is likely to play a vital role.

  4. Aristotelian-Inspired Model for Curtailing Academic Dishonesty in the United States

    ERIC Educational Resources Information Center

    Sanders, Maria A.

    2012-01-01

    This dissertation explores the growing epidemic of academic dishonesty in the United States in order to propose an Aristotelian-inspired model for developing moral character to curtail this epidemic. The task is laid out in four parts. Chapter one responds to the problem of "akrasia," adopting a modified version of Devin Henry's…

  5. A brief histone in time: understanding the combinatorial functions of histone PTMs in the nucleosome context.

    PubMed

    Ng, Marlee K; Cheung, Peter

    2016-02-01

    It has been over 50 years since Allfrey et al. proposed that histone acetylation regulates RNA synthesis, and the study of histone modifications has progressed at an extraordinary pace for the past two decades. In this review, we provide a perspective on some key events and advances in our understanding of histone modifications. We also highlight reagents and tools from past to present that facilitated progress in this research field. Using histone H3 phosphorylation as an underlying thread, we review the rationale that led to the proposal of the histone code hypothesis, as well as examples that illustrate the concepts of combinatorial histone modifications and cross-talk pathways. We further highlight the importance of investigating these mechanisms in the context of nucleosomes rather than just at the histone level and present current and developing approaches for such studies. Overall, research on histone modifications has yielded great mechanistic insights into the regulation of genomic functions, and extending these studies using nucleosomes will further elucidate the complexity of these pathways in a more physiologically relevant context.

  6. Genome wide nucleosome mapping for HSV-1 shows nucleosomes are deposited at preferred positions during lytic infection.

    PubMed

    Oh, Jaewook; Sanders, Iryna F; Chen, Eric Z; Li, Hongzhe; Tobias, John W; Isett, R Benjamin; Penubarthi, Sindura; Sun, Hao; Baldwin, Don A; Fraser, Nigel W

    2015-01-01

    HSV is a large double stranded DNA virus, capable of causing a variety of diseases from the common cold sore to devastating encephalitis. Although DNA within the HSV virion does not contain any histone protein, within 1 h of infecting a cell and entering its nucleus the viral genome acquires some histone protein (nucleosomes). During lytic infection, partial micrococcal nuclease (MNase) digestion does not give the classic ladder band pattern, seen on digestion of cell DNA or latent viral DNA. However, complete digestion does give a mono-nucleosome band, strongly suggesting that there are some nucleosomes present on the viral genome during the lytic infection, but that they are not evenly positioned, with a 200 bp repeat pattern, like cell DNA. Where then are the nucleosomes positioned? Here we perform HSV-1 genome wide nucleosome mapping, at a time when viral replication is in full swing (6 hr PI), using a microarray consisting of 50mer oligonucleotides, covering the whole viral genome (152 kb). Arrays were probed with MNase-protected fragments of DNA from infected cells. Cells were not treated with crosslinking agents, thus we are only mapping tightly bound nucleosomes. The data show that nucleosome deposition is not random. The distribution of signal on the arrays suggest that nucleosomes are located at preferred positions on the genome, and that there are some positions that are not occupied (nucleosome free regions -NFR or Nucleosome depleted regions -NDR), or occupied at frequency below our limit of detection in the population of genomes. Occupancy of only a fraction of the possible sites may explain the lack of a typical MNase partial digestion band ladder pattern for HSV DNA during lytic infection. On average, DNA encoding Immediate Early (IE), Early (E) and Late (L) genes appear to have a similar density of nucleosomes.

  7. Histone acetylation in neurodevelopment.

    PubMed

    Contestabile, Antonio; Sintoni, Silvia

    2013-01-01

    Post-translational modification of histones is a primary mechanism through which epigenetic regulation of DNA transcription does occur. Among these modifications, regulation of histone acetylation state is an important tool to influence gene expression. Epigenetic regulation of neurodevelopment contributes to the structural and functional shaping of the brain during neurogenesis and continues to impact on neural plasticity lifelong. Alterations of these mechanisms during neurodevelopment may result in later occurrence of neuropsychatric disorders. The present paper reviews and discusses available data on histone modifications, in particular histone acetylation, in neurogenesis considering results obtained in culture systems of neural progenitors as well as in in vivo studies. Possible teratogenic effects of altered histone acetylation state during development are also considered. The use during pregnancy of drugs such as valproic acid, which acts as a histone deacetylase inhibitor, may result during postnatal development in autistic-like symptoms. The effect of gestational administration of the drug has been, therefore, tested on adult hippocampal neurogenesis in animals showing behavioral impairment as a consequence of the drug administration at a specific stage of pregnancy. These experimental results show that adult neurogenesis in the hippocampal dentate gyrus is not quantitatively altered by gestational valproic acid administration. Future steps and goals of research on the role and mechanisms of histone acetylation in neurodevelopment are briefly discussed.

  8. Choreography for nucleosomes: the conformational freedom of the nucleosomal filament and its limitations.

    PubMed

    Engelhardt, Mogens

    2007-01-01

    Eukaryotic DNA is organized into nucleosomes by coiling around core particles of histones, forming a nucleosomal filament. The significance for the conformation of the filament of the DNA entry/exit angle (alpha) at the nucleosome, the angle of rotation (beta) of nucleosomes around their interconnecting DNA (linker DNA) and the length of the linker DNA, has been studied by means of wire models with straight linkers. It is shown that variations in alpha and beta endow the filament with an outstanding conformational freedom when alpha is increased beyond 60-90 degrees, owing to the ability of the filament to change between forward right-handed and backward left-handed coiling. A wealth of different helical and looped conformations are formed in response to repeated beta sequences, and helical conformations are shown to be able to contract to a high density and to associate pairwise into different types of double fibers. Filaments with random beta sequences are characterized by relatively stable loop clusters connected by segments of higher flexibility. Displacement of core particles along the DNA in such fibers, combined with limited twisting of the linkers, can generate the beta sequence necessary for compaction into a regular helix, thus providing a model for heterochromatinization.

  9. Histone Acetylation Induced Transformation of B-DNA to Z-DNA in Cells Probed through FT-IR Spectroscopy.

    PubMed

    Zhang, Fengqiu; Huang, Qing; Yan, Jingwen; Chen, Zhu

    2016-04-19

    A nucleosome is made up of DNA and histones, and acetylation of histones perturbs the interaction of DNA and histones and thus affects the chromatin conformation and function. However, whether or how acetylation induces DNA conformation changes is still elusive. In this work, we applied FT-IR spectroscopy to monitor the DNA signals in cells as the histone acetylation was regulated by trichostatin A (TSA), a reversible inhibitor to histone deacetylases (HDACs). Our results unambiguously demonstrate the significant transformation of B-DNA to Z-DNA upon histone acetylation in the TSA treated HeLa cells. This is the first report providing the explicit experimental evidence for such a B-Z transformation of DNA in the epigenetic states of cells.

  10. Tension-dependent free energies of nucleosome unwrapping

    DOE PAGES

    Lequieu, Joshua; Cordoba, Andres; Schwartz, David C.; ...

    2016-08-23

    Here, nucleosomes form the basic unit of compaction within eukaryotic genomes, and their locations represent an important, yet poorly understood, mechanism of genetic regulation. Quantifying the strength of interactions within the nucleosome is a central problem in biophysics and is critical to understanding how nucleosome positions influence gene expression. By comparing to single-molecule experiments, we demonstrate that a coarse-grained molecular model of the nucleosome can reproduce key aspects of nucleosome unwrapping. Using detailed simulations of DNA and histone proteins, we calculate the tension-dependent free energy surface corresponding to the unwrapping process. The model reproduces quantitatively the forces required to unwrapmore » the nucleosome and reveals the role played by electrostatic interactions during this process. We then demonstrate that histone modifications and DNA sequence can have significant effects on the energies of nucleosome formation. Most notably, we show that histone tails contribute asymmetrically to the stability of the outer and inner turn of nucleosomal DNA and that depending on which histone tails are modified, the tension-dependent response is modulated differently.« less

  11. Training-free atomistic prediction of nucleosome occupancy.

    PubMed

    Minary, Peter; Levitt, Michael

    2014-04-29

    Nucleosomes alter gene expression by preventing transcription factors from occupying binding sites along DNA. DNA methylation can affect nucleosome positioning and so alter gene expression epigenetically (without changing DNA sequence). Conventional methods to predict nucleosome occupancy are trained on observed DNA sequence patterns or known DNA oligonucleotide structures. They are statistical and lack the physics needed to predict subtle epigenetic changes due to DNA methylation. The training-free method presented here uses physical principles and state-of-the-art all-atom force fields to predict both nucleosome occupancy along genomic sequences as well as binding to known positioning sequences. Our method calculates the energy of both nucleosomal and linear DNA of the given sequence. Based on the DNA deformation energy, we accurately predict the in vitro occupancy profile observed experimentally for a 20,000-bp genomic region as well as the experimental locations of nucleosomes along 13 well-established positioning sequence elements. DNA with all C bases methylated at the 5 position shows less variation of nucleosome binding: Strong binding is weakened and weak binding is strengthened compared with normal DNA. Methylation also alters the preference of nucleosomes for some positioning sequences but not others.

  12. Tension-dependent free energies of nucleosome unwrapping

    SciTech Connect

    Lequieu, Joshua; Cordoba, Andres; Schwartz, David C.; de Pablo, Juan J.

    2016-08-23

    Here, nucleosomes form the basic unit of compaction within eukaryotic genomes, and their locations represent an important, yet poorly understood, mechanism of genetic regulation. Quantifying the strength of interactions within the nucleosome is a central problem in biophysics and is critical to understanding how nucleosome positions influence gene expression. By comparing to single-molecule experiments, we demonstrate that a coarse-grained molecular model of the nucleosome can reproduce key aspects of nucleosome unwrapping. Using detailed simulations of DNA and histone proteins, we calculate the tension-dependent free energy surface corresponding to the unwrapping process. The model reproduces quantitatively the forces required to unwrap the nucleosome and reveals the role played by electrostatic interactions during this process. We then demonstrate that histone modifications and DNA sequence can have significant effects on the energies of nucleosome formation. Most notably, we show that histone tails contribute asymmetrically to the stability of the outer and inner turn of nucleosomal DNA and that depending on which histone tails are modified, the tension-dependent response is modulated differently.

  13. Nucleosome geometry and internucleosomal interactions control the chromatin fiber conformation.

    PubMed

    Kepper, Nick; Foethke, Dietrich; Stehr, Rene; Wedemann, Gero; Rippe, Karsten

    2008-10-01

    Based on model structures with atomic resolution, a coarse-grained model for the nucleosome geometry was implemented. The dependence of the chromatin fiber conformation on the spatial orientation of nucleosomes and the path and length of the linker DNA was systematically explored by Monte Carlo simulations. Two fiber types were analyzed in detail that represent nucleosome chains without and with linker histones, respectively: two-start helices with crossed-linker DNA (CL conformation) and interdigitated one-start helices (ID conformation) with different nucleosome tilt angles. The CL conformation was derived from a tetranucleosome crystal structure that was extended into a fiber. At thermal equilibrium, the fiber shape persisted but relaxed into a structure with a somewhat lower linear mass density of 3.1 +/- 0.1 nucleosomes/11 nm fiber. Stable ID fibers required local nucleosome tilt angles between 40 degrees and 60 degrees. For these configurations, much higher mass densities of up to 7.9 +/- 0.2 nucleosomes/11 nm fiber were obtained. A model is proposed, in which the transition between a CL and ID fiber is mediated by relatively small changes of the local nucleosome geometry. These were found to be in very good agreement with changes induced by linker histone H1 binding as predicted from the high resolution model structures.

  14. Drosophila Brahma complex remodels nucleosome organizations in multiple aspects.

    PubMed

    Shi, Jiejun; Zheng, Meizhu; Ye, Youqiong; Li, Min; Chen, Xiaolong; Hu, Xinjie; Sun, Jin; Zhang, Xiaobai; Jiang, Cizhong

    2014-09-01

    ATP-dependent chromatin remodeling complexes regulate nucleosome organizations. In Drosophila, gene Brm encodes the core Brahma complex, the ATPase subunit of SWI/SNF class of chromatin remodelers. Its role in modulating the nucleosome landscape in vivo is unclear. In this study, we knocked down Brm in Drosophila third instar larvae to explore the changes in nucleosome profiles and global gene transcription. The results show that Brm knockdown leads to nucleosome occupancy changes throughout the entire genome with a bias in occupancy decrease. In contrast, the knockdown has limited impacts on nucleosome position shift. The knockdown also alters another important physical property of nucleosome positioning, fuzziness. Nucleosome position shift, gain or loss and fuzziness changes are all enriched in promoter regions. Nucleosome arrays around the 5' ends of genes are reorganized in five patterns as a result of Brm knockdown. Intriguingly, the concomitant changes in the genes adjacent to the Brahma-dependent remodeling regions have important roles in development and morphogenesis. Further analyses reveal abundance of AT-rich motifs for transcription factors in the remodeling regions.

  15. The split personality of CENP-A nucleosomes.

    PubMed

    Westhorpe, Frederick G; Straight, Aaron F

    2012-07-20

    The composition and structure of centromeric nucleosomes, which contain the histone H3 variant CENP-A, is intensely debated. Two independent studies in this issue, in yeast and human cells, now suggest that CENP-A nucleosomes adopt different structures depending on the stage of the cell cycle.

  16. Using deformation energy to analyze nucleosome positioning in genomes.

    PubMed

    Chen, Wei; Feng, Pengmian; Ding, Hui; Lin, Hao; Chou, Kuo-Chen

    2016-03-01

    By modulating the accessibility of genomic regions to regulatory proteins, nucleosome positioning plays important roles in cellular processes. Although intensive efforts have been made, the rules for determining nucleosome positioning are far from satisfaction yet. In this study, we developed a biophysical model to predict nucleosomal sequences based on the deformation energy of DNA sequences, and validated it against the experimentally determined nucleosome positions in the Saccharomyces cerevisiae genome, achieving very high success rates. Furthermore, using the deformation energy model, we analyzed the distribution of nucleosomes around the following three types of DNA functional sites: (1) double strand break (DSB), (2) single nucleotide polymorphism (SNP), and (3) origin of replication (ORI). We have found from the analyzed energy spectra that a remarkable "trough" or "valley" occurs around each of these functional sites, implying a depletion of nucleosome density, fully in accordance with experimental observations. These findings indicate that the deformation energy may play a key role for accurately predicting nucleosome positions, and that it can also provide a quantitative physical approach for in-depth understanding the mechanism of nucleosome positioning.

  17. Nucleosomal arrangement affects single-molecule transcription dynamics

    PubMed Central

    Fitz, Veronika; Shin, Jaeoh; Ehrlich, Christoph; Farnung, Lucas; Cramer, Patrick; Zaburdaev, Vasily; Grill, Stephan W.

    2016-01-01

    In eukaryotes, gene expression depends on chromatin organization. However, how chromatin affects the transcription dynamics of individual RNA polymerases has remained elusive. Here, we use dual trap optical tweezers to study single yeast RNA polymerase II (Pol II) molecules transcribing along a DNA template with two nucleosomes. The slowdown and the changes in pausing behavior within the nucleosomal region allow us to determine a drift coefficient, χ, which characterizes the ability of the enzyme to recover from a nucleosomal backtrack. Notably, χ can be used to predict the probability to pass the first nucleosome. Importantly, the presence of a second nucleosome changes χ in a manner that depends on the spacing between the two nucleosomes, as well as on their rotational arrangement on the helical DNA molecule. Our results indicate that the ability of Pol II to pass the first nucleosome is increased when the next nucleosome is turned away from the first one to face the opposite side of the DNA template. These findings help to rationalize how chromatin arrangement affects Pol II transcription dynamics. PMID:27791062

  18. Tension-Dependent Free Energies of Nucleosome Unwrapping

    PubMed Central

    2016-01-01

    Nucleosomes form the basic unit of compaction within eukaryotic genomes, and their locations represent an important, yet poorly understood, mechanism of genetic regulation. Quantifying the strength of interactions within the nucleosome is a central problem in biophysics and is critical to understanding how nucleosome positions influence gene expression. By comparing to single-molecule experiments, we demonstrate that a coarse-grained molecular model of the nucleosome can reproduce key aspects of nucleosome unwrapping. Using detailed simulations of DNA and histone proteins, we calculate the tension-dependent free energy surface corresponding to the unwrapping process. The model reproduces quantitatively the forces required to unwrap the nucleosome and reveals the role played by electrostatic interactions during this process. We then demonstrate that histone modifications and DNA sequence can have significant effects on the energies of nucleosome formation. Most notably, we show that histone tails contribute asymmetrically to the stability of the outer and inner turn of nucleosomal DNA and that depending on which histone tails are modified, the tension-dependent response is modulated differently. PMID:27725965

  19. Final report on the safety assessment of acetyl triethyl citrate, acetyl tributyl citrate, acetyl trihexyl citrate, and acetyl trioctyl citrate.

    PubMed

    Johnson, Wilbur

    2002-01-01

    Acetyl Triethyl Citrate, Acetyl Tributyl Citrate, Acetyl Trihexyl Citrate, and Acetyl Trioctyl Citrate all function as plasticizers in cosmetics. Additionally, the Trihexyl and Trioctyl forms are described as skin-conditioning agents-emollients, although there are currently no reported uses of Acetyl Trihexyl Citrate or Acetyl Trioctyl Citrate. Acetyl Triethyl Citrate and Acetyl Tributyl Citrate are used in nail products at concentrations up to 7%. Recognizing that there are no reported uses of Acetyl Trihexyl or Trioctyl Citrate, if they were to be used in the future, their concentration of use is expected to be no higher than that reported for Acetyl Triethyl and Tributyl Citrate. These ingredients were sufficiently similar in structure that safety test data on one were considered applicable to all. Approximately 99% of orally administered Acetyl Tributyl Citrate is excreted-intermediate metabolites include acetyl citrate, monobutyl citrate, acetyl monobutyl citrate, dibutyl citrate, and acetyl dibutyl citrate. In acute, short-term, subchronic, and chronic feeding studies, these ingredients were relatively nontoxic. Differences from controls were either not statistically significant or not related to any organ toxicity. Ocular exposures produced moderate reactions that cleared by 48 hours after instillation. Dermal application was not toxic in rabbits. In a guinea pig maximization test, Acetyl Triethyl Citrate was a sensitizer whereas Acetyl Tributyl Citrate was not. Limited clinical testing of Acetyl Triethyl Citrate and Acetyl Tributyl Citrate was negative for both skin irritation and sensitization. These clinical data were considered more relevant than the guinea pig maximization data, suggesting to the Cosmetic Ingredient Review Expert Panel that none of these ingredients would be a sensitizer. Physiologic effects noted with intravenous delivery of Acetyl Triethyl Citrate or Acetyl Tributyl Citrate include dose-related decreases in blood pressure and

  20. Structural insight into the sequence dependence of nucleosome positioning.

    PubMed

    Wu, Bin; Mohideen, Kareem; Vasudevan, Dileep; Davey, Curt A

    2010-03-14

    Nucleosome positioning displays sequence dependency and contributes to genomic regulation in a site-specific manner. We solved the structures of nucleosome core particle composed of strong positioning TTTAA elements flanking the nucleosome center. The positioning strength of the super flexible TA dinucleotide is consistent with its observed central location within minor groove inward regions, where it can contribute maximally to energetically challenging minor groove bending, kinking and compression. The marked preference for TTTAA and positioning power of the site 1.5 double helix turns from the nucleosome center relates to a unique histone protein motif at this location, which enforces a sustained, extremely narrow minor groove via a hydrophobic "sugar clamp." Our analysis sheds light on the basis of nucleosome positioning and indicates that the histone octamer has evolved not to fully minimize sequence discrimination in DNA binding.

  1. A synthetic biology approach to probing nucleosome symmetry.

    PubMed

    Ichikawa, Yuichi; Connelly, Caitlin F; Appleboim, Alon; Miller, Thomas Cr; Jacobi, Hadas; Abshiru, Nebiyu A; Chou, Hsin-Jung; Chen, Yuanyuan; Sharma, Upasna; Zheng, Yupeng; Thomas, Paul M; Chen, Hsuiyi V; Bajaj, Vineeta; Müller, Christoph W; Kelleher, Neil L; Friedman, Nir; Bolon, Daniel Na; Rando, Oliver J; Kaufman, Paul D

    2017-09-12

    The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read , we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read by effector proteins in the cell.

  2. Modeling the dynamics of the nucleosome at various levels.

    NASA Astrophysics Data System (ADS)

    Onufriev, Alexey; Fenley, Andrew; Zmuda-Ruscio, Jory; Adams, David

    2007-03-01

    The primary level of DNA compaction in eukaryotic organisms is the nucleosome, yet details of its dynamics are not fully understood. While the whole nucleosome must be highly stable, protective of its genetic material, at the same time its tightly wrapped DNA should be highly accessible, easily revealing its information content. A combination of atom-level classical molecular dynamics and a course-grained continuum description provide insights into the functioning of the system. In particular, the nucleosomal DNA appears to be considerably more flexible than what can be expected based on its canonical persistence length. A coarse-grained electrostatic model of the nucleosome explains how its stability can be modulated with small environmental changes as well as post-translational modifications. Implications for the nucleosome assembly process in vivo are discussed.

  3. Specific glucocorticoid receptor binding to DNA reconstituted in a nucleosome.

    PubMed Central

    Perlmann, T; Wrange, O

    1988-01-01

    We have reconstituted a nucleosome with core histones from rat liver using a restriction fragment containing a sequence from the mouse mammary tumour virus (MTV) long terminal repeat (LTR). This sequence harbours glucocorticoid responsive elements (GREs) which mediate glucocorticoid hormone induction of transcription from the MTV promoter via glucocorticoid receptor (GR) binding. Exonuclease III and DNase I footprinting demonstrated that the reconstituted nucleosome was specifically located between positions -219 and -76. A nucleosome was previously shown to be located at a similar or identical position in the MTV promoter in situ and to be structurally altered upon glucocorticoid hormone induction. We demonstrated, by DNase I footprinting, that GR is able to bind sequence specifically to the DNA in the in vitro assembled nucleosome. No evidence for unfolding of the nucleosome was obtained, but the DNase I footprinting pattern demonstrated GR induced local alterations in the DNA. Images PMID:2846275

  4. Statistical physics of nucleosome positioning and chromatin structure

    NASA Astrophysics Data System (ADS)

    Morozov, Alexandre

    2012-02-01

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

  5. p53 binding to nucleosomes within the p21 promoter in vivo leads to nucleosome loss and transcriptional activation.

    PubMed

    Laptenko, Oleg; Beckerman, Rachel; Freulich, Ella; Prives, Carol

    2011-06-28

    It is well established that p53 contacts DNA in a sequence-dependent manner in order to transactivate its myriad target genes. Yet little is known about how p53 interacts with its binding site/response element (RE) within such genes in vivo in the context of nucleosomal DNA. In this study we demonstrate that both distal (5') and proximal (3') p53 REs within the promoter of the p21 gene in unstressed HCT116 colon carcinoma cells are localized within a region of relatively high nucleosome occupancy. In the absence of cellular stress, p53 is prebound to both p21 REs within nucleosomal DNA in these cells. Treatment of cells with the DNA-damaging drug doxorubicin or the p53 stabilizing agent Nutlin-3, however, is accompanied by p53-dependent subsequent loss of nucleosomes associated with such p53 REs. We show that in vitro p53 can bind to mononucleosomal DNA containing the distal p21 RE, provided the binding site is not close to the diad center of the nucleosome. In line with this, our data indicate that the p53 distal RE within the p21 gene is located close to the end of the nucleosome. Thus, low- and high-resolution mapping of nucleosome boundaries around p53 REs within the p21 promoter have provided insight into the mechanism of p53 binding to its sites in cells and the consequent changes in nucleosome occupancy at such sites.

  6. Single-cell nucleosome mapping reveals the molecular basis of gene expression heterogeneity.

    PubMed

    Small, Eliza C; Xi, Liqun; Wang, Ji-Ping; Widom, Jonathan; Licht, Jonathan D

    2014-06-17

    Nucleosomes, the basic unit of chromatin, have a critical role in the control of gene expression. Nucleosome positions have generally been determined by examining bulk populations of cells and then correlated with overall gene expression. Here, we describe a technique to determine nucleosome positioning in single cells by virtue of the ability of the nucleosome to protect DNA from GpC methylation. In the acid phosphatase inducible PHO5 gene, we find that there is significant cell-to-cell variation in nucleosome positions and shifts in nucleosome positioning correlate with changes in gene expression. However, nucleosome positioning is not absolute, and even with major shifts in gene expression, some cells fail to change nucleosome configuration. Mutations of the PHO5 promoter that introduce a poly(dA:dT) tract-stimulated gene expression under nonpermissive conditions led to shifts of positioned nucleosomes similar to induction of PHO5. By contrast, mutations that altered AA/TT/AT periodicity reduced gene expression upon PHO5 induction and stabilized nucleosomes in most cells, suggesting that enhanced nucleosome affinity for DNA antagonizes chromatin remodelers. Finally, we determined nucleosome positioning in two regions described as "fuzzy" or nucleosome-free when examined in a bulk assay. These regions consisted of distinct nucleosomes with a larger footprint for potential location and an increase population of cells lacking a nucleosome altogether. These data indicate an underlying complexity of nucleosome positioning that may contribute to the flexibility and heterogeneity of gene expression.

  7. Intrinsic histone-DNA interactions are not the major determinant of nucleosome positions in vivo.

    PubMed

    Zhang, Yong; Moqtaderi, Zarmik; Rattner, Barbara P; Euskirchen, Ghia; Snyder, Michael; Kadonaga, James T; Liu, X Shirley; Struhl, Kevin

    2009-08-01

    We assess the role of intrinsic histone-DNA interactions by mapping nucleosomes assembled in vitro on genomic DNA. Nucleosomes strongly prefer yeast DNA over Escherichia coli DNA, indicating that the yeast genome evolved to favor nucleosome formation. Many yeast promoter and terminator regions intrinsically disfavor nucleosome formation, and nucleosomes assembled in vitro show strong rotational positioning. Nucleosome arrays generated by the ACF assembly factor have fewer nucleosome-free regions, reduced rotational positioning and less translational positioning than obtained by intrinsic histone-DNA interactions. Notably, nucleosomes assembled in vitro have only a limited preference for specific translational positions and do not show the pattern observed in vivo. Our results argue against a genomic code for nucleosome positioning, and they suggest that the nucleosomal pattern in coding regions arises primarily from statistical positioning from a barrier near the promoter that involves some aspect of transcriptional initiation by RNA polymerase II.

  8. Z curve theory-based analysis of the dynamic nature of nucleosome positioning in Saccharomyces cerevisiae.

    PubMed

    Wu, Xueting; Liu, Hui; Liu, Hongbo; Su, Jianzhong; Lv, Jie; Cui, Ying; Wang, Fang; Zhang, Yan

    2013-11-01

    Nucleosome is the elementary structural unit of eukaryotic chromatin. Instability of nucleosome positioning plays critical roles in chromatin remodeling in differentiation and disease. In this study, we investigated nucleosome dynamics in the Saccharomyces cerevisiae genome using a geometric model based on Z curve theory. We identified 52,941 stable nucleosomes and 7607 dynamic nucleosomes, compiling them into a genome-wide nucleosome dynamic positioning map and constructing a user-friendly visualization platform (http://bioinfo.hrbmu.edu.cn/nucleosome). Our approach achieved a sensitivity of 90.31% and a specificity of 87.76% for S. cerevisiae. Analysis revealed transcription factor binding sites (TFBSs) were enriched in linkers. And among the sparse nucleosomes around TFBSs, dynamic nucleosomes were slightly preferred. Gene Ontology (GO) enrichment analysis indicated that stable and dynamic nucleosomes were enriched on genes involved in different biological processes and functions. This study provides an approach for comprehending chromatin remodeling and transcriptional regulation of genes.

  9. Structure of a RSC-nucleosome complex and insights into chromatin remodeling.

    PubMed

    Chaban, Yuriy; Ezeokonkwo, Chukwudi; Chung, Wen-Hsiang; Zhang, Fan; Kornberg, Roger D; Maier-Davis, Barbara; Lorch, Yahli; Asturias, Francisco J

    2008-12-01

    ATP-dependent chromatin-remodeling complexes, such as RSC, can reposition, evict or restructure nucleosomes. A structure of a RSC-nucleosome complex with a nucleosome determined by cryo-EM shows the nucleosome bound in a central RSC cavity. Extensive interaction of RSC with histones and DNA seems to destabilize the nucleosome and lead to an overall ATP-independent rearrangement of its structure. Nucleosomal DNA appears disordered and largely free to bulge out into solution as required for remodeling, but the structure of the RSC-nucleosome complex indicates that RSC is unlikely to displace the octamer from the nucleosome to which it is bound. Consideration of the RSC-nucleosome structure and published biochemical information suggests that ATP-dependent DNA translocation by RSC may result in the eviction of histone octamers from adjacent nucleosomes.

  10. DNA-histone interactions in nucleosomes

    SciTech Connect

    Van Holde, K.E.; Allen, J.R.; Tatchell, K.; Weischet, W.O.; Lohr, D.

    1980-10-01

    We have utilized micrococcal nuclease digestion and thermal denaturation studies to investigate the binding of DNA to the histone core of the nucleosome. We conclude that a total of approx. 168 base pairs (bp) of DNA can interact with the histone core under appropriate solution conditions, even in the absence of lysine-rich histones. The interactions in this total length of DNA can be divided into three classes: (a) approx. 22 bp at the ends is bound only at moderate ionic strength. It is easily displaced, and its removal yields the 146 bp core particle; (b) approx. 46 bp near the ends of the core DNA are quite weakly bound to the core, and are displaced at quite moderate temperatures; (c) the remaining central 100 bp are strongly bound, and interact with all of the sites on the histones which strongly protect DNA against DNAse I digestion. A theoretical analysis of the cleavage of nucleosomal DNA by DNAse I has been used to develop evidence that the pattern of protection offered by the histone core is very similar in nuclei to that in isolated core particles.

  11. Aggregation of nucleosomes by divalent cations.

    PubMed Central

    de Frutos, M; Raspaud, E; Leforestier, A; Livolant, F

    2001-01-01

    Conditions of precipitation of nucleosome core particles (NCP) by divalent cations (Ca(2+) and Mg(2+)) have been explored over a large range of nucleosome and cation concentrations. Precipitation of NCP occurs for a threshold of divalent cation concentration, and redissolution is observed for further addition of salt. The phase diagram looks similar to those obtained with DNA and synthetic polyelectrolytes in the presence of multivalent cations, which supports the idea that NCP/NCP interactions are driven by cation condensation. In the phase separation domain the effective charge of the aggregates was determined by measurements of their electrophoretic mobility. Aggregates formed in the presence of divalent cations (Mg(2+)) remain negatively charged over the whole concentration range. They turn positively charged when aggregation is induced by trivalent (spermidine) or tetravalent (spermine) cations. The higher the valency of the counterions, the more significant is the reversal of the effective charge of the aggregates. The sign of the effective charge has no influence on the aspect of the phase diagram. We discuss the possible reasons for this charge reversal in the light of actual theoretical approaches. PMID:11463653

  12. The effects of histone H4 tail acetylations on cation-induced chromatin folding and self-association.

    PubMed

    Allahverdi, Abdollah; Yang, Renliang; Korolev, Nikolay; Fan, Yanping; Davey, Curt A; Liu, Chuan-Fa; Nordenskiöld, Lars

    2011-03-01

    Understanding the molecular mechanisms behind regulation of chromatin folding through covalent modifications of the histone N-terminal tails is hampered by a lack of accessible chromatin containing precisely modified histones. We study the internal folding and intermolecular self-association of a chromatin system consisting of saturated 12-mer nucleosome arrays containing various combinations of completely acetylated lysines at positions 5, 8, 12 and 16 of histone H4, induced by the cations Na(+), K(+), Mg(2+), Ca(2+), cobalt-hexammine(3+), spermidine(3+) and spermine(4+). Histones were prepared using a novel semi-synthetic approach with native chemical ligation. Acetylation of H4-K16, but not its glutamine mutation, drastically reduces cation-induced folding of the array. Neither acetylations nor mutations of all the sites K5, K8 and K12 can induce a similar degree of array unfolding. The ubiquitous K(+), (as well as Rb(+) and Cs(+)) showed an unfolding effect on unmodified arrays almost similar to that of H4-K16 acetylation. We propose that K(+) (and Rb(+)/Cs(+)) binding to a site on the H2B histone (R96-L99) disrupts H4K16 ε-amino group binding to this specific site, thereby deranging H4 tail-mediated nucleosome-nucleosome stacking and that a similar mechanism operates in the case of H4-K16 acetylation. Inter-array self-association follows electrostatic behavior and is largely insensitive to the position or nature of the H4 tail charge modification.

  13. A chemical approach to mapping nucleosomes at base pair resolution in yeast.

    PubMed

    Brogaard, Kristin R; Xi, Liqun; Wang, Ji-Ping; Widom, Jonathan

    2012-01-01

    Most eukaryotic DNA exists in DNA-protein complexes known as nucleosomes. The exact locations of nucleosomes along the genome play a critical role in chromosome functions and gene regulation. However, the current methods for nucleosome mapping do not provide the necessary accuracy to identify the precise nucleosome locations. Here we describe a new experimental approach that directly maps nucleosome center locations in vivo genome-wide at single base pair resolution.

  14. Preferential Nucleosome Occupancy at High Values of DNA Helical Rise

    PubMed Central

    Pedone, Francesco; Santoni, Daniele

    2012-01-01

    Nucleosomes are the basic structural units of eukaryotic chromatin and play a key role in the regulation of gene expression. Nucleosome formation depends on several factors, including properties of the sequence itself, but also physical constraints and epigenetic factors such as chromatin-remodelling enzymes. In this view, a sequence-dependent approach is able to capture a general tendency of a region to bind a histone octamer. A reference data set of positioned nucleosomes of Saccharomyces cerevisiae was used to study the role of DNA helical rise in histone–DNA interaction. Genomic sequences were transformed into arrays of helical rise values by a tetranucleotide code and then turned into profiles of mean helical rise values. These profiles resemble maps of nucleosome occupancy, suggesting that intrinsic histone–DNA interactions are linked to helical rise. The obtained results show that preferential nucleosome occupancy occurs where the mean helical rise reaches its largest values. Mean helical rise profiles obtained by using maps of positioned nucleosomes of the Drosophila melanogaster and Plasmodium falciparum genomes, as well as Homo sapiens chromosome 20 confirm that nucleosomes are mainly located where the mean helical rise reaches its largest values. PMID:22233711

  15. Sequence structure of Lowary/Widom clones forming strong nucleosomes.

    PubMed

    Trifonov, Edward N

    2016-01-01

    Lowary and Widom selected from random sequences those which form exceptionally stable nucleosomes, including clone 601, the current champion of strong nucleosome (SN) sequences. This unique sequence database (LW sequences) carries sequence elements which confer stability on the nucleosomes formed on the sequences, and, thus, may serve as source of information on the structure of "ideal" or close to ideal nucleosome DNA sequence. An important clue is also provided by crystallographic study of Vasudevan and coauthors on clone 601 nucleosomes. It demonstrated that YR·YR dinucleotide stacks (primarily TA·TA) follow one another at distances 10 or 11 bases or multiples thereof, such that they all are located on the interface between DNA and histone octamer. Combining this important information with alignment of the YR-containing 10-mers and 11-mers from LW sequences, the bendability matrices of the stable nucleosome DNA are derived. The matrices suggest that the periodically repeated TA (YR), RR, and YY dinucleotides are the main sequence features of the SNs. This consensus coincides with the one for recently discovered SNs with visibly periodic DNA sequences. Thus, the experimentally observed stable LW nucleosomes and SNs derived computationally appear to represent the same entity - exceptionally stable SNs.

  16. Predicting nucleosome positioning based on geometrically transformed Tsallis entropy.

    PubMed

    Wu, Jing; Zhang, Yusen; Mu, Zengchao

    2014-01-01

    As the fundamental unit of eukaryotic chromatin structure, nucleosome plays critical roles in gene expression and regulation by controlling physical access to transcription factors. In this paper, based on the geometrically transformed Tsallis entropy and two index-vectors, a valid nucleosome positioning information model is developed to describe the distribution of A/T-riched and G/C-riched dimeric and trimeric motifs along the DNA duplex. When applied to train the support vector machine, the model achieves high AUCs across five organisms, which have significantly outperformed the previous studies. Besides, we adopt the concept of relative distance to describe the probability of arbitrary DNA sequence covered by nucleosome. Thus, the average nucleosome occupancy profile over the S.cerevisiae genome is calculated. With our peak detection model, the isolated nucleosomes along genome sequence are located. When compared with some published results, it shows that our model is effective for nucleosome positioning. The index-vector component [Formula in text] is identified to be an important influencing factor of nucleosome organizations.

  17. Mechanisms for enhanced protein dissociation driven by nucleosomes

    NASA Astrophysics Data System (ADS)

    Bundschuh, Ralf; Chen, Cai

    2013-03-01

    When a transcription factor binding site is located within a nucleosome, the DNA in the nucleosome has to unwrap in order for the transcription factor to bind. Thus, it is not surprising that the rate of transcription factor binding is slowed significantly in the presence of a nucleosome. The resulting change in transcription factor binding site occupancy has been known for quite a while as a mechanism for regulation of gene expression via chromatin structure. More surprisingly, recent single molecule experiments have pointed out that not only is the on-rate of transcription factors reduced by the presence of a nucleosome but also is the off-rate increased. There are two possible explanations short of an active role of the nucleosome in pushing the transcription factor off the DNA: (i) the nucleosome can change the equilibrium between binding at the specific binding site and nonspecific binding to the surrounding DNA or (ii) for dimeric transcription factors the nucleosome can change the equilibrium between monomeric and dimeric binding. We explicitly model both scenarios and find that the first mechanism cannot be reconciled with experimental findings. However, we show that the second mechanism can indeed explain increases in off-rate by a factor as high as 100. This material is based upon work supported by the National Science Foundation under Grant No. 1105458.

  18. Probing Nucleosome Remodeling by Unzipping Single DNA Molecules

    NASA Astrophysics Data System (ADS)

    Wang, Michelle

    2006-03-01

    At the core of eukaryotic chromatin is the nucleosome, which consists of 147 bp of DNA wrapped 1.65 turns around an octamer of histone proteins. Even this lowest level of genomic compaction presents a strong barrier to DNA-binding cellular factors that are required for essential processes such as transcription, DNA replication, recombination and repair. Chromatin remodeling enzymes use the energy of ATP hydrolysis to regulate accessibility of the genetic code by altering chromatin structure. While remodeling enzymes have been the subject of extensive research in recent years, their precise mechanism remains unclear. In order to probe the structure of individual nucleosomes and their remodeling, we assembled a histone octamer onto a DNA segment containing a strong nucleosome positioning sequence. As the DNA double helix was unzipped through the nucleosome using a feedback-enhanced optical trap, the presence of the nucleosome was detected as a series of dramatic increases in the tension in the DNA, followed by sudden tension reductions. Analysis of the unzipping force throughout the disruption accurately revealed the spatial location and fine structure of the nucleosome to near base pair precision. Using this approach, we investigate how remodeling enzymes may alter the location and structure of a nucleosome.

  19. Heterochromatin assembly by interrupted Sir3 bridges across neighboring nucleosomes

    PubMed Central

    Behrouzi, Reza; Lu, Chenning; Currie, Mark A; Jih, Gloria; Iglesias, Nahid; Moazed, Danesh

    2016-01-01

    Heterochromatin is a conserved feature of eukaryotic chromosomes with central roles in regulation of gene expression and maintenance of genome stability. Heterochromatin formation involves spreading of chromatin-modifying factors away from initiation points over large DNA domains by poorly understood mechanisms. In Saccharomyces cerevisiae, heterochromatin formation requires the SIR complex, which contains subunits with histone-modifying, histone-binding, and self-association activities. Here, we analyze binding of the Sir proteins to reconstituted mono-, di-, tri-, and tetra-nucleosomal chromatin templates and show that key Sir-Sir interactions bridge only sites on different nucleosomes but not sites on the same nucleosome, and are therefore 'interrupted' with respect to sites on the same nucleosome. We observe maximal binding affinity and cooperativity to unmodified di-nucleosomes and propose that nucleosome pairs bearing unmodified histone H4-lysine16 and H3-lysine79 form the fundamental units of Sir chromatin binding and that cooperative binding requiring two appropriately modified nucleosomes mediates selective Sir recruitment and spreading. DOI: http://dx.doi.org/10.7554/eLife.17556.001 PMID:27835568

  20. Kinetics and thermodynamics of phenotype: unwinding and rewinding the nucleosome.

    PubMed

    Mack, Andrew H; Schlingman, Daniel J; Ilagan, Robielyn P; Regan, Lynne; Mochrie, Simon G J

    2012-11-09

    Chromatin "remodeling" is widely accepted as the mechanism that permits access to DNA by the transcription machinery. To date, however, there has been no experimental measurement of the changes in the kinetics and thermodynamics of the DNA-histone octamer association that are required to remodel chromatin so that transcription may occur. Here, we present the results of optical tweezer measurements that compare the kinetic and thermodynamic properties of nucleosomes composed of unmodified histones with those of nucleosomes that contain a mutant histone H4 (H4-R45H), which has been shown to allow SWI/SNF remodeling factor-independent transcription from the yeast HO promoter in vivo. Our measurements, carried out in a force-clamp mode, determine the force-dependent unwinding and rewinding rates of the nucleosome inner turn. At each force studied, nucleosomes containing H4-R45H unwind more rapidly and rewind more slowly than nucleosomes containing unmodified H4, indicating that the latter are the more stable. Extrapolation to forces at which the winding and unwinding rates are equal determines the absolute free energy of the nucleosome inner turn to be -32k(B)T for nucleosomes containing unmodified H4 and -27k(B)T for nucleosomes containing H4-R45H. Thus, the "loosening" or "remodeling" caused by this point mutation, which is demonstrated to be sufficient to allow transcriptional machinery access to the HO promoter (in the absence of other remodeling factors), is 5k(B)T. The correlation between the free energy of the nucleosome inner turn and the sin (SWI/SNF-independent) transcription suggests that, beyond partial unwinding, complete histone unwinding may play a role in transcriptional activation.

  1. Strategies implemented by the textile industry in response to natural-gas curtailments

    SciTech Connect

    Schreibeis, R.L.

    1980-01-01

    An examination is made of specific activities undertaken by textile firms in North and South Carolina and Georgia to insulate themselves against production losses resulting from natural gas curtailments. Results of the research effort focusing on investigating patterns or trends of precautionary activities undertaken by the textile industry in response to fuel interruptions are presented. Chapter II delineates the scope of the project, research design, and nature of the textile industry. One hundred candidate firms for detailed study were identified and 34 discussed their alternate fuel strategies. Information obtained was analyzed by means of two statistical analysis techniques. Methods employed and results are described in Chapter III. Overall results are presented in Chapter IV. Variations in the strategies implemented by various concerns were accounted for in terms of geographic location, plant size, plant type, and the duration and extent of curtailment impacts. Ranges of expenditures for short- and long-term strategies are identified.

  2. Estimation after classification using lot quality assurance sampling: corrections for curtailed sampling with application to evaluating polio vaccination campaigns.

    PubMed

    Olives, Casey; Valadez, Joseph J; Pagano, Marcello

    2014-03-01

    To assess the bias incurred when curtailment of Lot Quality Assurance Sampling (LQAS) is ignored, to present unbiased estimators, to consider the impact of cluster sampling by simulation and to apply our method to published polio immunization data from Nigeria. We present estimators of coverage when using two kinds of curtailed LQAS strategies: semicurtailed and curtailed. We study the proposed estimators with independent and clustered data using three field-tested LQAS designs for assessing polio vaccination coverage, with samples of size 60 and decision rules of 9, 21 and 33, and compare them to biased maximum likelihood estimators. Lastly, we present estimates of polio vaccination coverage from previously published data in 20 local government authorities (LGAs) from five Nigerian states. Simulations illustrate substantial bias if one ignores the curtailed sampling design. Proposed estimators show no bias. Clustering does not affect the bias of these estimators. Across simulations, standard errors show signs of inflation as clustering increases. Neither sampling strategy nor LQAS design influences estimates of polio vaccination coverage in 20 Nigerian LGAs. When coverage is low, semicurtailed LQAS strategies considerably reduces the sample size required to make a decision. Curtailed LQAS designs further reduce the sample size when coverage is high. Results presented dispel the misconception that curtailed LQAS data are unsuitable for estimation. These findings augment the utility of LQAS as a tool for monitoring vaccination efforts by demonstrating that unbiased estimation using curtailed designs is not only possible but these designs also reduce the sample size. © 2014 John Wiley & Sons Ltd.

  3. DPNuc: Identifying Nucleosome Positions Based on the Dirichlet Process Mixture Model.

    PubMed

    Chen, Huidong; Guan, Jihong; Zhou, Shuigeng

    2015-01-01

    Nucleosomes and the free linker DNA between them assemble the chromatin. Nucleosome positioning plays an important role in gene transcription regulation, DNA replication and repair, alternative splicing, and so on. With the rapid development of ChIP-seq, it is possible to computationally detect the positions of nucleosomes on chromosomes. However, existing methods cannot provide accurate and detailed information about the detected nucleosomes, especially for the nucleosomes with complex configurations where overlaps and noise exist. Meanwhile, they usually require some prior knowledge of nucleosomes as input, such as the size or the number of the unknown nucleosomes, which may significantly influence the detection results. In this paper, we propose a novel approach DPNuc for identifying nucleosome positions based on the Dirichlet process mixture model. In our method, Markov chain Monte Carlo (MCMC) simulations are employed to determine the mixture model with no need of prior knowledge about nucleosomes. Compared with three existing methods, our approach can provide more detailed information of the detected nucleosomes and can more reasonably reveal the real configurations of the chromosomes; especially, our approach performs better in the complex overlapping situations. By mapping the detected nucleosomes to a synthetic benchmark nucleosome map and two existing benchmark nucleosome maps, it is shown that our approach achieves a better performance in identifying nucleosome positions and gets a higher F-score. Finally, we show that our approach can more reliably detect the size distribution of nucleosomes.

  4. Effectiveness of building energy performance standards to curtail household energy demand: a theoretical analysis

    SciTech Connect

    Mathur, V.K.

    1984-01-01

    The main purposes of Titles III and IV of the Energy Conservation and Production Act of 1976 are to curtail energy use in households and commercial buildings. The authors analyze the effectiveness of various policies emerging from these Titles, and compares them with alternate, though traditional, policies of pricing, taxes, and subsidies aimed at reducing energy demand. The study concludes that a pricing policy is more efficient than a direct regulatory policy that requires more bureaucracy. 10 references, 1 figure.

  5. Genome-wide characterization of chromatin binding and nucleosome spacing activity of the nucleosome remodelling ATPase ISWI

    PubMed Central

    Sala, Anna; Toto, Maria; Pinello, Luca; Gabriele, Alessandra; Di Benedetto, Valeria; Ingrassia, Antonia M R; Lo Bosco, Giosuè; Di Gesù, Vito; Giancarlo, Raffaele; Corona, Davide F V

    2011-01-01

    The evolutionarily conserved ATP-dependent nucleosome remodelling factor ISWI can space nucleosomes affecting a variety of nuclear processes. In Drosophila, loss of ISWI leads to global transcriptional defects and to dramatic alterations in higher-order chromatin structure, especially on the male X chromosome. In order to understand if chromatin condensation and gene expression defects, observed in ISWI mutants, are directly correlated with ISWI nucleosome spacing activity, we conducted a genome-wide survey of ISWI binding and nucleosome positioning in wild-type and ISWI mutant chromatin. Our analysis revealed that ISWI binds both genic and intergenic regions. Remarkably, we found that ISWI binds genes near their promoters causing specific alterations in nucleosome positioning at the level of the Transcription Start Site, providing an important insights in understanding ISWI role in higher eukaryote transcriptional regulation. Interestingly, differences in nucleosome spacing, between wild-type and ISWI mutant chromatin, tend to accumulate on the X chromosome for all ISWI-bound genes analysed. Our study shows how in higher eukaryotes the activity of the evolutionarily conserved nucleosome remodelling factor ISWI regulates gene expression and chromosome organization genome-wide. PMID:21448136

  6. STAT5 acetylation

    PubMed Central

    Kosan, Christian; Ginter, Torsten; Heinzel, Thorsten; Krämer, Oliver H

    2013-01-01

    The cytokine-inducible transcription factors signal transducer and activator of transcription 5A and 5B (STAT5A and STAT5B) are important for the proper development of multicellular eukaryotes. Disturbed signaling cascades evoking uncontrolled expression of STAT5 target genes are associated with cancer and immunological failure. Here, we summarize how STAT5 acetylation is integrated into posttranslational modification networks within cells. Moreover, we focus on how inhibitors of deacetylases and tyrosine kinases can correct leukemogenic signaling nodes involving STAT5. Such small molecules can be exploited in the fight against neoplastic diseases and immunological disorders. PMID:24416653

  7. The H3-H4 N-Terminal Tail Domains Are the Primary Mediators of Transcription Factor IIIA Access to 5S DNA within a Nucleosome

    PubMed Central

    Vitolo, Joseph M.; Thiriet, Christophe; Hayes, Jeffrey J.

    2000-01-01

    Reconstitution of a DNA fragment containing a Xenopus borealis somatic type 5S rRNA gene into a nucleosome greatly restricts the binding of transcription factor IIIA (TFIIIA) to its cognate DNA sequence within the internal promoter of the gene. Removal of all core histone tail domains by limited trypsin proteolysis or acetylation of the core histone tails significantly relieves this inhibition and allows TFIIIA to exhibit high-affinity binding to nucleosomal DNA. Since only a single tail or a subset of tails may be primarily responsible for this effect, we determined whether removal of the individual tail domains of the H2A-H2B dimer or the H3-H4 tetramer affects TFIIIA binding to its cognate DNA site within the 5S nucleosome in vitro. The results show that the tail domains of H3 and H4, but not those of H2A and/or H2B, directly modulate the ability of TFIIIA to bind nucleosomal DNA. In vitro transcription assays carried out with nucleosomal templates lacking individual tail domains show that transcription efficiency parallels the binding of TFIIIA. In addition, we show that the stoichiometry of core histones within the 5S DNA-core histone-TFIIIA triple complex is not changed upon TFIIIA association. Thus, TFIIIA binding occurs by displacement of H2A-H2B–DNA contacts but without complete loss of the dimer from the nucleoprotein complex. These data, coupled with previous reports (M. Vettese-Dadey, P. A. Grant, T. R. Hebbes, C. Crane-Robinson, C. D. Allis, and J. L. Workman, EMBO J. 15:2508–2518, 1996; L. Howe, T. A. Ranalli, C. D. Allis, and J. Ausio, J. Biol. Chem. 273:20693–20696, 1998), suggest that the H3/H4 tails are the primary arbiters of transcription factor access to intranucleosomal DNA. PMID:10688663

  8. Histone acetylation in insect chromosomes.

    PubMed

    Allfrey, V G; Pogo, B G; Littau, V C; Gershey, E L; Mirsky, A E

    1968-01-19

    Acetylation of histones takes place along the salivary gland chromosomes of Chironomus thummi when RNA synthesis is active. It can be observed but not measured quantitatively by autoradiography of chromosome squashes. The "fixatives" commonly used in preparing squashes of insect chromosomes preferentially extract the highly acetylated "arginine-rich" histone fractions; the use of such fixatives may explain the reported absence of histone acetylation in Drosophila melanogaster.

  9. Multiscale modelling of nucleosome core particle aggregation

    NASA Astrophysics Data System (ADS)

    Lyubartsev, Alexander P.; Korolev, Nikolay; Fan, Yanping; Nordenskiöld, Lars

    2015-02-01

    The nucleosome core particle (NCP) is the basic building block of chromatin. Under the influence of multivalent cations, isolated mononucleosomes exhibit a rich phase behaviour forming various columnar phases with characteristic NCP-NCP stacking. NCP stacking is also a regular element of chromatin structure in vivo. Understanding the mechanism of nucleosome stacking and the conditions leading to self-assembly of NCPs is still incomplete. Due to the complexity of the system and the need to describe electrostatics properly by including the explicit mobile ions, novel modelling approaches based on coarse-grained (CG) methods at the multiscale level becomes a necessity. In this work we present a multiscale CG computer simulation approach to modelling interactions and self-assembly of solutions of NCPs induced by the presence of multivalent cations. Starting from continuum simulations including explicit three-valent cobalt(III)hexammine (CoHex3+) counterions and 20 NCPs, based on a previously developed advanced CG NCP model with one bead per amino acid and five beads per two DNA base pair unit (Fan et al 2013 PLoS One 8 e54228), we use the inverse Monte Carlo method to calculate effective interaction potentials for a ‘super-CG’ NCP model consisting of seven beads for each NCP. These interaction potentials are used in large-scale simulations of up to 5000 NCPs, modelling self-assembly induced by CoHex3+. The systems of ‘super-CG’ NCPs form a single large cluster of stacked NCPs without long-range order in agreement with experimental data for NCPs precipitated by the three-valent polyamine, spermidine3+.

  10. Repertoires of the nucleosome-positioning dinucleotides.

    PubMed

    Bettecken, Thomas; Trifonov, Edward N

    2009-11-02

    It is generally accepted that the organization of eukaryotic DNA into chromatin is strongly governed by a code inherent in the genomic DNA sequence. This code, as well as other codes, is superposed on the triplets coding for amino acids. The history of the chromatin code started three decades ago with the discovery of the periodic appearance of certain dinucleotides, with AA/TT and RR/YY giving the strongest signals, all with a period of 10.4 bases. Every base-pair stack in the DNA duplex has specific deformation properties, thus favoring DNA bending in a specific direction. The appearance of the corresponding dinucleotide at the distance 10.4 xn bases will facilitate DNA bending in that direction, which corresponds to the minimum energy of DNA folding in the nucleosome. We have analyzed the periodic appearances of all 16 dinucleotides in the genomes of thirteen different eukaryotic organisms. Our data show that a large variety of dinucleotides (if not all) are, apparently, contributing to the nucleosome positioning code. The choice of the periodical dinucleotides differs considerably from one organism to another. Among other 10.4 base periodicities, a strong and very regular 10.4 base signal was observed for CG dinucleotides in the genome of the honey bee A. mellifera. Also, the dinucleotide CG appears as the only periodical component in the human genome. This observation seems especially relevant since CpG methylation is well known to modulate chromatin packing and regularity. Thus, the selection of the dinucleotides contributing to the chromatin code is species specific, and may differ from region to region, depending on the sequence context.

  11. Multiplexing Genetic and Nucleosome Positioning Codes: A Computational Approach

    PubMed Central

    Eslami-Mossallam, Behrouz; Schram, Raoul D.; Tompitak, Marco; van Noort, John; Schiessel, Helmut

    2016-01-01

    Eukaryotic DNA is strongly bent inside fundamental packaging units: the nucleosomes. It is known that their positions are strongly influenced by the mechanical properties of the underlying DNA sequence. Here we discuss the possibility that these mechanical properties and the concomitant nucleosome positions are not just a side product of the given DNA sequence, e.g. that of the genes, but that a mechanical evolution of DNA molecules might have taken place. We first demonstrate the possibility of multiplexing classical and mechanical genetic information using a computational nucleosome model. In a second step we give evidence for genome-wide multiplexing in Saccharomyces cerevisiae and Schizosacharomyces pombe. This suggests that the exact positions of nucleosomes play crucial roles in chromatin function. PMID:27272176

  12. A translational signature for nucleosome positioning in vivo

    PubMed Central

    Caserta, Micaela; Agricola, Eleonora; Churcher, Mark; Hiriart, Edwige; Verdone, Loredana; Di Mauro, Ernesto; Travers, Andrew

    2009-01-01

    In vivo nucleosomes often occupy well-defined preferred positions on genomic DNA. An important question is to what extent these preferred positions are directly encoded by the DNA sequence itself. We derive here from in vivo positions, accurately mapped by partial micrococcal nuclease digestion, a translational positioning signal that identifies the approximate midpoint of DNA bound by a histone octamer. This midpoint is, on average, highly A/T rich (∼73%) and, in particular, the dinucleotide TpA occurs preferentially at this and other outward-facing minor grooves. We conclude that in this set of sequences the sequence code for DNA bending and nucleosome positioning differs from the other described sets and we suggest that the enrichment of AT-containing dinucleotides at the centre is required for local untwisting. We show that this signature is preferentially associated with nucleosomes flanking promoter regions and suggest that it contributes to the establishment of gene-specific nucleosome arrays. PMID:19596807

  13. Histones and nucleosomes in Archaea and Eukarya: a comparative analysis.

    PubMed

    Pereira, S L; Reeve, J N

    1998-08-01

    Archaeal histones from mesophilic, thermophilic, and hyperthermophilic members of the Euryarchaeota have primary sequences, the histone fold, tertiary structures, and dimer formation in common with the eukaryal nucleosome core histones H2A, H2B, H3, and H4. Archaeal histones form nucleoprotein complexes in vitro and in vivo, designated archaeal nucleosomes, that contain histone tetramers and protect approximately 60 base pairs of DNA from nuclease digestion. Based on the sequence and structural homologies and experimental data reviewed here, archaeal nucleosomes appear similar, and may be homologous in evolutionary terms and function, to the structure at the center of the eukaryal nucleosome formed by the histone (H3 + H4)2 tetramer.

  14. Nucleosome functions in spindle assembly and nuclear envelope formation

    PubMed Central

    Zierhut, Christian; Funabiki, Hironori

    2016-01-01

    Summary Chromosomes are not only carriers of the genetic material, but also actively regulate the assembly of complex intracellular architectures. During mitosis, chromosome-induced microtubule polymerisation ensures spindle assembly in cells without centrosomes and plays a supportive role in centrosome-containing cells. Chromosomal signals also mediate post-mitotic nuclear envelope (NE) re-formation. Recent studies using novel approaches to manipulate histones in oocytes, where functions can be analysed in the absence of transcription, have established that nucleosomes, but not DNA alone, mediate the chromosomal regulation of spindle assembly and NE formation. Both processes require the generation of RanGTP by RCC1 recruited to nucleosomes but nucleosomes also acquire cell cycle stage specific regulators, Aurora B in mitosis and ELYS, the initiator of nuclear pore complex assembly, at mitotic exit. Here, we review the mechanisms by which nucleosomes control assembly and functions of the spindle and the NE, and discuss their implications for genome maintenance. PMID:26222742

  15. DNA Shape Dominates Sequence Affinity in Nucleosome Formation

    NASA Astrophysics Data System (ADS)

    Freeman, Gordon S.; Lequieu, Joshua P.; Hinckley, Daniel M.; Whitmer, Jonathan K.; de Pablo, Juan J.

    2014-10-01

    Nucleosomes provide the basic unit of compaction in eukaryotic genomes, and the mechanisms that dictate their position at specific locations along a DNA sequence are of central importance to genetics. In this Letter, we employ molecular models of DNA and proteins to elucidate various aspects of nucleosome positioning. In particular, we show how DNA's histone affinity is encoded in its sequence-dependent shape, including subtle deviations from the ideal straight B-DNA form and local variations of minor groove width. By relying on high-precision simulations of the free energy of nucleosome complexes, we also demonstrate that, depending on DNA's intrinsic curvature, histone binding can be dominated by bending interactions or electrostatic interactions. More generally, the results presented here explain how sequence, manifested as the shape of the DNA molecule, dominates molecular recognition in the problem of nucleosome positioning.

  16. Role of DNA sequence in chromatin remodeling and the formation of nucleosome-free regions.

    PubMed

    Lorch, Yahli; Maier-Davis, Barbara; Kornberg, Roger D

    2014-11-15

    AT-rich DNA is concentrated in the nucleosome-free regions (NFRs) associated with transcription start sites of most genes. We tested the hypothesis that AT-rich DNA engenders NFR formation by virtue of its rigidity and consequent exclusion of nucleosomes. We found that the AT-rich sequences present in many NFRs have little effect on the stability of nucleosomes. Rather, these sequences facilitate the removal of nucleosomes by the RSC chromatin remodeling complex. RSC activity is stimulated by AT-rich sequences in nucleosomes and inhibited by competition with AT-rich DNA. RSC may remove NFR nucleosomes without effect on adjacent ORF nucleosomes. Our findings suggest that many NFRs are formed and maintained by an active mechanism involving the ATP-dependent removal of nucleosomes rather than a passive mechanism due to the intrinsic instability of nucleosomes on AT-rich DNA sequences. © 2014 Lorch et al.; Published by Cold Spring Harbor Laboratory Press.

  17. Altered promoter nucleosome positioning is an early event in gene silencing.

    PubMed

    Hesson, Luke B; Sloane, Mathew A; Wong, Jason Wh; Nunez, Andrea C; Srivastava, Sameer; Ng, Benedict; Hawkins, Nicholas J; Bourke, Michael J; Ward, Robyn L

    2014-10-01

    Gene silencing in cancer frequently involves hypermethylation and dense nucleosome occupancy across promoter regions. How a promoter transitions to this silent state is unclear. Using colorectal adenomas, we investigated nucleosome positioning, DNA methylation, and gene expression in the early stages of gene silencing. Genome-wide gene expression correlated with highly positioned nucleosomes upstream and downstream of a nucleosome-depleted transcription start site (TSS). Hypermethylated promoters displayed increased nucleosome occupancy, specifically at the TSS. We investigated 2 genes, CDH1 and CDKN2B, which were silenced in adenomas but lacked promoter hypermethylation. Instead, silencing correlated with loss of nucleosomes from the -2 position upstream of the TSS relative to normal mucosa. In contrast, permanent CDH1 silencing in carcinoma cells was characterized by promoter hypermethylation and dense nucleosome occupancy. Our findings suggest that silenced genes transition through an intermediary stage involving altered promoter nucleosome positioning, before permanent silencing by hypermethylation and dense nucleosome occupancy.

  18. Foxa2 and H2A.Z Mediate Nucleosome Depletion during Embryonic Stem Cell Differentiation

    PubMed Central

    Li, Zhaoyu; Gadue, Paul; Chen, Kaifu; Jiao, Yang; Tuteja, Geetu; Schug, Jonathan; Li, Wei; Kaestner, Klaus H.

    2012-01-01

    SUMMARY Nucleosome occupancy is fundamental for establishing chromatin architecture. However, little is known about the relationship between nucleosome dynamics and initial cell lineage specification. Here, we determine the mechanisms that control global nucleosome dynamics during embryonic stem (ES) cell differentiation into endoderm. Both nucleosome depletion and de novo occupation occur during the differentiation process, with higher overall nucleosome density after differentiation. The variant histone H2A.Z and the winged helix transcription factor Foxa2 both act to regulate nucleosome depletion and gene activation, thus promoting ES cell differentiation, while DNA methylation promotes nucleosome occupation and suppresses gene expression. Nucleosome depletion during ES cell differentiation is dependent on Nap1l1-coupled SWI/SNF and INO80 chromatin remodeling complexes. Thus, both epigenetic and genetic regulators cooperate to control nucleosome dynamics during ES cell fate decisions. PMID:23260146

  19. Nucleotide excision repair and photolyase repair of UV photoproducts in nucleosomes: assessing the existence of nucleosome and non-nucleosome rDNA chromatin in vivo.

    PubMed

    Tremblay, Maxime; Toussaint, Martin; D'Amours, Annie; Conconi, Antonio

    2009-02-01

    The genome is organized into nuclear domains, which create microenvironments that favor distinct chromatin structures and functions (e.g., highly repetitive sequences, centromeres, telomeres, noncoding sequences, inactive genes, RNA polymerase II and III transcribed genes, and the nucleolus). Correlations have been drawn between gene silencing and proximity to a heterochromatic compartment. At the other end of the scale are ribosomal genes, which are transcribed at a very high rate by RNA polymerase I (~60% of total transcription), have a loose chromatin structure, and are clustered in the nucleolus. The rDNA sequences have 2 distinct structures: active rRNA genes, which have no nucleosomes; and inactive rRNA genes, which have nucleosomes. Like DNA transcription and replication, DNA repair is modulated by the structure of chromatin, and the kinetics of DNA repair vary among the nuclear domains. Although research on DNA repair in all chromosomal contexts is important to understand the mechanisms of genome maintenance, this review focuses on nucleotide excision repair and photolyase repair of UV photoproducts in the first-order packing of DNA in chromatin: the nucleosome. In addition, it summarizes the studies that have demonstrated the existence of the 2 rDNA chromatins, and the way this feature of the rDNA locus allows for direct comparison of DNA repair in 2 very different structures: nucleosome and non-nucleosome DNA.

  20. Nucleosome alterations caused by mutations at modifiable histone residues in Saccharomyces cerevisiae

    PubMed Central

    Liu, Hongde; Wang, Pingyan; Liu, Lingjie; Min, Zhu; Luo, Kun; Wan, Yakun

    2015-01-01

    Nucleosome organization exhibits dynamic properties depending on the cell state and environment. Histone proteins, fundamental components of nucleosomes, are subject to chemical modifications on particular residues. We examined the effect of substituting modifiable residues of four core histones with the non-modifiable residue alanine on nucleosome dynamics. We mapped the genome-wide nucleosomes in 22 histone mutants of Saccharomyces cerevisiae and compared the nucleosome alterations relative to the wild-type strain. Our results indicated that different types of histone mutation resulted in different phenotypes and a distinct reorganization of nucleosomes. Nucleosome occupancy was altered at telomeres, but not at centromeres. The first nucleosomes upstream (−1) and downstream (+1) of the transcription start site (TSS) were more dynamic than other nucleosomes. Mutations in histones affected the nucleosome array downstream of the TSS. Highly expressed genes, such as ribosome genes and genes involved in glycolysis, showed increased nucleosome occupancy in many types of histone mutant. In particular, the H3K56A mutant exhibited a high percentage of dynamic genomic regions, decreased nucleosome occupancy at telomeres, increased occupancy at the +1 and −1 nucleosomes, and a slow growth phenotype under stress conditions. Our findings provide insight into the influence of histone mutations on nucleosome dynamics. PMID:26498326

  1. Nucleosome alterations caused by mutations at modifiable histone residues in Saccharomyces cerevisiae.

    PubMed

    Liu, Hongde; Wang, Pingyan; Liu, Lingjie; Min, Zhu; Luo, Kun; Wan, Yakun

    2015-10-26

    Nucleosome organization exhibits dynamic properties depending on the cell state and environment. Histone proteins, fundamental components of nucleosomes, are subject to chemical modifications on particular residues. We examined the effect of substituting modifiable residues of four core histones with the non-modifiable residue alanine on nucleosome dynamics. We mapped the genome-wide nucleosomes in 22 histone mutants of Saccharomyces cerevisiae and compared the nucleosome alterations relative to the wild-type strain. Our results indicated that different types of histone mutation resulted in different phenotypes and a distinct reorganization of nucleosomes. Nucleosome occupancy was altered at telomeres, but not at centromeres. The first nucleosomes upstream (-1) and downstream (+1) of the transcription start site (TSS) were more dynamic than other nucleosomes. Mutations in histones affected the nucleosome array downstream of the TSS. Highly expressed genes, such as ribosome genes and genes involved in glycolysis, showed increased nucleosome occupancy in many types of histone mutant. In particular, the H3K56A mutant exhibited a high percentage of dynamic genomic regions, decreased nucleosome occupancy at telomeres, increased occupancy at the +1 and -1 nucleosomes, and a slow growth phenotype under stress conditions. Our findings provide insight into the influence of histone mutations on nucleosome dynamics.

  2. Acetylation of Transition Protein 2 (TP2) by KAT3B (p300) Alters Its DNA Condensation Property and Interaction with Putative Histone Chaperone NPM3*

    PubMed Central

    Pradeepa, Madapura M.; Nikhil, Gupta; Hari Kishore, Annavarapu; Bharath, Giriyapura N.; Kundu, Tapas K.; Rao, Manchanahalli R. Satyanarayana

    2009-01-01

    The hallmark of mammalian spermiogenesis is the dramatic chromatin remodeling process wherein the nucleosomal histones are replaced by the transition proteins TP1, TP2, and TP4. Subsequently these transition proteins are replaced by the protamines P1 and P2. Hyperacetylation of histone H4 is linked to their replacement by transition proteins. Here we report that TP2 is acetylated in vivo as detected by anti-acetylated lysine antibody and mass spectrometric analysis. Further, recombinant TP2 is acetylated in vitro by acetyltransferase KAT3B (p300) more efficiently than by KAT2B (PCAF). In vivo p300 was demonstrated to acetylate TP2. p300 acetylates TP2 in its C-terminal domain, which is highly basic in nature and possesses chromatin-condensing properties. Mass spectrometric analysis showed that p300 acetylates four lysine residues in the C-terminal domain of TP2. Acetylation of TP2 by p300 leads to significant reduction in its DNA condensation property as studied by circular dichroism and atomic force microscopy analysis. TP2 also interacts with a putative histone chaperone, NPM3, wherein expression is elevated in haploid spermatids. Interestingly, acetylation of TP2 impedes its interaction with NPM3. Thus, acetylation of TP2 adds a new dimension to its role in the dynamic reorganization of chromatin during mammalian spermiogenesis. PMID:19710011

  3. DNA damage may drive nucleosomal reorganization to facilitate damage detection

    NASA Astrophysics Data System (ADS)

    LeGresley, Sarah E.; Wilt, Jamie; Antonik, Matthew

    2014-03-01

    One issue in genome maintenance is how DNA repair proteins find lesions at rates that seem to exceed diffusion-limited search rates. We propose a phenomenon where DNA damage induces nucleosomal rearrangements which move lesions to potential rendezvous points in the chromatin structure. These rendezvous points are the dyad and the linker DNA between histones, positions in the chromatin which are more likely to be accessible by repair proteins engaged in a random search. The feasibility of this mechanism is tested by considering the statistical mechanics of DNA containing a single lesion wrapped onto the nucleosome. We consider lesions which make the DNA either more flexible or more rigid by modeling the lesion as either a decrease or an increase in the bending energy. We include this energy in a partition function model of nucleosome breathing. Our results indicate that the steady state for a breathing nucleosome will most likely position the lesion at the dyad or in the linker, depending on the energy of the lesion. A role for DNA binding proteins and chromatin remodelers is suggested based on their ability to alter the mechanical properties of the DNA and DNA-histone binding, respectively. We speculate that these positions around the nucleosome potentially serve as rendezvous points where DNA lesions may be encountered by repair proteins which may be sterically hindered from searching the rest of the nucleosomal DNA. The strength of the repositioning is strongly dependent on the structural details of the DNA lesion and the wrapping and breathing of the nucleosome. A more sophisticated evaluation of this proposed mechanism will require detailed information about breathing dynamics, the structure of partially wrapped nucleosomes, and the structural properties of damaged DNA.

  4. Identification of autoreactive B cells with labeled nucleosomes.

    PubMed

    Gies, Vincent; Wagner, Alain; Seifert, Cécile; Guffroy, Aurélien; Fauny, Jean-D; Knapp, Anne-M; Pasquali, Jean-L; Martin, Thierry; Dumortier, Hélène; Korganow, Anne-S; Soulas-Sprauel, Pauline

    2017-04-04

    The pathogenesis of autoimmune diseases has not been completely elucidated yet, and only a few specific treatments have been developed so far. In autoimmune diseases mediated by pathogenic autoantibodies, such as systemic lupus erythematosus, the specific detection and analysis of autoreactive B cells is crucial for a better understanding of the physiopathology. Biological characterization of these cells may help to define new therapeutic targets. Very few techniques allowing the precise detection of autoreactive B cells have been described so far. Herein we propose a new flow cytometry technique for specific detection of anti-nucleosome B cells, which secrete autoantibodies in systemic lupus erythematosus, using labeled nucleosomes. We produced different fluorochrome-labeled nucleosomes, characterized them, and finally tested them in flow cytometry. Nucleosomes labeled via the cysteines present in H3 histone specifically bind to autoreactive B cells in the anti-DNA transgenic B6.56R mice model. The present work validates the use of fluorochrome-labeled nucleosomes via cysteines to identify anti-nucleosome B cells and offers new opportunities for the description of autoreactive B cell phenotype.

  5. The universality of nucleosome organization: from yeast to human

    NASA Astrophysics Data System (ADS)

    Chereji, Razvan

    The basic units of DNA packaging are called nucleosomes. Their locations on the chromosomes play an essential role in gene regulation. We study nucleosome positioning in yeast, fly, mouse, and human, and build biophysical models in order to explain the genome-wide nucleosome organization. We show that DNA sequence alone is not able to generate the phased arrays of nucleosomes observed in vivo near the transcription start sites. We discuss simple models which can account for the formation of nucleosome depleted regions and nucleosome phasing at the gene promoters. We show that the same principles apply to different organisms. References: [1] RV Chereji, D Tolkunov, G Locke, AV Morozov - Phys. Rev. E 83, 050903 (2011) [2] RV Chereji, AV Morozov - J. Stat. Phys. 144, 379 (2011) [3] RV Chereji, AV Morozov - Proc. Natl. Acad. Sci. U.S.A. 111, 5236 (2014) [4] RV Chereji, T-W Kan, et al. - Nucleic Acids Res. (2015) doi: 10.1093/nar/gkv978 [5] RV Chereji, AV Morozov - Brief. Funct. Genomics 14, 50 (2015) [6] HA Cole, J Ocampo, JR Iben, RV Chereji, DJ Clark - Nucleic Acids Res. 42, 12512 (2014) [7] D Ganguli, RV Chereji, J Iben, HA Cole, DJ Clark - Genome Res. 24, 1637 (2014)

  6. Nucleosome Positioning and NDR Structure at RNA Polymerase III Promoters

    NASA Astrophysics Data System (ADS)

    Helbo, Alexandra Søgaard; Lay, Fides D.; Jones, Peter A.; Liang, Gangning; Grønbæk, Kirsten

    2017-02-01

    Chromatin is structurally involved in the transcriptional regulation of all genes. While the nucleosome positioning at RNA polymerase II (pol II) promoters has been extensively studied, less is known about the chromatin structure at pol III promoters in human cells. We use a high-resolution analysis to show substantial differences in chromatin structure of pol II and pol III promoters, and between subtypes of pol III genes. Notably, the nucleosome depleted region at the transcription start site of pol III genes extends past the termination sequences, resulting in nucleosome free gene bodies. The +1 nucleosome is located further downstream than at pol II genes and furthermore displays weak positioning. The variable position of the +1 location is seen not only within individual cell populations and between cell types, but also between different pol III promoter subtypes, suggesting that the +1 nucleosome may be involved in the transcriptional regulation of pol III genes. We find that expression and DNA methylation patterns correlate with distinct accessibility patterns, where DNA methylation associates with the silencing and inaccessibility at promoters. Taken together, this study provides the first high-resolution map of nucleosome positioning and occupancy at human pol III promoters at specific loci and genome wide.

  7. Mechanisms that specify promoter nucleosome location and identity

    PubMed Central

    Hartley, Paul D.; Madhani, Hiten D.

    2009-01-01

    SUMMARY The chromatin architecture of eukaryotic gene promoters is generally characterized by a nucleosome-free region (NFR) flanked by at least one H2A.Z variant nucleosome. Computational predictions of nucleosome positions based on thermodynamic properties of DNA-histone interactions have met with limited success. Here we show that the action of the essential RSC remodeling complex in S. cerevisiae helps explain the discrepancy between theory and experiment. In RSC-depleted cells, NFRs shrink such that the average positions of flanking nucleosomes move toward predicted sites. Nucleosome positioning at distinct subsets of promoters additionally requires the essential Myb family proteins Abf1 and Reb1, whose binding sites are enriched in NFRs. In contrast, H2A.Z deposition is dispensable for nucleosome positioning. By regulating H2A.Z deposition using a steroid-inducible protein splicing strategy, we show that NFR establishment is necessary for H2A.Z deposition. These studies suggest an ordered pathway for the assembly of promoter chromatin architecture. PMID:19410542

  8. Nucleosomes determine their own patch size in base excision repair

    PubMed Central

    Meas, Rithy; Smerdon, Michael J.

    2016-01-01

    Base excision repair (BER) processes non-helix distorting lesions (e.g., uracils and gaps) and is composed of two subpathways that differ in the number of nucleotides (nts) incorporated during the DNA synthesis step: short patch (SP) repair incorporates 1 nt and long patch (LP) repair incorporates 2–12 nts. This choice for either LP or SP repair has not been analyzed in the context of nucleosomes. Initial studies with uracil located in nucleosome core DNA showed a distinct DNA polymerase extension profile in cell-free extracts that specifically limits extension to 1 nt, suggesting a preference for SP BER. Therefore, we developed an assay to differentiate long and short repair patches in ‘designed’ nucleosomes containing a single-nucleotide gap at specific locations relative to the dyad center. Using cell-free extracts or purified enzymes, we found that DNA lesions in the nucleosome core are preferentially repaired by DNA polymerase β and there is a significant reduction in BER polymerase extension beyond 1 nt, creating a striking bias for incorporation of short patches into nucleosomal DNA. These results show that nucleosomes control the patch size used by BER. PMID:27265863

  9. Nucleosome Positioning and NDR Structure at RNA Polymerase III Promoters

    PubMed Central

    Helbo, Alexandra Søgaard; Lay, Fides D.; Jones, Peter A.; Liang, Gangning; Grønbæk, Kirsten

    2017-01-01

    Chromatin is structurally involved in the transcriptional regulation of all genes. While the nucleosome positioning at RNA polymerase II (pol II) promoters has been extensively studied, less is known about the chromatin structure at pol III promoters in human cells. We use a high-resolution analysis to show substantial differences in chromatin structure of pol II and pol III promoters, and between subtypes of pol III genes. Notably, the nucleosome depleted region at the transcription start site of pol III genes extends past the termination sequences, resulting in nucleosome free gene bodies. The +1 nucleosome is located further downstream than at pol II genes and furthermore displays weak positioning. The variable position of the +1 location is seen not only within individual cell populations and between cell types, but also between different pol III promoter subtypes, suggesting that the +1 nucleosome may be involved in the transcriptional regulation of pol III genes. We find that expression and DNA methylation patterns correlate with distinct accessibility patterns, where DNA methylation associates with the silencing and inaccessibility at promoters. Taken together, this study provides the first high-resolution map of nucleosome positioning and occupancy at human pol III promoters at specific loci and genome wide. PMID:28176797

  10. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

    PubMed Central

    Isaac, R Stefan; Jiang, Fuguo; Doudna, Jennifer A; Lim, Wendell A; Narlikar, Geeta J; Almeida, Ricardo

    2016-01-01

    The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo. DOI: http://dx.doi.org/10.7554/eLife.13450.001 PMID:27130520

  11. Protein acetylation sites mediated by Schistosoma mansoni GCN5

    SciTech Connect

    Moraes Maciel, Renata de; Furtado Madeiro da Costa, Rodrigo; Meirelles Bastosde Oliveira, Francisco; Rumjanek, Franklin David; Fantappie, Marcelo Rosado

    2008-05-23

    The transcriptional co-activator GCN5, a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcription activation. As in other eukaryotes, the DNA from the parasite Schistosome mansoni is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Using a series of synthetic peptides we determined that Lys-14 of histone H3 was acetylated by the recombinant SmGCN5-HAT domain. SmGCN5 was also able to acetylate schistosome non-histone proteins, such as the nuclear receptors SmRXR1 and SmNR1, and the co-activator SmNCoA-62. Electron microscopy revealed the presence of SmGCN5 protein in the nuclei of vitelline cells. Within the nucleus, SmGCN5 was found to be located in interchromatin granule clusters (IGCs), which are transcriptionally active structures. The data suggest that SmGCN5 is involved in transcription activation.

  12. Relationship between periodic dinucleotides and the nucleosome structure revealed by alpha shape modeling

    NASA Astrophysics Data System (ADS)

    Zhou, Weiqiang; Yan, Hong

    2010-04-01

    As the fundamental repeating units in eukaryotic chromatin, nucleosomes play an important role in many biological processes. For this reason, the study of the structure of nucleosomes may help to reveal some of the crucial principals of these processes. In our research, we have used alpha shapes to model nucleosome structure and discovered that the periodic DNA dinucleotides AA, TT and GC occupy special positions in nucleosome structure with one nucleotide inside and the other outside the nucleosome surface. This structural feature and other dinucleotide characteristics can provide useful information for the study of nucleosome positioning.

  13. Binding of NF-κB to nucleosomes: effect of translational positioning, nucleosome remodeling and linker histone H1.

    PubMed

    Lone, Imtiaz Nisar; Shukla, Manu Shubhdarshan; Charles Richard, John Lalith; Peshev, Zahary Yordanov; Dimitrov, Stefan; Angelov, Dimitar

    2013-01-01

    NF-κB is a key transcription factor regulating the expression of inflammatory responsive genes. How NF-κB binds to naked DNA templates is well documented, but how it interacts with chromatin is far from being clear. Here we used a combination of UV laser footprinting, hydroxyl footprinting and electrophoretic mobility shift assay to investigate the binding of NF-κB to nucleosomal templates. We show that NF-κB p50 homodimer is able to bind to its recognition sequence, when it is localized at the edge of the core particle, but not when the recognition sequence is at the interior of the nucleosome. Remodeling of the nucleosome by the chromatin remodeling machine RSC was not sufficient to allow binding of NF-κB to its recognition sequence located in the vicinity of the nucleosome dyad, but RSC-induced histone octamer sliding allowed clearly detectable binding of NF-κB with the slid particle. Importantly, nucleosome dilution-driven removal of H2A-H2B dimer led to complete accessibility of the site located close to the dyad to NF-κB. Finally, we found that NF-κB was able to displace histone H1 and prevent its binding to nucleosome. These data provide important insight on the role of chromatin structure in the regulation of transcription of NF-κB dependent genes.

  14. Binding of NF-κB to Nucleosomes: Effect of Translational Positioning, Nucleosome Remodeling and Linker Histone H1

    PubMed Central

    Lone, Imtiaz Nisar; Shukla, Manu Shubhdarshan; Charles Richard, John Lalith; Peshev, Zahary Yordanov; Dimitrov, Stefan; Angelov, Dimitar

    2013-01-01

    NF-κB is a key transcription factor regulating the expression of inflammatory responsive genes. How NF-κB binds to naked DNA templates is well documented, but how it interacts with chromatin is far from being clear. Here we used a combination of UV laser footprinting, hydroxyl footprinting and electrophoretic mobility shift assay to investigate the binding of NF-κB to nucleosomal templates. We show that NF-κB p50 homodimer is able to bind to its recognition sequence, when it is localized at the edge of the core particle, but not when the recognition sequence is at the interior of the nucleosome. Remodeling of the nucleosome by the chromatin remodeling machine RSC was not sufficient to allow binding of NF-κB to its recognition sequence located in the vicinity of the nucleosome dyad, but RSC-induced histone octamer sliding allowed clearly detectable binding of NF-κB with the slid particle. Importantly, nucleosome dilution-driven removal of H2A–H2B dimer led to complete accessibility of the site located close to the dyad to NF-κB. Finally, we found that NF-κB was able to displace histone H1 and prevent its binding to nucleosome. These data provide important insight on the role of chromatin structure in the regulation of transcription of NF-κB dependent genes. PMID:24086160

  15. Reduced physical activity in adults at risk for type 2 diabetes who curtail their sleep.

    PubMed

    Booth, John N; Bromley, Lindsay E; Darukhanavala, Amy P; Whitmore, Harry R; Imperial, Jacqueline G; Penev, Pamen D

    2012-02-01

    Adults with parental history of type 2 diabetes have high metabolic morbidity, which is exacerbated by physical inactivity. Self-reported sleep <6 h/day is associated with increased incidence of obesity and diabetes, which may be mediated in part by sleep-loss-related reduction in physical activity. We examined the relationship between habitual sleep curtailment and physical activity in adults with parental history of type 2 diabetes. Forty-eight young urban adults with parental history of type 2 diabetes (27 F/21 M; mean (s.d.) age 26 (4) years; BMI 23.8 (2.5) kg/m(2)) each completed 13 (2) days of sleep and physical activity monitoring by wrist actigraphy and waist accelerometry while following their usual lifestyle at home. Laboratory polysomnography was used to screen for sleep disorders. The primary outcome of the study was the comparison of total daily activity counts between participants with habitual sleep <6 vs. ≥6 h/night. Secondary measures included daily time spent sedentary and in light, moderate, and vigorous physical activity. Short sleepers had no sleep abnormalities and showed signs of increased sleep pressure consistent with a behavioral pattern of habitual sleep curtailment. Compared to participants who slept ≥6 h/night, short sleepers had 27% fewer daily activity counts (P = 0.042), spent less time in moderate-plus-vigorous physical activity (-43 min/day; P = 0.010), and remained more sedentary (+69 min/day; P = 0.026). Our results indicate that young urban adults with parental history of type 2 diabetes who habitually curtail their sleep have less daily physical activity and more sedentary living, which may enhance their metabolic risk.

  16. Nucleosomal stability and dynamics vary significantly when viewed by internal versus terminal labels.

    PubMed

    Kelbauskas, Laimonas; Sun, Jenny; Woodbury, Neal; Lohr, D

    2008-09-09

    Nucleosomes are a major impediment to regulatory factor activities and therefore to the operation of genomic processes in eukaryotes. One suggested mechanism for overcoming in vivo nucleosomal repression is factor-mediated removal of H2A/H2B from nucleosomes. Using nucleosomes labeled internally with FRET fluorophores, we previously observed significant, DNA sequence-dependent variation in stability and dynamics under conditions (subnanomolar concentrations) reported to produce H2A/H2B release from nucleosomes. Here, the same analytical approaches are repeated using 5S and MMTV-B nucleosomes containing FRET labels that monitor the terminal regions. The results show that stability and dynamics vary significantly within the nucleosome; terminally labeled constructs report significantly reduced stability and enhanced DNA dynamics compared to internally labeled constructs. The data also strongly support previous suggestions (1) that subnanomolar concentrations cause H2A/H2B release from nucleosomes, including the 5S, and (2) that stabilities in the internal regions of 5S and two promoter-derived nucleosomes (MMTV-B, GAL10) differ. Sequence-dependent nucleosome stability/dynamics differences could produce inherent variations in the accessibility of histone-associated DNA in vivo. Such intrinsic variation could also provide a mechanism for producing enhanced effects on specific nucleosomes by processes affecting large chromatin regions, thus facilitating the localized targeting of alterations to nucleosomes on crucial regulatory sequences. The results demonstrate clearly the importance of studying physiologically relevant nucleosomes.

  17. Intracellular Hmgb1 Inhibits Inflammatory Nucleosome Release and Limits Acute Pancreatitis in Mice

    PubMed Central

    Kang, Rui; Zhang, Qiuhong; Hou, Wen; Yan, Zhenwen; Chen, Ruochan; Bonaroti, Jillian; Bansal, Preeti; Billiar, Timothy R.; Tsung, Allan; Wang, Qingde; Bartlett, David L.; Whitcomb, David C; Chang, Eugene B.; Zhu, Xiaorong; Wang, Haichao; Lu, Ben; Tracey, Kevin J.; Cao, Lizhi; Fan, Xue-Gong; Lotze, Michael T.; Zeh, Herbert J.; Tang, Daolin

    2014-01-01

    BACKGROUND & AIMS: High mobility group box 1 (HMGB1) is an abundant protein that regulates chromosome architecture and also functions as a damage-associated molecular pattern molecule. Little is known about its intracellular roles in response to tissue injury or during subsequent local and systemic inflammatory responses. We investigated the function of Hmgb1 in mice following induction of acute pancreatitis. METHODS: We utilized a Cre/LoxP system to create mice with pancreas-specific disruption in Hmbg1 (Pdx1-Cre; HMGB1flox/flox mice). Acute pancreatitis was induced in these mice (HMGB1flox/flox mice served as controls) following injection of L-arginine or cerulein. Pancreatic tissues and acinar cells were collected and analyzed by histologic, immunoblot, and immunohistochemical analyses. RESULTS: Following injection of L-arginine or cerulein, Pdx1-Cre; HMGB1flox/flox mice developed acute pancreatitis more rapidly than controls, with increased mortality. Pancreatic tissues of these mice also had higher levels of serum amylase, acinar cell death, leukocyte infiltration, and interstitial edema than controls. Pancreatic tissues and acinar cells collected from the Pdx1-Cre; HMGB1flox/flox mice following L-arginine- or cerulein injection demonstrated nuclear catastrophe with greater nucleosome release when compared with controls, along with increased phosphorylation/activation of RELA Nfκb, degradation of Iκb, and phosphorylation of Mapk. Inhibitors of reactive oxygen species (N-acetyl-L-cysteine) blocked L-arginine–induced DNA damage, necrosis, apoptosis, release of nucleosomes, and activation of Nfκb in pancreatic tissues and acinar cells from Pdx1-Cre; HMGB1flox/flox and control mice. Exogenous genomic DNA and recombinant histone H3 proteins significantly induced release of HMGB1 from mouse macrophages; administration of antibodies against H3 to mice reduced serum levels of HMGB1 and increased survival following L-arginine injection. CONCLUSIONS: In 2 mouse

  18. Structure of the CENP-A nucleosome and its implications for centromeric chromatin architecture.

    PubMed

    Tachiwana, Hiroaki; Kurumizaka, Hitoshi

    2011-01-01

    Centromeres are dictated by the epigenetic inheritance of the centromeric nucleosome containing the centromere-specific histone H3 variant, CENP-A. The structure of the CENP-A nucleosome has been considered to be the fundamental architecture of the centromeric chromatin. Controversy exists in the literature regarding the CENP-A nucleosome structures, with octasome, hemisome, compact octasome, hexasome, and tetrasome models being reported. Some of these CENP-A nucleosome models may correspond to transient intermediates for the assembly of the mature CENP-A nucleosome; however, their significances are still unclear. Therefore, the structure of the mature CENP-A nucleosome has been eagerly awaited. We reconstituted the human CENP-A nucleosome with its cognate centromeric DNA fragment, and determined its crystal structure. In this review, we describe the structure and the physical properties of the CENP-A nucleosome, and discuss their implications for centromeric chromatin architecture.

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

    PubMed

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

    2015-03-12

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

  20. Evolutionary analysis of nucleosome positioning sequences based on New Symmetric Relative Entropy.

    PubMed

    Meng, Hu; Li, Hong; Zheng, Yan; Yang, Zhenhua; Jia, Yun; Bo, Suling

    2017-09-14

    New Symmetric Relative Entropy (NSRE) was applied innovatively to analyze the nucleosome sequences in S. cerevisiae, S. pombe and Drosophila. NSRE distributions could well reflect the characteristic differences of nucleosome sequences among three organisms, and the differences indicate a concerted evolution in the sequence usage of nucleosome. Further analysis about the nucleosomes around TSS shows that the constitutive property of +1/-1 nucleosomes in S. cerevisiae is different from that in S. pombe and Drosophila, which indicates that S. cerevisiae has a different transcription regulation mechanism based on nucleosome. However, in either case, the nucleosome dyad region is conserved and always has a higher NSRE. Base composition analysis shows that this conservative property in nucleosome dyad region is mainly determined by base A and T, and the dependence degrees on base A and T are consistent in three organisms. Copyright © 2017. Published by Elsevier Inc.

  1. Acetylation of histones in neocortex and hippocampus of rats exposed to different modes of hypobaric hypoxia: Implications for brain hypoxic injury and tolerance.

    PubMed

    Samoilov, Mikhail; Churilova, Anna; Gluschenko, Tatjana; Vetrovoy, Oleg; Dyuzhikova, Natalia; Rybnikova, Elena

    2016-03-01

    Acetylation of nucleosome histones results in relaxation of DNA and its availability for the transcriptional regulators, and is generally associated with the enhancement of gene expression. Although it is well known that activation of a variety of pro-adaptive genes represents a key event in the development of brain hypoxic/ischemic tolerance, the role of epigenetic mechanisms, in particular histone acetylation, in this process is still unexplored. The aim of the present study was to investigate changes in acetylation of histones in vulnerable brain neurons using original well-standardized model of hypobaric hypoxia and preconditioning-induced tolerance of the brain. Using quantitative immunohistochemistry and Western blot, effects of severe injurious hypobaric hypoxia (SH, 180mm Hg, 3h) and neuroprotective preconditioning mode (three episodes of 360mm Hg for 2h spaced at 24h) on the levels of the acetylated proteins and acetylated H3 Lys24 (H3K24ac) in the neocortex and hippocampus of rats were studied. SH caused global repression of the acetylation processes in the neocortex (layers II-III, V) and hippocampus (CA1, CA3) by 3-24h, and this effect was prevented by the preconditioning. Moreover, hypoxic preconditioning remarkably increased the acetylation of H3K24 in response to SH in the brain areas examined. The preconditioning hypoxia without subsequent SH also stimulated acetylation processes in the neocortex and hippocampus. The moderately enhanced expression of the acetylated proteins in the preconditioned rats was maintained for 24h, whereas acetylation of H3K24 was intense but transient, peaked at 3h. The novel data obtained in the present study indicate that large activation of the acetylation processes, in particular acetylation of histones might be essential for the development of brain hypoxic tolerance.

  2. Roadmap of retail electricity market reform in China: assisting in mitigating wind energy curtailment

    NASA Astrophysics Data System (ADS)

    Yu, Dezhao; Qiu, Huadong; Yuan, Xiang; Li, Yuan; Shao, Changzheng; Lin, You; Ding, Yi

    2017-01-01

    Among the renewable energies, wind energy has gained the rapidest development in China. Moreover wind power generation has been penetrated into power system in a large scale. However, the high level wind curtailment also indicates a low efficiency of wind energy utilization over the last decade in China. One of the primary constraints on the utilization of wind energy is the lack of an electricity market, in which renewable energies can compete equally with traditional fossil fuel generation. Thus the new round electric power industry reform is essential in China. The reform involves implementing new pricing mechanism, introducing retail-side competition, promoting the consumption of renewable energy. The new round reform can be a promising solution for promoting the development and consumption of wind energy generation in China. Based on proposed reform policies of electric power industry, this paper suggests a roadmap for retail electricity market reform of China, which consists of three stages. Barriers to the efficient utilization of wind energy are also analysed. Finally, this paper introduces several efficient measures for mitigating wind curtailment in each stage of reform.

  3. Regulatory analysis for review and establishment of natural gas curtailment priorities

    SciTech Connect

    1980-05-01

    Technical appendices in Volume 4 provide data and detailed explanations for each of the seven components into which policy evaluation has been divided: (1) delineation of alternatives and then simulation of shortages under each alternative in each prototype company, (2) supplier decisions and costs, including allocation rules, (3) user fuel substitution costs, (4) calculation of user shortage costs, supplier operating cost, and non-user pollution cost, (5) institutional factors affecting implementation, (6) annual demand and supply trends, and (7) national projections for economic and environmental summaries. In essence, there is increasing detail and more intricate discussion from Volume 1 to Volumes 2 and 3 and then to the Technical Appendices in Volume 4. Volume 4 should be used to gain understanding of the many different curtailment plans across the nation and to better appreciate the types and levels of costs that occur to both users and suppliers. The diagrams and discussions in Volume 4 will help the reader interpret the important concepts in designing and evaluating curtailment alternatives. The scenarios on demand and supply in Volume 4 can provide background for other studies.

  4. Regulatory analysis for review and establishment of natural gas curtailment priorities. Volume 2

    SciTech Connect

    1980-05-01

    The overall perspective for policy review is in Volume 1. It contains a concise statement of the problem, a list of policy alternatives, all study findings, and an outline of the evaluations which produced the study findings. Volume 2 is a detailed evaluation of each policy alternative, except for the environmental impact assessment in Volume 3. Both Volumes 2 and 3 are designed to add details for the reader who has already studied Volume 1; background in Volume 1 is not repeated in other volumes. Volume 2 should be used to understand the basic causes for the differences in curtailment alternatives presented in Volume 1. It should also be used to gain understanding of how any additional curtailment alternative must be evaluated before it can be safely implemented. This volume also discusses the following basic policy goals which should guide the selection of any policy change: (1) efficiency as measured by shortage costs and supplier costs, (2) equity as measured by fairness among groups, (3) administrative burden as measured by record keeping and implementation problems, and (4) contribution toward special goals such as oil import reduction.

  5. mChIP-KAT-MS, a method to map protein interactions and acetylation sites for lysine acetyltransferases

    PubMed Central

    Mitchell, Leslie; Huard, Sylvain; Cotrut, Michael; Pourhanifeh-Lemeri, Roghayeh; Steunou, Anne-Lise; Hamza, Akil; Lambert, Jean-Philippe; Zhou, Hu; Ning, Zhibin; Basu, Amrita; Côté, Jacques; Figeys, Daniel A.; Baetz, Kristin

    2013-01-01

    Recent global proteomic and genomic studies have determined that lysine acetylation is a highly abundant posttranslational modification. The next challenge is connecting lysine acetyltransferases (KATs) to their cellular targets. We hypothesize that proteins that physically interact with KATs may not only predict the cellular function of the KATs but may be acetylation targets. We have developed a mass spectrometry-based method that generates a KAT protein interaction network from which we simultaneously identify both in vivo acetylation sites and in vitro acetylation sites. This modified chromatin-immunopurification coupled to an in vitro KAT assay with mass spectrometry (mChIP-KAT-MS) was applied to the Saccharomyces cerevisiae KAT nucleosome acetyltransferase of histone H4 (NuA4). Using mChIP-KAT-MS, we define the NuA4 interactome and in vitro-enriched acetylome, identifying over 70 previously undescribed physical interaction partners for the complex and over 150 acetyl lysine residues, of which 108 are NuA4-specific in vitro sites. Through this method we determine NuA4 acetylation of its own subunit Epl1 is a means of self-regulation and identify a unique link between NuA4 and the spindle pole body. Our work demonstrates that this methodology may serve as a valuable tool in connecting KATs with their cellular targets. PMID:23572591

  6. Chemical map of Schizosaccharomyces pombe reveals species-specific features in nucleosome positioning

    PubMed Central

    Moyle-Heyrman, Georgette; Zaichuk, Tetiana; Xi, Liqun; Zhang, Quanwei; Uhlenbeck, Olke C.; Holmgren, Robert; Widom, Jonathan; Wang, Ji-Ping

    2013-01-01

    Using a recently developed chemical approach, we have generated a genome-wide map of nucleosomes in vivo in Schizosaccharomyces pombe (S. pombe) at base pair resolution. The shorter linker length previously identified in S. pombe is due to a preponderance of nucleosomes separated by ∼4/5 bp, placing nucleosomes on opposite faces of the DNA. The periodic dinucleotide feature thought to position nucleosomes is equally strong in exons as in introns, demonstrating that nucleosome positioning information can be superimposed on coding information. Unlike the case in Saccharomyces cerevisiae, A/T-rich sequences are enriched in S. pombe nucleosomes, particularly at ±20 bp around the dyad. This difference in nucleosome binding preference gives rise to a major distinction downstream of the transcription start site, where nucleosome phasing is highly predictable by A/T frequency in S. pombe but not in S. cerevisiae, suggesting that the genomes and DNA binding preferences of nucleosomes have coevolved in different species. The poly (dA-dT) tracts affect but do not deplete nucleosomes in S. pombe, and they prefer special rotational positions within the nucleosome, with longer tracts enriched in the 10- to 30-bp region from the dyad. S. pombe does not have a well-defined nucleosome-depleted region immediately upstream of most transcription start sites; instead, the −1 nucleosome is positioned with the expected spacing relative to the +1 nucleosome, and its occupancy is negatively correlated with gene expression. Although there is generally very good agreement between nucleosome maps generated by chemical cleavage and micrococcal nuclease digestion, the chemical map shows consistently higher nucleosome occupancy on DNA with high A/T content. PMID:24277842

  7. Chemical map of Schizosaccharomyces pombe reveals species-specific features in nucleosome positioning.

    PubMed

    Moyle-Heyrman, Georgette; Zaichuk, Tetiana; Xi, Liqun; Zhang, Quanwei; Uhlenbeck, Olke C; Holmgren, Robert; Widom, Jonathan; Wang, Ji-Ping

    2013-12-10

    Using a recently developed chemical approach, we have generated a genome-wide map of nucleosomes in vivo in Schizosaccharomyces pombe (S. pombe) at base pair resolution. The shorter linker length previously identified in S. pombe is due to a preponderance of nucleosomes separated by ∼4/5 bp, placing nucleosomes on opposite faces of the DNA. The periodic dinucleotide feature thought to position nucleosomes is equally strong in exons as in introns, demonstrating that nucleosome positioning information can be superimposed on coding information. Unlike the case in Saccharomyces cerevisiae, A/T-rich sequences are enriched in S. pombe nucleosomes, particularly at ±20 bp around the dyad. This difference in nucleosome binding preference gives rise to a major distinction downstream of the transcription start site, where nucleosome phasing is highly predictable by A/T frequency in S. pombe but not in S. cerevisiae, suggesting that the genomes and DNA binding preferences of nucleosomes have coevolved in different species. The poly (dA-dT) tracts affect but do not deplete nucleosomes in S. pombe, and they prefer special rotational positions within the nucleosome, with longer tracts enriched in the 10- to 30-bp region from the dyad. S. pombe does not have a well-defined nucleosome-depleted region immediately upstream of most transcription start sites; instead, the -1 nucleosome is positioned with the expected spacing relative to the +1 nucleosome, and its occupancy is negatively correlated with gene expression. Although there is generally very good agreement between nucleosome maps generated by chemical cleavage and micrococcal nuclease digestion, the chemical map shows consistently higher nucleosome occupancy on DNA with high A/T content.

  8. Integrated molecular mechanism directing nucleosome reorganization by human FACT.

    PubMed

    Tsunaka, Yasuo; Fujiwara, Yoshie; Oyama, Takuji; Hirose, Susumu; Morikawa, Kosuke

    2016-03-15

    Facilitates chromatin transcription (FACT) plays essential roles in chromatin remodeling during DNA transcription, replication, and repair. Our structural and biochemical studies of human FACT-histone interactions present precise views of nucleosome reorganization, conducted by the FACT-SPT16 (suppressor of Ty 16) Mid domain and its adjacent acidic AID segment. AID accesses the H2B N-terminal basic region exposed by partial unwrapping of the nucleosomal DNA, thereby triggering the invasion of FACT into the nucleosome. The crystal structure of the Mid domain complexed with an H3-H4 tetramer exhibits two separate contact sites; the Mid domain forms a novel intermolecular β structure with H4. At the other site, the Mid-H2A steric collision on the H2A-docking surface of the H3-H4 tetramer within the nucleosome induces H2A-H2B displacement. This integrated mechanism results in disrupting the H3 αN helix, which is essential for retaining the nucleosomal DNA ends, and hence facilitates DNA stripping from histone.

  9. Structural Mechanisms of Nucleosome Recognition by Linker Histones.

    PubMed

    Zhou, Bing-Rui; Jiang, Jiansheng; Feng, Hanqiao; Ghirlando, Rodolfo; Xiao, T Sam; Bai, Yawen

    2015-08-20

    Linker histones bind to the nucleosome and regulate the structure of chromatin and gene expression. Despite more than three decades of effort, the structural basis of nucleosome recognition by linker histones remains elusive. Here, we report the crystal structure of the globular domain of chicken linker histone H5 in complex with the nucleosome at 3.5 Å resolution, which is validated using nuclear magnetic resonance spectroscopy. The globular domain sits on the dyad of the nucleosome and interacts with both DNA linkers. Our structure integrates results from mutation analyses and previous cross-linking and fluorescence recovery after photobleach experiments, and it helps resolve the long debate on structural mechanisms of nucleosome recognition by linker histones. The on-dyad binding mode of the H5 globular domain is different from the recently reported off-dyad binding mode of Drosophila linker histone H1. We demonstrate that linker histones with different binding modes could fold chromatin to form distinct higher-order structures.

  10. Chemical physics of DNA packaging in a nucleosome core particle

    NASA Astrophysics Data System (ADS)

    Spakowitz, Andrew; Sudhanshu, Bariz

    2008-03-01

    The fundamental unit of packaged DNA, the nucleosome core particle, contains 146 base pairs of DNA wrapped 1.7 times around a cationic protein complex called the histone octamer. A string of nucleosomes is organized into higher-order structures at several hierarchical levels to form chromatin, a remarkable complex that is compact yet maintains accessibility for gene expression. We develop a theoretical model of the nucleosome core particle in order to extract detailed quantitative information from single-molecule measurements of a single nucleosome under tension. We employ the wormlike chain model to describe the DNA strand as a thermally fluctuating polymer chain. The chain adsorbs on a spool that represents the histone octamer. This model is directly compared to single-molecule experiments conducted in Carlos Bustamante's lab; we find good agreement between our theory and the experimental data. Our model reveals the mechanism that underlies structural transitions that are apparent in the experimental measurements and predicts the conditions where these transitions occur. We proceed to construct a free energy surface to predict the dynamic response in a single-molecule experiment with a time-dependent rate of unwinding the nucleosome. The combination of single-molecule experiments and our theoretical modeling gives detailed information about the specific interactions between DNA and histone proteins.

  11. Integrated molecular mechanism directing nucleosome reorganization by human FACT

    PubMed Central

    Tsunaka, Yasuo; Fujiwara, Yoshie; Oyama, Takuji; Hirose, Susumu; Morikawa, Kosuke

    2016-01-01

    Facilitates chromatin transcription (FACT) plays essential roles in chromatin remodeling during DNA transcription, replication, and repair. Our structural and biochemical studies of human FACT–histone interactions present precise views of nucleosome reorganization, conducted by the FACT-SPT16 (suppressor of Ty 16) Mid domain and its adjacent acidic AID segment. AID accesses the H2B N-terminal basic region exposed by partial unwrapping of the nucleosomal DNA, thereby triggering the invasion of FACT into the nucleosome. The crystal structure of the Mid domain complexed with an H3–H4 tetramer exhibits two separate contact sites; the Mid domain forms a novel intermolecular β structure with H4. At the other site, the Mid–H2A steric collision on the H2A-docking surface of the H3–H4 tetramer within the nucleosome induces H2A–H2B displacement. This integrated mechanism results in disrupting the H3 αN helix, which is essential for retaining the nucleosomal DNA ends, and hence facilitates DNA stripping from histone. PMID:26966247

  12. Unique yeast histone sequences influence octamer and nucleosome stability.

    PubMed

    Leung, Andrew; Cheema, Manjinder; González-Romero, Rodrigo; Eirin-Lopez, Jose M; Ausió, Juan; Nelson, Christopher J

    2016-08-01

    Yeast nucleosomes are known to be intrinsically less stable than those from higher eukaryotes. This difference presents significant challenges for the production of yeast nucleosome core particles (NCPs) and chromatin for in vitro analyses. Using recombinant yeast, human, and chimeric histone proteins, we demonstrate that three divergent amino acids in histone H3 (Q120 K121 K125 ) are responsible for the poor reconstitution of yeast histones into octamers. This QKK motif is only found in Fungi, and is located at the nucleosome dyad axis. Yeast-to-human changes at these positions render yeast histones amenable to well-established octamer reconstitution and salt dialysis methods for generating nucleosomal and longer chromatin templates. By contrast, the most divergent yeast core histones, H2A and H2B, affect the biophysical properties of NCP but not their stability. An evolutionary analysis of H3 sequences shows that a gradual divergence in H3 sequences occurred in Fungi to yield QKK in budding yeast. This likely facilitates the highly euchromatic nature of yeast genomes. Our results provide an explanation for the long recognized difference in yeast nucleosome stability and they offer a simple method to generate yeast chromatin templates for in vitro studies. © 2016 Federation of European Biochemical Societies.

  13. Organization and roles of nucleosomes at mouse meiotic recombination hotspots

    PubMed Central

    Getun, Irina V.; Wu, Zhen K.; Bois, Philippe R.J.

    2012-01-01

    Meiotic double strand breaks (DSBs) occur at discrete regions in the genome coined hotspots. Precisely what directs site selection of these DSBs is hotly debated and in particular it is unclear which chromatin features, and regulatory factors are necessary for a genomic region to initiate and resolve DSBs as a crossover (CO) event. In human and mouse, one layer of hotspot selection control is a recognition sequence element present at these sites that is bound by the Prdm9 zinc-finger protein. Furthermore, an overall open chromatin structure is thought to be required to allow access of the recombination machinery, and this is often dictated by the packaging of DNA around nucleosomes. We recently defined the nucleosome occupancy maps of four mouse recombination hotspots throughout meiosis. These analyses revealed no obvious dynamic changes in nucleosome occupancy, suggesting an intrinsic nature of recombinogenic sites, yet they also revealed that nucleosomes define zones of exclusion for CO resolution. Here, we discuss new evidence implicating nucleosome occupancy in recombinogenic repair and its potential roles in controlling chromatin structure at mouse meiotic hotspots. PMID:22572955

  14. Measuring genome-wide nucleosome turnover using CATCH-IT.

    PubMed

    Teves, Sheila S; Deal, Roger B; Henikoff, Steven

    2012-01-01

    The dynamic interplay between DNA-binding proteins and nucleosomes underlies essential nuclear processes such as transcription, replication, and DNA repair. Manifestations of this interplay include the assembly, eviction, and replacement of nucleosomes. Hence, measurements of nucleosome turnover kinetics can lead to insights into the regulation of dynamic chromatin processes. In this chapter, we describe a genome-wide method for measuring nucleosome turnover that uses metabolic labeling followed by capture of newly synthesized histones, which we have termed Covalent Attachment of Tagged Histones to Capture and Identify Turnover (CATCH-IT). Although CATCH-IT can be used with any genome-wide mapping procedure, high-resolution profiling is attainable using paired-end sequencing of native chromatin. Our protocol also includes an efficient Solexa DNA sequencing library preparation protocol that can be used for single base-pair resolution mapping of both nucleosome and subnucleosomal particles. We not only describe the use of these protocols in the context of a Drosophila cell line but also provide the necessary changes for adaptation to other model systems. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Effects of DNA methylation on the structure of nucleosomes.

    PubMed

    Lee, Ju Yeon; Lee, Tae-Hee

    2012-01-11

    Nucleosomes are the fundamental packing units of the eukaryotic genome. Understanding the dynamic structure of a nucleosome is a key to the elucidation of genome packaging in eukaryotes, which is tied to the mechanisms of gene regulation. CpG methylation of DNA is an epigenetic modification associated with the inactivation of transcription and the formation of a repressive chromatin structure. Unraveling the changes in the structure of nucleosomes upon CpG methylation is an essential step toward the understanding of the mechanisms of gene repression and silencing by CpG methylation. Here we report single-molecule and ensemble fluorescence studies showing how the structure of a nucleosome is affected by CpG methylation. The results indicate that CpG methylation induces tighter wrapping of DNA around the histone core accompanied by a topology change. These findings suggest that changes in the physical properties of nucleosomes induced upon CpG methylation may contribute directly to the formation of a repressive chromatin structure.

  16. Flexible and dynamic nucleosome fiber in living mammalian cells.

    PubMed

    Nozaki, Tadasu; Kaizu, Kazunari; Pack, Chan-Gi; Tamura, Sachiko; Tani, Tomomi; Hihara, Saera; Nagai, Takeharu; Takahashi, Koichi; Maeshima, Kazuhiro

    2013-01-01

    Genomic DNA is organized three dimensionally within cells as chromatin and is searched and read by various proteins by an unknown mechanism; this mediates diverse cell functions. Recently, several pieces of evidence, including our cryomicroscopy and synchrotron X-ray scattering analyses, have demonstrated that chromatin consists of irregularly folded nucleosome fibers without a 30-nm chromatin fiber (i.e., a polymer melt-like structure). This melt-like structure implies a less physically constrained and locally more dynamic state, which may be crucial for protein factors to scan genomic DNA. Using a combined approach of fluorescence correlation spectroscopy, Monte Carlo computer simulations, and single nucleosome imaging, we demonstrated the flexible and dynamic nature of the nucleosome fiber in living mammalian cells. We observed local nucleosome fluctuation (~50 nm movement/30 ms) caused by Brownian motion. Our in vivo/in silico results suggest that local nucleosome dynamics facilitate chromatin accessibility and play a critical role in the scanning of genome information.

  17. Electrostatic mechanism of nucleosomal array folding revealed by computer simulation

    NASA Astrophysics Data System (ADS)

    Sun, Jian; Zhang, Qing; Schlick, Tamar

    2005-06-01

    Although numerous experiments indicate that the chromatin fiber displays salt-dependent conformations, the associated molecular mechanism remains unclear. Here, we apply an irregular Discrete Surface Charge Optimization (DiSCO) model of the nucleosome with all histone tails incorporated to describe by Monte Carlo simulations salt-dependent rearrangements of a nucleosomal array with 12 nucleosomes. The ensemble of nucleosomal array conformations display salt-dependent condensation in good agreement with hydrodynamic measurements and suggest that the array adopts highly irregular 3D zig-zag conformations at high (physiological) salt concentrations and transitions into the extended "beads-on-a-string" conformation at low salt. Energy analyses indicate that the repulsion among linker DNA leads to this extended form, whereas internucleosome attraction drives the folding at high salt. The balance between these two contributions determines the salt-dependent condensation. Importantly, the internucleosome and linker DNA-nucleosome attractions require histone tails; we find that the H3 tails, in particular, are crucial for stabilizing the moderately folded fiber at physiological monovalent salt. chromatin modeling | irregular 3D zig-zag | Discrete Surface Charge Optimization model

  18. Electrostatic mechanism of nucleosomal array folding revealed by computer simulation

    PubMed Central

    Sun, Jian; Zhang, Qing; Schlick, Tamar

    2005-01-01

    Although numerous experiments indicate that the chromatin fiber displays salt-dependent conformations, the associated molecular mechanism remains unclear. Here, we apply an irregular Discrete Surface Charge Optimization (DiSCO) model of the nucleosome with all histone tails incorporated to describe by Monte Carlo simulations salt-dependent rearrangements of a nucleosomal array with 12 nucleosomes. The ensemble of nucleosomal array conformations display salt-dependent condensation in good agreement with hydrodynamic measurements and suggest that the array adopts highly irregular 3D zig-zag conformations at high (physiological) salt concentrations and transitions into the extended “beads-on-a-string” conformation at low salt. Energy analyses indicate that the repulsion among linker DNA leads to this extended form, whereas internucleosome attraction drives the folding at high salt. The balance between these two contributions determines the salt-dependent condensation. Importantly, the internucleosome and linker DNA–nucleosome attractions require histone tails; we find that the H3 tails, in particular, are crucial for stabilizing the moderately folded fiber at physiological monovalent salt. PMID:15919827

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

    PubMed

    Agarwal, Poonam; Miller, Kyle M

    2016-10-01

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

  20. Histone acetylation in heterochromatin assembly

    PubMed Central

    Kim, Jeong-Hoon; Workman, Jerry L.

    2010-01-01

    Histone acetylation is generally considered a mark involved in activating gene expression by making chromatin structures less compact. In the April 1, 2010, issue of Genes & Development, Xhemalce and Kouzarides (pp. 647–652) demonstrate that the acetylation of histone H3 at Lys 4 (H3K4) plays a role in the formation of repressive heterochromatin in Schizosaccharomyces pombe. H3K4 acetylation mediates a switch of chromodomain proteins associated with methylated H3K9 during heterochromatin assembly. PMID:20395362

  1. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae.

    PubMed

    Weinert, Brian T; Iesmantavicius, Vytautas; Moustafa, Tarek; Schölz, Christian; Wagner, Sebastian A; Magnes, Christoph; Zechner, Rudolf; Choudhary, Chunaram

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation accumulated in growth-arrested cells in a manner that depended on acetyl-CoA generation in distinct subcellular compartments. Mitochondrial acetylation levels correlated with acetyl-CoA concentration in vivo and acetyl-CoA acetylated lysine residues nonenzymatically in vitro. We developed a method to estimate acetylation stoichiometry and found that the vast majority of mitochondrial and cytoplasmic acetylation had a very low stoichiometry. However, mitochondrial acetylation occurred at a significantly higher basal level than cytoplasmic acetylation, consistent with the distinct acetylation dynamics and higher acetyl-CoA concentration in mitochondria. High stoichiometry acetylation occurred mostly on histones, proteins present in histone acetyltransferase and deacetylase complexes, and on transcription factors. These data show that a majority of acetylation occurs at very low levels in exponentially growing yeast and is uniformly affected by exposure to acetyl-CoA.

  2. Socioeconomic impacts of natural gas curtailments: a study of the textile industry in the southeastern United States. Final report

    SciTech Connect

    Jennings, D.M.

    1980-01-01

    A study was undertaken to identify the effects of fuel curtailments in the textile industry in North and South Carolina. Regional economic and social structures were affected with natural gas curtailments in 1976 and 1977. This document presents results of the effects of production shutdown resulting from the curtailments. Chapter II presents background information on the pipelines that service the region. Chapters III and IV describe the affected communities and the observed increase in government expenditures to counteract the impacts. Chapter V contains a complete list of textile plants in the study area that had to either work under abbreviated schedules or close entirely during the winter of 1976-1977. Attention was given to economic impacts at the industrial level that may have been attributable to the curtailment. Chapter VI covers these topics. In some instances, textile mills have relocated their plant facilities because they could not be guaranteed continuous fuel service at their original site. These data are the main concern of Chapter VII. Chapter VIII concentrates on social impacts; many facilities which provide services essential to human needs were subjected to gas curtailments so that the critical energy supplies could be diverted to industry. Chapter VIII also discusses an interesting geographic separation between social and economic impacts.

  3. Interaction of Transcriptional Regulators with Specific Nucleosomes Across the Saccharomyces Genome

    PubMed Central

    Koerber, R. Thomas; Rhee, Ho Sung; Jiang, Cizhong; Pugh, B. Franklin

    2009-01-01

    SUMMARY A canonical nucleosome architecture around promoters establishes the context in which proteins regulate gene expression. Whether gene regulatory proteins that interact with nucleosomes are selective for individual nucleosome positions across the genome is not known. Here we examine in Saccharomyces several protein-nucleosome interactions, including those that 1) bind histones (Bdf1/SWR1 and Srm1), 2) bind specific DNA sequences (Rap1 and Reb1), and 3) potentially collide with nucleosomes during transcription (RNA polymerase II). We find that the Bdf1/SWR1 complex forms a di-nucleosome complex that is selective for the +1 and +2 nucleosomes of active genes. Rap1 selectively binds to its cognate site on the rotationally exposed first and second helical turn of DNA inside either border of the −1 nucleosome, whereas Reb1 is selective for a single NFR-proximal border of the −1 nucleosome. We find that a transcribing RNA polymerase creates a delocalized state of resident nucleosomes. These findings suggest that nucleosomes around promoter regions have position-specific functions, and that some gene regulators have position-specific nucleosomal interactions. PMID:19782036

  4. Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response.

    PubMed

    Sexton, Brittany S; Druliner, Brooke R; Vera, Daniel L; Avey, Denis; Zhu, Fanxiu; Dennis, Jonathan H

    2016-02-09

    Nucleosome occupancy is critically important in regulating access to the eukaryotic genome. Few studies in human cells have measured genome-wide nucleosome distributions at high temporal resolution during a response to a common stimulus. We measured nucleosome distributions at high temporal resolution following Kaposi's-sarcoma-associated herpesvirus (KSHV) reactivation using our newly developed mTSS-seq technology, which maps nucleosome distribution at the transcription start sites (TSS) of all human genes. Nucleosomes underwent widespread changes in organization 24 hours after KSHV reactivation and returned to their basal nucleosomal architecture 48 hours after KSHV reactivation. The widespread changes consisted of an indiscriminate remodeling event resulting in the loss of nucleosome rotational phasing signals. Additionally, one in six TSSs in the human genome possessed nucleosomes that are translationally remodeled. 72% of the loci with translationally remodeled nucleosomes have nucleosomes that moved to positions encoded by the underlying DNA sequence. Finally we demonstrated that these widespread alterations in nucleosomal architecture potentiated regulatory factor binding. These descriptions of nucleosomal architecture changes provide a new framework for understanding the role of chromatin in the genomic response, and have allowed us to propose a hierarchical model for chromatin-based regulation of genome response.

  5. Widespread signatures of recent selection linked to nucleosome positioning in the human lineage.

    PubMed

    Prendergast, James G D; Semple, Colin A M

    2011-11-01

    In this study we investigated the strengths and modes of selection associated with nucleosome positioning in the human lineage through the comparison of interspecies and intraspecies rates of divergence. We identify significant evidence for both positive and negative selection linked to human nucleosome positioning for the first time, implicating a widespread and important role for DNA sequence in the location of well-positioned nucleosomes. Selection appears to be acting on particular base substitutions to maintain optimum GC compositions in core and linker regions, with, e.g., unexpectedly elevated rates of C→T substitutions during recent human evolution at linker regions 60-90 bp from the nucleosome dyad but significant depletion of the same substitutions within nucleosome core regions. These patterns are strikingly consistent with the known relationships between genomic sequence composition and nucleosome assembly. By stratifying nucleosomes according to the GC content of their genomic neighborhood, we also show that the strength and direction of selection detected is dictated by local GC content. Intriguingly these signatures of selection are not restricted to nucleosomes in close proximity to exons, suggesting the correct positioning of nucleosomes is not only important in and around coding regions. This analysis provides strong evidence that the genomic sequences associated with nucleosomes are not evolving neutrally, and suggests that underlying DNA sequence is an important factor in nucleosome positioning. Recent signatures of selection linked to genomic features as ubiquitous as the nucleosome have important implications for human genome evolution and disease.

  6. A Novel Wavelet-Based Approach for Predicting Nucleosome Positions Using DNA Structural Information.

    PubMed

    Gan, Yanglan; Zou, Guobing; Guan, Jihong; Xu, Guangwei

    2014-01-01

    Nucleosomes are basic elements of chromatin structure. The positioning of nucleosomes along a genome is very important to dictate eukaryotic DNA compaction and access. Current computational methods have focused on the analysis of nucleosome occupancy and the positioning of well-positioned nucleosomes. However, fuzzy nucleosomes require more complex configurations and are more difficult to predict their positions. We analyzed the positioning of well-positioned and fuzzy nucleosomes from a novel structural perspective, and proposed WaveNuc, a computational approach for inferring their positions based on continuous wavelet transformation. The comparative analysis demonstrates that these two kinds of nucleosomes exhibit different propeller twist structural characteristics. Well-positioned nucleosomes tend to locate at sharp peaks of the propeller twist profile, whereas fuzzy nucleosomes correspond to broader peaks. The sharpness of these peaks shows that the propeller twist profile may contain nucleosome positioning information. Exploiting this knowledge, we applied WaveNuc to detect the two different kinds of peaks of the propeller twist profile along the genome. We compared the performance of our method with existing methods on real data sets. The results show that the proposed method can accurately resolve complex configurations of fuzzy nucleosomes, which leads to better performance of nucleosome positioning prediction on the whole genome.

  7. Genome-wide profiling of nucleosome sensitivity and chromatin accessibility in Drosophila melanogaster.

    PubMed

    Chereji, Răzvan V; Kan, Tsung-Wai; Grudniewska, Magda K; Romashchenko, Alexander V; Berezikov, Eugene; Zhimulev, Igor F; Guryev, Victor; Morozov, Alexandre V; Moshkin, Yuri M

    2016-02-18

    Nucleosomal DNA is thought to be generally inaccessible to DNA-binding factors, such as micrococcal nuclease (MNase). Here, we digest Drosophila chromatin with high and low concentrations of MNase to reveal two distinct nucleosome types: MNase-sensitive and MNase-resistant. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C-containing dinucleotides, whereas MNase-sensitive nucleosomes form on A/T-rich sequences found at transcription start and termination sites, enhancers and DNase I hypersensitive sites. Estimates of nucleosome formation energies indicate that MNase-sensitive nucleosomes tend to be less stable than MNase-resistant ones. Strikingly, a decrease in cell growth temperature of about 10°C makes MNase-sensitive nucleosomes less accessible, suggesting that observed variations in MNase sensitivity are related to either thermal fluctuations of chromatin fibers or the activity of enzymatic machinery. In the vicinity of active genes and DNase I hypersensitive sites nucleosomes are organized into periodic arrays, likely due to 'phasing' off potential barriers formed by DNA-bound factors or by nucleosomes anchored to their positions through external interactions. The latter idea is substantiated by our biophysical model of nucleosome positioning and energetics, which predicts that nucleosomes immediately downstream of transcription start sites are anchored and recapitulates nucleosome phasing at active genes significantly better than sequence-dependent models.

  8. RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

    PubMed Central

    Khan, Dilshad H.; Gonzalez, Carolina; Cooper, Charlton; Sun, Jian-Min; Chen, Hou Yu; Healy, Shannon; Xu, Wayne; Smith, Karen T.; Workman, Jerry L.; Leygue, Etienne; Davie, James R.

    2014-01-01

    Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA. PMID:24234443

  9. Molecular basis for the autoregulation of the protein acetyl transferase Rtt109

    PubMed Central

    Stavropoulos, Pete; Nagy, Vivien; Blobel, Günter; Hoelz, André

    2008-01-01

    Rtt109 is a protein acetyltransferase (PAT) that is responsible for the acetylation of lysine-56 of histone 3 (H3K56) in yeast. H3K56 acetylation has been implicated in the weakening of the interaction between the histone core and the surrounding DNA in the nucleosomal particle. Rtt109, in cooperation with various histone chaperones, promotes genomic stability and is required for resistance to DNA damaging agents. Here, we present the crystal structure of Rtt109 in complex with acetyl-CoA at a 2.0-Å resolution. Rtt109 consists of a core PAT domain, which binds the acetyl-CoA cofactor. A second domain, the activation domain, is tightly associated with the PAT domain. Autoacetylation of lysine-290 within the activation domain is required for stabilizing the interaction between the two domains and is essential for catalysis. Biochemical analysis demonstrates the requirement of a loop within the PAT domain for the binding of the histone chaperone Vps75, and mutational analysis identifies key residues for catalysis. We propose a model in which the autoacetylation of Rtt109 is crucial for the regulation of its catalytic activity. PMID:18719104

  10. Histone acetylation characterizes chromatin presetting by NF1 and Oct1 and enhances glucocorticoid receptor binding to the MMTV promoter

    SciTech Connect

    Astrand, Carolina; Belikov, Sergey; Wrange, Orjan

    2009-09-10

    Transcription from the mouse mammary tumor virus (MMTV) promoter is induced by the glucocorticoid receptor (GR). This switch was reconstituted in Xenopus oocytes. Previously, we showed that Nuclear Factor 1 (NF1) and Octamer Transcription Factor 1 (Oct1) bind constitutively to the MMTV promoter and thereby induce translational nucleosome positioning representing an intermediary, i.e. preset, state of nucleosome organization. Here we further characterize this NF1 and Oct1 induced preset chromatin in relation to the inactive and the hormone-activated state. The preset chromatin exhibits increased histone acetylation but does not cause dissociation of histone H1 as oppose to the hormone-activated state. Furthermore, upon hormone induction the preset MMTV chromatin displays an enhanced and prolonged GR binding capacity and transcription during an intrinsic and time-dependent silencing of the injected template. The silencing process correlates with a reduced histone acetylation. However, a histone deacetylase inhibitor, trichostatin A (TSA), does not counteract silencing in spite of its distinct stimulation of GR-DNA binding. The latter indicates the importance of histone acetylation to maintain DNA access for inducible factor binding. We discuss how constitutively bound factors such as NF1 and Oct1 may participate in the maintenance of tissue specificity of hormone responsive genes.

  11. Rapid Histone-Catalyzed DNA Lesion Excision and Accompanying Protein Modification in Nucleosomes and Nucleosome Core Particles.

    PubMed

    Weng, Liwei; Greenberg, Marc M

    2015-09-02

    C5'-Hydrogen atoms are frequently abstracted during DNA oxidation. The oxidized abasic lesion 5'-(2-phosphoryl-1,4-dioxobutane) (DOB) is an electrophilic product of the C5'-radical. DOB is a potent irreversible inhibitor of DNA polymerase β, and forms interstrand cross-links in free DNA. We examined the reactivity of DOB within nucleosomes and nucleosome core particles (NCPs), the monomeric component of chromatin. Depending upon the position at which DOB is generated within a NCP, it is excised from nucleosomal DNA at a rate 275-1500-fold faster than that in free DNA. The half-life of DOB (7.0-16.8 min) in NCPs is shorter than any other abasic lesion. DOB's lifetime in NCPs is also significantly shorter than the estimated lifetime of an abasic site within a cell, suggesting that the observed chemistry would occur intracellularly. Histones also catalyze DOB excision when the lesion is present in the DNA linker region of a nucleosome. Schiff-base formation between DOB and histone proteins is detected in nucleosomes and NCPs, resulting in pyrrolone formation at the lysine residues. The lysines modified by DOB are often post-translationally modified. Consequently, the histone modifications described herein could affect the regulation of gene expression and may provide a chemical basis for the cytotoxicity of the DNA damaging agents that produce this lesion.

  12. Mechanism(s) of SWI/SNF-induced nucleosome mobilization.

    PubMed

    Liu, Ning; Balliano, Angela; Hayes, Jeffrey J

    2011-01-24

    Impediments to DNA access due to assembly of the eukaryotic genome into chromatin are in part overcome by the activity of ATP-dependent chromatin-remodeling complexes. These complexes employ energy derived from ATP hydrolysis to destabilize histone-DNA interactions and alter nucleosome positions, thereby increasing the accessibility of DNA-binding factors to their targets. However, the mechanism by which theses complexes accomplish this task remains unresolved. We review aspects of nucleosome alteration by the SWI/SNF complex, the archetypal remodeling enzyme. We focus on experiments that provide insights into how SWI/SNF induces nucleosome movement along DNA. Numerous biochemical activities have been characterized for this complex, all likely providing clues as to the molecular mechanism of translocation.

  13. Twist Neutrality and the Diameter of the Nucleosome Core Particle

    NASA Astrophysics Data System (ADS)

    Bohr, Jakob; Olsen, Kasper

    2012-03-01

    The diameter of the nucleosome core particle is the same for all the eukaryotes. Here we discuss the possibility that this selectiveness is consistent with a propensity for twist neutrality, in particular, for the double helical DNA to stay rotationally neutral when strained. Reorganization of DNA cannot be done without some level of temporal tensile stress, and as a consequence chiral molecules, such as helices, will twist under strain. The requirement that the nucleosome, constituting the nucleosome core particle and linker DNA, has a vanishing strain-twist coupling leads to a requirement for the amount of bending. For the diameter of the coiled DNA we obtain the relatively accurate numerical estimate of 2R=82Å.

  14. Nucleosomes and neutrophil activation in sickle cell disease painful crisis.

    PubMed

    Schimmel, Marein; Nur, Erfan; Biemond, Bart J; van Mierlo, Gerard J; Solati, Shabnam; Brandjes, Dees P; Otten, Hans-Martin; Schnog, John-John; Zeerleder, Sacha

    2013-11-01

    Activated polymorphonuclear neutrophils play an important role in the pathogenesis of vaso-occlusive painful sickle cell crisis. Upon activation, polymorphonuclear neutrophils can form neutrophil extracellular traps. Neutrophil extracellular traps consist of a meshwork of extracellular DNA, nucleosomes, histones and neutrophil proteases. Neutrophil extracellular traps have been demonstrated to be toxic to endothelial and parenchymal cells. This prospective cohort study was conducted to determine neutrophil extracellular trap formation in sickle cell patients during steady state and painful crisis. As a measure of neutrophil extracellular traps, plasma nucleosomes levels were determined and polymorphonuclear neutrophil activation was assessed measuring plasma levels of elastase-α1-antitrypsin complexes in 74 patients in steady state, 70 patients during painful crisis, and 24 race-matched controls using Enzyme Linked Immunosorbent Assay. Nucleosome levels in steady state sickle cell patients were significantly higher than levels in controls. During painful crisis levels of both nucleosomes and elastase-α1-antitrypsin complexes increased significantly. Levels of nucleosomes correlated significantly to elastase-α1-antitrypsin complex levels during painful crisis, (Sr = 0.654, P<0.001). This was seen in both HbSS/HbSβ(0)-thalassemia (Sr=0.55, P<0.001) and HbSC/HbSβ(+-)thalassemia patients (Sr=0.90, P<0.001) during painful crisis. Levels of nucleosomes showed a correlation with length of hospital stay and were highest in patients with acute chest syndrome. These data support the concept that neutrophil extracellular trap formation and neutrophil activation may play a role in the pathogenesis of painful sickle cell crisis and acute chest syndrome.

  15. Hierarchical looping of zigzag nucleosome chains in metaphase chromosomes

    PubMed Central

    Grigoryev, Sergei A.; Bascom, Gavin; Buckwalter, Jenna M.; Schubert, Michael B.; Woodcock, Christopher L.; Schlick, Tamar

    2016-01-01

    The architecture of higher-order chromatin in eukaryotic cell nuclei is largely unknown. Here, we use electron microscopy-assisted nucleosome interaction capture (EMANIC) cross-linking experiments in combination with mesoscale chromatin modeling of 96-nucleosome arrays to investigate the internal organization of condensed chromatin in interphase cell nuclei and metaphase chromosomes at nucleosomal resolution. The combined data suggest a novel hierarchical looping model for chromatin higher-order folding, similar to rope flaking used in mountain climbing and rappelling. Not only does such packing help to avoid tangling and self-crossing, it also facilitates rope unraveling. Hierarchical looping is characterized by an increased frequency of higher-order internucleosome contacts for metaphase chromosomes compared with chromatin fibers in vitro and interphase chromatin, with preservation of a dominant two-start zigzag organization associated with the 30-nm fiber. Moreover, the strong dependence of looping on linker histone concentration suggests a hierarchical self-association mechanism of relaxed nucleosome zigzag chains rather than longitudinal compaction as seen in 30-nm fibers. Specifically, concentrations lower than one linker histone per nucleosome promote self-associations and formation of these looped networks of zigzag fibers. The combined experimental and modeling evidence for condensed metaphase chromatin as hierarchical loops and bundles of relaxed zigzag nucleosomal chains rather than randomly coiled threads or straight and stiff helical fibers reconciles aspects of other models for higher-order chromatin structure; it constitutes not only an efficient storage form for the genomic material, consistent with other genome-wide chromosome conformation studies that emphasize looping, but also a convenient organization for local DNA unraveling and genome access. PMID:26787893

  16. Nonuniform distribution of excision repair synthesis in nucleosome core DNA

    SciTech Connect

    Lan, S.Y.; Smerdon, M.J.

    1985-12-17

    We have studied the distribution in nucleosome core DNA of nucleotides incorporated by excision repair synthesis occurring immediately after UV irradiation in human cells. The differences previously observed for whole nuclei between the DNase I digestion profiles of repaired DNA (following its refolding into a nucleosome structure) and bulk DNA are obtained for isolated nucleosome core particles. Analysis of the differences obtained indicates that they could reflect a significant difference in the level of repair-incorporated nucleotides at different sites within the core DNA region. To test this possibility directly, we have used exonuclease III digestion of very homogeneous sized core particle DNA to map the distribution of repair synthesis in these regions. Results indicate that in a significant fraction of the nucleosomes the 5' and 3' ends of the core DNA are markedly enhanced in repair-incorporated nucleotides relative to the central region of the core particle. A best fit analysis indicates that a good approximation of the data is obtained for a distribution where the core DNA is uniformly labeled from the 5' end to position 62 and from position 114 to the 3' end, with the 52-base central region being devoid of repair-incorporated nucleotides. This distribution accounts for all of the quantitative differences observed previously between repaired DNA and bulk DNA following the rapid phase of nucleosome rearrangement when it is assumed that linker DNA and the core DNA ends are repaired with equal efficiency and the nucleosome structure of newly repaired DNA is identical with that of bulk chromatin. The 52-base central region that is devoid of repair synthesis contains the lowest frequency cutting sites for DNase I in vitro, as well as the only internal locations where two (rather than one) histones interact with a 10-base segment of each DNA strand.

  17. Hierarchical looping of zigzag nucleosome chains in metaphase chromosomes.

    PubMed

    Grigoryev, Sergei A; Bascom, Gavin; Buckwalter, Jenna M; Schubert, Michael B; Woodcock, Christopher L; Schlick, Tamar

    2016-02-02

    The architecture of higher-order chromatin in eukaryotic cell nuclei is largely unknown. Here, we use electron microscopy-assisted nucleosome interaction capture (EMANIC) cross-linking experiments in combination with mesoscale chromatin modeling of 96-nucleosome arrays to investigate the internal organization of condensed chromatin in interphase cell nuclei and metaphase chromosomes at nucleosomal resolution. The combined data suggest a novel hierarchical looping model for chromatin higher-order folding, similar to rope flaking used in mountain climbing and rappelling. Not only does such packing help to avoid tangling and self-crossing, it also facilitates rope unraveling. Hierarchical looping is characterized by an increased frequency of higher-order internucleosome contacts for metaphase chromosomes compared with chromatin fibers in vitro and interphase chromatin, with preservation of a dominant two-start zigzag organization associated with the 30-nm fiber. Moreover, the strong dependence of looping on linker histone concentration suggests a hierarchical self-association mechanism of relaxed nucleosome zigzag chains rather than longitudinal compaction as seen in 30-nm fibers. Specifically, concentrations lower than one linker histone per nucleosome promote self-associations and formation of these looped networks of zigzag fibers. The combined experimental and modeling evidence for condensed metaphase chromatin as hierarchical loops and bundles of relaxed zigzag nucleosomal chains rather than randomly coiled threads or straight and stiff helical fibers reconciles aspects of other models for higher-order chromatin structure; it constitutes not only an efficient storage form for the genomic material, consistent with other genome-wide chromosome conformation studies that emphasize looping, but also a convenient organization for local DNA unraveling and genome access.

  18. Nucleosome remodelers in double-strand break repair.

    PubMed

    Seeber, Andrew; Hauer, Michael; Gasser, Susan M

    2013-04-01

    ATP-dependent nucleosome remodelers use ATP hydrolysis to shift, evict and exchange histone dimers or octamers and have well-established roles in transcription. Earlier work has suggested a role for nucleosome remodelers such as INO80 in double-strand break (DSB) repair. This review will begin with an update on recent studies that explore how remodelers are recruited to DSBs. We then examine their impact on various steps of repair, focusing on resection and the formation of the Rad51-ssDNA nucleofilament. Finally, we will explore new studies that implicate remodelers in the physical movement of chromatin in response to damage.

  19. Mapping post-translational modifications of mammalian testicular specific histone variant TH2B in tetraploid and haploid germ cells and their implications on the dynamics of nucleosome structure.

    PubMed

    Pentakota, Satya Krishna; Sandhya, Sankaran; P Sikarwar, Arun; Chandra, Nagasuma; Satyanarayana Rao, Manchanahalli R

    2014-12-05

    Histones regulate a variety of chromatin templated events by their post-translational modifications (PTMs). Although there are extensive reports on the PTMs of canonical histones, the information on the histone variants remains very scanty. Here, we report the identification of different PTMs, such as acetylation, methylation, and phosphorylation of a major mammalian histone variant TH2B. Our mass spectrometric analysis has led to the identification of both conserved and unique modifications across tetraploid spermatocytes and haploid spermatids. We have also computationally derived the 3-dimensional model of a TH2B containing nucleosome in order to study the spatial orientation of the PTMs identified and their effect on nucleosome stability and DNA binding potential. From our nucleosome model, it is evident that substitution of specific amino acid residues in TH2B results in both differential histone-DNA and histone-histone contacts. Furthermore, we have also observed that acetylation on the N-terminal tail of TH2B weakens the interactions with the DNA. These results provide direct evidence that, similar to somatic H2B, the testis specific histone TH2B also undergoes multiple PTMs, suggesting the possibility of chromatin regulation by such covalent modifications in mammalian male germ cells.

  20. Phosphorylated Pol II CTD recruits multiple HDACs, including Rpd3C(S), for methylation-dependent deacetylation of ORF nucleosomes

    PubMed Central

    Govind, Chhabi K.; Qiu, Hongfang; Ginsburg, Daniel S.; Ruan, Chun; Hofmeyer, Kimberly; Hu, Cuihua; Swaminathan, Venkatesh; Workman, Jerry L.; Li, Bing; Hinnebusch, Alan G.

    2010-01-01

    Methylation of histone H3 by Set1 and Set2 is required for deacetylation of nucleosomes in coding regions by histone deacetylase complexes (HDACs) Set3C and Rpd3C(S), respectively. We report that Set3C and Rpd3C(S) are co-transcriptionally recruited in the absence of Set1 and Set2, but in a manner stimulated by Pol II CTD kinase Cdk7/Kin28. Consistently, Rpd3C(S) and Set3C interact with Ser5-phosphorylated Pol II and histones in extracts, but only the histone interactions require H3 methylation. Moreover, reconstituted Rpd3C(S) binds specifically to Ser5-phosphorylated CTD peptides in vitro. Hence, whereas interaction with methylated H3 residues is required for Rpd3C(S) and Set3C deacetylation activities, their co-transcriptional recruitment is stimulated by the phosphorylated CTD. We further demonstrate that Rpd3, Hos2, and Hda1 have overlapping functions in deacetylating histones and suppressing co-transcriptional histone eviction. A strong correlation between increased acetylation and lower histone occupancy in HDA mutants implies that histone acetylation is a key determinant of nucleosome eviction. PMID:20670892

  1. Phosphorylated Pol II CTD recruits multiple HDACs, including Rpd3C(S), for methylation-dependent deacetylation of ORF nucleosomes.

    PubMed

    Govind, Chhabi K; Qiu, Hongfang; Ginsburg, Daniel S; Ruan, Chun; Hofmeyer, Kimberly; Hu, Cuihua; Swaminathan, Venkatesh; Workman, Jerry L; Li, Bing; Hinnebusch, Alan G

    2010-07-30

    Methylation of histone H3 by Set1 and Set2 is required for deacetylation of nucleosomes in coding regions by histone deacetylase complexes (HDACs) Set3C and Rpd3C(S), respectively. We report that Set3C and Rpd3C(S) are cotranscriptionally recruited in the absence of Set1 and Set2, but in a manner stimulated by Pol II CTD kinase Cdk7/Kin28. Consistently, Rpd3C(S) and Set3C interact with Ser5-phosphorylated Pol II and histones in extracts, but only the histone interactions require H3 methylation. Moreover, reconstituted Rpd3C(S) binds specifically to Ser5-phosphorylated CTD peptides in vitro. Hence, whereas interaction with methylated H3 residues is required for Rpd3C(S) and Set3C deacetylation activities, their cotranscriptional recruitment is stimulated by the phosphorylated CTD. We further demonstrate that Rpd3, Hos2, and Hda1 have overlapping functions in deacetylating histones and suppressing cotranscriptional histone eviction. A strong correlation between increased acetylation and lower histone occupancy in HDA mutants implies that histone acetylation is important for nucleosome eviction. Copyright 2010 Elsevier Inc. All rights reserved.

  2. The chromatin-binding protein HMGN3 stimulates histone acetylation and transcription across the Glyt1 gene

    PubMed Central

    Barkess, Gráinne; Postnikov, Yuri; Campos, Chrisanne D.; Mishra, Shivam; Mohan, Gokula; Verma, Sakshi; Bustin, Michael; West, Katherine L.

    2013-01-01

    HMGNs are nucleosome-binding proteins that alter the pattern of histone modifications and modulate the binding of linker histones to chromatin. The HMGN3 family member exists as two splice forms, HMGN3a which is full-length and HMGN3b which lacks the C-terminal RD (regulatory domain). In the present study, we have used the Glyt1 (glycine transporter 1) gene as a model system to investigate where HMGN proteins are bound across the locus in vivo, and to study how the two HMGN3 splice variants affect histone modifications and gene expression. We demonstrate that HMGN1, HMGN2, HMGN3a and HMGN3b are bound across the Glyt1 gene locus and surrounding regions, and are not enriched more highly at the promoter or putative enhancer. We conclude that the peaks of H3K4me3 (trimethylated Lys4 of histone H3) and H3K9ac (acetylated Lys9 of histone H3) at the active Glyt1a promoter do not play a major role in recruiting HMGN proteins. HMGN3a/b binding leads to increased H3K14 (Lys14 of histone H3) acetylation and stimulates Glyt1a expression, but does not alter the levels of H3K4me3 or H3K9ac enrichment. Acetylation assays show that HMGN3a stimulates the ability of PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] to acetylate nucleosomal H3 in vitro, whereas HMGN3b does not. We propose a model where HMGN3a/b-stimulated H3K14 acetylation across the bodies of large genes such as Glyt1 can lead to more efficient transcription elongation and increased mRNA production. PMID:22150271

  3. The effects of histone H4 tail acetylations on cation-induced chromatin folding and self-association

    PubMed Central

    Allahverdi, Abdollah; Yang, Renliang; Korolev, Nikolay; Fan, Yanping; Davey, Curt A.; Liu, Chuan-Fa; Nordenskiöld, Lars

    2011-01-01

    Understanding the molecular mechanisms behind regulation of chromatin folding through covalent modifications of the histone N-terminal tails is hampered by a lack of accessible chromatin containing precisely modified histones. We study the internal folding and intermolecular self-association of a chromatin system consisting of saturated 12-mer nucleosome arrays containing various combinations of completely acetylated lysines at positions 5, 8, 12 and 16 of histone H4, induced by the cations Na+, K+, Mg2+, Ca2+, cobalt-hexammine3+, spermidine3+ and spermine4+. Histones were prepared using a novel semi-synthetic approach with native chemical ligation. Acetylation of H4-K16, but not its glutamine mutation, drastically reduces cation-induced folding of the array. Neither acetylations nor mutations of all the sites K5, K8 and K12 can induce a similar degree of array unfolding. The ubiquitous K+, (as well as Rb+ and Cs+) showed an unfolding effect on unmodified arrays almost similar to that of H4-K16 acetylation. We propose that K+ (and Rb+/Cs+) binding to a site on the H2B histone (R96-L99) disrupts H4K16 ε-amino group binding to this specific site, thereby deranging H4 tail-mediated nucleosome–nucleosome stacking and that a similar mechanism operates in the case of H4-K16 acetylation. Inter-array self-association follows electrostatic behavior and is largely insensitive to the position or nature of the H4 tail charge modification. PMID:21047799

  4. Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM.

    PubMed

    Rodriguez, Jairo; McKnight, Jeffrey N; Tsukiyama, Toshio

    2014-10-01

    Because histones bind DNA very tightly, the location on DNA and the level of occupancy of a given DNA sequence by nucleosomes can profoundly affect accessibility of non-histone proteins to chromatin, affecting virtually all DNA-dependent processes, such as transcription, DNA repair, DNA replication and recombination. Therefore, it is often necessary to determine positions and occupancy of nucleosomes to understand how DNA-dependent processes are regulated. Recent technological advances made such analyses feasible on a genome-wide scale at high resolution. In addition, we have recently developed a method to measure nuclease accessibility of nucleosomes on a global scale. This unit describes methods to map nucleosome positions, to determine nucleosome density, and to determine nuclease accessibility of nucleosomes using deep sequencing.

  5. Positioning of a nucleosome on mouse satellite DNA inserted into a yeast plasmid is determined by its DNA sequence and an adjacent nucleosome.

    PubMed

    Kiryanov, G I; Kintsurashvili, L N; Isaeva, L V; Zakharova, M G

    2004-09-01

    It has earlier been shown that multiple positioning of nucleosomes on mouse satellite DNA is determined by its nucleotide sequence. To clarify whether other factors, such as boundary ones, can affect the positionings, we modified the environment of satellite DNA monomer by inserting it into a yeast plasmid between inducible GalCyc promoter and a structural region of the yeast FLP gene. We have revealed that the positions of nucleosomes on satellite DNA are identical to those detected upon reconstruction in vitro. The positioning signal (GAAAAA sequence) of satellite DNA governs nucleosome location at the adjacent nucleotide sequence as well. Upon promoter induction the nucleosome, translationally positioned on the GalCyc promoter, transfers to the satellite DNA and its location follows the positioning signal of the latter. Thus, the alternatives of positioning of a nucleosome on satellite DNA are controlled by its nucleotide sequence, though the choice of one of them is determined by the adjacent nucleosome.

  6. Myeloid Growth Factors Promote Resistance to Mycobacterial Infection by Curtailing Granuloma Necrosis through Macrophage Replenishment

    PubMed Central

    Pagán, Antonio J.; Yang, Chao-Tsung; Cameron, James; Swaim, Laura E.; Ellett, Felix; Lieschke, Graham J.; Ramakrishnan, Lalita

    2015-01-01

    Summary The mycobacterial ESX-1 virulence locus accelerates macrophage recruitment to the forming tuberculous granuloma. Newly recruited macrophages phagocytose previously infected apoptotic macrophages to become new bacterial growth niches. Granuloma macrophages can then necrose, releasing mycobacteria into the extracellular milieu, which potentiates their growth even further. Using zebrafish with genetic or pharmacologically induced macrophage deficiencies, we find that global macrophage deficits increase susceptibility to mycobacterial infection by accelerating granuloma necrosis. This is because reduction in the macrophage supply below a critical threshold decreases granuloma macrophage replenishment to the point where apoptotic infected macrophages, failing to get engulfed, necrose. Reducing macrophage demand by removing bacterial ESX-1 offsets the susceptibility of macrophage deficits. Conversely, increasing macrophage supply in wild-type fish by overexpressing myeloid growth factors induces resistance by curtailing necrosis. These findings may explain the susceptibility of humans with mononuclear cytopenias to mycobacterial infections and highlight the therapeutic potential of myeloid growth factors in tuberculosis. PMID:26159717

  7. Use of a geomembrane steel sheet pile verticle barrier to curtail organic seepage

    SciTech Connect

    Guglielmetti, J.L.; Butler, P.B.

    1997-12-31

    At a Superfund site in Delaware, contaminated groundwater, seeping out of a riverbank, produced a visible sheen on the river. As part of an emergency response action, a geomembrane steel sheet pile vertical barrier system was installed to contain the sheen and contaminated soil and sediments. The response action presented an engineering challenge due to the close proximity manufacturing facilities, steep riverbank slopes, tidal fluctuations, high velocity river flow, and underground and overhead interferences. A unique vertical containment barrier was developed to stabilize the riverbank slope, curtail sheens on the river, and prevent groundwater mounding behind the vertical barrier. In addition, the cost-effective vertical barrier enables natural chemical and biological processes to contain the organic seepage without requiring a groundwater extraction system.

  8. Myeloid Growth Factors Promote Resistance to Mycobacterial Infection by Curtailing Granuloma Necrosis through Macrophage Replenishment.

    PubMed

    Pagán, Antonio J; Yang, Chao-Tsung; Cameron, James; Swaim, Laura E; Ellett, Felix; Lieschke, Graham J; Ramakrishnan, Lalita

    2015-07-08

    The mycobacterial ESX-1 virulence locus accelerates macrophage recruitment to the forming tuberculous granuloma. Newly recruited macrophages phagocytose previously infected apoptotic macrophages to become new bacterial growth niches. Granuloma macrophages can then necrose, releasing mycobacteria into the extracellular milieu, which potentiates their growth even further. Using zebrafish with genetic or pharmacologically induced macrophage deficiencies, we find that global macrophage deficits increase susceptibility to mycobacterial infection by accelerating granuloma necrosis. This is because reduction in the macrophage supply below a critical threshold decreases granuloma macrophage replenishment to the point where apoptotic infected macrophages, failing to get engulfed, necrose. Reducing macrophage demand by removing bacterial ESX-1 offsets the susceptibility of macrophage deficits. Conversely, increasing macrophage supply in wild-type fish by overexpressing myeloid growth factors induces resistance by curtailing necrosis. These findings may explain the susceptibility of humans with mononuclear cytopenias to mycobacterial infections and highlight the therapeutic potential of myeloid growth factors in tuberculosis.

  9. Fish oil curtails the human action potential dome in a heterogeneous manner: implication for arrhythmogenesis.

    PubMed

    Verkerk, Arie O; den Ruijter, Hester M; de Jonge, Nicolaas; Coronel, Ruben

    2009-02-06

    Omega-3 polyunsaturated fatty acids (omega 3-PUFAs) from fish oil modulate various ion channels, including the L-type calcium current (I(Ca,L)). As a result, fish oil shortens the cardiac action potential and may cause a loss of the dome of the action potential (AP). Under conditions of increased preexisting heterogeneity in repolarization this may aggravate dispersion in action potential duration. We isolated ventricular myocytes of explanted hearts from patients with cardiomyopathy at the time of cardiac transplantation, and characterized spike-and-dome morphology in the presence of acutely administered fish oil. Fish oil omega 3-PUFA eicosapentaenoic acid (EPA), but not the control omega 9-PUFA oleic acid (OA), curtails the AP-dome in a heterogeneous manner and may even result in loss of the AP-dome in some but not all myocytes.

  10. From the Cover: Mechanical disruption of individual nucleosomes reveals a reversible multistage release of DNA

    NASA Astrophysics Data System (ADS)

    Brower-Toland, Brent D.; Smith, Corey L.; Yeh, Richard C.; Lis, John T.; Peterson, Craig L.; Wang, Michelle D.

    2002-02-01

    The dynamic structure of individual nucleosomes was examined by stretching nucleosomal arrays with a feedback-enhanced optical trap. Forced disassembly of each nucleosome occurred in three stages. Analysis of the data using a simple worm-like chain model yields 76 bp of DNA released from the histone core at low stretching force. Subsequently, 80 bp are released at higher forces in two stages: full extension of DNA with histones bound, followed by detachment of histones. When arrays were relaxed before the dissociated state was reached, nucleosomes were able to reassemble and to repeat the disassembly process. The kinetic parameters for nucleosome disassembly also have been determined.

  11. The relationship between periodic dinucleotides and the nucleosomal DNA deformation revealed by normal mode analysis

    NASA Astrophysics Data System (ADS)

    Wang, Debby D.; Yan, Hong

    2011-12-01

    Nucleosomes, which contain DNA and proteins, are the basic unit of eukaryotic chromatins. Polymers such as DNA and proteins are dynamic, and their conformational changes can lead to functional changes. Periodic dinucleotide patterns exist in nucleosomal DNA chains and play an important role in the nucleosome structure. In this paper, we use normal mode analysis to detect significant structural deformations of nucleosomal DNA and investigate the relationship between periodic dinucleotides and DNA motions. We have found that periodic dinucleotides are usually located at the peaks or valleys of DNA and protein motions, revealing that they dominate the nucleosome dynamics. Also, a specific dinucleotide pattern CA/TG appears most frequently.

  12. A map of nucleosome positions in yeast at base-pair resolution.

    PubMed

    Brogaard, Kristin; Xi, Liqun; Wang, Ji-Ping; Widom, Jonathan

    2012-06-28

    The exact positions of nucleosomes along genomic DNA can influence many aspects of chromosome function. However, existing methods for mapping nucleosomes do not provide the necessary single-base-pair accuracy to determine these positions. Here we develop and apply a new approach for direct mapping of nucleosome centres on the basis of chemical modification of engineered histones. The resulting map locates nucleosome positions genome-wide in unprecedented detail and accuracy. It shows new aspects of the in vivo nucleosome organization that are linked to transcription factor binding, RNA polymerase pausing and the higher-order structure of the chromatin fibre.

  13. Water-Related Power Plant Curtailments: An Overview of Incidents and Contributing Factors

    SciTech Connect

    McCall, James; Macknick, Jordan; Macknick, Jordan

    2016-12-01

    Water temperatures and water availability can affect the reliable operations of power plants in the United States. Data on water-related impacts on the energy sector are not consolidated and are reported by multiple agencies. This study provides an overview of historical incidents where water resources have affected power plant operations, discusses the various data sources providing information, and creates a publicly available and open access database that contains consolidated information about water-related power plant curtailment and shut down incidents. Power plants can be affected by water resources if incoming water temperatures are too high, water discharge temperatures are too high, or if there is not enough water available to operate. Changes in climate have the potential to exacerbate uncertainty over water resource availability and temperature. Power plant impacts from water resources include curtailment of generation, plant shut downs, and requests for regulatory variances. In addition, many power plants have developed adaptation approaches to reducing the potential risks of water-related issues by investing in new technologies or developing and implementing plans to undertake during droughts or heatwaves. This study identifies 42 incidents of water-related power plant issues from 2000-2015, drawing from a variety of different datasets. These incidents occur throughout the U.S. and affect coal and nuclear plants that use once-through, recirculating, and pond cooling systems. In addition, water temperature violations reported to the Environmental Protection Agency are also considered, with 35 temperature violations noted from 2012-2015. In addition to providing some background information on incidents, this effort has also created an open access database on the Open Energy Information platform that contains information about water-related power plant issues that can be updated by users.

  14. Coupling between Histone Conformations and DNA Geometry in Nucleosomes on a Microsecond Timescale: Atomistic Insights into Nucleosome Functions.

    PubMed

    Shaytan, Alexey K; Armeev, Grigoriy A; Goncearenco, Alexander; Zhurkin, Victor B; Landsman, David; Panchenko, Anna R

    2016-01-16

    An octamer of histone proteins wraps about 200bp of DNA into two superhelical turns to form nucleosomes found in chromatin. Although the static structure of the nucleosomal core particle has been solved, details of the dynamic interactions between histones and DNA remain elusive. We performed extensively long unconstrained, all-atom microsecond molecular dynamics simulations of nucleosomes including linker DNA segments and full-length histones in explicit solvent. For the first time, we were able to identify and characterize the rearrangements in nucleosomes on a microsecond timescale including the coupling between the conformation of the histone tails and the DNA geometry. We found that certain histone tail conformations promoted DNA bulging near its entry/exit sites, resulting in the formation of twist defects within the DNA. This led to a reorganization of histone-DNA interactions, suggestive of the formation of initial nucleosome sliding intermediates. We characterized the dynamics of the histone tails upon their condensation on the core and linker DNA and showed that tails may adopt conformationally constrained positions due to the insertion of "anchoring" lysines and arginines into the DNA minor grooves. Potentially, these phenomena affect the accessibility of post-translationally modified histone residues that serve as important sites for epigenetic marks (e.g., at H3K9, H3K27, H4K16), suggesting that interactions of the histone tails with the core and linker DNA modulate the processes of histone tail modifications and binding of the effector proteins. We discuss the implications of the observed results on the nucleosome function and compare our results to different experimental studies.

  15. A computational approach to map nucleosome positions and alternative chromatin states with base pair resolution

    PubMed Central

    Zhou, Xu; Blocker, Alexander W; Airoldi, Edoardo M; O'Shea, Erin K

    2016-01-01

    Understanding chromatin function requires knowing the precise location of nucleosomes. MNase-seq methods have been widely applied to characterize nucleosome organization in vivo, but generally lack the accuracy to determine the precise nucleosome positions. Here we develop a computational approach leveraging digestion variability to determine nucleosome positions at a base-pair resolution from MNase-seq data. We generate a variability template as a simple error model for how MNase digestion affects the mapping of individual nucleosomes. Applied to both yeast and human cells, this analysis reveals that alternatively positioned nucleosomes are prevalent and create significant heterogeneity in a cell population. We show that the periodic occurrences of dinucleotide sequences relative to nucleosome dyads can be directly determined from genome-wide nucleosome positions from MNase-seq. Alternatively positioned nucleosomes near transcription start sites likely represent different states of promoter nucleosomes during transcription initiation. Our method can be applied to map nucleosome positions in diverse organisms at base-pair resolution. DOI: http://dx.doi.org/10.7554/eLife.16970.001 PMID:27623011

  16. Nucleosome distribution and linker DNA: connecting nuclear function to dynamic chromatin structure.

    PubMed

    Szerlong, Heather J; Hansen, Jeffrey C

    2011-02-01

    Genetic information in eukaryotes is managed by strategic hierarchical organization of chromatin structure. Primary chromatin structure describes an unfolded nucleosomal array, often referred to as "beads on a string". Chromatin is compacted by the nonlinear rearrangement of nucleosomes to form stable secondary chromatin structures. Chromatin conformational transitions between primary and secondary structures are mediated by both nucleosome-stacking interactions and the intervening linker DNA. Chromatin model system studies find that the topography of secondary structures is sensitive to the spacing of nucleosomes within an array. Understanding the relationship between nucleosome spacing and higher order chromatin structure will likely yield important insights into the dynamic nature of secondary chromatin structure as it occurs in vivo. Genome-wide nucleosome mapping studies find the distance between nucleosomes varies, and regions of uniformly spaced nucleosomes are often interrupted by regions of nonuniform spacing. This type of organization is found at a subset of actively transcribed genes in which a nucleosome-depleted region near the transcription start site is directly adjacent to uniformly spaced nucleosomes in the coding region. Here, we evaluate secondary chromatin structure and discuss the structural and functional implications of variable nucleosome distributions in different organisms and at gene regulatory junctions.

  17. Structure and Scm3-mediated assembly of budding yeast centromeric nucleosomes.

    PubMed

    Dechassa, Mekonnen Lemma; Wyns, Katharina; Li, Ming; Hall, Michael A; Wang, Michelle D; Luger, Karolin

    2011-01-01

    Much controversy exists regarding the structural organization of the yeast centromeric nucleosome and the role of the nonhistone protein, Scm3, in its assembly and architecture. Here we show that the substitution of H3 with its centromeric variant Cse4 results in octameric nucleosomes that organize DNA in a left-handed superhelix. We demonstrate by single-molecule approaches, micrococcal nuclease digestion and small-angle X-ray scattering that Cse4-nucleosomes exhibit an open conformation with weakly bound terminal DNA segments. The Cse4-octamer does not preferentially form nucleosomes on its cognate centromeric DNA. We show that Scm3 functions as a Cse4-specific nucleosome assembly factor, and that the resulting octameric nucleosomes do not contain Scm3 as a stably bound component. Taken together, our data provide insights into the assembly and structural features of the budding yeast centromeric nucleosome.

  18. Nucleosome Presence at AML-1 Binding Sites Inversely Correlates with Ly49 Expression: Revelations from an Informatics Analysis of Nucleosomes and Immune Cell Transcription Factors.

    PubMed

    Wight, Andrew; Yang, Doo; Ioshikhes, Ilya; Makrigiannis, Andrew P

    2016-04-01

    Beyond its role in genomic organization and compaction, the nucleosome is believed to participate in the regulation of gene transcription. Here, we report a computational method to evaluate the nucleosome sensitivity for a transcription factor over a given stretch of the genome. Sensitive factors are predicted to be those with binding sites preferentially contained within nucleosome boundaries and lacking 10 bp periodicity. Based on these criteria, the Acute Myeloid Leukemia-1a (AML-1a) transcription factor, a regulator of immune gene expression, was identified as potentially sensitive to nucleosomal regulation within the mouse Ly49 gene family. This result was confirmed in RMA, a cell line with natural expression of Ly49, using MNase-Seq to generate a nucleosome map of chromosome 6, where the Ly49 gene family is located. Analysis of this map revealed a specific depletion of nucleosomes at AML-1a binding sites in the expressed Ly49A when compared to the other, silent Ly49 genes. Our data suggest that nucleosome-based regulation contributes to the expression of Ly49 genes, and we propose that this method of predicting nucleosome sensitivity could aid in dissecting the regulatory role of nucleosomes in general.

  19. Genetic modifiers of chromatin acetylation antagonize the reprogramming of epi-polymorphisms.

    PubMed

    Abraham, Anne-Laure; Nagarajan, Muniyandi; Veyrieras, Jean-Baptiste; Bottin, Hélène; Steinmetz, Lars M; Yvert, Gaël

    2012-09-01

    Natural populations are known to differ not only in DNA but also in their chromatin-associated epigenetic marks. When such inter-individual epigenomic differences (or "epi-polymorphisms") are observed, their stability is usually not known: they may or may not be reprogrammed over time or upon environmental changes. In addition, their origin may be purely epigenetic, or they may result from regulatory variation encoded in the DNA. Studying epi-polymorphisms requires, therefore, an assessment of their nature and stability. Here we estimate the stability of yeast epi-polymorphisms of chromatin acetylation, and we provide a genome-by-epigenome map of their genetic control. A transient epi-drug treatment was able to reprogram acetylation variation at more than one thousand nucleosomes, whereas a similar amount of variation persisted, distinguishing "labile" from "persistent" epi-polymorphisms. Hundreds of genetic loci underlied acetylation variation at 2,418 nucleosomes either locally (in cis) or distantly (in trans), and this genetic control overlapped only partially with the genetic control of gene expression. Trans-acting regulators were not necessarily associated with genes coding for chromatin modifying enzymes. Strikingly, "labile" and "persistent" epi-polymorphisms were associated with poor and strong genetic control, respectively, showing that genetic modifiers contribute to persistence. These results estimate the amount of natural epigenomic variation that can be lost after transient environmental exposures, and they reveal the complex genetic architecture of the DNA-encoded determinism of chromatin epi-polymorphisms. Our observations provide a basis for the development of population epigenetics.

  20. Structural features based genome-wide characterization and prediction of nucleosome organization

    PubMed Central

    2012-01-01

    Background Nucleosome distribution along chromatin dictates genomic DNA accessibility and thus profoundly influences gene expression. However, the underlying mechanism of nucleosome formation remains elusive. Here, taking a structural perspective, we systematically explored nucleosome formation potential of genomic sequences and the effect on chromatin organization and gene expression in S. cerevisiae. Results We analyzed twelve structural features related to flexibility, curvature and energy of DNA sequences. The results showed that some structural features such as DNA denaturation, DNA-bending stiffness, Stacking energy, Z-DNA, Propeller twist and free energy, were highly correlated with in vitro and in vivo nucleosome occupancy. Specifically, they can be classified into two classes, one positively and the other negatively correlated with nucleosome occupancy. These two kinds of structural features facilitated nucleosome binding in centromere regions and repressed nucleosome formation in the promoter regions of protein-coding genes to mediate transcriptional regulation. Based on these analyses, we integrated all twelve structural features in a model to predict more accurately nucleosome occupancy in vivo than the existing methods that mainly depend on sequence compositional features. Furthermore, we developed a novel approach, named DLaNe, that located nucleosomes by detecting peaks of structural profiles, and built a meta predictor to integrate information from different structural features. As a comparison, we also constructed a hidden Markov model (HMM) to locate nucleosomes based on the profiles of these structural features. The result showed that the meta DLaNe and HMM-based method performed better than the existing methods, demonstrating the power of these structural features in predicting nucleosome positions. Conclusions Our analysis revealed that DNA structures significantly contribute to nucleosome organization and influence chromatin structure and gene

  1. Comparative analysis of methods for genome-wide nucleosome cartography.

    PubMed

    Quintales, Luis; Vázquez, Enrique; Antequera, Francisco

    2015-07-01

    Nucleosomes contribute to compacting the genome into the nucleus and regulate the physical access of regulatory proteins to DNA either directly or through the epigenetic modifications of the histone tails. Precise mapping of nucleosome positioning across the genome is, therefore, essential to understanding the genome regulation. In recent years, several experimental protocols have been developed for this purpose that include the enzymatic digestion, chemical cleavage or immunoprecipitation of chromatin followed by next-generation sequencing of the resulting DNA fragments. Here, we compare the performance and resolution of these methods from the initial biochemical steps through the alignment of the millions of short-sequence reads to a reference genome to the final computational analysis to generate genome-wide maps of nucleosome occupancy. Because of the lack of a unified protocol to process data sets obtained through the different approaches, we have developed a new computational tool (NUCwave), which facilitates their analysis, comparison and assessment and will enable researchers to choose the most suitable method for any particular purpose. NUCwave is freely available at http://nucleosome.usal.es/nucwave along with a step-by-step protocol for its use. © The Author 2014. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  2. Activation domains drive nucleosome eviction by SWI/SNF

    PubMed Central

    Gutiérrez, José L; Chandy, Mark; Carrozza, Michael J; Workman, Jerry L

    2007-01-01

    ATP-dependent chromatin remodeling complexes play a critical role in chromatin dynamics. A large number of in vitro studies have pointed towards nucleosome sliding as the principal remodeling outcome of SWI/SNF action, whereas few have described histone octamer transfer as the principal outcome. In contrast, recent in vivo studies have linked the activity of SWI/SNF to histone eviction in trans from gene promoters. In this study, we have found that the chimeric transcription factor Gal4-VP16 can enhance SWI/SNF histone octamer transfer activity, resulting in targeted histone eviction from a nucleosome probe. This effect is dependent on the presence of the activation domain. We observed that under conditions mimicking the in vivo relative abundance of SWI/SNF with respect to the total number of nucleosomes in a cell nucleus, the accessibility of the transcription factor binding site is the first determinant in the sequence of events leading to nucleosome remodeling. We propose a model mechanism for this transcription factor-mediated enhancement of SWI/SNF octamer transfer activity. PMID:17235287

  3. Effect of the Spiroiminodihydantoin Lesion on Nucleosome Stability and Positioning.

    PubMed

    Norabuena, Erika M; Barnes Williams, Sara; Klureza, Margaret A; Goehring, Liana J; Gruessner, Brian; Radhakrishnan, Mala L; Jamieson, Elizabeth R; Núñez, Megan E

    2016-04-26

    DNA is constantly under attack by oxidants, generating a variety of potentially mutagenic covalently modified species, including oxidized guanine base products. One such product is spiroiminodihydantoin (Sp), a chiral, propeller-shaped lesion that strongly destabilizes the DNA helix in its vicinity. Despite its unusual shape and thermodynamic effect on double-stranded DNA structure, DNA duplexes containing the Sp lesion form stable nucleosomes upon being incubated with histone octamers. Indeed, among six different combinations of lesion location and stereochemistry, only two duplexes display a diminished ability to form nucleosomes, and these only by ∼25%; the other four are statistically indistinguishable from the control. Nonetheless, kinetic factors also play a role: when the histone proteins have less time during assembly of the core particle to sample both lesion-containing and normal DNA strands, they are more likely to bind the Sp lesion DNA than during slower assembly processes that better approximate thermodynamic equilibrium. Using DNase I footprinting and molecular modeling, we discovered that the Sp lesion causes only a small perturbation (±1-2 bp) on the translational position of the DNA within the nucleosome. Each diastereomeric pair of lesions has the same effect on nucleosome positioning, but lesions placed at different locations behave differently, illustrating that the location of the lesion and not its shape serves as the primary determinant of the most stable DNA orientation.

  4. Perturbations in nucleosome structure from heavy metal association

    PubMed Central

    Mohideen, Kareem; Muhammad, Reyhan; Davey, Curt A.

    2010-01-01

    Heavy metals have the potential to engage in strong bonding interactions and can thus function in essential as well as toxic or therapeutic capacities. We conducted crystallographic analyses of heavy cation binding to the nucleosome core particle and found that Co2+ and Ni2+ preferentially associate with the DNA major groove, in a sequence- and conformation-dependent manner. Conversely, Rb+ and Cs+ are found to bind only opportunistically to minor groove elements of the DNA, in particular at narrow AT dinucleotide sites. Furthermore, relative to Mn2+ the aggressive coordination of Co2+ and Ni2+ to guanine bases is observed to induce a shift in histone–DNA register around the nucleosome center by stabilizing DNA stretching over one region accompanied by expulsion of two bases at an opposing location. These ‘softer’ transition metals also associate with multiple histone protein sites, including inter-nucleosomal cross-linking, and display a proclivity for coordination to histidine. Sustained binding and the ability to induce structural perturbations at specific locations in the nucleosome may contribute to genetic and epigenetic mechanisms of carcinogenesis mediated by Co2+ and Ni2+. PMID:20494975

  5. Promoter nucleosome dynamics regulated by signalling through the CTD code

    PubMed Central

    Materne, Philippe; Anandhakumar, Jayamani; Migeot, Valerie; Soriano, Ignacio; Yague-Sanz, Carlo; Hidalgo, Elena; Mignion, Carole; Quintales, Luis; Antequera, Francisco; Hermand, Damien

    2015-01-01

    The phosphorylation of the RNA polymerase II C-terminal domain (CTD) plays a key role in delineating transcribed regions within chromatin by recruiting histone methylases and deacetylases. Using genome-wide nucleosome mapping, we show that CTD S2 phosphorylation controls nucleosome dynamics in the promoter of a subset of 324 genes, including the regulators of cell differentiation ste11 and metabolic adaptation inv1. Mechanistic studies on these genes indicate that during gene activation a local increase of phospho-S2 CTD nearby the promoter impairs the phospho-S5 CTD-dependent recruitment of Set1 and the subsequent recruitment of specific HDACs, which leads to nucleosome depletion and efficient transcription. The early increase of phospho-S2 results from the phosphorylation of the CTD S2 kinase Lsk1 by MAP kinase in response to cellular signalling. The artificial tethering of the Lsk1 kinase at the ste11 promoter is sufficient to activate transcription. Therefore, signalling through the CTD code regulates promoter nucleosomes dynamics. DOI: http://dx.doi.org/10.7554/eLife.09008.001 PMID:26098123

  6. Chromosomal protein poly(ADP-ribosyl)ation in pancreatic nucleosomes.

    PubMed

    Aubin, R J; Dam, V T; Miclette, J; Brousseau, Y; Poirier, G G

    1982-03-01

    When pancreatic chromatin fragments were prepared and resolved in the presence of 80 mM NaCl, endogenous poly(ADP-ribose) polymerase activity was found to be maximal in nucleosome periodicities of four to five units and did not respond to any further increases in nucleosomal architecture. Furthermore, in nucleosome complexities spanning 1 through 14 and over unit lengths, polyacrylamide gel electrophoresis on acid-urea and acid-urea-Triton gels has shown pancreatic histone H1 to be the only actively ADP-ribosylated histone species. The extent of ADP-ribosylation of histone H1 was also demonstrated to retard the protein's mobility in acid-urea, acid-urea-Triton, and lithium dodecyl sulfate polyacrylamide gels and to consist of at least 12 distinct ADP-ribosylated species extractable in all nucleosome complexities studied. Finally, extraction and subsequent electrophoresis of total chromosomal proteins in the presence of lithium dodecyl sulfate also evidenced heavy ADP-ribosylation at the level of nonhistone chromosomal proteins of the high mobility group comigrating in the core histone region, as well as in the topmost region of the gels where poly(ADP-ribose) polymerase was found to form a poly(ADP-ribosyl)ated aggregate.

  7. Interrogating Nucleosome Positioning Through Coarse-Grain Molecular Simulation

    NASA Astrophysics Data System (ADS)

    Freeman, Gordon S.; Hinckley, Daniel M.; Ortiz, Vanessa; de Pablo, Juan J.

    2012-02-01

    Nucleosome positioning plays a crucial role in biology. As the fundamental unit in chromosome structure, the nucleosome core particle (NCP) binds to approximately 147 DNA base pairs. The location of bound NCPs in the genome, therefore, affects gene expression. The specific positioning of NCPs has been experimentally probed and competing viewpoints have been presented in the literature. Models for nucleosome positioning based on sequence-dependent flexibility (a genomic ``code" for nucleosome positioning) have been demonstrated to explain available experimental data. However, so do statistical models with no built-in sequence preference; the driving force for NCP positioning therefore remains an open question. We use a coarse-grain model for the NCP in combination with advanced sampling techniques to probe the sequence preference of NCPs. We present a method for determining the relative affinity of two DNA sequences for the NCP and use this method to compare high- and low-affinity sequences. We discuss several coarse-grain protein models with varying level of detail to examine the impact of model resolution on the agreement of our results with experimental evidence. We also investigate the dynamics of the NCP-DNA complex and their dependence on system characteristics.

  8. BRCA 1-Mediated Histone Monoubiquitylation: Effect on Nucleosome Dynamics

    DTIC Science & Technology

    2008-02-01

    nucleosome positioning sequence from sea urchin 5s rRNA gene; perform and analyze the ubiquitylation reaction (month 4). Done Task 2: Use single...sea urchin 5S rDNA, and oligonucleosomes were reconstituted on the same sequence repeated in tandem (208-12) (Simpson et al., 1985). Isolation of

  9. Propagation of thrombosis by neutrophils and extracellular nucleosome networks

    PubMed Central

    Pfeiler, Susanne; Stark, Konstantin; Massberg, Steffen; Engelmann, Bernd

    2017-01-01

    Neutrophils, early mediators of the innate immune defense, are recruited to developing thrombi in different types of thrombosis. They amplify intravascular coagulation by stimulating the tissue factor-dependent extrinsic pathway via inactivation of endogenous anticoagulants, enhancing factor XII activation or decreasing plasmin generation. Neutrophil-dependent prothrombotic mechanisms are supported by the externalization of decondensed nucleosomes and granule proteins that together form neutrophil extracellular traps. These traps, either in intact or fragmented form, are causally involved in various forms of experimental thrombosis as first indicated by their role in the enhancement of both microvascular thrombosis during bacterial infection and carotid artery thrombosis. Neutrophil extracellular traps can be induced by interactions of neutrophils with activated platelets; vice versa, these traps enhance adhesion of platelets via von Willebrand factor. Neutrophil-induced microvascular thrombus formation can restrict the dissemination and survival of blood-borne bacteria and thereby sustain intravascular immunity. Dysregulation of this innate immune pathway may support sepsis-associated coagulopathies. Notably, neutrophils and extracellular nucleosomes, together with platelets, critically promote fibrin formation during flow restriction-induced deep vein thrombosis. Neutrophil extracellular traps/extracellular nucleosomes are increased in thrombi and in the blood of patients with different vaso-occlusive pathologies and could be therapeutically targeted for the prevention of thrombosis. Thus, during infections and in response to blood vessel damage, neutrophils and externalized nucleosomes are major promoters of intravascular blood coagulation and thrombosis. PMID:27927771

  10. Nucleosome interactions in chromatin: Fiber stiffening and hairpin formation

    NASA Astrophysics Data System (ADS)

    Mergell, Boris; Everaers, Ralf; Schiessel, Helmut

    2004-07-01

    We use Monte Carlo simulations to study attractive and excluded volume interactions between nucleosome core particles in 30-nm chromatin fibers. The nucleosomes are treated as disklike objects having an excluded volume and short-range attraction modeled by a variant of the Gay-Berne potential. The nucleosomes are connected via bendable and twistable linker DNA in the crossed linker fashion. We investigate the influence of the nucleosomal excluded volume on the stiffness of the fiber. For parameter values that correspond to chicken erythrocyte chromatin, we find that the persistence length is governed to a large extent by that excluded volume whereas the soft linker backbone elasticity plays only a minor role. We further find that internucleosomal attraction can induce the formation of hairpin configurations. Tension-induced opening of such configurations into straight fibers manifests itself in a quasiplateau in the force-extension curve that resembles results from recent micromanipulation experiments. Such hairpins may play a role in the formation of higher-order structures in chromosomes like chromonema fibers.

  11. Routes to DNA accessibility: alternative pathways for nucleosome unwinding.

    PubMed

    Schlingman, Daniel J; Mack, Andrew H; Kamenetska, Masha; Mochrie, Simon G J; Regan, Lynne

    2014-07-15

    The dynamic packaging of DNA into chromatin is a key determinant of eukaryotic gene regulation and epigenetic inheritance. Nucleosomes are the basic unit of chromatin, and therefore the accessible states of the nucleosome must be the starting point for mechanistic models regarding these essential processes. Although the existence of different unwound nucleosome states has been hypothesized, there have been few studies of these states. The consequences of multiple states are far reaching. These states will behave differently in all aspects, including their interactions with chromatin remodelers, histone variant exchange, and kinetic properties. Here, we demonstrate the existence of two distinct states of the unwound nucleosome, which are accessible at physiological forces and ionic strengths. Using optical tweezers, we measure the rates of unwinding and rewinding for these two states and show that the rewinding rates from each state are different. In addition, we show that the probability of unwinding into each state is dependent on the applied force and ionic strength. Our results demonstrate not only that multiple unwound states exist but that their accessibility can be differentially perturbed, suggesting possible roles for these states in gene regulation. For example, different histone variants or modifications may facilitate or suppress access to DNA by promoting unwinding into one state or the other. We anticipate that the two unwound states reported here will be the basis for future models of eukaryotic transcriptional control.

  12. Fatal Intoxication with Acetyl Fentanyl.

    PubMed

    Cunningham, Susan M; Haikal, Nabila A; Kraner, James C

    2016-01-01

    Among the new psychoactive substances encountered in forensic investigations is the opioid, acetyl fentanyl. The death of a 28-year-old man from recreational use of this compound is reported. The decedent was found in the bathroom of his residence with a tourniquet secured around his arm and a syringe nearby. Postmortem examination findings included marked pulmonary and cerebral edema and needle track marks. Toxicological analysis revealed acetyl fentanyl in subclavian blood, liver, vitreous fluid, and urine at concentrations of 235 ng/mL, 2400 ng/g, 131 ng/mL, and 234 ng/mL, respectively. Acetyl fentanyl was also detected in the accompanying syringe. Death was attributed to recreational acetyl fentanyl abuse, likely through intravenous administration. The blood acetyl fentanyl concentration is considerably higher than typically found in fatal fentanyl intoxications. Analysis of this case underscores the need for consideration of a wide range of compounds with potential opioid-agonist activity when investigating apparent recreational drug-related deaths. © 2015 American Academy of Forensic Sciences.

  13. Structure of the Rtt109-AcCoA/Vps75 Complex and Implications for Chaperone-Mediated Histone Acetylation

    PubMed Central

    Tang, Yong; Holbert, Marc A.; Delgoshaie, Neda; Wurtele, Hugo; Guillemette, Benoît; Meeth, Katrina; Yuan, Hua; Drogaris, Paul; Lee, Eun-Hye; Durette, Chantal; Thibault, Pierre; Verreault, Alain; Cole, Philip A.; Marmorstein, Ronen

    2011-01-01

    Yeast Rtt109 promotes nucleosome assembly and genome stability by acetylating K9, K27 and K56 of histone H3 through interaction with either of two distinct histone chaperones, Vps75 or Asf1. We report the crystal structure of an Rtt109-AcCoA/Vps75 complex revealing an elongated Vps75 homodimer bound to two globular Rtt109 molecules to form a symmetrical holoenzyme with a ~12 Å diameter central hole. Vps75 and Rtt109 residues that mediate complex formation in the crystals are also important for Rtt109-Vps75 interaction and H3K9/K27 acetylation both in vitro and in yeast cells. The same Rtt109 residues do not participate in Asf1-mediated Rtt109 acetylation in vitro or H3K56 acetylation in yeast cells, demonstrating that Asf1 and Vps75 dictate Rtt109 substrate specificity through distinct mechanisms. These studies also suggest that Vps75 binding stimulates Rtt109 catalytic activity by appropriately presenting the H3–H4 substrate within the central cavity of the holoenzyme to promote H3K9/K27 acetylation of new histones prior to deposition. PMID:21256037

  14. Dynamic Competing Histone H4 K5K8 Acetylation and Butyrylation Are Hallmarks of Highly Active Gene Promoters

    PubMed Central

    Goudarzi, Afsaneh; Zhang, Di; Huang, He; Barral, Sophie; Kwon, Oh Kwang; Qi, Shankang; Tang, Zhanyun; Buchou, Thierry; Vitte, Anne-Laure; He, Tieming; Cheng, Zhongyi; Montellier, Emilie; Gaucher, Jonathan; Curtet, Sandrine; Debernardi, Alexandra; Charbonnier, Guillaume; Puthier, Denis; Petosa, Carlo; Panne, Daniel; Rousseaux, Sophie; Roeder, Robert G.; Zhao, Yingming; Khochbin, Saadi

    2016-01-01

    Summary Recently discovered histone lysine acylation marks increase the functional diversity of nucleosomes well beyond acetylation. Here, we focus on histone butyrylation in the context of sperm cell differentiation. Specifically, we investigate the butyrylation of histone H4 lysine 5 and 8 at gene promoters where acetylation guides the binding of Brdt, a bromodomain-containing protein, thereby mediating stage-specific gene expression programs and post-meiotic chromatin reorganization. Genome-wide mapping data show that highly active Brdt-bound gene promoters systematically harbor competing histone acetylation and butyrylation marks at H4 K5 and H4 K8. Despite acting as a direct stimulator of transcription, histone butyrylation competes with acetylation, especially at H4 K5, to prevent Brdt binding. Additionally, H4 K5K8 butyrylation also marks retarded histone removal during late spermatogenesis. Hence, alternating H4 acetylation and butyrylation, while sustaining direct gene activation and dynamic bromodomain binding, could impact the final male epigenome features. PMID:27105113

  15. DNA sequence-dependent variation in nucleosome structure, stability, and dynamics detected by a FRET-based analysis.

    PubMed

    Kelbauskas, L; Woodbury, N; Lohr, D

    2009-02-01

    Förster resonance energy transfer (FRET) techniques provide powerful and sensitive methods for the study of conformational features in biomolecules. Here, we review FRET-based studies of nucleosomes, focusing particularly on our work comparing the widely used nucleosome standard, 5S rDNA, and 2 promoter-derived regulatory element-containing nucleosomes, mouse mammary tumor virus (MMTV)-B and GAL10. Using several FRET approaches, we detected significant DNA sequence-dependent structure, stability, and dynamics differences among the three. In particular, 5S nucleosomes and 5S H2A/H2B-depleted nucleosomal particles have enhanced stability and diminished DNA dynamics, compared with MMTV-B and GAL10 nucleosomes and particles. H2A/H2B-depleted nucleosomes are of interest because they are produced by the activities of many transcription-associated complexes. Significant location-dependent (intranucleosomal) stability and dynamics variations were also observed. These also vary among nucleosome types. Nucleosomes restrict regulatory factor access to DNA, thereby impeding genetic processes. Eukaryotic cells possess mechanisms to alter nucleosome structure, to generate DNA access, but alterations often must be targeted to specific nucleosomes on critical regulatory DNA elements. By endowing specific nucleosomes with intrinsically higher DNA accessibility and (or) enhanced facility for conformational transitions, DNA sequence-dependent nucleosome dynamics and stability variations have the potential to facilitate nucleosome recognition and, thus, aid in the crucial targeting process. This and other nucleosome structure and function conclusions from FRET analyses are discussed.

  16. 25 CFR 36.112 - Can a homeliving program be closed, transferred, consolidated, or substantially curtailed for...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ..., consolidated, or substantially curtailed for failure to meet these standards? 36.112 Section 36.112 Indians... EDUCATION OF INDIAN CHILDREN AND NATIONAL CRITERIA FOR DORMITORY SITUATIONS Homeliving Programs Waivers and... for failure to meet these standards? No, a homeliving program cannot be closed, transferred to...

  17. Structure and nucleosome interaction of the yeast NuA4 and Piccolo-NuA4 histone acetyltransferase complexes

    PubMed Central

    Chittuluru, Johnathan R.; Chaban, Yuriy; Monnet-Saksouk, Julie; Carrozza, Michael J.; Sapountzi, Vasileia; Selleck, William; Huang, Jiehuan; Utley, Rhea T.; Cramet, Myriam; Allard, Stephane; Cai, Gang; Workman, Jerry L.; Fried, Michael G.; Tan, Song; Côté, Jacques; Asturias, Francisco J.

    2011-01-01

    We have used electron microscopy (EM) and biochemistry to characterize the structure and nucleosome core particle (NCP) interaction of NuA4, an essential yeast histone acetyltransferase (HAT) complex conserved throughout eukaryotes. The ATM-related Tra1 subunit, shared with the SAGA coactivator, forms a large domain joined to a second portion that accommodates the Piccolo catalytic subcomplex and other NuA4 subunits. EM analysis of an NuA4–NCP complex shows the NCP bound at NuA4's periphery. EM characterization of Piccolo and Piccolo–NCP provided further information about subunit organization and confirmed that histone acetylation requires minimal contact with the NCP. A small conserved region at the N-terminus of Piccolo subunit Epl1 is essential for NCP interaction, whereas subunit Yng2 apparently positions Piccolo for efficient acetylation of H4 or H2A tails. Taken together, these results provide an understanding of NuA4 subunit organization and NCP interactions. PMID:21984211

  18. BRG1 Governs Nanog Transcription in Early Mouse Embryos and Embryonic Stem Cells via Antagonism of Histone H3 Lysine 9/14 Acetylation

    PubMed Central

    Carey, Timothy S.; Cao, Zubing; Choi, Inchul; Ganguly, Avishek; Wilson, Catherine A.; Paul, Soumen

    2015-01-01

    During mouse preimplantation development, the generation of the inner cell mass (ICM) and trophoblast lineages comprises upregulation of Nanog expression in the ICM and its silencing in the trophoblast. However, the underlying epigenetic mechanisms that differentially regulate Nanog in the first cell lineages are poorly understood. Here, we report that BRG1 (Brahma-related gene 1) cooperates with histone deacetylase 1 (HDAC1) to regulate Nanog expression. BRG1 depletion in preimplantation embryos and Cdx2-inducible embryonic stem cells (ESCs) revealed that BRG1 is necessary for Nanog silencing in the trophoblast lineage. Conversely, in undifferentiated ESCs, loss of BRG1 augmented Nanog expression. Analysis of histone H3 within the Nanog proximal enhancer revealed that H3 lysine 9/14 (H3K9/14) acetylation increased in BRG1-depleted embryos and ESCs. Biochemical studies demonstrated that HDAC1 was present in BRG1-BAF155 complexes and BRG1-HDAC1 interactions were enriched in the trophoblast lineage. HDAC1 inhibition triggered an increase in H3K9/14 acetylation and a corresponding rise in Nanog mRNA and protein, phenocopying BRG1 knockdown embryos and ESCs. Lastly, nucleosome-mapping experiments revealed that BRG1 is indispensable for nucleosome remodeling at the Nanog enhancer during trophoblast development. In summary, our data suggest that BRG1 governs Nanog expression via a dual mechanism involving histone deacetylation and nucleosome remodeling. PMID:26416882

  19. BRG1 Governs Nanog Transcription in Early Mouse Embryos and Embryonic Stem Cells via Antagonism of Histone H3 Lysine 9/14 Acetylation.

    PubMed

    Carey, Timothy S; Cao, Zubing; Choi, Inchul; Ganguly, Avishek; Wilson, Catherine A; Paul, Soumen; Knott, Jason G

    2015-12-01

    During mouse preimplantation development, the generation of the inner cell mass (ICM) and trophoblast lineages comprises upregulation of Nanog expression in the ICM and its silencing in the trophoblast. However, the underlying epigenetic mechanisms that differentially regulate Nanog in the first cell lineages are poorly understood. Here, we report that BRG1 (Brahma-related gene 1) cooperates with histone deacetylase 1 (HDAC1) to regulate Nanog expression. BRG1 depletion in preimplantation embryos and Cdx2-inducible embryonic stem cells (ESCs) revealed that BRG1 is necessary for Nanog silencing in the trophoblast lineage. Conversely, in undifferentiated ESCs, loss of BRG1 augmented Nanog expression. Analysis of histone H3 within the Nanog proximal enhancer revealed that H3 lysine 9/14 (H3K9/14) acetylation increased in BRG1-depleted embryos and ESCs. Biochemical studies demonstrated that HDAC1 was present in BRG1-BAF155 complexes and BRG1-HDAC1 interactions were enriched in the trophoblast lineage. HDAC1 inhibition triggered an increase in H3K9/14 acetylation and a corresponding rise in Nanog mRNA and protein, phenocopying BRG1 knockdown embryos and ESCs. Lastly, nucleosome-mapping experiments revealed that BRG1 is indispensable for nucleosome remodeling at the Nanog enhancer during trophoblast development. In summary, our data suggest that BRG1 governs Nanog expression via a dual mechanism involving histone deacetylation and nucleosome remodeling.

  20. Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions

    PubMed Central

    Schep, Alicia N.; Buenrostro, Jason D.; Denny, Sarah K.; Schwartz, Katja; Sherlock, Gavin; Greenleaf, William J.

    2015-01-01

    Transcription factors canonically bind nucleosome-free DNA, making the positioning of nucleosomes within regulatory regions crucial to the regulation of gene expression. Using the assay of transposase accessible chromatin (ATAC-seq), we observe a highly structured pattern of DNA fragment lengths and positions around nucleosomes in Saccharomyces cerevisiae, and use this distinctive two-dimensional nucleosomal “fingerprint” as the basis for a new nucleosome-positioning algorithm called NucleoATAC. We show that NucleoATAC can identify the rotational and translational positions of nucleosomes with up to base-pair resolution and provide quantitative measures of nucleosome occupancy in S. cerevisiae, Schizosaccharomyces pombe, and human cells. We demonstrate the application of NucleoATAC to a number of outstanding problems in chromatin biology, including analysis of sequence features underlying nucleosome positioning, promoter chromatin architecture across species, identification of transient changes in nucleosome occupancy and positioning during a dynamic cellular response, and integrated analysis of nucleosome occupancy and transcription factor binding. PMID:26314830

  1. Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions.

    PubMed

    Schep, Alicia N; Buenrostro, Jason D; Denny, Sarah K; Schwartz, Katja; Sherlock, Gavin; Greenleaf, William J

    2015-11-01

    Transcription factors canonically bind nucleosome-free DNA, making the positioning of nucleosomes within regulatory regions crucial to the regulation of gene expression. Using the assay of transposase accessible chromatin (ATAC-seq), we observe a highly structured pattern of DNA fragment lengths and positions around nucleosomes in Saccharomyces cerevisiae, and use this distinctive two-dimensional nucleosomal "fingerprint" as the basis for a new nucleosome-positioning algorithm called NucleoATAC. We show that NucleoATAC can identify the rotational and translational positions of nucleosomes with up to base-pair resolution and provide quantitative measures of nucleosome occupancy in S. cerevisiae, Schizosaccharomyces pombe, and human cells. We demonstrate the application of NucleoATAC to a number of outstanding problems in chromatin biology, including analysis of sequence features underlying nucleosome positioning, promoter chromatin architecture across species, identification of transient changes in nucleosome occupancy and positioning during a dynamic cellular response, and integrated analysis of nucleosome occupancy and transcription factor binding.

  2. Genome-scale identification of nucleosome organization by using 1000 porcine oocytes at different developmental stages

    PubMed Central

    Tao, Chenyu; Li, Juan; Chen, Baobao; Chi, Daming; Zeng, Yaqiong

    2017-01-01

    The nucleosome is the basic structural unit of chromosomes, and its occupancy and distribution in promoters are crucial for the regulation of gene expression. During the growth process of porcine oocytes, the “growing” oocytes (SF) have a much higher transcriptional activity than the “fully grown” oocytes (BF). However, the chromosome status of the two kinds of oocytes remains poorly understood. In this study, we profiled the nucleosome distributions of SF and BF with as few as 1000 oocytes. By comparing the altered regions, we found that SF tended toward nucleosome loss and more open chromosome architecture than BF did. BF had decreased nucleosome occupancy in the coding region and increased nucleosome occupancy in the promoter compared to SF. The nucleosome occupancy of SF was higher than that of BF in the GC-poor regions, but lower than that of BF in the GC-rich regions. The nucleosome distribution around the transcriptional start site (TSS) of all the genes of the two samples was basically the same, but the nucleosome occupancy around the TSS of SF was lower than that of BF. GO functional annotation of genes with different nucleosome occupancy in promoter showed the genes were mainly involved in cell, cellular process, and metabolic process biological process. The results of this study revealed the dynamic reorganization of porcine oocytes in different developmental stages and the critical role of nucleosome arrangement during the oocyte growth process. PMID:28333987

  3. High mobility group protein 1: A collaborator in nucleosome dynamics and estrogen-responsive gene expression

    PubMed Central

    Scovell, William M

    2016-01-01

    High mobility group protein 1 (HMGB1) is a multifunctional protein that interacts with DNA and chromatin to influence the regulation of transcription, DNA replication and repair and recombination. We show that HMGB1 alters the structure and stability of the canonical nucleosome (N) in a nonenzymatic, adenosine triphosphate-independent manner. As a result, the canonical nucleosome is converted to two stable, physically distinct nucleosome conformers. Although estrogen receptor (ER) does not bind to its consensus estrogen response element within a nucleosome, HMGB1 restructures the nucleosome to facilitate strong ER binding. The isolated HMGB1-restructured nucleosomes (N’ and N’’) remain stable and exhibit a number of characteristics that are distinctly different from the canonical nucleosome. These findings complement previous studies that showed (1) HMGB1 stimulates in vivo transcriptional activation at estrogen response elements and (2) knock down of HMGB1 expression by siRNA precipitously reduced transcriptional activation. The findings indicate that a major facet of the mechanism of HMGB1 action involves a restructuring of aspects of the nucleosome that appear to relax structural constraints within the nucleosome. The findings are extended to reveal the differences between ER and the other steroid hormone receptors. A working proposal outlines mechanisms that highlight the multiple facets that HMGB1 may utilize in restructuring the nucleosome. PMID:27247709

  4. Using DNA mechanics to predict intrinsic and extrinsic nucleosome positioning signals

    NASA Astrophysics Data System (ADS)

    Morozov, Alexandre

    2008-03-01

    In eukaryotic genomes, nucleosomes function to compact DNA and to regulate access to it both by simple physical occlusion and by providing the substrate for numerous covalent epigenetic tags. While nucleosome positions in vitro are determined by sequence alone, in vivo competition with other DNA-binding factors and action of chromatin remodeling enzymes play a role that needs to be quantified. We developed a biophysical, DNA mechanics-based model for the sequence dependence of DNA bending energies, and validated it against a collection of in vitro free energies of nucleosome formation and a nucleosome crystal structure; we also successfully designed both strong and poor histone binding sequences ab initio. For in vivo data from S.cerevisiae, the strongest positioning signal came from the competition with other factors rather than intrinsic nucleosome sequence preferences. Based on sequence alone, our model predicts that functional transcription factor binding sites tend to be covered by nucleosomes, yet are uncovered in vivo because functional sites cluster within a single nucleosome footprint and thus make transcription factors bind cooperatively. Similarly a weak enhancement of nucleosome binding in the TATA region becomes a strong depletion when the TATA-binding protein is included, in quantitative agreement with experiment. Our model distinguishes multiple ways in which genomic sequence influences nucleosome positions, and thus provides alternative explanations for several genome-wide experimental findings. In the future our approach will be used to rationally alter gene expression levels in model systems through redesign of nucleosome occupancy profiles.

  5. Active nucleosome positioning beyond intrinsic biophysics is revealed by in vitro reconstitution.

    PubMed

    Korber, Philipp

    2012-04-01

    Genome-wide nucleosome maps revealed well-positioned nucleosomes as a major theme in eukaryotic genome organization. Promoter regions often show a conserved pattern with an NDR (nucleosome-depleted region) from which regular nucleosomal arrays emanate. Three mechanistic contributions to such NDR-array-organization and nucleosome positioning in general are discussed: DNA sequence, DNA binders and DNA-templated processes. Especially, intrinsic biophysics of DNA sequence preferences for nucleosome formation was prominently suggested to explain the majority of nucleosome positions ('genomic code for nucleosome positioning'). Nonetheless, non-histone factors that bind DNA with high or low specificity, such as transcription factors or remodelling enzymes respectively and processes such as replication, transcription and the so-called 'statistical positioning' may be involved too. Recently, these models were tested for yeast by genome-wide reconstitution. DNA sequence preferences as probed by SGD (salt gradient dialysis) reconstitution generated many NDRs, but only few individual nucleosomes, at their proper positions, and no arrays. Addition of a yeast extract and ATP led to dramatically more in vivo-like nucleosome positioning, including regular arrays for the first time. This improvement depended essentially on the extract and ATP but not on transcription or replication. Nucleosome occupancy and close spacing were maintained around promoters, even at lower histone density, arguing for active packing of nucleosomes against the 5' ends of genes rather than statistical positioning. A first extract fractionation identified a direct, specific, necessary, but not sufficient role for the RSC (remodels the structure of chromatin) remodelling enzyme. Collectively, nucleosome positioning in yeast is actively determined by factors beyond intrinsic biophysics, and in steady-state rather than at equilibrium.

  6. Differential cofactor requirements for histone eviction from two nucleosomes at the yeast PHO84 promoter are determined by intrinsic nucleosome stability.

    PubMed

    Wippo, Christian J; Krstulovic, Bojana Silic; Ertel, Franziska; Musladin, Sanja; Blaschke, Dorothea; Stürzl, Sabrina; Yuan, Guo-Cheng; Hörz, Wolfram; Korber, Philipp; Barbaric, Slobodan

    2009-06-01

    We showed previously that the strong PHO5 promoter is less dependent on chromatin cofactors than the weaker coregulated PHO8 promoter. In this study we asked if chromatin remodeling at the even stronger PHO84 promoter was correspondingly less cofactor dependent. The repressed PHO84 promoter showed a short hypersensitive region that was flanked upstream and downstream by a positioned nucleosome and contained two transactivator Pho4 sites. Promoter induction generated an extensive hypersensitive and histone-depleted region, yielding two more Pho4 sites accessible. This remodeling was strictly Pho4 dependent, strongly dependent on the remodelers Snf2 and Ino80 and on the histone acetyltransferase Gcn5, and more weakly on the acetyltransferase Rtt109. Importantly, remodeling of each of the two positioned nucleosomes required Snf2 and Ino80 to different degrees. Only remodeling of the upstream nucleosome was strictly dependent on Snf2. Further, remodeling of the upstream nucleosome was more dependent on Ino80 than remodeling of the downstream nucleosome. Both nucleosomes differed in their intrinsic stabilities as predicted in silico and measured in vitro. The causal relationship between the different nucleosome stabilities and the different cofactor requirements was shown by introducing destabilizing mutations in vivo. Therefore, chromatin cofactor requirements were determined by intrinsic nucleosome stabilities rather than correlated to promoter strength.

  7. N-ACETYL GROUPS IN VITELLENIN,

    DTIC Science & Technology

    The presence of acetyl groups in vitellenin was confirmed by hydrazinolysis according to the DNP method of Phillips. After hydrazinolysis of 10-30...hydrazinolysis at room temperature for 1 hour, vitellenin contains N- acetyl , but no Oacetyl, groups. (Author)

  8. Modelling the impact of vaccination on curtailing Haemophilus influenzae serotype 'a'.

    PubMed

    Konini, Angjelina; Moghadas, Seyed M

    2015-12-21

    Haemophilus influenzae serotype a (Hia) is a human-restricted bacterial pathogen transmitted via direct contacts with an infectious individual. Currently, there is no vaccine available for prevention of Hia, and the disease is treated with antibiotics upon diagnosis. With ongoing efforts for the development of an anti-Hia protein-polysaccharide conjugated vaccine, we sought to investigate the effect of vaccination on curtailing Hia infection. We present the first stochastic model of Hia transmission and control dynamics, and parameterize it using available estimates in the literature. Since both naturally acquired and vaccine-induced immunity wane with time, model simulations show three important results. First, vaccination of only newborns cannot eliminate the pathogen from the population, even when a booster program is implemented with a high coverage. Second, achieving and maintaining a sufficiently high level of herd immunity for pathogen elimination requires vaccination of susceptible individuals in addition to a high vaccination coverage of newborns. Third, for a low vaccination rate of susceptible individuals, a high coverage of booster dose may be needed to raise the level of herd immunity for Hia eradication. Our findings highlight the importance of vaccination and timely boosting of the individual׳s immunity within the expected duration of vaccine-induced protection against Hia. When an anti-Hia vaccine becomes available, enhanced surveillance of Hia incidence and herd immunity could help determine vaccination rates and timelines for booster doses necessary to eliminate Hia from affected populations. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Hairpin Ribozyme Genes Curtail Alcohol Drinking: from Rational Design to in vivo Effects in the Rat

    PubMed Central

    Sapag, Amalia; Irrazábal, Thergiory; Lobos-González, Lorena; Muñoz-Brauning, Carlos R; Quintanilla, María Elena; Tampier, Lutske

    2016-01-01

    Ribozyme genes were designed to reduce voluntary alcohol drinking in a rat model of alcohol dependence. Acetaldehyde generated from alcohol in the liver is metabolized by the mitochondrial aldehyde dehydrogenase (ALDH2) such that diminishing ALDH2 activity leads to the aversive effects of blood acetaldehyde upon alcohol intake. A stepwise approach was followed to design genes encoding ribozymes targeted to the rat ALDH2 mRNA. In vitro studies of accessibility to oligonucleotides identified suitable target sites in the mRNA, one of which fulfilled hammerhead and hairpin ribozyme requirements (CGGUC). Ribozyme genes delivered in plasmid constructs were tested in rat cells in culture. While the hairpin ribozyme reduced ALDH2 activity 56% by cleavage and blockade (P < 0.0001), the hammerhead ribozyme elicited minor effects by blockade. The hairpin ribozyme was tested in vivo by adenoviral gene delivery to UChB alcohol drinker rats. Ethanol intake was curtailed 47% for 34 days (P < 0.0001), while blood acetaldehyde more than doubled upon ethanol administration and ALDH2 activity dropped 25% in liver homogenates, not affecting other ALDH isoforms. Thus, hairpin ribozymes targeted to 16 nt in the ALDH2 mRNA provide durable and specific effects in vivo, representing an improvement on previous work and encouraging development of gene therapy for alcoholism. PMID:27404720

  10. Modelling the impact of curtailing antibiotic usage in food animals on antibiotic resistance in humans.

    PubMed

    van Bunnik, B A D; Woolhouse, M E J

    2017-04-01

    Consumption of antibiotics in food animals is increasing worldwide and is approaching, if not already surpassing, the volume consumed by humans. It is often suggested that reducing the volume of antibiotics consumed by food animals could have public health benefits. Although this notion is widely regarded as intuitively obvious there is a lack of robust, quantitative evidence to either support or contradict the suggestion. As a first step towards addressing this knowledge gap, we develop a simple mathematical model for exploring the generic relationship between antibiotic consumption by food animals and levels of resistant bacterial infections in humans. We investigate the impact of restricting antibiotic consumption by animals and identify which model parameters most strongly determine that impact. Our results suggest that, for a wide range of scenarios, curtailing the volume of antibiotics consumed by food animals has, as a stand-alone measure, little impact on the level of resistance in humans. We also find that reducing the rate of transmission of resistance from animals to humans may be more effective than an equivalent reduction in the consumption of antibiotics in food animals. Moreover, the response to any intervention is strongly determined by the rate of transmission from humans to animals, an aspect which is rarely considered.

  11. A Model for Optimizing the Combination of Solar Electricity Generation, Supply Curtailment, Transmission and Storage

    NASA Astrophysics Data System (ADS)

    Perez, Marc J. R.

    With extraordinary recent growth of the solar photovoltaic industry, it is paramount to address the biggest barrier to its high-penetration across global electrical grids: the inherent variability of the solar resource. This resource variability arises from largely unpredictable meteorological phenomena and from the predictable rotation of the earth around the sun and about its own axis. To achieve very high photovoltaic penetration, the imbalance between the variable supply of sunlight and demand must be alleviated. The research detailed herein consists of the development of a computational model which seeks to optimize the combination of 3 supply-side solutions to solar variability that minimizes the aggregate cost of electricity generated therefrom: Storage (where excess solar generation is stored when it exceeds demand for utilization when it does not meet demand), interconnection (where solar generation is spread across a large geographic area and electrically interconnected to smooth overall regional output) and smart curtailment (where solar capacity is oversized and excess generation is curtailed at key times to minimize the need for storage.). This model leverages a database created in the context of this doctoral work of satellite-derived photovoltaic output spanning 10 years at a daily interval for 64,000 unique geographic points across the globe. Underpinning the model's design and results, the database was used to further the understanding of solar resource variability at timescales greater than 1-day. It is shown that--as at shorter timescales--cloud/weather-induced solar variability decreases with geographic extent and that the geographic extent at which variability is mitigated increases with timescale and is modulated by the prevailing speed of clouds/weather systems. Unpredictable solar variability up to the timescale of 30 days is shown to be mitigated across a geographic extent of only 1500km if that geographic extent is oriented in a north

  12. How and why are calcium currents curtailed in the skeletal muscle voltage‐gated calcium channels?

    PubMed Central

    Tuluc, Petronel

    2017-01-01

    Abstract Voltage‐gated calcium channels represent the sole mechanism converting electrical signals of excitable cells into cellular functions such as contraction, secretion and gene regulation. Specific voltage‐sensing domains detect changes in membrane potential and control channel gating. Calcium ions entering through the channel function as second messengers regulating cell functions, with the exception of skeletal muscle, where CaV1.1 essentially does not function as a channel but activates calcium release from intracellular stores. It has long been known that calcium currents are dispensable for skeletal muscle contraction. However, the questions as to how and why the channel function of CaV1.1 is curtailed remained obscure until the recent discovery of a developmental CaV1.1 splice variant with normal channel functions. This discovery provided new means to study the molecular mechanisms regulating the channel gating and led to the understanding that in skeletal muscle, calcium currents need to be restricted to allow proper regulation of fibre type specification and to prevent mitochondrial damage. PMID:27896815

  13. Stem bromelain-induced macrophage apoptosis and activation curtail Mycobacterium tuberculosis persistence.

    PubMed

    Mahajan, Sahil; Chandra, Vemika; Dave, Sandeep; Nanduri, Ravikanth; Gupta, Pawan

    2012-08-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, has a remarkable ability to usurp its host's innate immune response, killing millions of infected people annually. One approach to manage infection is prevention through the use of natural agents. In this regard, stem bromelain (SBM), a pharmacologically active member of the sulfhydryl proteolytic enzyme family, obtained from Ananas comosus and possessing a remarkable ability to induce the innate and acquired immune systems, is important. We evaluated SBM's ability to induce apoptosis and free-radical generation in macrophages. We also studied antimycobacterial properties of SBM and its effect on foamy macrophages. SBM treatment of peritoneal macrophages resulted in the upregulation of proapoptotic proteins and downregulation of antiapoptotic proteins. Additionally, SBM treatment activated macrophages, curtailed the levels of free glutathione, and augmented the production of hydrogen peroxide, superoxide anion, peroxynitrite, and nitric oxide. SBM cleaves CD36 and reduced the formation of foam cells, the hallmark of M. tuberculosis infection. These conditions created an environment for the increased clearance of M. tuberculosis. Together these data provide a mechanism for antimycobacterial activity of SBM and provide important insights for the use of cysteine proteases as immunomodulatory agents.

  14. Hairpin Ribozyme Genes Curtail Alcohol Drinking: from Rational Design to in vivo Effects in the Rat.

    PubMed

    Sapag, Amalia; Irrazábal, Thergiory; Lobos-González, Lorena; Muñoz-Brauning, Carlos R; Quintanilla, María Elena; Tampier, Lutske

    2016-07-12

    Ribozyme genes were designed to reduce voluntary alcohol drinking in a rat model of alcohol dependence. Acetaldehyde generated from alcohol in the liver is metabolized by the mitochondrial aldehyde dehydrogenase (ALDH2) such that diminishing ALDH2 activity leads to the aversive effects of blood acetaldehyde upon alcohol intake. A stepwise approach was followed to design genes encoding ribozymes targeted to the rat ALDH2 mRNA. In vitro studies of accessibility to oligonucleotides identified suitable target sites in the mRNA, one of which fulfilled hammerhead and hairpin ribozyme requirements (CGGUC). Ribozyme genes delivered in plasmid constructs were tested in rat cells in culture. While the hairpin ribozyme reduced ALDH2 activity 56% by cleavage and blockade (P < 0.0001), the hammerhead ribozyme elicited minor effects by blockade. The hairpin ribozyme was tested in vivo by adenoviral gene delivery to UChB alcohol drinker rats. Ethanol intake was curtailed 47% for 34 days (P < 0.0001), while blood acetaldehyde more than doubled upon ethanol administration and ALDH2 activity dropped 25% in liver homogenates, not affecting other ALDH isoforms. Thus, hairpin ribozymes targeted to 16 nt in the ALDH2 mRNA provide durable and specific effects in vivo, representing an improvement on previous work and encouraging development of gene therapy for alcoholism.

  15. Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106

    SciTech Connect

    Su, Dan; Hu, Qi; Li, Qing; Thompson, James R; Cui, Gaofeng; Fazly, Ahmed; Davies, Brian A; Botuyan, Maria Victoria; Zhang, Zhiguo; Mer, Georges

    2013-04-08

    Dynamic variations in the structure of chromatin influence virtually all DNA-related processes in eukaryotes and are controlled in part by post-translational modifications of histones. One such modification, the acetylation of lysine 56 (H3K56ac) in the amino-terminal α-helix (αN) of histone H3, has been implicated in the regulation of nucleosome assembly during DNA replication and repair, and nucleosome disassembly during gene transcription. In Saccharomyces cerevisiae, the histone chaperone Rtt106 contributes to the deposition of newly synthesized H3K56ac-carrying H3-H4 complex on replicating DNA, but it is unclear how Rtt106 binds H3-H4 and specifically recognizes H3K56ac as there is no apparent acetylated lysine reader domain in Rtt106. Here, we show that two domains of Rtt106 are involved in a combinatorial recognition of H3-H4. An N-terminal domain homodimerizes and interacts with H3-H4 independently of acetylation while a double pleckstrin-homology (PH) domain binds the K56-containing region of H3. Affinity is markedly enhanced upon acetylation of K56, an effect that is probably due to increased conformational entropy of the αN helix of H3. Our data support a mode of interaction where the N-terminal homodimeric domain of Rtt106 intercalates between the two H3-H4 components of the (H3-H4)2 tetramer while two double PH domains in the Rtt106 dimer interact with each of the two H3K56ac sites in (H3-H4)2. We show that the Rtt106-(H3-H4)2 interaction is important for gene silencing and the DNA damage response.

  16. MOF Acetylates the Histone Demethylase LSD1 to Suppress Epithelial-to-Mesenchymal Transition.

    PubMed

    Luo, Huacheng; Shenoy, Anitha K; Li, Xuehui; Jin, Yue; Jin, Lihua; Cai, Qingsong; Tang, Ming; Liu, Yang; Chen, Hao; Reisman, David; Wu, Lizi; Seto, Edward; Qiu, Yi; Dou, Yali; Casero, Robert A; Lu, Jianrong

    2016-06-21

    The histone demethylase LSD1 facilitates epithelial-to-mesenchymal transition (EMT) and tumor progression by repressing epithelial marker expression. However, little is known about how its function may be modulated. Here, we report that LSD1 is acetylated in epithelial but not mesenchymal cells. Acetylation of LSD1 reduces its association with nucleosomes, thus increasing histone H3K4 methylation at its target genes and activating transcription. The MOF acetyltransferase interacts with LSD1 and is responsible for its acetylation. MOF is preferentially expressed in epithelial cells and is downregulated by EMT-inducing signals. Expression of exogenous MOF impedes LSD1 binding to epithelial gene promoters and histone demethylation, thereby suppressing EMT and tumor invasion. Conversely, MOF depletion enhances EMT and tumor metastasis. In human cancer, high MOF expression correlates with epithelial markers and a favorable prognosis. These findings provide insight into the regulation of LSD1 and EMT and identify MOF as a critical suppressor of EMT and tumor progression. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  17. Bacterial protein acetylation: new discoveries unanswered questions.

    PubMed

    Wolfe, Alan J

    2016-05-01

    Nε-acetylation is emerging as an abundant post-translational modification of bacterial proteins. Two mechanisms have been identified: one is enzymatic, dependent on an acetyltransferase and acetyl-coenzyme A; the other is non-enzymatic and depends on the reactivity of acetyl phosphate. Some, but not most, of those acetylations are reversed by deacetylases. This review will briefly describe the current status of the field and raise questions that need answering.

  18. Protein acetylation in archaea, bacteria, and eukaryotes.

    PubMed

    Soppa, Jörg

    2010-09-16

    Proteins can be acetylated at the alpha-amino group of the N-terminal amino acid (methionine or the penultimate amino acid after methionine removal) or at the epsilon-amino group of internal lysines. In eukaryotes the majority of proteins are N-terminally acetylated, while this is extremely rare in bacteria. A variety of studies about N-terminal acetylation in archaea have been reported recently, and it was revealed that a considerable fraction of proteins is N-terminally acetylated in haloarchaea and Sulfolobus, while this does not seem to apply for methanogenic archaea. Many eukaryotic proteins are modified by differential internal acetylation, which is important for a variety of processes. Until very recently, only two bacterial proteins were known to be acetylation targets, but now 125 acetylation sites are known for E. coli. Knowledge about internal acetylation in archaea is extremely limited; only two target proteins are known, only one of which--Alba--was used to study differential acetylation. However, indications accumulate that the degree of internal acetylation of archaeal proteins might be underestimated, and differential acetylation has been shown to be essential for the viability of haloarchaea. Focused proteomic approaches are needed to get an overview of the extent of internal protein acetylation in archaea.

  19. Poly-dA:dT tracts form an in vivo nucleosomal turnstile.

    PubMed

    de Boer, Carl G; Hughes, Timothy R

    2014-01-01

    Nucleosomes regulate many DNA-dependent processes by controlling the accessibility of DNA, and DNA sequences such as the poly-dA:dT element are known to affect nucleosome binding. We demonstrate that poly-dA:dT tracts form an asymmetric barrier to nucleosome movement in vivo, mediated by ATP-dependent chromatin remodelers. We theorize that nucleosome transit over poly-A elements is more energetically favourable in one direction, leading to an asymmetric arrangement of nucleosomes around these sequences. We demonstrate that different arrangements of poly-A and poly-T tracts result in very different outcomes for nucleosome occupancy in yeast, mouse, and human, and show that yeast takes advantage of this phenomenon in its promoter architecture.

  20. Linker histones: novel insights into structure-specific recognition of the nucleosome.

    PubMed

    Cutter, Amber R; Hayes, Jeffrey J

    2017-04-01

    Linker histones (H1s) are a primary component of metazoan chromatin, fulfilling numerous functions, both in vitro and in vivo, including stabilizing the wrapping of DNA around the nucleosome, promoting folding and assembly of higher order chromatin structures, influencing nucleosome spacing on DNA, and regulating specific gene expression. However, many molecular details of how H1 binds to nucleosomes and recognizes unique structural features on the nucleosome surface remain undefined. Numerous, confounding studies are complicated not only by experimental limitations, but the use of different linker histone isoforms and nucleosome constructions. This review summarizes the decades of research that has resulted in several models of H1 association with nucleosomes, with a focus on recent advances that suggest multiple modes of H1 interaction in chromatin, while highlighting the remaining questions.

  1. The structure of nucleosomal core particles within transcribed and repressed gene regions.

    PubMed Central

    Studitsky, V M; Belyavsky, A V; Melnikova, A F; Mirzabekov, A D

    1988-01-01

    The arrangement of histones along DNA in nucleosomal core particles within transcribed heat shock gene (hsp 70) region and repressed insertion within ribosomal genes of Drosophila was analysed by using protein-DNA crosslinking methods combined with hybridization tests. In addition, two-dimensional gel electrophoresis was employed to compare the overall nucleosomal shape and the nucleosomal DNA size. The arrangement of histones along DNA and general compactness of nucleosomes were shown to be rather similar in transcriptionally active and inactive genomic regions. On the other hand, nucleosomes within transcriptionally active chromatin are characterized by a larger size of nucleosomal DNA produced by micrococcal nuclease digestion and some peculiarity in electrophoretic mobility. Images PMID:3144704

  2. Structure of RCC1 chromatin factor bound to the nucleosome core particle

    SciTech Connect

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

    2010-11-11

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

  3. Site-specific DNA repair at the nucleosome level in a yeast minichromosome

    SciTech Connect

    Smerdon, M.J.; Thoma, F. )

    1990-05-18

    The rate of excision repair of UV-induced pyrimidine dimers (PDs) was measured at specific sites in each strand of a yeast minichromosome containing an active gene (URA3), a replication origin (ARS1), and positioned nucleosomes. All six PD sites analyzed in the transcribed URA3 strand were repaired more rapidly (greater than 5-fold on average) than any of the nine PD sites analyzed in the nontranscribed strand. Efficient repair also occurred in both strands of a disrupted TRP1 gene (ten PD sites), containing four unstable nucleosomes, and in a nucleosome gap at the 5' end of URA3 (two PD sites). Conversely, slow repair occurred in both strands immediately downstream of the URA3 gene (12 of 14 PD sites). This region contains the ARS1 consensus sequence, a nucleosome gap, and two stable nucleosomes. Thus, modulation of DNA repair occurs in a simple yeast minichromosome and correlates with gene expression, nucleosome stability, and (possibly) control of replication.

  4. Crystal structure of the nucleosome containing ultraviolet light-induced cyclobutane pyrimidine dimer.

    PubMed

    Horikoshi, Naoki; Tachiwana, Hiroaki; Kagawa, Wataru; Osakabe, Akihisa; Matsumoto, Syota; Iwai, Shigenori; Sugasawa, Kaoru; Kurumizaka, Hitoshi

    2016-02-26

    The cyclobutane pyrimidine dimer (CPD) is induced in genomic DNA by ultraviolet (UV) light. In mammals, this photolesion is primarily induced within nucleosomal DNA, and repaired exclusively by the nucleotide excision repair (NER) pathway. However, the mechanism by which the CPD is accommodated within the nucleosome has remained unknown. We now report the crystal structure of a nucleosome containing CPDs. In the nucleosome, the CPD induces only limited local backbone distortion, and the affected bases are accommodated within the duplex. Interestingly, one of the affected thymine bases is located within 3.0 Å from the undamaged complementary adenine base, suggesting the formation of complementary hydrogen bonds in the nucleosome. We also found that UV-DDB, which binds the CPD at the initial stage of the NER pathway, also efficiently binds to the nucleosomal CPD. These results provide important structural and biochemical information for understanding how the CPD is accommodated and recognized in chromatin.

  5. Statistical mechanics of nucleosome ordering by chromatin-structure-induced two-body interactions.

    PubMed

    Chereji, Răzvan V; Tolkunov, Denis; Locke, George; Morozov, Alexandre V

    2011-05-01

    One-dimensional arrays of nucleosomes (DNA-bound histone octamers separated by stretches of linker DNA) fold into higher-order chromatin structures which ultimately make up eukaryotic chromosomes. Chromatin structure formation leads to 10-11 base pair (bp) discretization of linker lengths caused by the smaller free energy cost of packaging nucleosomes into regular chromatin fibers if their rotational setting (defined by the DNA helical twist) is conserved. We describe nucleosome positions along the fiber using a thermodynamic model of finite-size particles with both intrinsic histone-DNA interactions and an effective two-body potential. We infer one- and two-body energies directly from high-throughput maps of nucleosome positions. We show that higher-order chromatin structure helps explains in vitro and in vivo nucleosome ordering in transcribed regions, and plays a leading role in establishing well-known 10-11 bp genome-wide periodicity of nucleosome positions.

  6. Statistical mechanics of nucleosome ordering by chromatin-structure-induced two-body interactions

    NASA Astrophysics Data System (ADS)

    Chereji, Răzvan V.; Tolkunov, Denis; Locke, George; Morozov, Alexandre V.

    2011-05-01

    One-dimensional arrays of nucleosomes (DNA-bound histone octamers separated by stretches of linker DNA) fold into higher-order chromatin structures which ultimately make up eukaryotic chromosomes. Chromatin structure formation leads to 10-11 base pair (bp) discretization of linker lengths caused by the smaller free energy cost of packaging nucleosomes into regular chromatin fibers if their rotational setting (defined by the DNA helical twist) is conserved. We describe nucleosome positions along the fiber using a thermodynamic model of finite-size particles with both intrinsic histone-DNA interactions and an effective two-body potential. We infer one- and two-body energies directly from high-throughput maps of nucleosome positions. We show that higher-order chromatin structure helps explains in vitro and in vivo nucleosome ordering in transcribed regions, and plays a leading role in establishing well-known 10-11 bp genome-wide periodicity of nucleosome positions.

  7. Regulation of V(D)J recombination by nucleosome positioning at recombination signal sequences

    PubMed Central

    Baumann, Matthias; Mamais, Adamantios; McBlane, Fraser; Xiao, Hua; Boyes, Joan

    2003-01-01

    A key component in the regulation of V(D)J recombination is control of the accessibility of RAG proteins to recombination signal sequences (RSS). Nucleosomes are known to inhibit this accessibility. We show here that the signal sequence itself represses accessibility by causing nucleosome positioning over the RSS. This positioning is mediated, in vitro and in vivo, by the conserved nonamer of the RSS. Consistent with this strong positioning, nucleosomes at RSSs are resistant to remodelling by nucleosome sliding. In vivo we find that consensus RSSs are preferentially protected, whereas those that lack a consensus nonamer, including some cryptic RSSs, fail to position nucleosomes. Decreased protection of these non-consensus RSSs correlates with their increased use in recombination assays. We therefore suggest that nucleosome positioning by RSSs provides a previously unanticipated level of protection and regulation of V(D)J recombination. PMID:14517257

  8. Regulation of V(D)J recombination by nucleosome positioning at recombination signal sequences.

    PubMed

    Baumann, Matthias; Mamais, Adamantios; McBlane, Fraser; Xiao, Hua; Boyes, Joan

    2003-10-01

    A key component in the regulation of V(D)J recombination is control of the accessibility of RAG proteins to recombination signal sequences (RSS). Nucleosomes are known to inhibit this accessibility. We show here that the signal sequence itself represses accessibility by causing nucleosome positioning over the RSS. This positioning is mediated, in vitro and in vivo, by the conserved nonamer of the RSS. Consistent with this strong positioning, nucleosomes at RSSs are resistant to remodelling by nucleosome sliding. In vivo we find that consensus RSSs are preferentially protected, whereas those that lack a consensus nonamer, including some cryptic RSSs, fail to position nucleosomes. Decreased protection of these non-consensus RSSs correlates with their increased use in recombination assays. We therefore suggest that nucleosome positioning by RSSs provides a previously unanticipated level of protection and regulation of V(D)J recombination.

  9. Thermal fluctuation spectroscopy in histone and nucleosomes during denaturation

    NASA Astrophysics Data System (ADS)

    Raychaudhuri, Arup; Nagapriya, K. S.

    2007-03-01

    Thermal stability of biomolecules is an important issue. We have studied thermal denaturation of histone and nucleosome using precision thermal fluctuation spectroscopy (TFS) . - a problem that we believe has not been studied experimentally before. TFS uses a very sensitive noise calorimeter which can detect thermal fluctuations of micro Kelvin at around room temperature. We find that the thermal denaturation of histones (in particular H1) as well as that of the nucleosome are associated with large fluctuations, which are few orders higher than those away from the denaturation temperature. It involves large energy exchange which can be few tens of kBT0 (T0=300K). It appears that the denaturation occurs in three distinct steps 1. breaking of bonds leading to the cooling jumps, 2. the change in its secondary, tertiary structure leading to slow dynamics and 3. formation of bonds as it is unfolding and in the newly folded high temperature phase which accounts for the heating jumps.

  10. Anisotropic flexibility of DNA and the nucleosomal structure.

    PubMed Central

    Zhurkin, V B; Lysov, Y P; Ivanov, V I

    1979-01-01

    Potential energy calculations of the DNA duplex dimeric subunit show that the double helix may be bent in the direction of minor and major grooves much more easily than in other directions. It is found that the total winding angle of DNA decreases upon such bending. A new model for DNA folding in the nucleosome is proposed on the basis of these findings according to which the DNA molecule is kinked each fifth base pair to the side of the minor and major grooves alternatively. The model explains the known contradiction between a C-like circular dichroism for the nucleosomal DNA and the nuclease digestion data, which testify to the B-form of DNA. PMID:440969

  11. Regulation of Replication Fork Advance and Stability by Nucleosome Assembly.

    PubMed

    Prado, Felix; Maya, Douglas

    2017-01-24

    The advance of replication forks to duplicate chromosomes in dividing cells requires the disassembly of nucleosomes ahead of the fork and the rapid assembly of parental and de novo histones at the newly synthesized strands behind the fork. Replication-coupled chromatin assembly provides a unique opportunity to regulate fork advance and stability. Through post-translational histone modifications and tightly regulated physical and genetic interactions between chromatin assembly factors and replisome components, chromatin assembly: (1) controls the rate of DNA synthesis and adjusts it to histone availability; (2) provides a mechanism to protect the integrity of the advancing fork; and (3) regulates the mechanisms of DNA damage tolerance in response to replication-blocking lesions. Uncoupling DNA synthesis from nucleosome assembly has deleterious effects on genome integrity and cell cycle progression and is linked to genetic diseases, cancer, and aging.

  12. Regulation of Replication Fork Advance and Stability by Nucleosome Assembly

    PubMed Central

    Prado, Felix; Maya, Douglas

    2017-01-01

    The advance of replication forks to duplicate chromosomes in dividing cells requires the disassembly of nucleosomes ahead of the fork and the rapid assembly of parental and de novo histones at the newly synthesized strands behind the fork. Replication-coupled chromatin assembly provides a unique opportunity to regulate fork advance and stability. Through post-translational histone modifications and tightly regulated physical and genetic interactions between chromatin assembly factors and replisome components, chromatin assembly: (1) controls the rate of DNA synthesis and adjusts it to histone availability; (2) provides a mechanism to protect the integrity of the advancing fork; and (3) regulates the mechanisms of DNA damage tolerance in response to replication-blocking lesions. Uncoupling DNA synthesis from nucleosome assembly has deleterious effects on genome integrity and cell cycle progression and is linked to genetic diseases, cancer, and aging. PMID:28125036

  13. An Advanced Coarse-Grained Nucleosome Core Particle Model for Computer Simulations of Nucleosome-Nucleosome Interactions under Varying Ionic Conditions

    PubMed Central

    Fan, Yanping; Korolev, Nikolay; Lyubartsev, Alexander P.; Nordenskiöld, Lars

    2013-01-01

    In the eukaryotic cell nucleus, DNA exists as chromatin, a compact but dynamic complex with histone proteins. The first level of DNA organization is the linear array of nucleosome core particles (NCPs). The NCP is a well-defined complex of 147 bp DNA with an octamer of histones. Interactions between NCPs are of paramount importance for higher levels of chromatin compaction. The polyelectrolyte nature of the NCP implies that nucleosome-nucleosome interactions must exhibit a great influence from both the ionic environment as well as the positively charged and highly flexible N-terminal histone tails, protruding out from the NCP. The large size of the system precludes a modelling analysis of chromatin at an all-atom level and calls for coarse-grained approximations. Here, a model of the NCP that include the globular histone core and the flexible histone tails described by one particle per each amino acid and taking into account their net charge is proposed. DNA wrapped around the histone core was approximated at the level of two base pairs represented by one bead (bases and sugar) plus four beads of charged phosphate groups. Computer simulations, using a Langevin thermostat, in a dielectric continuum with explicit monovalent (K+), divalent (Mg2+) or trivalent (Co(NH3)63+) cations were performed for systems with one or ten NCPs. Increase of the counterion charge results in a switch from repulsive NCP-NCP interaction in the presence of K+, to partial aggregation with Mg2+ and to strong mutual attraction of all 10 NCPs in the presence of CoHex3+. The new model reproduced experimental results and the structure of the NCP-NCP contacts is in agreement with available data. Cation screening, ion-ion correlations and tail bridging contribute to the NCP-NCP attraction and the new NCP model accounts for these interactions. PMID:23418426

  14. An advanced coarse-grained nucleosome core particle model for computer simulations of nucleosome-nucleosome interactions under varying ionic conditions.

    PubMed

    Fan, Yanping; Korolev, Nikolay; Lyubartsev, Alexander P; Nordenskiöld, Lars

    2013-01-01

    In the eukaryotic cell nucleus, DNA exists as chromatin, a compact but dynamic complex with histone proteins. The first level of DNA organization is the linear array of nucleosome core particles (NCPs). The NCP is a well-defined complex of 147 bp DNA with an octamer of histones. Interactions between NCPs are of paramount importance for higher levels of chromatin compaction. The polyelectrolyte nature of the NCP implies that nucleosome-nucleosome interactions must exhibit a great influence from both the ionic environment as well as the positively charged and highly flexible N-terminal histone tails, protruding out from the NCP. The large size of the system precludes a modelling analysis of chromatin at an all-atom level and calls for coarse-grained approximations. Here, a model of the NCP that include the globular histone core and the flexible histone tails described by one particle per each amino acid and taking into account their net charge is proposed. DNA wrapped around the histone core was approximated at the level of two base pairs represented by one bead (bases and sugar) plus four beads of charged phosphate groups. Computer simulations, using a Langevin thermostat, in a dielectric continuum with explicit monovalent (K(+)), divalent (Mg(2+)) or trivalent (Co(NH(3))(6) (3+)) cations were performed for systems with one or ten NCPs. Increase of the counterion charge results in a switch from repulsive NCP-NCP interaction in the presence of K(+), to partial aggregation with Mg(2+) and to strong mutual attraction of all 10 NCPs in the presence of CoHex(3+). The new model reproduced experimental results and the structure of the NCP-NCP contacts is in agreement with available data. Cation screening, ion-ion correlations and tail bridging contribute to the NCP-NCP attraction and the new NCP model accounts for these interactions.

  15. Multiple functions of nucleosomes and regulatory factors in transcription.

    PubMed

    Workman, J L; Buchman, A R

    1993-03-01

    The in vivo packaging of DNA with histone proteins to form chromatin makes its transcription a difficult process. Biochemical and genetic studies are beginning to reveal mechanistic details of how transcriptional regulatory factors confront at least two hurdles created by nucleosomes, the primary structural unit of chromatin. Regulatory factors must gain access to their respective binding sites and activate the formation of transcription complexes at core promoter elements. Distinct regulatory factors may be specialized to perform these functions.

  16. Identification of the amino acid residues responsible for stable nucleosome formation by histone H3.Y.

    PubMed

    Kujirai, Tomoya; Horikoshi, Naoki; Xie, Yan; Taguchi, Hiroyuki; Kurumizaka, Hitoshi

    2017-05-04

    Histone H3.Y is conserved among primates. We previously reported that exogenously produced H3.Y accumulates around transcription start sites, suggesting that it may play a role in transcription regulation. The H3.Y nucleosome forms a relaxed chromatin conformation with flexible DNA ends. The H3.Y-specific Lys42 residue is partly responsible for enhancing the flexibility of the nucleosomal DNA. To our surprise, we found that H3.Y stably associates with chromatin and nucleosomes in vivo and in vitro. However, the H3.Y residues responsible for its stable nucleosome incorporation have not been identified yet. In the present study, we performed comprehensive mutational analyses of H3.Y, and determined that the H3.Y C-terminal region including amino acid residues 124-135 is responsible for its stable association with DNA. Among the H3.Y C-terminal residues, the H3.Y Met124 residue significantly contributed to the stable DNA association with the H3.Y-H4 tetramer. The H3.Y M124I mutation substantially reduced the H3.Y-H4 association in the nucleosome. In contrast, the H3.Y K42R mutation affected the nucleosome stability less, although it contributes to the flexible DNA ends of the nucleosome. Therefore, these H3.Y-specific residues, Lys42 and Met124, play different and specific roles in nucleosomal DNA relaxation and stable nucleosome formation, respectively, in chromatin.

  17. Stabilization of Nucleosomes by Histone Tails and by FACT Revealed by spFRET Microscopy

    PubMed Central

    Valieva, Maria E.; Gerasimova, Nadezhda S.; Kudryashova, Kseniya S.; Kozlova, Anastasia L.; Kirpichnikov, Mikhail P.; Hu, Qi; Botuyan, Maria Victoria; Mer, Georges; Feofanov, Alexey V.; Studitsky, Vasily M.

    2017-01-01

    A correct chromatin structure is important for cell viability and is tightly regulated by numerous factors. Human protein complex FACT (facilitates chromatin transcription) is an essential factor involved in chromatin transcription and cancer development. Here FACT-dependent changes in the structure of single nucleosomes were studied with single-particle Förster resonance energy transfer (spFRET) microscopy using nucleosomes labeled with a donor-acceptor pair of fluorophores, which were attached to the adjacent gyres of DNA near the contact between H2A-H2B dimers. Human FACT and its version without the C-terminal domain (CTD) and the high mobility group (HMG) domain of the structure-specific recognition protein 1 (SSRP1) subunit did not change the structure of the nucleosomes, while FACT without the acidic C-terminal domains of the suppressor of Ty 16 (Spt16) and the SSRP1 subunits caused nucleosome aggregation. Proteolytic removal of histone tails significantly disturbed the nucleosome structure, inducing partial unwrapping of nucleosomal DNA. Human FACT reduced DNA unwrapping and stabilized the structure of tailless nucleosomes. CTD and/or HMG domains of SSRP1 are required for this FACT activity. In contrast, previously it has been shown that yeast FACT unfolds (reorganizes) nucleosomes using the CTD domain of SSRP1-like Pol I-binding protein 3 subunit (Pob3). Thus, yeast and human FACT complexes likely utilize the same domains for nucleosome reorganization and stabilization, respectively, and these processes are mechanistically similar. PMID:28067802

  18. Crystal structures of heterotypic nucleosomes containing histones H2A.Z and H2A

    PubMed Central

    Horikoshi, Naoki; Arimura, Yasuhiro; Taguchi, Hiroyuki; Kurumizaka, Hitoshi

    2016-01-01

    H2A.Z is incorporated into nucleosomes located around transcription start sites and functions as an epigenetic regulator for the transcription of certain genes. During transcriptional regulation, the heterotypic H2A.Z/H2A nucleosome containing one each of H2A.Z and H2A is formed. However, previous homotypic H2A.Z nucleosome structures suggested that the L1 loop region of H2A.Z would sterically clash with the corresponding region of canonical H2A in the heterotypic nucleosome. To resolve this issue, we determined the crystal structures of heterotypic H2A.Z/H2A nucleosomes. In the H2A.Z/H2A nucleosome structure, the H2A.Z L1 loop structure was drastically altered without any structural changes of the canonical H2A L1 loop, thus avoiding the steric clash. Unexpectedly, the heterotypic H2A.Z/H2A nucleosome is more stable than the homotypic H2A.Z nucleosome. These data suggested that the flexible character of the H2A.Z L1 loop plays an essential role in forming the stable heterotypic H2A.Z/H2A nucleosome. PMID:27358293

  19. Crystal structures of heterotypic nucleosomes containing histones H2A.Z and H2A.

    PubMed

    Horikoshi, Naoki; Arimura, Yasuhiro; Taguchi, Hiroyuki; Kurumizaka, Hitoshi

    2016-06-01

    H2A.Z is incorporated into nucleosomes located around transcription start sites and functions as an epigenetic regulator for the transcription of certain genes. During transcriptional regulation, the heterotypic H2A.Z/H2A nucleosome containing one each of H2A.Z and H2A is formed. However, previous homotypic H2A.Z nucleosome structures suggested that the L1 loop region of H2A.Z would sterically clash with the corresponding region of canonical H2A in the heterotypic nucleosome. To resolve this issue, we determined the crystal structures of heterotypic H2A.Z/H2A nucleosomes. In the H2A.Z/H2A nucleosome structure, the H2A.Z L1 loop structure was drastically altered without any structural changes of the canonical H2A L1 loop, thus avoiding the steric clash. Unexpectedly, the heterotypic H2A.Z/H2A nucleosome is more stable than the homotypic H2A.Z nucleosome. These data suggested that the flexible character of the H2A.Z L1 loop plays an essential role in forming the stable heterotypic H2A.Z/H2A nucleosome. © 2016 The Authors.

  20. Chromatin gels are auxetic due to cooperative nucleosome assembly and disassembly dynamics

    NASA Astrophysics Data System (ADS)

    Yamamoto, Tetsuya; Schiessel, Helmut

    2017-04-01

    We study “chromatin gels”, model systems for chromatin, to theoretically predict the conditions, under which such gels show negative Poisson's ratios. A chromatin gel shows phase separation due to an instability arising from the disassembly of nucleosomes by RNA polymerases during transcription. We predict a negative Poisson's ratio near a miscibility threshold due to the cooperative assembly and disassembly of nucleosomes. The Poisson's ratio becomes more negative with an increasing number of RNAP because the disassembly rate of nucleosomes increases. In contrast, the chromatin gel shows a positive Poisson's ratio far from the miscibility threshold because the assembly of nucleosomes is arrested by the expiration of freely diffusing histone proteins.

  1. Preferential Nucleosome Assembly at DNA Triplet Repeats from the Myotonic Dystrophy Gene

    NASA Astrophysics Data System (ADS)

    Wang, Yuh-Hwa; Amirhaeri, Sorour; Kang, Seongman; Wells, Robert D.; Griffith, Jack D.

    1994-07-01

    The expansion of CTG repeats in DNA occurs in or near genes involved in several human diseases, including myotonic dystrophy and Huntington's disease. Nucleosomes, the basic structural element of chromosomes, consist of 146 base pairs of DNA coiled about an octamer of histone proteins and mediate general transcriptional repression. Electron microscopy was used to examine in vitro the nucleosome assembly of DNA containing repeating CTG triplets. The efficiency of nucleosome formation increased with expanded triplet blocks, suggesting that such blocks may repress transcription through the creation of stable nucleosomes.

  2. Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure.

    PubMed

    North, Justin A; Šimon, Marek; Ferdinand, Michelle B; Shoffner, Matthew A; Picking, Jonathan W; Howard, Cecil J; Mooney, Alex M; van Noort, John; Poirier, Michael G; Ottesen, Jennifer J

    2014-04-01

    Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Posttranslational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and atomic force microscopy measurements of two DNA-histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA molecules wrapped around two histone octamers, and an altosome complex that contains one DNA molecule wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA molecules, whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA molecules. These results are the first report of a histone PTM significantly altering the nucleosome structure.

  3. The nucleosome landscape of Plasmodium falciparum reveals chromatin architecture and dynamics of regulatory sequences.

    PubMed

    Kensche, Philip Reiner; Hoeijmakers, Wieteke Anna Maria; Toenhake, Christa Geeke; Bras, Maaike; Chappell, Lia; Berriman, Matthew; Bártfai, Richárd

    2016-03-18

    In eukaryotes, the chromatin architecture has a pivotal role in regulating all DNA-associated processes and it is central to the control of gene expression. For Plasmodium falciparum, a causative agent of human malaria, the nucleosome positioning profile of regulatory regions deserves particular attention because of their extreme AT-content. With the aid of a highly controlled MNase-seq procedure we reveal how positioning of nucleosomes provides a structural and regulatory framework to the transcriptional unit by demarcating landmark sites (transcription/translation start and end sites). In addition, our analysis provides strong indications for the function of positioned nucleosomes in splice site recognition. Transcription start sites (TSSs) are bordered by a small nucleosome-depleted region, but lack the stereotypic downstream nucleosome arrays, highlighting a key difference in chromatin organization compared to model organisms. Furthermore, we observe transcription-coupled eviction of nucleosomes on strong TSSs during intraerythrocytic development and demonstrate that nucleosome positioning and dynamics can be predictive for the functionality of regulatory DNA elements. Collectively, the strong nucleosome positioning over splice sites and surrounding putative transcription factor binding sites highlights the regulatory capacity of the nucleosome landscape in this deadly human pathogen.

  4. DNA-guided establishment of nucleosome patterns within coding regions of a eukaryotic genome.

    PubMed

    Beh, Leslie Y; Müller, Manuel M; Muir, Tom W; Kaplan, Noam; Landweber, Laura F

    2015-11-01

    A conserved hallmark of eukaryotic chromatin architecture is the distinctive array of well-positioned nucleosomes downstream from transcription start sites (TSS). Recent studies indicate that trans-acting factors establish this stereotypical array. Here, we present the first genome-wide in vitro and in vivo nucleosome maps for the ciliate Tetrahymena thermophila. In contrast with previous studies in yeast, we find that the stereotypical nucleosome array is preserved in the in vitro reconstituted map, which is governed only by the DNA sequence preferences of nucleosomes. Remarkably, this average in vitro pattern arises from the presence of subsets of nucleosomes, rather than the whole array, in individual Tetrahymena genes. Variation in GC content contributes to the positioning of these sequence-directed nucleosomes and affects codon usage and amino acid composition in genes. Given that the AT-rich Tetrahymena genome is intrinsically unfavorable for nucleosome formation, we propose that these "seed" nucleosomes--together with trans-acting factors--may facilitate the establishment of nucleosome arrays within genes in vivo, while minimizing changes to the underlying coding sequences.

  5. DNA methylation determines nucleosome occupancy in the 5'-CpG islands of tumor suppressor genes.

    PubMed

    Portela, A; Liz, J; Nogales, V; Setién, F; Villanueva, A; Esteller, M

    2013-11-21

    Promoter CpG island hypermethylation of tumor suppressor genes is an epigenetic hallmark of human cancer commonly associated with nucleosome occupancy and the transcriptional silencing of the neighboring gene. Nucleosomes can determine the underlying DNA methylation status. Herein, we show that the opposite is also true: DNA methylation can determine nucleosome positioning. Using a cancer model and digital nucleosome positioning techniques, we demonstrate that the induction of DNA hypomethylation events by genetic (DNMT1/DNMT3B deficient cells) or drug (a DNA demethylating agent) approaches is associated with the eviction of nucleosomes from previously hypermethylated CpG islands of tumor suppressor genes. Most importantly, the establishment of a stable cell line that restores DNMT1/DNMT3B deficiency shows that nucleosomes reoccupy their positions in de novo methylated CpG islands. Finally, we extend these results to the genomic level, combining a DNA methylation microarray and the nucleosome positioning technique. Using this global approach, we observe the dependency of nucleosome occupancy upon the DNA methylation status. Thus, our results suggest that there is a close association between hypermethylated CpG islands and the presence of nucleosomes, such that each of these epigenetic mechanisms can determine the recruitment of the other.

  6. ISWI proteins participate in the genome-wide nucleosome distribution in Arabidopsis.

    PubMed

    Li, Guang; Liu, Shujing; Wang, Jiawei; He, Jianfeng; Huang, Hai; Zhang, Yijing; Xu, Lin

    2014-05-01

    Chromatin is a highly organized structure with repetitive nucleosome subunits. Nucleosome distribution patterns, which contain information on epigenetic controls, are dynamically affected by ATP-dependent chromatin remodeling factors (remodelers). However, whether plants have specific nucleosome distribution patterns and how plant remodelers contribute to the pattern formation are not clear. In this study we used the micrococcal nuclease digestion followed by deep sequencing (MNase-seq) assay to show the genome-wide nucleosome pattern in Arabidopsis thaliana. We demonstrated that the nucleosome distribution patterns of Arabidopsis are associated with the gene expression level, and have several specific characteristics that are different from those of animals and yeast. In addition, we found that remodelers in the A. thaliana imitation switch (AtISWI) subfamily are important for the formation of the nucleosome distribution pattern. Double mutations in the AtISWI genes, CHROMATIN REMODELING 11 (CHR11) and CHR17, resulted in the loss of the evenly spaced nucleosome pattern in gene bodies, but did not affect nucleosome density, supporting a previous idea that the primary role of ISWI is to slide nucleosomes in gene bodies for pattern formation.

  7. Nonhistone nuclear high mobility group proteins 14 and 17 stabilize nucleosome core particles

    SciTech Connect

    Paton, A.E.; Wilkinson-Singley, E.; Olins, D.W.

    1983-11-10

    Nucleosome core particles form well defined complexes with the nuclear nonhistone proteins HMG 14 or 17. The binding of HMG 14 or 17 to nucleosomes results in greater stability of the nucleosomal DNA as shown by circular dichroism and thermal denaturation. Under appropriate conditions the binding is cooperative, and cooperativity is ionic strength dependent. The specificity and cooperative transitions of high mobility group (HMG) binding are preserved in 1 M urea. Specificity is lost in 4 M urea. Thermal denaturation and circular dichroism show a dramatic reversal of the effects of urea on nucleosomes when HMG 14 or 17 is bound, indicating stabilization of the nucleosome by HMG proteins. Complexes formed between reconstructed nucleosomes containing purified inner histones plus poly (dA-dT) and HMG 14 or 17 demonstrate that the HMG binding site requires only DNA and histones. Electron microscopy reveals no major structural alterations in the nucleosome upon binding of HMG 14 or 17. Cross-linking the nucleosome extensively with formaldehyde under cooperative HMG binding conditions does not prevent the ionic strength-dependent shift to noncooperative binding. This suggests mechanisms other than internal nucleosome conformational changes may be involved in cooperative HMG binding.

  8. Kinetic Control of Nucleosome Displacement by ISWI/ACF Chromatin Remodelers

    NASA Astrophysics Data System (ADS)

    Florescu, Ana-Maria; Schiessel, Helmut; Blossey, Ralf

    2012-09-01

    Chromatin structure is dynamically organized by chromatin remodelers, motor protein complexes which move and remove nucleosomes. The regulation of remodeler action has recently been proposed to underlie a kinetic proofreading scheme which combines the recognition of histone-tail states and the ATP-dependent loosening of DNA around nucleosomes. Members of the ISWI-family of remodelers additionally recognize linker length between nucleosomes. Here, we show that the additional proofreading step involving linker length alone is sufficient to promote the formation of regular arrays of nucleosomes. ATP-dependent remodeling by bidirectional motors is shown to reinforce positioning as compared to statistical positioning.

  9. Activator control of nucleosome occupancy in activation and repression of transcription.

    PubMed

    Bryant, Gene O; Prabhu, Vidya; Floer, Monique; Wang, Xin; Spagna, Dan; Schreiber, David; Ptashne, Mark

    2008-12-23

    The relationship between chromatin structure and gene expression is a subject of intense study. The universal transcriptional activator Gal4 removes promoter nucleosomes as it triggers transcription, but how it does so has remained obscure. The reverse process, repression of transcription, has often been correlated with the presence of nucleosomes. But it is not known whether nucleosomes are required for that effect. A new quantitative assay describes, for any given location, the fraction of DNA molecules in the population that bears a nucleosome at any given instant. This allows us to follow the time courses of nucleosome removal and reformation, in wild-type and mutant cells, upon activation (by galactose) and repression (by glucose) of the GAL genes of yeast. We show that upon being freed of its inhibitor Gal80 by the action of galactose, Gal4 quickly recruits SWI/SNF to the genes, and that nucleosome "remodeler" rapidly removes promoter nucleosomes. In the absence of SWI/SNF, Gal4's action also results in nucleosome removal and the activation of transcription, but both processes are significantly delayed. Addition of glucose to cells growing in galactose represses transcription. But if galactose remains present, Gal4 continues to work, recruiting SWI/SNF and maintaining the promoter nucleosome-free despite it being repressed. This requirement for galactose is obviated in a mutant in which Gal4 works constitutively. These results show how an activator's recruiting function can control chromatin structure both during gene activation and repression. Thus, both under activating and repressing conditions, the activator can recruit an enzymatic machine that removes promoter nucleosomes. Our results show that whereas promoter nucleosome removal invariably accompanies activation, reformation of nucleosomes is not required for repression. The finding that there are two routes to nucleosome removal and activation of transcription-one that requires the action of SWI

  10. H2A.Z nucleosomes enriched over active genes are homotypic.

    PubMed

    Weber, Christopher M; Henikoff, Jorja G; Henikoff, Steven

    2010-12-01

    Nucleosomes that contain the histone variant H2A.Z are enriched around transcriptional start sites, but the mechanistic basis for this enrichment is unknown. A single octameric nucleosome can contain two H2A.Z histones (homotypic) or one H2A.Z and one canonical H2A (heterotypic). To elucidate the function of H2A.Z, we generated high-resolution maps of homotypic and heterotypic Drosophila H2A.Z (H2Av) nucleosomes. Although homotypic and heterotypic H2A.Z nucleosomes mapped throughout most of the genome, homotypic nucleosomes were enriched and heterotypic nucleosomes were depleted downstream of active promoters and intron-exon junctions. The distribution of homotypic H2A.Z nucleosomes resembled that of classical active chromatin and showed evidence of disruption during transcriptional elongation. Both homotypic H2A.Z nucleosomes and classical active chromatin were depleted downstream of paused polymerases. Our results suggest that H2A.Z enrichment patterns result from intrinsic structural differences between heterotypic and homotypic H2A.Z nucleosomes that follow disruption during transcriptional elongation.

  11. Structure of human nucleosome containing the testis-specific histone variant TSH2B

    SciTech Connect

    Urahama, Takashi; Horikoshi, Naoki; Osakabe, Akihisa; Tachiwana, Hiroaki; Kurumizaka, Hitoshi

    2014-03-25

    The crystal structure of human nucleosome containing the testis-specific TSH2B variant has been determined. The TSH2B Ser85 residue does not interact with H4 in the nucleosome, and induces a local structural difference between TSH2B and H2B in nucleosomes. The human histone H2B variant TSH2B is highly expressed in testis and may function in the chromatin transition during spermatogenesis. In the present study, the crystal structure of the human testis-specific nucleosome containing TSH2B was determined at 2.8 Å resolution. A local structural difference between TSH2B and canonical H2B in nucleosomes was detected around the TSH2B-specific amino-acid residue Ser85. The TSH2B Ser85 residue does not interact with H4 in the nucleosome, but in the canonical nucleosome the H2B Asn84 residue (corresponding to the TSH2B Ser85 residue) forms water-mediated hydrogen bonds with the H4 Arg78 residue. In contrast, the other TSH2B-specific amino-acid residues did not induce any significant local structural changes in the TSH2B nucleosome. These findings may provide important information for understanding how testis-specific histone variants form nucleosomes during spermatogenesis.

  12. Dynamics of the Competition Between Nucleosome Unwrapping and DNA Binding Proteins

    NASA Astrophysics Data System (ADS)

    Bundschuh, Ralf

    2015-03-01

    In eukaryotic organisms DNA is tightly wrapped into nucleosomes. This bears the question how this DNA can be accessed in order to be copied, transcribed, or repaired. A process that allows access to the DNA is transient unwrapping of the DNA from the histone proteins. We have developed a quantitative model of this unwrapping process which we calibrate by comparison to nucleosome unzipping experiments by the Wang group. We then apply this model to quantitatively explain the dynamics of transcription factor binding within nucleosomal DNA. In this context, it has been well known that nucleosomes reduce the affinity for transcription factors to binding sites covered by the nucleosome. It has been assumed that this is due to a reduction in on-rate since a transcription factor can only bind when a rare thermal fluctuation of the nucleosome makes the DNA accessible. However, recent experimental data surprisingly shows that the off-rate of transcription factors is also strongly affected in the presence of a nucleosome. The application of our nucleosome unwrapping free energy landscape demonstrates that this increase in off-rate by several orders of magnitude is a consequence of a competition between partial binding events of dimeric transcription factors and the nucleosome. This material is based upon work supported by the National Science Foundation under Grant Nos. 1105458 and 1410172.

  13. Cracking the chromatin code: precise rule of nucleosome positioning.

    PubMed

    Trifonov, Edward N

    2011-03-01

    Various aspects of packaging DNA in eukaryotic cells are outlined in physical rather than biological terms. The informational and physical nature of packaging instructions encoded in DNA sequences is discussed with the emphasis on signal processing difficulties--very low signal-to-noise ratio and high degeneracy of the nucleosome positioning signal. As the author has been contributing to the field from its very onset in 1980, the review is mostly focused at the works of the author and his colleagues. The leading concept of the overview is the role of deformational properties of DNA in the nucleosome positioning. The target of the studies is to derive the DNA bendability matrix describing where along the DNA various dinucleotide elements should be positioned, to facilitate its bending in the nucleosome. Three different approaches are described leading to derivation of the DNA deformability sequence pattern, which is a simplified linear presentation of the bendability matrix. All three approaches converge to the same unique sequence motif CGRAAATTTYCG or, in binary form, YRRRRRYYYYYR, both representing the chromatin code.

  14. Naturally occurring nucleosome positioning signals in human exons and introns.

    PubMed

    Baldi, P; Brunak, S; Chauvin, Y; Krogh, A

    1996-11-08

    We describe the structural implications of a periodic pattern found in human exons and introns by hidden Markov models. We show that exons (besides the reading frame) have a specific sequential structure in the form of a pattern with triplet consensus non-T(A/T)G, and a minimal periodicity of roughly ten nucleotides. The periodic pattern is also present in intron sequences, although the strength per nucleotide is weaker. Using two independent profile methods based on triplet bendability parameters from DNase I experiments and nucleosome positioning data, we show that the pattern in multiple alignments of internal exon and intron sequences corresponds to a periodic "in phase" bending potential towards the major groove of the DNA. The nucleosome positioning data show that the consensus triplets (and their complements) have a preference for locations on a bent double helix where the major groove faces inward and is compressed. The in-phase triplets are located adjacent to GCC/GGC triplets known to have the strongest bias in their positioning on the nuclesome. Analysis of mRNA sequences encoding proteins with known tertiary structure exclude the possibility that the pattern is a consequence of the previously well-known periodicity caused by the encoding of alpha-helices in proteins. Finally, we discuss the relation between the bending potential of coding and non-coding regions and its impact on the translational positioning of nucleosomes and the recognition of genes by the transcriptional machinery.

  15. Nucleosomes and neutrophil extracellular traps in septic and burn patients.

    PubMed

    Kaufman, Tomás; Magosevich, Débora; Moreno, María Carolina; Guzman, María Alejandra; D'Atri, Lina Paola; Carestia, Agostina; Fandiño, María Eugenia; Fondevila, Carlos; Schattner, Mirta

    2017-08-29

    NETosis is a host defense mechanism associated with inflammation and tissue damage. Experimental models show that platelets and von Willebrand factor (VWF) are key elements for intravascular NETosis. We determined NETosis in septic and burn patients at 1 and 4days post-admission (dpa). Nucleosomes were elevated in patients. In septics, they correlated with Human Neutrophil Elastase (HNE)-DNA complexes and SOFA score at 1dpa, and were associated with mortality. Patient's neutrophils had spontaneous NETosis and were unresponsive to stimulation. Although platelet P-selectin and TNF-α were increased in both groups, higher platelet TLR4 expression, VWF levels and IL-6 were found in septics at 1dpa. Neither platelet activation markers nor cytokines correlated with nucleosomes or HNE-DNA. Nucleosomes could be indicators of organ damage and predictors of mortality in septic but not in burn patients. Platelet activation, VWF and cytokines do not appear to be key mediators of NETosis in these patient groups. Copyright © 2017. Published by Elsevier Inc.

  16. Curtailing patient-specific IMRT QA procedures from 2D dose error distribution.

    PubMed

    Kurosu, Keita; Sumida, Iori; Mizuno, Hirokazu; Otani, Yuki; Oda, Michio; Isohashi, Fumiaki; Seo, Yuji; Suzuki, Osamu; Ogawa, Kazuhiko

    2016-06-01

    A patient-specific quality assurance (QA) test is conducted to verify the accuracy of dose delivery. It generally consists of three verification processes: the absolute point dose difference, the planar dose differences at each gantry angle, and the planar dose differences by 3D composite irradiation. However, this imposes a substantial workload on medical physicists. The objective of this study was to determine whether our novel method that predicts the 3D delivered dose allows certain patient-specific IMRT QAs to be curtailed. The object was IMRT QA for the pelvic region with regard to point dose and composite planar dose differences. We compared measured doses, doses calculated in the treatment planning system, and doses predicted by in-house software. The 3D predicted dose was reconstructed from the per-field measurement by incorporating the relative dose error distribution into the original dose grid of each beam. All point dose differences between the measured and the calculated dose were within ±3%, whereas 93.3% of them between the predicted and the calculated dose were within ±3%. As for planar dose differences, the gamma passing rates between the calculated and the predicted dose were higher than those between the calculated and the measured dose. Comparison and statistical analysis revealed a correlation between the predicted and the measured dose with regard to both point dose and planar dose differences. We concluded that the prediction-based approach is an accurate substitute for the conventional measurement-based approach in IMRT QA for the pelvic region. Our novel approach will help medical physicists save time on IMRT QA.

  17. Assessing the efficacy of melatonin to curtail benzodiazepine/Z drug abuse.

    PubMed

    Cardinali, Daniel P; Golombek, Diego A; Rosenstein, Ruth E; Brusco, Luis I; Vigo, Daniel E

    2016-07-01

    The abuse of benzodiazepine (BZP) and Z drugs has become, due to the tolerance and dependence they produce, a serious public health problem. Thirty years ago, we demonstrated in experimental animals the interaction of melatonin with central BZD receptors, and in 1997 we published the first series of elderly patients who reduced BZP consumption after melatonin treatment. Almost every single neuron in the hypothalamic suprachiasmatic nuclei (SCN), the central pacemaker of the circadian system, contains γ-aminobutyric acid (GABA) and many results in animals point out to a melatonin interaction with GABA-containing neurons. In addition, central-type BZD antagonism, that obliterates GABAA receptor function, blunted most behavioral effects of melatonin including sleep. Melatonin is involved in the regulation of human sleep. This is supported by the temporal relationship between the rise of plasma melatonin levels and sleep propensity as well as by the sleep-promoting effects of exogenously administered melatonin. Both meta-analyses and consensus agreements give support to the therapeutic use of melatonin in sleep disorders. This action is attributed to MT1 and MT2 melatoninergic receptors localized in the SCN, as well as in other brain areas. This review discusses available data on the efficacy of melatonin to curtail chronic BZD/Z drug use in insomnia patients. A major advantage is that melatonin has a very safe profile, it is usually remarkably well tolerated and, in some studies, it has been administered to patients at very large doses and for long periods of time, without any potentiality of abuse. Further studies on this application of melatonin are warranted.

  18. Curtailing patient-specific IMRT QA procedures from 2D dose error distribution

    PubMed Central

    Kurosu, Keita; Sumida, Iori; Mizuno, Hirokazu; Otani, Yuki; Oda, Michio; Isohashi, Fumiaki; Seo, Yuji; Suzuki, Osamu; Ogawa, Kazuhiko

    2016-01-01

    A patient-specific quality assurance (QA) test is conducted to verify the accuracy of dose delivery. It generally consists of three verification processes: the absolute point dose difference, the planar dose differences at each gantry angle, and the planar dose differences by 3D composite irradiation. However, this imposes a substantial workload on medical physicists. The objective of this study was to determine whether our novel method that predicts the 3D delivered dose allows certain patient-specific IMRT QAs to be curtailed. The object was IMRT QA for the pelvic region with regard to point dose and composite planar dose differences. We compared measured doses, doses calculated in the treatment planning system, and doses predicted by in-house software. The 3D predicted dose was reconstructed from the per-field measurement by incorporating the relative dose error distribution into the original dose grid of each beam. All point dose differences between the measured and the calculated dose were within ±3%, whereas 93.3% of them between the predicted and the calculated dose were within ±3%. As for planar dose differences, the gamma passing rates between the calculated and the predicted dose were higher than those between the calculated and the measured dose. Comparison and statistical analysis revealed a correlation between the predicted and the measured dose with regard to both point dose and planar dose differences. We concluded that the prediction-based approach is an accurate substitute for the conventional measurement-based approach in IMRT QA for the pelvic region. Our novel approach will help medical physicists save time on IMRT QA. PMID:26661854

  19. Curtailing Agricultural Pumping in an Era of Extended Drought: Infusing Science and Leagality into a Common Hydrologic Framework

    NASA Astrophysics Data System (ADS)

    Carroll, R. W. H.; Pohll, G.; Benedict, J.; Felling, R.

    2016-12-01

    Many arid and semi-arid agricultural systems of the Great Basin in the western United States depend on supplemental groundwater pumping to augment diminished surface water flows during periods of drought. As droughts become longer and more severe in the region, unprecedented drawdown in these aquifer systems has occurred with legal and environmental implications on both surface and groundwater. The Walker River in the Great Basin supports extensive agriculture in the region and is the sole perennial stream to one of the few desert terminal lakes in North America. Continuous declines in the lake have spurred extensive research into management options to balance demands of agriculture and increase water deliveries to the lake. Smith and Mason Valleys are important agricultural centers within the Walker Basin. In 2015 the region entered its fifth year of drought and both valleys were the focus of curtailment orders to restrict the use of supplemental groundwater rights. To aid management decisions, hydrologic models were developed that simulate complex feedbacks between surface diversions, crop consumptive needs, groundwater recharge, return flow, and groundwater-surface water interactions. Demand-driven pumping that incorporates priority dates and maximum duty allocations are directly input to the hydrologic model to allow an assessment of groundwater curtailment options under a variety of drought scenarios to meet targeted water levels and downstream conveyance of surface water in a legally defensible framework. Hydrologic results using a sliding scale approach to priority based curtailment are presented in the arena of stakeholder participation and response.

  20. Enzymatic and chemical mapping of nucleosome distribution in purified micro- and macronuclei of the ciliated model organism, Tetrahymena thermophila.

    PubMed

    Chen, Xiao; Gao, Shan; Liu, Yifan; Wang, Yuanyuan; Wang, Yurui; Song, Weibo

    2016-09-01

    Genomic distribution of the nucleosome, the basic unit of chromatin, contains important epigenetic information. To map nucleosome distribution in structurally and functionally differentiated micronucleus (MIC) and macronucleus (MAC) of the ciliate Tetrahymena thermophila, we have purified MIC and MAC and performed micrococcal nuclease (MNase) digestion as well as hydroxyl radical cleavage. Different factors that may affect MNase digestion were examined, to optimize mono-nucleosome production. Mono-nucleosome purity was further improved by ultracentrifugation in a sucrose gradient. As MNase concentration increased, nucleosomal DNA sizes in MIC and MAC converged on 147 bp, as expected for the nucleosome core particle. Both MNase digestion and hydroxyl radical cleavage consistently showed a nucleosome repeat length of ~200 bp in MAC of Tetrahymena, supporting ~50 bp of linker DNA. Our work has systematically tested methods currently available for mapping nucleosome distribution in Tetrahymena, and provided a solid foundation for future epigenetic studies in this ciliated model organism.

  1. Nucleosome positioning and kinetics near transcription-start-site barriers are controlled by interplay between active remodeling and DNA sequence.

    PubMed

    Parmar, Jyotsana J; Marko, John F; Padinhateeri, Ranjith

    2014-01-01

    We investigate how DNA sequence, ATP-dependent chromatin remodeling and nucleosome-depleted 'barriers' co-operate to determine the kinetics of nucleosome organization, in a stochastic model of nucleosome positioning and dynamics. We find that 'statistical' positioning of nucleosomes against 'barriers', hypothesized to control chromatin structure near transcription start sites, requires active remodeling and therefore cannot be described using equilibrium statistical mechanics. We show that, unlike steady-state occupancy, DNA site exposure kinetics near a barrier is dominated by DNA sequence rather than by proximity to the barrier itself. The timescale for formation of positioning patterns near barriers is proportional to the timescale for active nucleosome eviction. We also show that there are strong gene-to-gene variations in nucleosome positioning near barriers, which are eliminated by averaging over many genes. Our results suggest that measurement of nucleosome kinetics can reveal information about sequence-dependent regulation that is not apparent in steady-state nucleosome occupancy.

  2. Establishment of a promoter-based chromatin architecture on recently replicated DNA can accommodate variable inter-nucleosome spacing

    PubMed Central

    Fennessy, Ross T.; Owen-Hughes, Tom

    2016-01-01

    Nucleosomes, the fundamental subunits of eukaryotic chromatin, are organized with respect to transcriptional start sites. A major challenge to the persistence of this organization is the disassembly of nucleosomes during DNA replication. Here, we use complimentary approaches to map the locations of nucleosomes on recently replicated DNA. We find that nucleosomes are substantially realigned with promoters during the minutes following DNA replication. As a result, the nucleosomal landscape is largely re-established before newly replicated chromosomes are partitioned into daughter cells and can serve as a platform for the re-establishment of gene expression programmes. When the supply of histones is disrupted through mutation of the chaperone Caf1, a promoter-based architecture is generated, but with increased inter-nucleosomal spacing. This indicates that the chromatin remodelling enzymes responsible for spacing nucleosomes are capable of organizing nucleosomes with a range of different linker DNA lengths. PMID:27106059

  3. DNase-seq predicts regions of rotational nucleosome stability across diverse human cell types

    PubMed Central

    Winter, Deborah R.; Song, Lingyun; Mukherjee, Sayan; Furey, Terrence S.; Crawford, Gregory E.

    2013-01-01

    DNase-seq is primarily used to identify nucleosome-depleted DNase I hypersensitive (DHS) sites genome-wide that correspond to active regulatory elements. However, ∼40 yr ago it was demonstrated that DNase I also digests with a ∼10-bp periodicity around nucleosomes matching the exposure of the DNA minor groove as it wraps around histones. Here, we use DNase-seq data from 49 samples representing diverse cell types to reveal this digestion pattern at individual loci and predict genomic locations where nucleosome rotational positioning, the orientation of DNA with respect to the histone surface, is stably maintained. We call these regions DNase I annotated regions of nucleosome stability (DARNS). Compared to MNase-seq experiments, we show DARNS correspond well to annotated nucleosomes. Interestingly, many DARNS are positioned over only one side of annotated nucleosomes, suggesting that the periodic digestion pattern attenuates over the nucleosome dyad. DARNS reproduce the arrangement of nucleosomes around transcription start sites and are depleted at ubiquitous DHS sites. We also generated DARNS from multiple lymphoblast cell line (LCL) samples. We found that LCL DARNS were enriched at DHS sites present in most of the original 49 samples but absent in LCLs, while multi-cell-type DARNS were enriched at LCL-specific DHS sites. This indicates that variably open DHS sites are often occupied by rotationally stable nucleosomes in cell types where the DHS site is closed. DARNS provide additional information about precise DNA orientation within individual nucleosomes not available from other nucleosome positioning assays and contribute to understanding the role of chromatin in gene regulation. PMID:23657885

  4. DNase-seq predicts regions of rotational nucleosome stability across diverse human cell types.

    PubMed

    Winter, Deborah R; Song, Lingyun; Mukherjee, Sayan; Furey, Terrence S; Crawford, Gregory E

    2013-07-01

    DNase-seq is primarily used to identify nucleosome-depleted DNase I hypersensitive (DHS) sites genome-wide that correspond to active regulatory elements. However, ≈ 40 yr ago it was demonstrated that DNase I also digests with a ≈ 10-bp periodicity around nucleosomes matching the exposure of the DNA minor groove as it wraps around histones. Here, we use DNase-seq data from 49 samples representing diverse cell types to reveal this digestion pattern at individual loci and predict genomic locations where nucleosome rotational positioning, the orientation of DNA with respect to the histone surface, is stably maintained. We call these regions DNase I annotated regions of nucleosome stability (DARNS). Compared to MNase-seq experiments, we show DARNS correspond well to annotated nucleosomes. Interestingly, many DARNS are positioned over only one side of annotated nucleosomes, suggesting that the periodic digestion pattern attenuates over the nucleosome dyad. DARNS reproduce the arrangement of nucleosomes around transcription start sites and are depleted at ubiquitous DHS sites. We also generated DARNS from multiple lymphoblast cell line (LCL) samples. We found that LCL DARNS were enriched at DHS sites present in most of the original 49 samples but absent in LCLs, while multi-cell-type DARNS were enriched at LCL-specific DHS sites. This indicates that variably open DHS sites are often occupied by rotationally stable nucleosomes in cell types where the DHS site is closed. DARNS provide additional information about precise DNA orientation within individual nucleosomes not available from other nucleosome positioning assays and contribute to understanding the role of chromatin in gene regulation.

  5. Determinants of nucleosome positioning and their influence on plant gene expression.

    PubMed

    Liu, Ming-Jung; Seddon, Alexander E; Tsai, Zing Tsung-Yeh; Major, Ian T; Floer, Monique; Howe, Gregg A; Shiu, Shin-Han

    2015-08-01

    Nucleosome positioning influences the access of transcription factors (TFs) to their binding sites and gene expression. Studies in plant, animal, and fungal models demonstrate similar nucleosome positioning patterns along genes and correlations between occupancy and expression. However, the relationships among nucleosome positioning, cis-regulatory element accessibility, and gene expression in plants remain undefined. Here we showed that plant nucleosome depletion occurs on specific 6-mer motifs and this sequence-specific nucleosome depletion is predictive of expression levels. Nucleosome-depleted regions in Arabidopsis thaliana tend to have higher G/C content, unlike yeast, and are centered on specific G/C-rich 6-mers, suggesting that intrinsic sequence properties, such as G/C content, cannot fully explain plant nucleosome positioning. These 6-mer motif sites showed higher DNase I hypersensitivity and are flanked by strongly phased nucleosomes, consistent with known TF binding sites. Intriguingly, this 6-mer-specific nucleosome depletion pattern occurs not only in promoter but also in genic regions and is significantly correlated with higher gene expression level, a phenomenon also found in rice but not in yeast. Among the 6-mer motifs enriched in genes responsive to treatment with the defense hormone jasmonate, there are no significant changes in nucleosome occupancy, suggesting that these sites are potentially preconditioned to enable rapid response without changing chromatin state significantly. Our study provides a global assessment of the joint contribution of nucleosome occupancy and motif sequences that are likely cis-elements to the control of gene expression in plants. Our findings pave the way for further understanding the impact of chromatin state on plant transcriptional regulatory circuits.

  6. The roles of the monomer length and nucleotide context of plant tandem repeats in nucleosome positioning.

    PubMed

    Levitsky, Victor G; Babenko, Vladimir N; Vershinin, Alexander V

    2014-01-01

    Similar to regularly spaced nucleosomes in chromatin, long tandem DNA arrays are composed of regularly alternating monomers that have almost identical primary DNA structures. Such a similarity in the structural organization makes these arrays especially interesting for studying the role of intrinsic DNA preferences in nucleosome positioning. We have studied the nucleosome formation potential of DNA tandem repeat families with different monomer lengths (ML). In total, 165 plant tandem repeat families from the PlantSat database (http://w3lamc.umbr.cas.cz/PlantSat/) were divided into two classes based on the number of nucleosome repeats in one DNA monomer. For predicting nucleosome formation potential, we developed the Phase method, which combines the advantages of multiple bioinformatics models. The Phase method was able to distinguish interfamily differences and intrafamily monomer variation and identify the influence of nucleotide context on nucleosome formation potential. Three main types of nucleosome arrangement in DNA tandem repeat arrays--regular, partially regular (partial), and flexible--were distinguished among a great variety of Phase profiles. The regular type, in which all nucleosomes of the monomer array are positioned in a context-dependent manner, is the most representative type of the class 1 families, with ML equal to or a multiple of the nucleosome repeat length (NRL). In the partially regular type, nucleotide context influences the positioning of only a subset of nucleosomes. The influence of the nucleotide context on nucleosome positioning has the least effect in the flexible type, which contains the greatest number of families (65). The majority of these families belong to class 2 and have nonmultiple ML to NRL ratios.

  7. Reactivity in ELISA with DNA-loaded nucleosomes in patients with proliferative lupus nephritis.

    PubMed

    Dieker, Jürgen; Schlumberger, Wolfgang; McHugh, Neil; Hamann, Philip; van der Vlag, Johan; Berden, Jo H

    2015-11-01

    Autoantibodies against nucleosomes are considered a hallmark of systemic lupus erythematosus (SLE). We compared in patients with proliferative lupus nephritis the diagnostic usefulness of a dsDNA-loaded nucleosome ELISA (anti-dsDNA-NcX) with ELISAs in which dsDNA or nucleosomes alone were coated. First, we analysed whether DNA loading on nucleosomes led to masking of epitopes by using defined monoclonal anti-DNA, anti-histone and nucleosome-specific autoantibodies to evaluate the accessibility of nucleosomal epitopes in the anti-dsDNA-NcX ELISA. Second, autoantibody levels were measured in these 3 ELISAs in 100 patients with proliferative lupus nephritis (LN) before immunosuppressive treatment and in 128 non-SLE disease controls. In patients with LN inter-assay comparisons and associations with clinical and serological parameters were analysed. The panel of monoclonal antibodies revealed that all epitopes were equally accessible in the anti-dsDNA-NcX ELISA as in the two other ELISAs. Patients with proliferative lupus nephritis were positive with dsDNA-loaded nucleosomes in 86%, with DNA in 66% and with nucleosomes in 85%. In the non-lupus disease control group these frequencies were 1.6% (2 out of 128) for both the anti-dsDNA-NcX and the anti-dsDNA ELISA and 0% in the anti-nucleosome ELISA. The levels in the anti-dsDNA-NcX ELISA were high in a group of patients with LN that showed absent reactivity in the anti-DNA or low levels in the anti-nucleosome ELISA. Anti-dsDNA-NcX positivity was associated with higher SLEDAI scores within this group. Within nucleosome-based ELISAs, we propose the anti-dsDNA-NcX ELISA as the preferred test system.

  8. Promoter-Targeted Histone Acetylation of Chromatinized Parvoviral Genome Is Essential for the Progress of Infection

    PubMed Central

    Mäntylä, Elina; Salokas, Kari; Oittinen, Mikko; Aho, Vesa; Mäntysaari, Pekka; Palmujoki, Lassi; Kalliolinna, Olli; Ihalainen, Teemu O.; Niskanen, Einari A.; Timonen, Jussi

    2016-01-01

    ABSTRACT The association of host histones with parvoviral DNA is poorly understood. We analyzed the chromatinization and histone acetylation of canine parvovirus DNA during infection by confocal imaging and in situ proximity ligation assay combined with chromatin immunoprecipitation and high-throughput sequencing. We found that during late infection, parvovirus replication bodies were rich in histones bearing modifications characteristic of transcriptionally active chromatin, i.e., histone H3 lysine 27 acetylation (H3K27ac). H3K27ac, in particular, was located in close proximity to the viral DNA-binding protein NS1. Importantly, our results show for the first time that in the chromatinized parvoviral genome, the two viral promoters in particular were rich in H3K27ac. Histone acetyltransferase (HAT) inhibitors efficiently interfered with the expression of viral proteins and infection progress. Altogether, our data suggest that the acetylation of histones on parvoviral DNA is essential for viral gene expression and the completion of the viral life cycle. IMPORTANCE Viral DNA introduced into cell nuclei is exposed to cellular responses to foreign DNA, including chromatinization and epigenetic silencing, both of which determine the outcome of infection. How the incoming parvovirus resists cellular epigenetic downregulation of its genes is not understood. Here, the critical role of epigenetic modifications in the regulation of parvovirus infection was demonstrated. We showed for the first time that a successful parvovirus infection is characterized by the deposition of nucleosomes with active histone acetylation on the viral promoter areas. The results provide new insights into the regulation of parvoviral gene expression, which is an important aspect of the development of parvovirus-based virotherapy. PMID:26842481

  9. Acetylation of H4 suppresses the repressive effects of the N-termini of histones H3/H4 and facilitates the formation of positively coiled DNA.

    PubMed

    Peterson, Sharon; Jackson, Vaughn

    2008-07-08

    We have studied the role of the N-termini of histones H3/H4 in the regulation of the conformational changes that occur in H3/H4 during their deposition on DNA by NAP1 (nucleosome assembly protein 1). Removal of the N-termini extensively increased the right-handed conformation of H3/H4 as assayed by the increased levels of positive coils that were formed on DNA. The osmolytes, TMAO, betaine, sarcosine, alanine, glycine, and proline to varying degrees, facilitated the formation of positive coils. The denaturant, urea (0.6 M), blocked the osmolyte effects, causing a preference of H3/H4 to form negative coils (the left-handed conformation). Acetylated H3/H4 also formed high levels of positive coils, and it is proposed that both the osmolytes and acetylation promote the formation of an alpha-helix in the N-termini. This structural change may ultimately explain a unique feature of transcription through nucleosomes, i.e., that H2A/H2B tends to be more mobile than H3/H4. By using combinations of H3 and H4 that were either acetylated or the N-termini removed, it was also determined that the N-terminus of H4 is primarily responsible for repressing the formation of positive coils. Additional gradient analyses indicate that NAP1 establishes an equilibrium with the H3/H4-DNA complexes. This equilibrium facilitates a histone saturation of the DNA, a unique state that promotes the right-handed conformation. NAP1 persists in the binding of the complexes through interaction with the N-terminus of H3, which may be a mechanism for subsequent remodeling of the nucleosome during transcription and replication.

  10. Genome-Wide Mapping of Nucleosome Positions in Yeast Using High-Resolution MNase ChIP-Seq

    PubMed Central

    Wal, Megha

    2016-01-01

    Eukaryotic DNA is packaged into chromatin where nucleosomes form the basic building unit. Knowing the precise positions of nucleosomes is important because they determine the accessibility of underlying regulatory DNA sequences. Here we describe a detailed method to map on a genomic scale the locations of nucleosomes with very high resolution. Micrococcal nuclease (MNase) digestion followed by chromatin immunoprecipitation and facilitated library construction for deep sequencing provides a simple and accurate map of nucleosome positions. PMID:22929772

  11. A positive role for nucleosome mobility in the transcriptional activity of chromatin templates: restriction by linker histones.

    PubMed Central

    Ura, K; Hayes, J J; Wolffe, A P

    1995-01-01

    Nucleosome mobility facilitates the transcription of chromatin templates containing only histone octamers. Inclusion of linker histones in chromatin inhibits nucleosome mobility, directs nucleosome positioning and represses transcription. Transcriptional repression by linker histone occurs preferentially on templates associated with histone octamers relative to naked DNA. Mobile nucleosomes and the restriction of mobility by linker histones might be expected to exert a major influence on the accessibility of chromatin to regulatory molecules. Images PMID:7641694

  12. Modulations of DNA Contacts by Linker Histones and Post-translational Modifications Determine the Mobility and Modifiability of Nucleosomal H3 Tails.

    PubMed

    Stützer, Alexandra; Liokatis, Stamatios; Kiesel, Anja; Schwarzer, Dirk; Sprangers, Remco; Söding, Johannes; Selenko, Philipp; Fischle, Wolfgang

    2016-01-21

    Post-translational histone modifications and linker histone incorporation regulate chromatin structure and genome activity. How these systems interface on a molecular level is unclear. Using biochemistry and NMR spectroscopy, we deduced mechanistic insights into the modification behavior of N-terminal histone H3 tails in different nucleosomal contexts. We find that linker histones generally inhibit modifications of different H3 sites and reduce H3 tail dynamics in nucleosomes. These effects are caused by modulations of electrostatic interactions of H3 tails with linker DNA and largely depend on the C-terminal domains of linker histones. In agreement, linker histone occupancy and H3 tail modifications segregate on a genome-wide level. Charge-modulating modifications such as phosphorylation and acetylation weaken transient H3 tail-linker DNA interactions, increase H3 tail dynamics, and, concomitantly, enhance general modifiability. We propose that alterations of H3 tail-linker DNA interactions by linker histones and charge-modulating modifications execute basal control mechanisms of chromatin function.

  13. Genome-wide nucleosome positioning is orchestrated by genomic regions associated with DNase I hypersensitivity in rice.

    PubMed

    Wu, Yufeng; Zhang, Wenli; Jiang, Jiming

    2014-05-01

    Nucleosome positioning dictates the DNA accessibility for regulatory proteins, and thus is critical for gene expression and regulation. It has been well documented that only a subset of nucleosomes are reproducibly positioned in eukaryotic genomes. The most prominent example of phased nucleosomes is the context of genes, where phased nucleosomes flank the transcriptional starts sites (TSSs). It is unclear, however, what factors determine nucleosome positioning in regions that are not close to genes. We mapped both nucleosome positioning and DNase I hypersensitive site (DHS) datasets across the rice genome. We discovered that DHSs located in a variety of contexts, both genic and intergenic, were flanked by strongly phased nucleosome arrays. Phased nucleosomes were also found to flank DHSs in the human genome. Our results suggest the barrier model may represent a general feature of nucleosome organization in eukaryote genomes. Specifically, regions bound with regulatory proteins, including intergenic regions, can serve as barriers that organize phased nucleosome arrays on both sides. Our results also suggest that rice DHSs often span a single, phased nucleosome, similar to the H2A.Z-containing nucleosomes observed in DHSs in the human genome.

  14. DNA-guided establishment of nucleosome patterns within coding regions of a eukaryotic genome

    PubMed Central

    Beh, Leslie Y.; Müller, Manuel M.; Muir, Tom W.; Kaplan, Noam; Landweber, Laura F.

    2015-01-01

    A conserved hallmark of eukaryotic chromatin architecture is the distinctive array of well-positioned nucleosomes downstream from transcription start sites (TSS). Recent studies indicate that trans-acting factors establish this stereotypical array. Here, we present the first genome-wide in vitro and in vivo nucleosome maps for the ciliate Tetrahymena thermophila. In contrast with previous studies in yeast, we find that the stereotypical nucleosome array is preserved in the in vitro reconstituted map, which is governed only by the DNA sequence preferences of nucleosomes. Remarkably, this average in vitro pattern arises from the presence of subsets of nucleosomes, rather than the whole array, in individual Tetrahymena genes. Variation in GC content contributes to the positioning of these sequence-directed nucleosomes and affects codon usage and amino acid composition in genes. Given that the AT-rich Tetrahymena genome is intrinsically unfavorable for nucleosome formation, we propose that these “seed” nucleosomes—together with trans-acting factors—may facilitate the establishment of nucleosome arrays within genes in vivo, while minimizing changes to the underlying coding sequences. PMID:26330564

  15. Novel nucleosomal particles containing core histones and linker DNA but no histone H1.

    PubMed

    Cole, Hope A; Cui, Feng; Ocampo, Josefina; Burke, Tara L; Nikitina, Tatiana; Nagarajavel, V; Kotomura, Naoe; Zhurkin, Victor B; Clark, David J

    2016-01-29

    Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these 'proto-chromatosomes' are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.

  16. Cse4 is Part of an Octameric Nucleosome in Budding Yeast

    PubMed Central

    Camahort, Raymond; Shivaraju, Manjunatha; Mattingly, Mark; Li, Bing; Nakanishi, Shima; Zhu, Dongxiao; Shilatifard, Ali; Workman, Jerry L.; Gerton, Jennifer L.

    2009-01-01

    The budding yeast CenH3 histone variant Cse4 localizes to centromeric nucleosomes and is required for kinetochore assembly and chromosome segregation. The exact composition of centromeric Cse4–containing nucleosomes is a subject of debate. ChIP-chip experiments and high resolution quantitative PCR confirm that there is a single Cse4 nucleosome at each centromere, and additional regions of the genome contain Cse4 nucleosomes at low levels. Using unbiased biochemical, cell biological, and genetic approaches we have tested the composition of Cse4-containing nucleosomes. Using micrococcal nuclease-treated chromatin, we find that Cse4 is associated with the histones H2A, H2B, and H4, but not H3 or the non-histone protein Scm3. Overexpression of Cse4 rescues the lethality of a scm3 deletion, indicating Scm3 is not essential for the formation of functional centromeric chromatin. Additionally, octameric Cse4 nucleosomes can be reconstituted in vitro. The Cse4-Cse4 interaction domain appears to be essential and interaction occurs in vivo in the centromeric nucleosome. Taken together, our experimental evidence supports the model that the Cse4-nucleosome is an octamer, containing two copies each of Cse4, H2A, H2B, and H4. PMID:19782029

  17. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells

    PubMed Central

    de Dieuleveult, Maud; Yen, Kuangyu; Hmitou, Isabelle; Depaux, Arnaud; Boussouar, Fayçal; Dargham, Daria Bou; Jounier, Sylvie; Humbertclaude, Hélène; Ribierre, Florence; Baulard, Céline; Farrell, Nina P.; Park, Bongsoo; Keime, Céline; Carrière, Lucie; Berlivet, Soizick; Gut, Marta; Gut, Ivo; Werner, Michel; Deleuze, Jean-François; Olaso, Robert; Aude, Jean-Christophe; Chantalat, Sophie; Pugh, B. Franklin; Gérard, Matthieu

    2015-01-01

    Summary ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers1–3 target specific nucleosomes to regulate transcription is unclear. Here, we present genome-wide remodeller-nucleosome interaction profiles for Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank MNase-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites (TSSs) are nevertheless chromatinized with non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and modifications (H3K4me3 and H3K27ac). RNA polymerase (pol) II therefore navigates hundreds of bp of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3′ end of the NFR. Transcriptome analysis upon remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers play either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs. PMID:26814966

  18. Asymmetric breathing motions of nucleosomal DNA and the role of histone tails

    NASA Astrophysics Data System (ADS)

    Chakraborty, Kaushik; Loverde, Sharon M.

    2017-08-01

    The most important packing unit of DNA in the eukaryotic cell is the nucleosome. It undergoes large-scale structural re-arrangements during different cell cycles. For example, the disassembly of the nucleosome is one of the key steps for DNA replication, whereas reassembly occurs after replication. Thus, conformational dynamics of the nucleosome is crucial for different DNA metabolic processes. We perform three different sets of atomistic molecular dynamics simulations of the nucleosome core particle at varying degrees of salt conditions for a total of 0.7 μs simulation time. We find that the conformational dynamics of the nucleosomal DNA tails are oppositely correlated from each other during the initial breathing motions. Furthermore, the strength of the interaction of the nucleosomal DNA tail with the neighboring H2A histone tail modulates the conformational state of the nucleosomal DNA tail. With increasing salt concentration, the degree of asymmetry in the conformation of the nucleosomal DNA tails decreases as both tails tend to unwrap. This direct correlation between the asymmetric breathing motions of the DNA tails and the H2A histone tails, and its decrease at higher salt concentrations, may play a significant role in the molecular pathway of unwrapping.

  19. Comparison of the Isw1a, Isw1b, and Isw2 nucleosome disrupting activities.

    PubMed

    Krajewski, Wladyslaw A

    2013-10-08

    The three Saccharomyces cerevisiae ISWI chromatin remodeling complexes, Isw1a, Isw1b, and Isw2, are implicated in the regularization of arrayed nucleosomes and regulation of gene activity. Although Isw1a and Isw1b are based on the same catalytic unit, in general, their functions in vivo do not overlap. To better understand the structural consequences of these complexes, we compared the putative nucleosome disrupting activities of the purified Isw1a, Isw1b, and Isw2. To account for the putative effects of nucleosomal environment, we employed reconstituted dinucleosomes in which the histone octamers were specifically positioned by the 146 base pair high-affinity nucleosome sequence "601". We have compared the MNase and deoxyribonuclease I protection patterns of remodeled nucleosome templates and evaluated the nucleosome destabilizing abilities of the Isw1a/b and Isw2 using restriction endonucleases. Although the Isw2 showed little evidence of nucleosome disassembly, the Isw1b remodeled dinucleosomes exhibited some common features with the ySwi-Snf remodeling products. The nuclease digestion data suggest that Isw1a can also promote ATP-dependent distortion of nucleosome structure, although less efficiently than the Isw1b complex.

  20. Nanopores suggest a negligible influence of CpG methylation on nucleosome packaging and stability.

    PubMed

    Langecker, Martin; Ivankin, Andrey; Carson, Spencer; Kinney, Shannon R M; Simmel, Friedrich C; Wanunu, Meni

    2015-01-14

    Nucleosomes are the fundamental repeating units of chromatin, and dynamic regulation of their positioning along DNA governs gene accessibility in eukaryotes. Although epigenetic factors have been shown to influence nucleosome structure and dynamics, the impact of DNA methylation on nucleosome packaging remains controversial. Further, all measurements to date have been carried out under zero-force conditions. In this paper, we present the first automated force measurements that probe the impact of CpG DNA methylation on nucleosome stability. In solid-state nanopore force spectroscopy, a nucleosomal DNA tail is captured into a pore and pulled on with a time-varying electrophoretic force until unraveling is detected. This is automatically repeated for hundreds of nucleosomes, yielding statistics of nucleosome lifetime vs electrophoretic force. The force geometry, which is similar to displacement forces exerted by DNA polymerases and helicases, reveals that nucleosome stability is sensitive to DNA sequence yet insensitive to CpG methylation. Our label-free method provides high-throughput data that favorably compares with other force spectroscopy experiments and is suitable for studying a variety of DNA-protein complexes.

  1. The impact of the HIRA histone chaperone upon global nucleosome architecture.

    PubMed

    Gal, Csenge; Moore, Karen M; Paszkiewicz, Konrad; Kent, Nicholas A; Whitehall, Simon K

    2015-01-01

    HIRA is an evolutionarily conserved histone chaperone that mediates replication-independent nucleosome assembly and is important for a variety of processes such as cell cycle progression, development, and senescence. Here we have used a chromatin sequencing approach to determine the genome-wide contribution of HIRA to nucleosome organization in Schizosaccharomyces pombe. Cells lacking HIRA experience a global reduction in nucleosome occupancy at gene sequences, consistent with the proposed role for HIRA in chromatin reassembly behind elongating RNA polymerase II. In addition, we find that at its target promoters, HIRA commonly maintains the full occupancy of the -1 nucleosome. HIRA does not affect global chromatin structure at replication origins or in rDNA repeats but is required for nucleosome occupancy in silent regions of the genome. Nucleosome organization associated with the heterochromatic (dg-dh) repeats located at the centromere is perturbed by loss of HIRA function and furthermore HIRA is required for normal nucleosome occupancy at Tf2 LTR retrotransposons. Overall, our data indicate that HIRA plays an important role in maintaining nucleosome architecture at both euchromatic and heterochromatic loci.

  2. Dynamic Nucleosome Movement Provides Structural Information of Topological Chromatin Domains in Living Human Cells

    PubMed Central

    Shinkai, Soya; Nozaki, Tadasu; Maeshima, Kazuhiro

    2016-01-01

    The mammalian genome is organized into submegabase-sized chromatin domains (CDs) including topologically associating domains, which have been identified using chromosome conformation capture-based methods. Single-nucleosome imaging in living mammalian cells has revealed subdiffusively dynamic nucleosome movement. It is unclear how single nucleosomes within CDs fluctuate and how the CD structure reflects the nucleosome movement. Here, we present a polymer model wherein CDs are characterized by fractal dimensions and the nucleosome fibers fluctuate in a viscoelastic medium with memory. We analytically show that the mean-squared displacement (MSD) of nucleosome fluctuations within CDs is subdiffusive. The diffusion coefficient and the subdiffusive exponent depend on the structural information of CDs. This analytical result enabled us to extract information from the single-nucleosome imaging data for HeLa cells. Our observation that the MSD is lower at the nuclear periphery region than the interior region indicates that CDs in the heterochromatin-rich nuclear periphery region are more compact than those in the euchromatin-rich interior region with respect to the fractal dimensions as well as the size. Finally, we evaluated that the average size of CDs is in the range of 100–500 nm and that the relaxation time of nucleosome movement within CDs is a few seconds. Our results provide physical and dynamic insights into the genome architecture in living cells. PMID:27764097

  3. The elongation factor Spt4/5 regulates RNA polymerase II transcription through the nucleosome

    PubMed Central

    Crickard, John B.; Lee, Jaehyoun; Lee, Tae-Hee

    2017-01-01

    Abstract RNA polymerase II (RNAPII) passes through the nucleosome in a coordinated manner, generating several intermediate nucleosomal states as it breaks and then reforms histone–DNA contacts ahead of and behind it, respectively. Several studies have defined transcription-induced nucleosome intermediates using only RNA Polymerase. However, RNAPII is decorated with elongation factors as it transcribes the genome. One such factor, Spt4/5, becomes an integral component of the elongation complex, making direct contact with the ‘jaws’ of RNAPII and nucleic acids in the transcription scaffold. We have characterized the effect of incorporating Spt4/5 into the elongation complex on transcription through the 601R nucleosome. Spt4/5 suppressed RNAPII pausing at the major H3/H4-induced arrest point, resulting in downstream re-positioning of RNAPII further into the nucleosome. Using a novel single molecule FRET system, we found that Spt4/5 affected the kinetics of DNA re-wrapping and stabilized a nucleosomal intermediate with partially unwrapped DNA behind RNAPII. Comparison of nucleosomes of different sequence polarities suggest that the strength of the DNA–histone interactions behind RNAPII specifies the Spt4/5 requirement. We propose that Spt4/5 may be important to coordinate the mechanical movement of RNAPII through the nucleosome with co-transcriptional chromatin modifications during transcription, which is affected by the strength of histone–DNA interactions. PMID:28379497

  4. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells.

    PubMed

    de Dieuleveult, Maud; Yen, Kuangyu; Hmitou, Isabelle; Depaux, Arnaud; Boussouar, Fayçal; Bou Dargham, Daria; Jounier, Sylvie; Humbertclaude, Hélène; Ribierre, Florence; Baulard, Céline; Farrell, Nina P; Park, Bongsoo; Keime, Céline; Carrière, Lucie; Berlivet, Soizick; Gut, Marta; Gut, Ivo; Werner, Michel; Deleuze, Jean-François; Olaso, Robert; Aude, Jean-Christophe; Chantalat, Sophie; Pugh, B Franklin; Gérard, Matthieu

    2016-02-04

    ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers target specific nucleosomes to regulate transcription is unclear. Here we present genome-wide remodeller-nucleosome interaction profiles for the chromatin remodellers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank micrococcal nuclease (MNase)-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites are nevertheless bound by non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and marked by H3K4me3 and H3K27ac modifications. RNA polymerase II therefore navigates hundreds of base pairs of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3' end of the NFR. Transcriptome analysis after remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers have either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs.

  5. Re-establishment of nucleosome occupancy during double-strand break repair in budding yeast.

    PubMed

    Tsabar, Michael; Hicks, Wade M; Tsaponina, Olga; Haber, James E

    2016-11-01

    Homologous recombination (HR) is an evolutionarily conserved pathway in eukaryotes that repairs a double-strand break (DSB) by copying homologous sequences from a sister chromatid, a homologous chromosome or an ectopic location. Recombination is challenged by the packaging of DNA into nucleosomes, which may impair the process at many steps, from resection of the DSB ends to the re-establishement of nucleosomes after repair. However, nucleosome dynamics during DSB repair have not been well described, primarily because of a lack of well-ordered nucleosomes around a DSB. We designed a system in budding yeast Saccharomyces cerevisiae to monitor nucleosome dynamics during repair of an HO endonuclease-induced DSB. Nucleosome occupancy around the break is lost following DSB formation, by 5'-3' resection of the DSB end. Soon after repair is complete, nucleosome occupancy is partially restored in a repair-dependent but cell cycle-independent manner. Full re-establishment of nucleosome protection back to the level prior to DSB induction is achieved when the cell cycle resumes following repair. These findings may have implications to the mechanisms by which cells sense the completion of repair.

  6. Genome-wide mapping of nucleosome positions in Saccharomyces cerevisiae in response to different nitrogen conditions

    PubMed Central

    Zhang, Peng; Du, Guocheng; Zou, Huijun; Xie, Guangfa; Chen, Jian; Shi, Zhongping; Zhou, Jingwen

    2016-01-01

    Well-organized chromatin is involved in a number of various transcriptional regulation and gene expression. We used genome-wide mapping of nucleosomes in response to different nitrogen conditions to determine both nucleosome profiles and gene expression events in Saccharomyces cerevisiae. Nitrogen conditions influence general nucleosome profiles and the expression of nitrogen catabolite repression (NCR) sensitive genes. The nucleosome occupancy of TATA-containing genes was higher compared to TATA-less genes. TATA-less genes in high or low nucleosome occupancy, showed a significant change in gene coding regions when shifting cells from glutamine to proline as the sole nitrogen resource. Furthermore, a correlation between the expression of nucleosome occupancy induced NCR sensitive genes or TATA containing genes in NCR sensitive genes, and nucleosome prediction were found when cells were cultured in proline or shifting from glutamine to proline as the sole nitrogen source compared to glutamine. These results also showed that variation of nucleosome occupancy accompany with chromatin-dependent transcription factor could influence the expression of a series of genes involved in the specific regulation of nitrogen utilization. PMID:27659668

  7. The role of a positioned nucleosome at the Drosophila melanogaster hsp26 promoter.

    PubMed Central

    Lu, Q; Wallrath, L L; Elgin, S C

    1995-01-01

    The regulatory region of Drosophila melanogaster hsp26 includes a positioned nucleosome located between the two DNase I hypersensitive (DH) sites that encompass the critical heat shock elements (HSEs). To test the role of this nucleosome in regulated expression, transgenic flies containing hsp26-lacZ fusion genes with alterations in the nucleosome-associated region have been generated. The positioned nucleosome is associated with a DNA sequence that does not itself contain any critical regulatory elements for heat shock-inducible expression. The nucleosome-associated sequence can be deleted, reversed, duplicated or replaced by a random sequence with no significant effect on DH site formation and gene expression. Analyses of hsp26 and hsp70 transgenes with spacing changes within the promoter region indicate that the location of the (CT)n.(GA)n elements dictates the location of DH site formation. Wrapping the DNA between the regulatory elements around a nucleosome is as effective for gene expression as placing the regulatory elements close to each other. A loss of inducible gene expression was observed when the nucleosome-associated DNA was replaced with sequences which appear to misdirect nucleosome placement. The results indicate considerable flexibility in the spacing between DH regulatory sites. Images PMID:7588603

  8. Singlet-singlet energy transfer studies of the internal organization of nucleosomes.

    PubMed

    Eshaghpour, H; Dieterich, A E; Cantor, C R; Crothers, D M

    1980-04-29

    We report the measurement of two specific protein to DNA distances in several conformational states of core nucleosomes by singlet-singlet energy transfer. A distance of 50-53 A separates each DNA terminus from cysteine-110 of chicken erythrocyte histone H3 in the native nucleosome. This cysteine residue must therefore be located very near the center of the nucleosome. The H3-DNA distance remained nearly constant in several unfolded forms of the core particles, as found in very low salt, in 0.6 M NaCl, and in high urea. Furthermore, it was shown that each DNA end lies within 32 A of cysteine-73 of Arbacia lixula sperm histone H4 in both the compact and the low-salt unfolded forms of the nucleosome. Because of the invariance of the two measured distances in the various conformational states of the nucleosome, we conclude that the cysteine-containing C-terminal segments of histones H3 and H4 maintain a very strong and close association with the terminal positions of the 146 base pair nucleosomal DNA. This binding may provide the primary interactions necessary for the folding of DNA into nucleosomes and for protection of 146 base pair nucleosomes from further nuclease digestion.

  9. Affinity, stoichiometry and cooperativity of heterochromatin protein 1 (HP1) binding to nucleosomal arrays

    NASA Astrophysics Data System (ADS)

    Teif, Vladimir B.; Kepper, Nick; Yserentant, Klaus; Wedemann, Gero; Rippe, Karsten

    2015-02-01

    Heterochromatin protein 1 (HP1) participates in establishing and maintaining heterochromatin via its histone-modification-dependent chromatin interactions. In recent papers HP1 binding to nucleosomal arrays was measured in vitro and interpreted in terms of nearest-neighbour cooperative binding. This mode of chromatin interaction could lead to the spreading of HP1 along the nucleosome chain. Here, we reanalysed previous data by representing the nucleosome chain as a 1D binding lattice and showed how the experimental HP1 binding isotherms can be explained by a simpler model without cooperative interactions between neighboring HP1 dimers. Based on these calculations and spatial models of dinucleosomes and nucleosome chains, we propose that binding stoichiometry depends on the nucleosome repeat length (NRL) rather than protein interactions between HP1 dimers. According to our calculations, more open nucleosome arrays with long DNA linkers are characterized by a larger number of binding sites in comparison to chains with a short NRL. Furthermore, we demonstrate by Monte Carlo simulations that the NRL dependent folding of the nucleosome chain can induce allosteric changes of HP1 binding sites. Thus, HP1 chromatin interactions can be modulated by the change of binding stoichiometry and the type of binding to condensed (methylated) and non-condensed (unmethylated) nucleosome arrays in the absence of direct interactions between HP1 dimers.

  10. The Arabidopsis Adh gene exhibits diverse nucleosome arrangements within a small DNase I-sensitive domain.