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Sample records for atp-dependent nucleosome translocation

  1. The ATP-dependent chromatin remodeling enzyme Fun30 represses transcription by sliding promoter-proximal nucleosomes.

    PubMed

    Byeon, Boseon; Wang, Wei; Barski, Artem; Ranallo, Ryan T; Bao, Kan; Schones, Dustin E; Zhao, Keji; Wu, Carl; Wu, Wei-Hua

    2013-08-09

    The evolutionarily conserved ATP-dependent chromatin remodeling enzyme Fun30 has recently been shown to play important roles in heterochromatin silencing and DNA repair. However, how Fun30 remodels nucleosomes is not clear. Here we report a nucleosome sliding activity of Fun30 and its role in transcriptional repression. We observed that Fun30 repressed the expression of genes involved in amino acid and carbohydrate metabolism, the stress response, and meiosis. In addition, Fun30 was localized at the 5' and 3' ends of genes and within the open reading frames of its targets. Consistent with its role in gene repression, we observed that Fun30 target genes lacked histone modifications often associated with gene activation and showed an increased level of ubiquitinated histone H2B. Furthermore, a genome-wide nucleosome mapping analysis revealed that the length of the nucleosome-free region at the 5' end of a subset of genes was changed in Fun30-depleted cells. In addition, the positions of the -1, +2, and +3 nucleosomes at the 5' end of target genes were shifted significantly, whereas the position of the +1 nucleosome remained largely unchanged in the fun30Δ mutant. Finally, we demonstrated that affinity-purified, single-component Fun30 exhibited a nucleosome sliding activity in an ATP-dependent manner. These results define a role for Fun30 in the regulation of transcription and indicate that Fun30 remodels chromatin at the 5' end of genes by sliding promoter-proximal nucleosomes.

  2. The dMi-2 chromodomains are DNA binding modules important for ATP-dependent nucleosome mobilization

    PubMed Central

    Bouazoune, Karim; Mitterweger, Angelika; Längst, Gernot; Imhof, Axel; Akhtar, Asifa; Becker, Peter B.; Brehm, Alexander

    2002-01-01

    Drosophila Mi-2 (dMi-2) is the ATPase subunit of a complex combining ATP-dependent nucleosome remodelling and histone deacetylase activities. dMi-2 contains an HMG box-like region, two PHD fingers, two chromodomains and a SNF2-type ATPase domain. It is not known which of these domains contribute to nucleosome remodelling. We have tested a panel of dMi-2 deletion mutants in ATPase, nucleosome mobilization and nucleosome binding assays. Deletion of the chromodomains impairs all three activities. A dMi-2 mutant lacking the chromodomains is incorporated into a functional histone deacetylase complex in vivo but has lost nucleosome-stimulated ATPase activity. In contrast to dHP1, dMi-2 does not bind methylated histone H3 tails and does not require histone tails for nucleosome binding. Instead, the dMi-2 chromodomains display DNA binding activity that is not shared by other chromodomains. Our results suggest that the chromodomains act at an early step of the remodelling process to bind the nucleosome substrate predominantly via protein–DNA interactions. Furthermore, we identify DNA binding as a novel chromodomain-associated activity. PMID:12006495

  3. 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

  4. 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.

  5. Phosphatidylserine translocation to the mitochondrion is an ATP-dependent process in permeabilized animal cells

    SciTech Connect

    Voelker, D.R. )

    1989-12-01

    Chinese hamster ovary (CHO-K1) cells were pulse labeled with ({sup 3}H)serine, and the synthesis of phosphatidyl({sup 3}H)ethanolamine from phosphatidyl({sup 3}H)serine during the subsequent chase was used as a measure of lipid translocation to the mitochondria. When the CHO-K1 cells were pulse labeled and subsequently permeabilized with 50 {mu}g of saponin per ml, there was no significant turnover of nascent phosphatidyl({sup 3}H)serine to form phosphatidyl({sup 3}H)ethanolamine during an ensuring chase. Supplementation of the permeabilized cells with 2 mM ATP resulted in significant phosphatidyl({sup 3}H)ethanolamine synthesis (83% of that found in intact cells) from phosphatidyl({sup 3}H)serine during a subsequent 2-hr chase. Phosphatidyl({sup 3}H)ethanolamine synthesis essentially ceased after 2 hr in the permeabilized cells. The translocation-dependent synthesis of phosphatidyl({sup 3}H)ethanolamine was a saturable process with respect to ATP concentration in permeabilized cells. The conversion of phosphatidyl({sup 3}H)serine to phosphatidyl({sup 3}H)ethanolamine did not occur in saponin-treated cultures supplemented with 2 mM AMP, 2 mM 5{prime}-adenylyl imidodiphosphate, or apyrase plus 2 mM ATP. ATP was the most effective nucleotide, but the addition of GTP, CTP, UTP, and ADP also supported the translocation-dependent synthesis of phosphatidyl({sup 3}H)ethanolamine albeit to a lesser extent. These data provide evidence that the interorganelle translocation of phosphatidylserine requires ATP and is largely independent of soluble cytosolic proteins.

  6. The ISW1 and CHD1 ATP-dependent chromatin remodelers compete to set nucleosome spacing in vivo

    PubMed Central

    Ocampo, Josefina; Chereji, Răzvan V.; Eriksson, Peter R.; Clark, David J.

    2016-01-01

    Adenosine triphosphate-dependent chromatin remodeling machines play a central role in gene regulation by manipulating chromatin structure. Most genes have a nucleosome-depleted region at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. In vitro, the three known yeast nucleosome spacing enzymes (CHD1, ISW1 and ISW2) form arrays with different spacing. We used genome-wide nucleosome sequencing to determine whether these enzymes space nucleosomes differently in vivo. We find that CHD1 and ISW1 compete to set the spacing on most genes, such that CHD1 dominates genes with shorter spacing and ISW1 dominates genes with longer spacing. In contrast, ISW2 plays a minor role, limited to transcriptionally inactive genes. Heavily transcribed genes show weak phasing and extreme spacing, either very short or very long, and are depleted of linker histone (H1). Genes with longer spacing are enriched in H1, which directs chromatin folding. We propose that CHD1 directs short spacing, resulting in eviction of H1 and chromatin unfolding, whereas ISW1 directs longer spacing, allowing H1 to bind and condense the chromatin. Thus, competition between the two remodelers to set the spacing on each gene may result in a highly dynamic chromatin structure. PMID:26861626

  7. Mechanisms of ATP Dependent Chromatin Remodeling

    PubMed Central

    Gangaraju, Vamsi K.; Bartholomew, Blaine

    2007-01-01

    The inter-relationship between DNA repair and ATP dependent chromatin remodeling has begun to become very apparent with recent discoveries. ATP dependent remodeling complexes mobilize nucleosomes along DNA, promote the exchange of histones, or completely displace nucleosomes from DNA. These remodeling complexes are often categorized based on the domain organization of their catalytic subunit. The biochemical properties and structural information of several of these remodeling complexes are reviewed. The different models for how these complexes are able to mobilize nucleosomes and alter nucleosome structure are presented incorporating several recent findings. Finally the role of histone tails and their respective modifications in ATP-dependent remodeling are discussed. PMID:17306844

  8. Evidence that transporters associated with antigen processing translocate a major histocompatibility complex class I-binding peptide into the endoplasmic reticulum in an ATP-dependent manner.

    PubMed Central

    Androlewicz, M J; Anderson, K S; Cresswell, P

    1993-01-01

    We have investigated the role of the putative peptide transporters associated with antigen processing (TAP) by using a permeabilized-cell system. The main objective was to determine whether these molecules, which bear homology to the ATP-binding cassette family of transporters, translocate antigenic peptides across the endoplasmic reticulum membrane for assembly with major histocompatibility complex (MHC) class I molecules and beta 2-microglobulin light chain. The pore-forming toxin streptolysin O was used to generate permeabilized cells, and peptide translocation was determined by measuring the amount of added radiolabeled peptide bound to endogenous class I molecules. No radiolabeled peptide was associated with MHC class I glycoproteins from unpermeabilized cells. We found that efficient peptide binding to MHC class I molecules in permeabilized cells is both transporter dependent and ATP dependent. In antigen-processing mutant cells lacking a functional transporter, uptake occurs only through a less-efficient transporter and ATP-independent pathway. In addition, short peptides (8-10 amino acids) known to bind MHC class I molecules compete efficiently with a radiolabeled peptide for TAP-dependent translocation, whereas longer peptides and a peptide derived from an endoplasmic reticulum signal sequence do not compete efficiently. This result indicates that the optimal substrates for TAP possess the characteristics of MHC-binding peptides. Images Fig. 2 Fig. 3 Fig. 4 PMID:8415666

  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. 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.

  11. 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.

  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. Kinetic Mechanism of DNA Translocation by the RSC Molecular Motor

    PubMed Central

    Eastlund, Allen; Malik, Shuja Shafi; Fischer, Christopher J.

    2013-01-01

    ATP-dependent nucleosome repositioning by chromatin remodeling enzymes requires the translocation of these enzymes along the nucleosomal DNA. Using a fluorescence stopped-flow assay we monitored DNA translocation by a minimal RSC motor and through global analysis of these time courses we have determined that this motor has a macroscopic translocation rate of 2.9 bp/s with a step size of 1.24 bp. From the complementary quantitative analysis of the associated time courses of ATP consumption during DNA translocation we have determined that this motor has an efficiency of 3.0 ATP/bp, which is slightly less that the efficiency observed for several genetically related DNA helicases and which likely results from random pausing by the motor during translocation. Nevertheless, this motor is able to exert enough force during translocation to displace streptavidin from biotinylated DNA. Taken together these results are the necessary first step for quantifying both the role of DNA translocation in nucleosome repositioning by RSC and the efficiency at which RSC couples ATP binding and hydrolysis to nucleosome repositioning. PMID:23399434

  14. ATP-dependent chromatin remodeling and DNA double-strand break repair.

    PubMed

    van Attikum, Haico; Gasser, Susan M

    2005-08-01

    The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic stability. Two pathways for the repair of DBSs, nonhomologous end-joining (NHEJ) and homologous recombination (HR), have evolved in eukaryotes. These pathways, like processes such as transcription and replication, act on DNA that is embedded in nucleosomes. Recent studies have shown that DNA repair, like transcription, is facilitated both by histone tail modification and by ATP-dependent chromatin remodeling. This review emphasizes recent reports that demonstrate a function for the ATP-dependent chromatin remodeling complexes INO80 and RSC in NHEJ and HR. We also discuss the possible role of SWR1- and TIP60-mediated nucleosomal histone exchange in DNA repair.

  15. Histone H4 tail mediates allosteric regulation of nucleosome remodelling by linker DNA.

    PubMed

    Hwang, William L; Deindl, Sebastian; Harada, Bryan T; Zhuang, Xiaowei

    2014-08-14

    Imitation switch (ISWI)-family remodelling enzymes regulate access to genomic DNA by mobilizing nucleosomes. These ATP-dependent chromatin remodellers promote heterochromatin formation and transcriptional silencing by generating regularly spaced nucleosome arrays. The nucleosome-spacing activity arises from the dependence of nucleosome translocation on the length of extranucleosomal linker DNA, but the underlying mechanism remains unclear. Here we study nucleosome remodelling by human ATP-dependent chromatin assembly and remodelling factor (ACF), an ISWI enzyme comprising a catalytic subunit, Snf2h, and an accessory subunit, Acf1 (refs 2, 11 - 13). We find that ACF senses linker DNA length through an interplay between its accessory and catalytic subunits mediated by the histone H4 tail of the nucleosome. Mutation of AutoN, an auto-inhibitory domain within Snf2h that bears sequence homology to the H4 tail, abolishes the linker-length sensitivity in remodelling. Addition of exogenous H4-tail peptide or deletion of the nucleosomal H4 tail also diminishes the linker-length sensitivity. Moreover, Acf1 binds both the H4-tail peptide and DNA in an amino (N)-terminal domain dependent manner, and in the ACF-bound nucleosome, lengthening the linker DNA reduces the Acf1-H4 tail proximity. Deletion of the N-terminal portion of Acf1 (or its homologue in yeast) abolishes linker-length sensitivity in remodelling and leads to severe growth defects in vivo. Taken together, our results suggest a mechanism for nucleosome spacing where linker DNA sensing by Acf1 is allosterically transmitted to Snf2h through the H4 tail of the nucleosome. For nucleosomes with short linker DNA, Acf1 preferentially binds to the H4 tail, allowing AutoN to inhibit the ATPase activity of Snf2h. As the linker DNA lengthens, Acf1 shifts its binding preference to the linker DNA, freeing the H4 tail to compete AutoN off the ATPase and thereby activating ACF.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. A Novel Mechanism of Antagonism between ATP-Dependent Chromatin Remodeling Complexes Regulates RNR3 Expression▿

    PubMed Central

    Tomar, Raghuvir S.; Psathas, James N.; Zhang, Hesheng; Zhang, Zhengjian; Reese, Joseph C.

    2009-01-01

    Gene expression depends upon the antagonistic actions of chromatin remodeling complexes. While this has been studied extensively for the enzymes that covalently modify the tails of histones, the mechanism of how ATP-dependent remodeling complexes antagonize each other to maintain the proper level of gene activity is not known. The gene encoding a large subunit of ribonucleotide reductase, RNR3, is regulated by ISW2 and SWI/SNF, complexes that repress and activate transcription, respectively. Here, we studied the functional interactions of these two complexes at RNR3. Deletion of ISW2 causes constitutive recruitment of SWI/SNF, and conditional reexpression of ISW2 causes the repositioning of nucleosomes and reduced SWI/SNF occupancy at RNR3. Thus, ISW2 is required for restriction of access of SWI/SNF to the RNR3 promoter under the uninduced condition. Interestingly, the binding of sequence-specific DNA binding factors and the general transcription machinery are unaffected by the status of ISW2, suggesting that disruption of nucleosome positioning does not cause a nonspecific increase in cross-linking of all factors to RNR3. We provide evidence that ISW2 does not act on SWI/SNF directly but excludes its occupancy by positioning nucleosomes over the promoter. Genetic disruption of nucleosome positioning by other means led to a similar phenotype, linking repressed chromatin structure to SWI/SNF exclusion. Thus, incorporation of promoters into a repressive chromatin structure is essential for prevention of the opportunistic actions of nucleosome-disrupting activities in vivo, providing a novel mechanism for maintaining tight control of gene expression. PMID:19349301

  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. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Molecular architecture of the ATP-dependent chromatin-remodeling complex SWR1.

    PubMed

    Nguyen, Vu Q; Ranjan, Anand; Stengel, Florian; Wei, Debbie; Aebersold, Ruedi; Wu, Carl; Leschziner, Andres E

    2013-09-12

    The ATP-dependent chromatin-remodeling complex SWR1 exchanges a variant histone H2A.Z/H2B dimer for a canonical H2A/H2B dimer at nucleosomes flanking histone-depleted regions, such as promoters. This localization of H2A.Z is conserved throughout eukaryotes. SWR1 is a 1 megadalton complex containing 14 different polypeptides, including the AAA+ ATPases Rvb1 and Rvb2. Using electron microscopy, we obtained the three-dimensional structure of SWR1 and mapped its major functional components. Our data show that SWR1 contains a single heterohexameric Rvb1/Rvb2 ring that, together with the catalytic subunit Swr1, brackets two independently assembled multisubunit modules. We also show that SWR1 undergoes a large conformational change upon engaging a limited region of the nucleosome core particle. Our work suggests an important structural role for the Rvbs and a distinct substrate-handling mode by SWR1, thereby providing a structural framework for understanding the complex dimer-exchange reaction.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. ATP-dependent potassium channels and type 2 diabetes mellitus.

    PubMed

    Bonfanti, Dianne Heloisa; Alcazar, Larissa Pontes; Arakaki, Priscila Akemi; Martins, Laysa Toschi; Agustini, Bruna Carla; de Moraes Rego, Fabiane Gomes; Frigeri, Henrique Ravanhol

    2015-05-01

    Diabetes mellitus is a public health problem, which affects a millions worldwide. Most diabetes cases are classified as type 2 diabetes mellitus, which is highly associated with obesity. Type 2 diabetes is considered a multifactorial disorder, with both environmental and genetic factors contributing to its development. An important issue linked with diabetes development is the failure of the insulin releasing mechanism involving abnormal activity of the ATP-dependent potassium channel, KATP. This channel is a transmembrane protein encoded by the KCNJ11 and ABCC8 genes. Furthermore, polymorphisms in these genes have been linked to type 2 diabetes because of the role of KATP in insulin release. While several genetic variations have been reported to be associated with this disease, the E23K polymorphism is most commonly associated with this pathology, as well as to obesity. Here, we review the molecular genetics of the potassium channel and discusses its most described polymorphisms and their associations with type 2 diabetes mellitus.

  14. ATP dependent chromatin remodeling enzymes in embryonic stem cells.

    PubMed

    Saladi, Srinivas Vinod; de la Serna, Ivana L

    2010-03-01

    Embryonic stem (ES) cells are pluripotent cells that can self renew or be induced to differentiate into multiple cell lineages, and thus have the potential to be utilized in regenerative medicine. Key pluripotency specific factors (Oct 4/Sox2/Nanog/Klf4) maintain the pluripotent state by activating expression of pluripotency specific genes and by inhibiting the expression of developmental regulators. Pluripotent ES cells are distinguished from differentiated cells by a specialized chromatin state that is required to epigenetically regulate the ES cell phenotype. Recent studies show that in addition to pluripotency specific factors, chromatin remodeling enzymes play an important role in regulating ES cell chromatin and the capacity to self-renew and to differentiate. Here we review recent studies that delineate the role of ATP dependent chromatin remodeling enzymes in regulating ES cell chromatin structure.

  15. Mechanisms of ATP-Dependent Chromatin Remodeling Motors.

    PubMed

    Zhou, Coral Y; Johnson, Stephanie L; Gamarra, Nathan I; Narlikar, Geeta J

    2016-07-05

    Chromatin remodeling motors play essential roles in all DNA-based processes. These motors catalyze diverse outcomes ranging from sliding the smallest units of chromatin, known as nucleosomes, to completely disassembling chromatin. The broad range of actions carried out by these motors on the complex template presented by chromatin raises many stimulating mechanistic questions. Other well-studied nucleic acid motors provide examples of the depth of mechanistic understanding that is achievable from detailed biophysical studies. We use these studies as a guiding framework to discuss the current state of knowledge of chromatin remodeling mechanisms and highlight exciting open questions that would continue to benefit from biophysical analyses.

  16. 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.

  17. 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.

  18. The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive

    PubMed Central

    Calapez, Alexandre; Pereira, Henrique M.; Calado, Angelo; Braga, José; Rino, José; Carvalho, Célia; Tavanez, João Paulo; Wahle, Elmar; Rosa, Agostinho C.; Carmo-Fonseca, Maria

    2002-01-01

    fAter being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22°C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes. PMID:12473688

  19. 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.

  20. 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.

  1. Preparation of Membrane Vesicles Enriched in ATP-Dependent Proton Transport from Suspension Cultures of Tomato Cells

    PubMed Central

    Dupont, Frances M.; Zabala, Maria De Gracia

    1985-01-01

    Membranes enriched in ATP-dependent proton transport were prepared from suspension cultures of tomato cells (Lycopersicon esculentum Mill cv VF36). Suspension cultures were a source of large quantities of membranes from rapidly growing, undifferentiated cells. Proton transport activity was assayed as quench of acridine orange fluorescence. The activity of the proton translocating ATPase and of several other membrane enzymes was measured as a function of the cell culture cycle. The relative distribution of the enzymes between the 3,000, 10,000, and 100,000g pellets remained the same throughout the cell culture cycle, but yield of total activity and activity per gram fresh weight with time had a unique profile for each enzyme tested. Maximal yield of the proton translocating ATPase activity was obtained from cells in the middle logarithmic phase of growth, and from 50 to 90% of the activity was found in the 10,000g pellet. The proton translocating ATPase activity was separable from NADPH cytochrome c reductase and cytochrome c oxidase on a sucrose gradient. Proton transport activity had a broad pH optimum (7.0-8.0), was stimulated by KCl with a Km of 5 to 10 millimolar, stimulation being due to the anion, Cl−, and not the cation, K+, and was not inhibited by vanadate, but was inhibited by NO3−. The activity is tentatively identified as the tonoplast ATPase. PMID:16664030

  2. Nucleosome recognition and spacing by chromatin remodelling factor ISW1a.

    PubMed

    Richmond, Timothy J

    2012-04-01

    Nucleosomes are actively positioned along DNA by ATP-dependent, chromatin remodelling factors. A structural model for the ISW1a chromatin remodelling factor from Saccharomyces cerevisiae in complex with a dinucleosome substrate was constructed from the X-ray structures of ISW1a (ΔATPase) with and without DNA bound, two different cryo-EM (cryo-electron microscopy) structures of ISW1a (ΔATPase) bound to a nucleosome, and site-directed photo-cross-linking analyses in solution. The X-ray structure of ISW1a (ΔATPase) with DNA bound suggests that DNA sequence may be involved in nucleosome recognition and thereby specificity of promoter interaction. The model suggests how the highly ordered nucleosome arrays observed by mapping nucleosomes in genes and their promoter regions could be generated by a chromatin remodelling factor.

  3. Recombinant expression and purification of an ATP-dependent DNA ligase from Aliivibrio salmonicida.

    PubMed

    Williamson, Adele; Pedersen, Hege

    2014-05-01

    The genome of the psychrophilic fish-pathogen Aliivibrio salmonicida encodes a putative ATP-dependent DNA ligase in addition to a housekeeping NAD-dependent enzyme. In order to study the structure and activity of the ATP dependent ligase in vitro we have undertaken its recombinant production and purification from an Escherichia coli based expression system. Expression and purification of this protein presented two significant challenges. First, the gene product was moderately toxic to E. coli cells, second it was necessary to remove the large amounts of E. coli DNA present in bacterial lysates without contamination of the protein preparation by nucleases which might interfere with future assaying. The toxicity problem was overcome by fusion of the putative ligase to large solubility tags such as maltose-binding protein (MBP) or Glutathione-S-transferase (GST), and DNA was removed by treatment with a nuclease which could be inhibited by reducing agents. As the A. salmonicida ATP-dependent DNA ligase gene encodes a predicted leader peptide, both the full-length and mature forms of the protein were produced. Both possessed ATP-dependent DNA ligase activity, but the truncated form was significantly more active. Here we detail the first reported production, purification and preliminary characterization of active A. salmonicida ATP-dependent DNA ligase.

  4. 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.

  5. Chromatin remodeller Fun30Fft3 induces nucleosome disassembly to facilitate RNA polymerase II elongation

    PubMed Central

    Lee, Junwoo; Shik Choi, Eun; David Seo, Hogyu; Kang, Keunsoo; Gilmore, Joshua M.; Florens, Laurence; Washburn, Michael P.; Choe, Joonho; Workman, Jerry L.; Lee, Daeyoup

    2017-01-01

    Previous studies have revealed that nucleosomes impede elongation of RNA polymerase II (RNAPII). Recent observations suggest a role for ATP-dependent chromatin remodellers in modulating this process, but direct in vivo evidence for this is unknown. Here using fission yeast, we identify Fun30Fft3 as a chromatin remodeller, which localizes at transcribing regions to promote RNAPII transcription. Fun30Fft3 associates with RNAPII and collaborates with the histone chaperone, FACT, which facilitates RNAPII elongation through chromatin, to induce nucleosome disassembly at transcribing regions during RNAPII transcription. Mutants, resulting in reduced nucleosome-barrier, such as deletion mutants of histones H3/H4 themselves and the genes encoding components of histone deacetylase Clr6 complex II suppress the defects in growth and RNAPII occupancy of cells lacking Fun30Fft3. These data suggest that RNAPII utilizes the chromatin remodeller, Fun30Fft3, to overcome the nucleosome barrier to transcription elongation. PMID:28218250

  6. 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.

  7. 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

  8. 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.

  9. Structural constraints in collaborative competition of transcription factors against the nucleosome.

    PubMed

    Moyle-Heyrman, Georgette; Tims, Hannah S; Widom, Jonathan

    2011-09-30

    Cooperativity in transcription factor (TF) binding is essential in eukaryotic gene regulation and arises through diverse mechanisms. Here, we focus on one mechanism, collaborative competition, which is of interest because it arises both automatically (with no requirement for TF coevolution) and spontaneously (with no requirement for ATP-dependent nucleosome remodeling factors). Previous experimental studies of collaborative competition analyzed cases in which target sites for pairs of cooperating TFs were contained within the same side of the nucleosome. Here, we utilize new assays to measure cooperativity in protein binding to pairs of nucleosomal DNA target sites. We focus on the cases that are of greatest in vivo relevance, in which one binding site is located close to the end of a nucleosome and the other binding site is located at diverse positions throughout the nucleosome. Our results reveal energetically significant positive (favorable) cooperativity for pairs of sites on the same side of the nucleosome but, for the cases examined, energetically insignificant cooperativity between sites on opposite sides of the nucleosome. These findings imply a special significance for TF binding sites that are spaced within one-half nucleosome length (74 bp) or less along the genome and may prove useful for prediction of cooperatively acting TFs genome wide.

  10. Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system.

    PubMed Central

    Ciechanover, A; Heller, H; Katz-Etzion, R; Hershko, A

    1981-01-01

    It had been shown previously that the heat-stable polypeptide of the ATP-dependent proteolytic system of reticulocytes, designated APF-1, forms covalent conjugates with protein substrates in an ATP-requiring process. We now describe an enzyme that carries out the activation by ATP of the polypeptide with pyrophosphate displacement. The formation of AMP-polypeptide and transfer of the polypeptide to a secondary acceptor are suggested by an APF-1 requirement for ATP-PPi and ATP-AMP exchange reactions, respectively. With radiolabeled polypeptide, an ATP-dependent labeling of the enzyme was shown to be by a linkage that is acid stable but is labile to treatment with mild alkali, hydroxylamine, borohydride, or mercuric salts. It therefore appears that the AMP-polypeptide undergoes attack by an -SH group of the enzyme to form a thiolester. PMID:6262770

  11. 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.

  12. Cloning, sequencing, and expression of Bacillus subtilis genes involved in ATP-dependent nuclease synthesis.

    PubMed Central

    Kooistra, J; Venema, G

    1991-01-01

    The genes encoding the subunits of the Bacillus subtilis ATP-dependent nuclease (add genes) have been cloned. The genes were located on an 8.8-kb SalI-SmaI chromosomal DNA fragment. Transformants of a recBCD deletion mutant of Escherichia coli with plasmid pGV1 carrying this DNA fragment showed ATP-dependent nuclease activity. Three open reading frames were identified on the 8.8-kb SalI-SmaI fragment, which could encode three proteins with molecular masses of 135 (AddB protein), 141 (AddA protein), and 28 kDa. Only the AddB and AddA proteins are required for ATP-dependent exonuclease activity. Both the AddB and AddA proteins contained a conserved amino acid sequence for ATP binding. In the AddA protein, a number of small regions were present showing a high degree of sequence similarity with regions in the E. coli RecB protein. The AddA protein contained six conserved motifs which were also present in the E. coli helicase II (UvrD protein) and the Rep helicase, suggesting that these motifs are involved in the DNA unwinding activity of the enzyme. When linked to the T7 promoter, a high level of expression was obtained in E. coli. Images PMID:1646786

  13. PICKLE is a CHD subfamily II ATP-dependent chromatin remodeling factor.

    PubMed

    Ho, Kwok Ki; Zhang, Heng; Golden, Barbara L; Ogas, Joe

    2013-02-01

    PICKLE plays a critical role in repression of genes that regulate development identity in Arabidopsis thaliana. PICKLE codes for a putative ATP-dependent chromatin remodeler that exhibits sequence similarity to members of subfamily II of animal CHD remodelers, which includes remodelers such as CHD3/Mi-2 that also restrict expression of developmental regulators. Whereas animal CHD3 remodelers are a component of the Mi-2/NuRD complex that promotes histone deacetylation, PICKLE promotes trimethylation of histone H3 lysine 27 suggesting that it acts via a distinct epigenetic pathway. Here, we examine whether PICKLE is also a member of a multisubunit complex and characterize the biochemical properties of recombinant PICKLE protein. Phylogenetic analysis indicates that PICKLE-related proteins in plants share a common ancestor with members of subfamily II of animal CHD remodelers. Biochemical characterization of PICKLE in planta, however, reveals that PICKLE primarily exists as a monomer. Recombinant PICKLE protein is an ATPase that is stimulated by ssDNA and mononucleosomes and binds to both naked DNA and mononucleosomes. Furthermore, recombinant PICKLE exhibits ATP-dependent chromatin remodeling activity. These studies demonstrate that subfamily II CHD proteins in plants, such as PICKLE, retain ATP-dependent chromatin remodeling activity but act through a mechanism that does not involve the ubiquitous Mi-2/NuRD complex.

  14. Role of transcription factor-mediated nucleosome disassembly in PHO5 gene expression.

    PubMed

    Kharerin, Hungyo; Bhat, Paike J; Marko, John F; Padinhateeri, Ranjith

    2016-02-04

    Studying nucleosome dynamics in promoter regions is crucial for understanding gene regulation. Nucleosomes regulate gene expression by sterically occluding transcription factors (TFs) and other non-histone proteins accessing genomic DNA. How the binding competition between nucleosomes and TFs leads to transcriptionally compatible promoter states is an open question. Here, we present a computational study of the nucleosome dynamics and organization in the promoter region of PHO5 gene in Saccharomyces cerevisiae. Introducing a model for nucleosome kinetics that takes into account ATP-dependent remodeling activity, DNA sequence effects, and kinetics of TFs (Pho4p), we compute the probability of obtaining different "promoter states" having different nucleosome configurations. Comparing our results with experimental data, we argue that the presence of local remodeling activity (LRA) as opposed to basal remodeling activity (BRA) is crucial in determining transcriptionally active promoter states. By modulating the LRA and Pho4p binding rate, we obtain different mRNA distributions-Poisson, bimodal, and long-tail. Through this work we explain many features of the PHO5 promoter such as sequence-dependent TF accessibility and the role of correlated dynamics between nucleosomes and TFs in opening/coverage of the TATA box. We also obtain possible ranges for TF binding rates and the magnitude of LRA.

  15. Role of transcription factor-mediated nucleosome disassembly in PHO5 gene expression

    NASA Astrophysics Data System (ADS)

    Kharerin, Hungyo; Bhat, Paike J.; Marko, John F.; Padinhateeri, Ranjith

    2016-02-01

    Studying nucleosome dynamics in promoter regions is crucial for understanding gene regulation. Nucleosomes regulate gene expression by sterically occluding transcription factors (TFs) and other non–histone proteins accessing genomic DNA. How the binding competition between nucleosomes and TFs leads to transcriptionally compatible promoter states is an open question. Here, we present a computational study of the nucleosome dynamics and organization in the promoter region of PHO5 gene in Saccharomyces cerevisiae. Introducing a model for nucleosome kinetics that takes into account ATP-dependent remodeling activity, DNA sequence effects, and kinetics of TFs (Pho4p), we compute the probability of obtaining different “promoter states” having different nucleosome configurations. Comparing our results with experimental data, we argue that the presence of local remodeling activity (LRA) as opposed to basal remodeling activity (BRA) is crucial in determining transcriptionally active promoter states. By modulating the LRA and Pho4p binding rate, we obtain different mRNA distributions—Poisson, bimodal, and long-tail. Through this work we explain many features of the PHO5 promoter such as sequence-dependent TF accessibility and the role of correlated dynamics between nucleosomes and TFs in opening/coverage of the TATA box. We also obtain possible ranges for TF binding rates and the magnitude of LRA.

  16. Sequence-targeted nucleosome sliding in vivo by a hybrid Chd1 chromatin remodeler

    PubMed Central

    Bowman, Gregory D.

    2016-01-01

    ATP-dependent chromatin remodelers regulate chromatin dynamics by modifying nucleosome positions and occupancy. DNA-dependent processes such as replication and transcription rely on chromatin to faithfully regulate DNA accessibility, yet how chromatin remodelers achieve well-defined nucleosome positioning in vivo is poorly understood. Here, we report a simple method for site-specifically altering nucleosome positions in live cells. By fusing the Chd1 remodeler to the DNA binding domain of the Saccharomyces cerevisiae Ume6 repressor, we have engineered a fusion remodeler that selectively positions nucleosomes on top of adjacent Ume6 binding motifs in a highly predictable and reproducible manner. Positioning of nucleosomes by the fusion remodeler recapitulates closed chromatin structure at Ume6-sensitive genes analogous to the endogenous Isw2 remodeler. Strikingly, highly precise positioning of single founder nucleosomes by either chimeric Chd1-Ume6 or endogenous Isw2 shifts phased chromatin arrays in cooperation with endogenous chromatin remodelers. Our results demonstrate feasibility of engineering precise nucleosome rearrangements through sequence-targeted chromatin remodeling and provide insight into targeted action and cooperation of endogenous chromatin remodelers in vivo. PMID:26993344

  17. Role of transcription factor-mediated nucleosome disassembly in PHO5 gene expression

    PubMed Central

    Kharerin, Hungyo; Bhat, Paike J.; Marko, John F.; Padinhateeri, Ranjith

    2016-01-01

    Studying nucleosome dynamics in promoter regions is crucial for understanding gene regulation. Nucleosomes regulate gene expression by sterically occluding transcription factors (TFs) and other non–histone proteins accessing genomic DNA. How the binding competition between nucleosomes and TFs leads to transcriptionally compatible promoter states is an open question. Here, we present a computational study of the nucleosome dynamics and organization in the promoter region of PHO5 gene in Saccharomyces cerevisiae. Introducing a model for nucleosome kinetics that takes into account ATP-dependent remodeling activity, DNA sequence effects, and kinetics of TFs (Pho4p), we compute the probability of obtaining different “promoter states” having different nucleosome configurations. Comparing our results with experimental data, we argue that the presence of local remodeling activity (LRA) as opposed to basal remodeling activity (BRA) is crucial in determining transcriptionally active promoter states. By modulating the LRA and Pho4p binding rate, we obtain different mRNA distributions—Poisson, bimodal, and long-tail. Through this work we explain many features of the PHO5 promoter such as sequence-dependent TF accessibility and the role of correlated dynamics between nucleosomes and TFs in opening/coverage of the TATA box. We also obtain possible ranges for TF binding rates and the magnitude of LRA. PMID:26843321

  18. 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

  19. 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.

  20. Acidification of endocytic vesicles by an ATP-dependent proton pump

    PubMed Central

    1983-01-01

    One of the early events in the pathway of receptor-mediated endocytosis is the acidification of the newly formed endocytic vesicle. To examine the mechanism of acidification, we used fluorescein-labeled alpha 2- macroglobulin (F-alpha 2M) as a probe for endocytic vesicle pH. Changes in pH were determined from the change in fluorescein fluorescence at 490-nm excitation as measured with a microscope spectrofluorometer. After endocytosis of F-alpha 2M, mouse fibroblast cells were permeabilized by brief exposure to the detergent digitonin. Treatment with the ionophore monensin or the protonophore carbonyl cyanide p- trifluoromethoxyphenylhydrazone (FCCP) caused a rapid increase in the pH of the endocytic vesicle. Upon removal of the ionophore, the endocytic vesicle rapidly acidified only when MgATP or MgGTP was added. Neither ADP nor the nonhydrolyzable analog, adenosine 5'-(beta, gamma- imido)triphosphate (AMP-PNP) could support acidification. The ATP- dependent acidification did not require a specific cation or anion in the external media. Acidification was insensitive to vanadate and amiloride but was inhibited by Zn2+ and the anion transport inhibitor diisothiocyanostilbene disulfonic acid (DIDS). We also examined the acidification of lysosomes with the permeabilized cell system, using fluorescein isothiocyanate dextran as probe. DIDS inhibited the ATP- dependent reacidification of lysosomes, although at a lower concentration than that for inhibition of endocytic vesicle reacidification. These results demonstrate that endocytic vesicles contain an ATP-dependent acidification mechanism that shares similar characteristics with the previously described lysosomal proton pump. PMID:6224803

  1. The Role of ATP-Dependent Machines in Regulating Genome Topology

    PubMed Central

    Hauk, Glenn; Berger, James M

    2016-01-01

    All cells must copy and express genes in accord with internal and external cues. The proper timing and response of such events relies on the active control of higher-order genomic organization. Cells use ATP-dependent molecular machines to alter the local and global topology of DNA so as to promote and counteract the persistent effects of transcription and replication. X-ray crystallography and electron microscopy, coupled with biochemical and single molecule methods are continuing to provide a wealth of mechanistic information on how DNA remodeling factors are employed to dynamically shape and organize the genome. PMID:26827284

  2. 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

  3. 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.

  4. The histone-fold protein complex CHRAC-15/17 enhances nucleosome sliding and assembly mediated by ACF.

    PubMed

    Kukimoto, Iwao; Elderkin, Sarah; Grimaldi, Margaret; Oelgeschläger, Thomas; Varga-Weisz, Patrick D

    2004-01-30

    The histone fold is a structural motif with which two related proteins interact and is found in complexes involved in wrapping DNA, the nucleosome, and transcriptional regulation, as in NC2. We reveal a novel function for histone-fold proteins: facilitation of nucleosome remodeling. ACF1-ISWI complex (ATP-dependent chromatin assembly and remodeling factor [ACF]) associates with histone-fold proteins (CHRAC-15 and CHRAC-17 in the human chromatin accessibility complex [CHRAC]) whose functional relevance has been unclear. We show that these histone-fold proteins facilitate ATP-dependent nucleosome sliding by ACF. Direct interaction of the CHRAC-15/17 complex with the ACF1 subunit is essential for this process. CHRAC-17 interacts with another histone-fold protein, p12, in DNA polymerase epsilon, but CHRAC-15 is essential for interaction with ACF and enhancement of nucleosome sliding. Surprisingly, CHRAC-15/17, p12/CHRAC-17, and NC2 complexes facilitate ACF-mediated chromatin assembly by a mechanism different from nucleosome sliding enhancement, suggesting a general activity of H2A/H2B type histone-fold complexes in chromatin assembly.

  5. Dietary protein deficiency reduces lysosomal and nonlysosomal ATP-dependent proteolysis in muscle

    NASA Technical Reports Server (NTRS)

    Tawa, N. E. Jr; Kettelhut, I. C.; Goldberg, A. L.

    1992-01-01

    When rats are fed a protein deficient (PD) diet for 7 days, rates of proteolysis in skeletal muscle decrease by 40-50% (N. E. Tawa, Jr., and A. L. Goldberg. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E317-325, 1992). To identify the underlying biochemical adaptations, we measured different proteolytic processes in incubated muscles. The capacity for intralysosomal proteolysis, as shown by sensitivity to methylamine or lysosomal protease inhibitors, fell 55-75% in muscles from PD rats. Furthermore, extracts of muscles of PD rats showed 30-70% lower activity of many lysosomal proteases, including cathepsins B, H, and C, and carboxypeptidases A and C, as well as other lysosomal hydrolases. The fall in cathepsin B and proteolysis was evident by 3 days on the PD diet, and both returned to control levels 3 days after refeeding of the normal diet. In muscles maintained under optimal conditions, 80-90% of protein breakdown occurs by nonlysosomal pathways. In muscles of PD rats, this ATP-dependent process was also 40-60% slower. Even though overall proteolysis decreased in muscles of PD rats, their capacity for Ca(2+)-dependent proteolysis increased (by 66%), as did the activity of the calpains (+150-250%). Thus the lysosomal and the ATP-dependent processes decrease coordinately and contribute to the fall in muscle proteolysis in PD animals.

  6. ATP-dependent calcium transport across basal plasma membranes of human placental trophoblast

    SciTech Connect

    Fisher, G.J.; Kelley, L.K.; Smith, C.H.

    1987-01-01

    As a first step in understanding the cellular basis of maternal-fetal calcium transfer, the authors examined the characteristics of calcium uptake by a highly purified preparation of the syncytiotrophoblast basal (fetal facing) plasma membrane. In the presence of nanomolar concentrations of free calcium, basal membranes demonstrated substantial ATP-dependent calcium uptake. This uptake required magnesium, was not significantly affected by Na/sup +/ or K/sup +/ (50 mM), or sodium azide (10 mM). Intravesicular calcium was rapidly and completely released by the calcium ionophore rapidly and completely released by the calcium ionophore A23187. Calcium transport was significantly stimulated by the calcium-dependent regulatory protein calmodulin. Placental membrane fractions enriched in endoplasmic reticulum (ER) and mitochondria also demonstrated ATP-dependent calcium uptake. In contrast to basal membrane, mitochondrial calcium uptake was completely inhibited by azide. The rate of calcium uptake was completely inhibited by azide. The rate of calcium uptake by the ER was only 20% of that of basal membranes. They conclude that the placental basal plasma membrane possesses a high-affinity calcium transport system similar to that found in plasma membranes of a variety of cell types. This transporter is situated to permit it to function in vivo in maternal-fetal calcium transfer.

  7. ATP Dependent Rotational Motion of Group II Chaperonin Observed by X-ray Single Molecule Tracking

    PubMed Central

    Sekiguchi, Hiroshi; Nakagawa, Ayumi; Moriya, Kazuki; Makabe, Koki; Ichiyanagi, Kouhei; Nozawa, Shunsuke; Sato, Tokushi; Adachi, Shin-ichi; Kuwajima, Kunihiro; Yohda, Masafumi; Sasaki, Yuji C.

    2013-01-01

    Group II chaperonins play important roles in protein homeostasis in the eukaryotic cytosol and in Archaea. These proteins assist in the folding of nascent polypeptides and also refold unfolded proteins in an ATP-dependent manner. Chaperonin-mediated protein folding is dependent on the closure and opening of a built-in lid, which is controlled by the ATP hydrolysis cycle. Recent structural studies suggest that the ring structure of the chaperonin twists to seal off the central cavity. In this study, we demonstrate ATP-dependent dynamics of a group II chaperonin at the single-molecule level with highly accurate rotational axes views by diffracted X-ray tracking (DXT). A UV light-triggered DXT study with caged-ATP and stopped-flow fluorometry revealed that the lid partially closed within 1 s of ATP binding, the closed ring subsequently twisted counterclockwise within 2–6 s, as viewed from the top to bottom of the chaperonin, and the twisted ring reverted to the original open-state with a clockwise motion. Our analyses clearly demonstrate that the biphasic lid-closure process occurs with unsynchronized closure and a synchronized counterclockwise twisting motion. PMID:23734192

  8. ATP-dependent specific binding of Tn3 transposase to Tn3 inverted repeats

    NASA Astrophysics Data System (ADS)

    Wishart, W. L.; Broach, J. R.; Ohtsubo, E.

    1985-04-01

    Transposons are discrete segments of DNA which are capable of moving from one site in a genome to many different sites1,2. Tn3 is a prokaryotic transposon which is 4,957 base pairs (bp) long and encodes a transposase protein which is essential for transposition3-7. We report here a simple method for purifying Tn3 transposase and demonstrate that the transposase protein binds specifically to the ends of the Tn3 transposon in an ATP-dependent manner. The transposase protein binds to linear double-stranded DNA both nonspecifically and specifically; the nonspecific DNA binding activity is sensitive to challenge with heparin. Site-specific DNA binding to the ends (inverted repeats) of Tn3 is observed only when binding is performed in the presence of ATP; this ATP-dependent site-specific DNA binding activity is resistant to heparin challenge. Our results indicate that ATP qualitatively alters the DNA binding activity of the transposase protein so that the protein is able to bind specifically to the ends of the Tn3 transposon.

  9. ATP-dependent transport of bile acid intermediates across rat liver peroxisomal membranes.

    PubMed

    Une, Mizuho; Iguchi, Yusuke; Sakamoto, Tomoko; Tomita, Takashi; Suzuki, Yasuyuki; Morita, Masashi; Imanaka, Tsuneo

    2003-08-01

    The bile acid intermediate 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid (THCA) is converted to cholic acid exclusively in peroxisomes by the oxidative cleavage of the side chain. To investigate the mechanism by which the biosynthetic intermediates of bile acids are transported into peroxisomes, we incubated THCA or its CoA ester (THC-CoA) with isolated intact rat liver peroxisomes and analyzed their oxidation products, cholic acid and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoic acid. The oxidation of both THCA and THC-CoA was dependent on incubation time and peroxisomal proteins, and was stimulated by ATP. THC-CoA was efficiently oxidized to cholic acid and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoic acid as compared with THCA, suggesting that THC-CoA is the preferred substrate for transport into peroxisomes. The oxidation of THC-CoA was significantly inhibited by sodium azide, verapamile, and N-ethylmaleimide. Furthermore, the stimulatory effect of ATP on the oxidation was not replaced by GTP or AMP. In addition, the ATP-dependent oxidation of THC-CoA was markedly inhibited by pretreatment of peroxisomes with proteinase K when peroxisomal matrix proteins were not degraded. These results suggest that an ATP-dependent transport system for THC-CoA exists on peroxisomal membranes.

  10. 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.

  11. 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

  12. Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1.

    PubMed

    Chang, Xiu-bao

    2010-01-01

    Millions of new cancer patients are diagnosed each year and over half of these patients die from this devastating disease. Thus, cancer causes a major public health problem worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Over-expression of ATP-binding cassette transporters, such as P-glycoprotein, breast cancer resistance protein and/or multidrug resistance-associated protein 1 (MRP1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs across the cell membrane barrier. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

  13. The chloroplast ATP-dependent Clp protease in vascular plants - new dimensions and future challenges.

    PubMed

    Clarke, Adrian K

    2012-05-01

    The ATP-dependent Clp protease is by far the most intricate protease in chloroplasts of vascular plants. Structurally, it is particularly complex with a proteolytic core complex containing 11 distinct subunits along with three potential chaperone partners. The Clp protease is also essential for chloroplast development and overall plant viability. Over the past decade, many of the important characteristics of this crucial protease have been revealed in the model plant species Arabidopsis thaliana. Despite this, challenges still remain in fully resolving certain key features, in particular, how the assembly of this multisubunit protease is regulated, the full range of native protein substrates and how they are targeted for degradation and how this complicated enzyme might have developed from simpler bacterial forms. This article focuses upon the recent advances in revealing the details underlying these important features. It also take the opportunity to speculate upon many of these findings in the hope of stimulating further investigation.

  14. Phosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism.

    PubMed

    Garcia-Alles, Luis F; Siebold, Christian; Nyffeler, Therese Lüthi; Flükiger-Brühwiler, Karin; Schneider, Philipp; Bürgi, Hans-Beat; Baumann, Ulrich; Erni, Bernhard

    2004-10-19

    Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants, and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) in most bacteria. Here, we compare the PTS-dependent kinase of Escherichia coli and the ATP-dependent kinase of Citrobacter freundii. They display 30% sequence identity. The binding constants of the E. coli kinase for eleven short-chain carbonyl compounds were determined by acetone precipitation of the enzyme-substrate complexes. They are 3.4 microM for Dha, 780 microM for Dha-phosphate (DhaP), 50 microM for D,L-glyceraldehyde (GA), and 90 microM for D,L-glyceraldehyde-3-phosphate. The k(cat) for Dha of the PTS-dependent kinase is 290 min(-1), and that of the ATP-dependent kinase is 1050 min(-1). The Km for Dha of both kinases is <6 microM. The X-ray structures of the enzyme-GA and the enzyme-DhaP complex show that substrates as well as products are bound in hemiaminal linkage to an active-site histidine. Quantum-mechanical calculations offer no indication for activation of the reacting hydroxyl group by the formation of the hemiaminal. However, the formation of the hemiaminal bond allows selection for short-chain carbonyl compounds and discrimination against structurally similar polyols. The Dha kinase remains fully active in the presence of 2 M glycerol, and phosphorylates trace impurities of carbonyl compounds present in glycerol.

  15. Conformational change opening the CFTR chloride channel pore coupled to ATP-dependent gating.

    PubMed

    Wang, Wuyang; Linsdell, Paul

    2012-03-01

    Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are controlled by ATP binding and hydrolysis by its nucleotide binding domains (NBDs). This is presumed to control opening of a single "gate" within the permeation pathway, however, the location of such a gate has not been described. We used patch clamp recording to monitor access of cytosolic cysteine reactive reagents to cysteines introduced into different transmembrane (TM) regions in a cysteine-less form of CFTR. The rate of modification of Q98C (TM1) and I344C (TM6) by both [2-sulfonatoethyl] methanethiosulfonate (MTSES) and permeant Au(CN)(2)(-) ions was reduced when ATP concentration was reduced from 1mM to 10μM, and modification by MTSES was accelerated when 2mM pyrophosphate was applied to prevent channel closure. Modification of K95C (TM1) and V345C (TM6) was not affected by these manoeuvres. We also manipulated gating by introducing the mutations K464A (in NBD1) and E1371Q (in NBD2). The rate of modification of Q98C and I344C by both MTSES and Au(CN)(2)(-) was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected. These results suggest that access from the cytoplasm to K95 and V345 is similar in open and closed channels. In contrast, modifying ATP-dependent channel gating alters access to Q98 and I344, located further into the pore. We propose that ATP-dependent gating of CFTR is associated with the opening and closing of a gate within the permeation pathway at the level of these pore-lining amino acids.

  16. Effect of ATP-dependent channel modulators on ischemia-induced arrhythmia change depending on age and gender.

    PubMed

    Bozdogan, Ömer; Kaya, Salih Tunç; Yasar, Selçuk; Orallar, Hayriye

    2013-10-01

    The number of ATP-dependent potassium channels in myocardial cells has been previously shown to change depending on gender and age. Different effects of the ATP-dependent potassium channel blocker, glybenclamide and ATP-dependent potassium channel opener, pinacidil on ischemia or reperfusion-induced arrhythmia observed in various research might depend on different ages and genders of the animals used. The aim of this study is to research the effect of ATP-dependent potassium channel modulators on ischemia-induced arrhythmia in animals of different ages and genders. Sprague-Dawley rats of different ages and genders were used in this study. Ischemia was produced by the ligation of the left coronary artery for 30 min. Electrocardiogram (ECG), blood pressure, infarct area and blood glucose were determined during the 30 min of ischemia. An arrhythmia score from an ECG recorded during 30 min of ischemia was determined by examining the duration and type of arrhythmia. Different effects of glybenclamide and pinacidil on the arrhythmias were observed in male and female young and middle-age rats. Pinacidil decreased the infarct zone in younger female rats, but differences in the type and length of ischemia-induced arrhythmias between females and males disappeared in older age. The results of this study showed that the effect of ATP-dependent potassium channel modulators on ischemia-induced arrhythmia changed due to the age and gender of rats.

  17. 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.

  18. Coordinated Action of Nap1 and RSC in Disassembly of Tandem Nucleosomes

    PubMed Central

    Prasad, Rashmi; D'Arcy, Sheena; Hada, Arjan; Luger, Karolin

    2016-01-01

    The SWI/SNF and RSC family of ATP-dependent chromatin remodelers disassembles nucleosomes by moving nucleosomes into the vicinity of adjoining nucleosomes. We found that the histone chaperone Nap1 efficiently promotes disassembly of adjacent nucleosomes with which RSC collides and not the disassembly of nucleosomes mobilized by RSC. Nap1 is specific to RSC, as it does not target SWI/SNF, its paralog in Saccharomyces cerevisiae. Extensive mutational analysis of Nap1 has revealed that Nap1 affinity for histones H2A-H2B and H3-H4 and its ability to displace histones from DNA are required for Nap1 to enhance RSC-mediated disassembly. Other histone chaperones, such as Vps75, that also bind histones are not able to enhance RSC-mediated disassembly. Our study suggests a mechanism by which Nap1 is recruited to actively transcribed regions and assists in the passage of the transcription complex through chromatin, and it provides a novel mechanism for the coordinated action of RSC and Nap1. PMID:27273866

  19. 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

  20. 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.

  1. The Chromatin Remodelling Enzymes SNF2H and SNF2L Position Nucleosomes adjacent to CTCF and Other Transcription Factors

    PubMed Central

    Wiechens, Nicola; Gkikopoulos, Triantaffyllos; Schofield, Pieta; Rocha, Sonia; Owen-Hughes, Tom

    2016-01-01

    Within the genomes of metazoans, nucleosomes are highly organised adjacent to the binding sites for a subset of transcription factors. Here we have sought to investigate which chromatin remodelling enzymes are responsible for this. We find that the ATP-dependent chromatin remodelling enzyme SNF2H plays a major role organising arrays of nucleosomes adjacent to the binding sites for the architectural transcription factor CTCF sites and acts to promote CTCF binding. At many other factor binding sites SNF2H and the related enzyme SNF2L contribute to nucleosome organisation. The action of SNF2H at CTCF sites is functionally important as depletion of CTCF or SNF2H affects transcription of a common group of genes. This suggests that chromatin remodelling ATPase’s most closely related to the Drosophila ISWI protein contribute to the function of many human gene regulatory elements. PMID:27019336

  2. The mechanism of ATP-dependent RNA unwinding by DEAD box proteins.

    PubMed

    Hilbert, Manuel; Karow, Anne R; Klostermeier, Dagmar

    2009-12-01

    DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecA-like domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysis- and unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

  3. ATP-dependent motor activity of the transcription termination factor Rho from Mycobacterium tuberculosis

    PubMed Central

    D'Heygère, François; Schwartz, Annie; Coste, Franck; Castaing, Bertrand; Boudvillain, Marc

    2015-01-01

    The bacterial transcription termination factor Rho—a ring-shaped molecular motor displaying directional, ATP-dependent RNA helicase/translocase activity—is an interesting therapeutic target. Recently, Rho from Mycobacterium tuberculosis (MtbRho) has been proposed to operate by a mechanism uncoupled from molecular motor action, suggesting that the manner used by Rho to dissociate transcriptional complexes is not conserved throughout the bacterial kingdom. Here, however, we demonstrate that MtbRho is a bona fide molecular motor and directional helicase which requires a catalytic site competent for ATP hydrolysis to disrupt RNA duplexes or transcription elongation complexes. Moreover, we show that idiosyncratic features of the MtbRho enzyme are conferred by a large, hydrophilic insertion in its N-terminal ‘RNA binding’ domain and by a non-canonical R-loop residue in its C-terminal ‘motor’ domain. We also show that the ‘motor’ domain of MtbRho has a low apparent affinity for the Rho inhibitor bicyclomycin, thereby contributing to explain why M. tuberculosis is resistant to this drug. Overall, our findings support that, in spite of adjustments of the Rho motor to specific traits of its hosting bacterium, the basic principles of Rho action are conserved across species and could thus constitute pertinent screening criteria in high-throughput searches of new Rho inhibitors. PMID:25999346

  4. ATP-dependent G-quadruplex unfolding by Bloom helicase exhibits low processivity

    PubMed Central

    Budhathoki, Jagat B.; Stafford, Edward J.; Yodh, Jaya G.; Balci, Hamza

    2015-01-01

    Various helicases and single stranded DNA (ssDNA) binding proteins unfold G-quadruplex (GQ) structures. However, the underlying mechanisms of this activity have only recently come to focus. We report kinetic studies on Bloom (BLM) helicase and human telomeric GQ interactions using single-molecule Förster resonance energy transfer (smFRET). Using partial duplex DNA (pdDNA) constructs with different 5′ ssDNA overhangs, we show that BLM localizes in the vicinity of ssDNA/double-stranded DNA (dsDNA) junction and reels in the ssDNA overhang in an ATP-dependent manner. A comparison of DNA constructs with or without GQ in the overhang shows that GQ unfolding is achieved in 50–70% of reeling attempts under physiological salt and pH conditions. The unsuccessful attempts often result in dissociation of BLM from DNA which slows down the overall BLM activity. BLM-mediated GQ unfolding is typically followed by refolding of the GQ, a pattern that is repeated several times before BLM dissociates from DNA. BLM is significantly less processive compared to the highly efficient GQ destabilizer Pif1 that can repeat GQ unfolding activity hundreds of times before dissociating from DNA. Despite the variations in processivity, our studies point to possible common patterns used by different helicases in minimizing the duration of stable GQ formation. PMID:25990739

  5. ATP-dependent motor activity of the transcription termination factor Rho from Mycobacterium tuberculosis.

    PubMed

    D'Heygère, François; Schwartz, Annie; Coste, Franck; Castaing, Bertrand; Boudvillain, Marc

    2015-07-13

    The bacterial transcription termination factor Rho-a ring-shaped molecular motor displaying directional, ATP-dependent RNA helicase/translocase activity-is an interesting therapeutic target. Recently, Rho from Mycobacterium tuberculosis (MtbRho) has been proposed to operate by a mechanism uncoupled from molecular motor action, suggesting that the manner used by Rho to dissociate transcriptional complexes is not conserved throughout the bacterial kingdom. Here, however, we demonstrate that MtbRho is a bona fide molecular motor and directional helicase which requires a catalytic site competent for ATP hydrolysis to disrupt RNA duplexes or transcription elongation complexes. Moreover, we show that idiosyncratic features of the MtbRho enzyme are conferred by a large, hydrophilic insertion in its N-terminal 'RNA binding' domain and by a non-canonical R-loop residue in its C-terminal 'motor' domain. We also show that the 'motor' domain of MtbRho has a low apparent affinity for the Rho inhibitor bicyclomycin, thereby contributing to explain why M. tuberculosis is resistant to this drug. Overall, our findings support that, in spite of adjustments of the Rho motor to specific traits of its hosting bacterium, the basic principles of Rho action are conserved across species and could thus constitute pertinent screening criteria in high-throughput searches of new Rho inhibitors.

  6. Calcium uptake in rat liver mitochondria accompanied by activation of ATP-dependent potassium channel.

    PubMed

    Akopova, O V; Nosar, V I; Mankovskaya, I N; Sagach, V F

    2008-10-01

    The influence of potassium ions on calcium uptake in rat liver mitochondria is studied. It is shown that an increase in K+ and Ca2+ concentrations in the incubation medium leads to a decrease in calcium uptake in mitochondria together with a simultaneous increase in potassium uptake due to the potential-dependent transport of K+ in the mitochondrial matrix. Both effects are more pronounced in the presence of an ATP-dependent K+-channel (K+(ATP)-channel) opener, diazoxide (Dz). Activation of the K+(ATP)-channel by Dz alters the functional state of mitochondria and leads to an increase in the respiration rate in state 2 and a decrease in the oxygen uptake and the rate of ATP synthesis in state 3. The effect of Dz on oxygen consumption in state 3 is mimicked by valinomycin, but it is opposite to that of the classical protonophore uncoupler CCCP. It is concluded that the potential-dependent uptake of potassium is closely coupled to calcium transport and is an important parameter of energy coupling responsible for complex changes in oxygen consumption and Ca2+-transport properties of mitochondria.

  7. Decomposition of Slide Helix Contributions to ATP-dependent Inhibition of Kir6.2 Channels*

    PubMed Central

    Li, Jenny B. W.; Huang, Xinyang; Zhang, Roger S.; Kim, Robin Y.; Yang, Runying; Kurata, Harley T.

    2013-01-01

    Regulation of inwardly rectifying potassium channels by intracellular ligands couples cell membrane excitability to important signaling cascades and metabolic pathways. We investigated the molecular mechanisms that link ligand binding to the channel gate in ATP-sensitive Kir6.2 channels. In these channels, the “slide helix” forms an interface between the cytoplasmic (ligand-binding) domain and the transmembrane pore, and many slide helix mutations cause loss of function. Using a novel approach to rescue electrically silent channels, we decomposed the contribution of each interface residue to ATP-dependent gating. We demonstrate that effective inhibition by ATP relies on an essential aspartate at residue 58. Characterization of the functional importance of this conserved aspartate, relative to other residues in the slide helix, has been impossible because of loss-of-function of Asp-58 mutant channels. The Asp-58 position exhibits an extremely stringent requirement for aspartate because even a highly conservative mutation to glutamate is insufficient to restore normal channel function. These findings reveal unrecognized slide helix elements that are required for functional channel expression and control of Kir6.2 gating by intracellular ATP. PMID:23798684

  8. ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled

    PubMed Central

    Singh, Himansha; Velamakanni, Saroj; Deery, Michael J.; Howard, Julie; Wei, Shen L.; van Veen, Hendrik W.

    2016-01-01

    ATP-binding cassette transporters mediate the transbilayer movement of a vast number of substrates in or out of cells in organisms ranging from bacteria to humans. Current alternating access models for ABC exporters including the multidrug and Lipid A transporter MsbA from Escherichia coli suggest a role for nucleotide as the fundamental source of free energy. These models involve cycling between conformations with inward- and outward-facing substrate-binding sites in response to engagement and hydrolysis of ATP at the nucleotide-binding domains. Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. These findings introduce ion coupling as a new parameter in the mechanism of this homodimeric ABC transporter. PMID:27499013

  9. ISWI ATP-dependent remodeling of nucleoplasmic ω-speckles in the brain of Drosophila melanogaster.

    PubMed

    Lo Piccolo, Luca; Attardi, Andrea; Bonaccorso, Rosa; Li Greci, Lorenzo; Giurato, Giorgio; Ingrassia, Antonia Maria Rita; Onorati, Maria Cristina

    2017-02-20

    Heterogeneous nuclear ribonucleoproteins (hnRNPs) belong to the RNA-binding proteins family. They are involved in processing heterogeneous nuclear RNAs (hnRNAs) into mature mRNAs. These proteins participate in every step of mRNA cycle, such as mRNA export, localization, translation, stability and alternative splicing. At least 14 major hnRNPs, which have structural and functional homologues in mammals, are expressed in Drosophila melanogaster. Until now, six of these hnRNPs are known to be nucleus-localized and associated with the long non-coding RNA (lncRNA) heat shock responsive ω (hsrω) in the omega speckle compartments (ω-speckles). The chromatin remodeler ISWI is the catalytic subunit of several ATP-dependent chromatin-remodeling complexes, and it is an essential factor for organization of ω-speckles. Indeed, in ISWI null mutant, severe defects in ω-speckles structure are detectable. Here, we clarify the role of ISWI in the hnRNPs‒hsrω interaction. Moreover, we describe how ISWI by its remodeling activity, controls hsrω and hnRNPs engagement in ω-speckles. Finally, we demonstrate that the sequestration of hnRNPs in ω-speckles nuclear compartment is a fundamental event in gene expression control and represents a key step in the regulation of several pathways.

  10. The E1 protein of bovine papilloma virus 1 is an ATP-dependent DNA helicase.

    PubMed Central

    Yang, L; Mohr, I; Fouts, E; Lim, D A; Nohaile, M; Botchan, M

    1993-01-01

    For efficient DNA replication of papillomaviruses, only two viral-encoded proteins, E1 and E2, are required. Other proteins and factors are provided by the host cell. E2 is an enhancer of both transcription and replication and is known to help E1 bind cooperatively to the origin of DNA replication. E1 is sufficient for replication in extracts prepared from permissive cells, but the activity is enhanced by E2. Here we show that purified E1 can act as an ATP-dependent DNA helicase. To measure this activity, we have used strand displacement, unwinding of topologically constrained DNA, denaturation of duplex fragments, and electron microscopy. The ability of E1 to unwind circular DNA is found to be independent of origin-specific viral DNA sequences under a variety of experimental conditions. In unfractionated cellular extracts, E1-dependent viral DNA replication is origin-dependent, but at elevated E1 concentrations, replication can occur on non-origin-containing DNA templates. This conversion from an origin-dependent replication system to a nonspecific initiator system is discussed in the context of the current understanding of the initiation of chromosomal DNA replication. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8389467

  11. 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.

  12. 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.

  13. 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.

  14. The ATP-Dependent RNA Helicase DDX3X Modulates Herpes Simplex Virus 1 Gene Expression.

    PubMed

    Khadivjam, Bita; Stegen, Camille; Hogue-Racine, Marc-Aurèle; El Bilali, Nabil; Döhner, Katinka; Sodeik, Beate; Lippé, Roger

    2017-04-15

    The human protein DDX3X is a DEAD box ATP-dependent RNA helicase that regulates transcription, mRNA maturation, and mRNA export and translation. DDX3X concomitantly modulates the replication of several RNA viruses and promotes innate immunity. We previously showed that herpes simplex virus 1 (HSV-1), a human DNA virus, incorporates DDX3X into its mature particles and that DDX3X is required for optimal HSV-1 infectivity. Here, we show that viral gene expression, replication, and propagation depend on optimal DDX3X protein levels. Surprisingly, DDX3X from incoming viral particles was not required for the early stages of the HSV-1 infection, but, rather, the protein controlled the assembly of new viral particles. This was independent of the previously reported ability of DDX3X to stimulate interferon type I production. Instead, both the lack and overexpression of DDX3X disturbed viral gene transcription and thus subsequent genome replication. This suggests that in addition to its effect on RNA viruses, DDX3X impacts DNA viruses such as HSV-1 by an interferon-independent pathway.IMPORTANCE Viruses interact with a variety of cellular proteins to complete their life cycle. Among them is DDX3X, an RNA helicase that participates in most aspects of RNA biology, including transcription, splicing, nuclear export, and translation. Several RNA viruses and a limited number of DNA viruses are known to manipulate DDX3X for their own benefit. In contrast, DDX3X is also known to promote interferon production to limit viral propagation. Here, we show that DDX3X, which we previously identified in mature HSV-1 virions, stimulates HSV-1 gene expression and, consequently, virion assembly by a process that is independent of its ability to promote the interferon pathway.

  15. The effect of intracellular anions on ATP-dependent potassium channels of rat skeletal muscle.

    PubMed Central

    McKillen, H C; Davies, N W; Stanfield, P R; Standen, N B

    1994-01-01

    1. We have used excised inside-out patches to study the effects of anions bathing the cytoplasmic surface of the membrane on ATP-dependent K+ channels of rat flexor digitorum brevis muscle. Channels were closed by ATP applied to the cytoplasmic face of the patch with a concentration for half-closure (Ki) of 14 microM, were highly selective for K+ and had unitary conductances of 62 pS in symmetrical 155 mM K+ and 27 pS in 5 mM [K+]o. 2. In 139 mM Cl- internal solution channel activity declined rapidly after excision of the patch. Inclusion of 40 mM potassium gluconate (substituted for KCl) in the solution both restored channel activity and greatly slowed its subsequent run-down. 3. The action of gluconate was concentration dependent. The effect did not involve a change in ATP binding, since the Ki for ATP was not significantly changed by gluconate, and was specific for the cytoplasmic face of the patch. 4. The anions pyruvate, lactate and acetate were all able to restore channel activity after run-down, though less well than gluconate, while sulphate and methylsulphate were without effect. 5. Analysis of single channel kinetics showed that gluconate did not affect mean open lifetime, but led to a decrease in the number and duration of long closings. 6. Anions are most likely to act by stabilizing the structure of the channel protein. Changes in the intracellular concentration of certain anions may play a role in regulating channel activity. PMID:7837093

  16. ATM mediates repression of DNA end-degradation in an ATP-dependent manner.

    PubMed

    Rahal, Elias A; Henricksen, Leigh A; Li, Yuling; Turchi, John J; Pawelczak, Katherine S; Dixon, Kathleen

    2008-03-01

    Ataxia telangiectasia mutated (ATM) is a PI3-kinase-like kinase (PIKK) associated with DNA double-strand break (DSB) repair and cell cycle control. We have previously reported comparable efficiencies of DSB repair in nuclear extracts from both ATM deficient (A-T) and control (ATM+) cells; however, the repair products from the A-T nuclear extracts contained deletions encompassing longer stretches of DNA compared to controls. These deletions appeared to result from end-joining at sites of microhomology. These data suggest that ATM hinders error-prone repair pathways that depend on degradation of DNA ends at a break. Such degradation may account for the longer deletions we formerly observed in A-T cell extracts. To address this possibility we assessed the degradation of DNA duplex substrates in A-T and control nuclear extracts under DSB repair conditions. We observed a marked shift in signal intensity from full-length products to shorter products in A-T nuclear extracts, and addition of purified ATM to A-T nuclear extracts restored full-length product detection. This repression of degradation by ATM was both ATP-dependent and inhibited by the PIKK inhibitors wortmannin and caffeine. Addition of pre-phosphorylated ATM to an A-T nuclear extract in the presence of PIKK inhibitors was insufficient in repressing degradation, indicating that kinase activities are required. These results demonstrate a role for ATM in preventing the degradation of DNA ends possibly through repressing nucleases implicated in microhomology-mediated end-joining.

  17. Role of ATP-dependent K channels in the effects of erythropoietin in renal ischaemia injury

    PubMed Central

    Yilmaz, Tonguç Utku; Yazihan, Nuray; Dalgic, Aydın; Kaya, Ezgi Ermis; Salman, Bulent; Kocak, Mehtap; Akcil, Ethem

    2015-01-01

    Background & objectives: Erythropoietin (EPO) has cytoprotective and anti-apoptotic effects in pathological conditions, including hypoxia and ischaemia-reperfusion injury. One of the targets to protect against injury is ATP-dependent potassium (KATP) channels. These channels could be involved in EPO induced ischaemic preconditoning like a protective effect. We evaluated the cell cytoprotective effects of EPO in relation to KATP channel activation in the renal tubular cell culture model under hypoxic/normoxic conditions. Methods: Dose and time dependent effects of EPO, KATP channel blocker glibenclamide and KATP channel opener diazoxide on cellular proliferation were evaluated by colorimetric assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] under normoxic and hypoxic conditions in human renal proximal tubular cell line (CRL-2830). Evaluation of the dose and time dependent effects of EPO, glibenclamide and diazoxide on apoptosis was done by caspase-3 activity levels. Hypoxia inducible factor-1 alpha (HIF-1 α) mRNA levels were measured by semi-quantative reverse transcription polymerase chain reaction (RT)-PCR. Kir 6.1 protein expresion was evalutaed by Western blot. Results: Glibenclamide treatment decreased the number of living cells in a time and dose dependent manner, whereas EPO and diazoxide treatments increased. Glibenclamide (100 μM) treatment significantly blocked the anti-apoptotic effects of EPO (10 IU/ml) under both normoxic and hypoxic conditions. EPO (10 IU/ml) and diazoxide (100 μM) treatments significantly increased (P<0.01) whereas glibenclamide decreased (P<0.05) HIF-1 α mRNA expression. Glibenclamide significantly (P<0.01) decreased EPO induced HIF-1 α mRNA expression when compared with the EPO alone group. Interpretation & conclusions: Our results showed that the cell proliferative, cytoprotective and anti-apoptotic effects of EPO were associated with KATP channels in the renal tubular cell culture model under hypoxic

  18. 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.

  19. 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

  20. Tension-dependent nucleosome remodeling at the pericentromere in yeast.

    PubMed

    Verdaasdonk, Jolien S; Gardner, Ryan; Stephens, Andrew D; Yeh, Elaine; Bloom, Kerry

    2012-07-01

    Nucleosome positioning is important for the structural integrity of chromosomes. During metaphase the mitotic spindle exerts physical force on pericentromeric chromatin. The cell must adjust the pericentromeric chromatin to accommodate the changing tension resulting from microtubule dynamics to maintain a stable metaphase spindle. Here we examine the effects of spindle-based tension on nucleosome dynamics by measuring the histone turnover of the chromosome arm and the pericentromere during metaphase in the budding yeast Saccharomyces cerevisiae. We find that both histones H2B and H4 exhibit greater turnover in the pericentromere during metaphase. Loss of spindle-based tension by treatment with the microtubule-depolymerizing drug nocodazole or compromising kinetochore function results in reduced histone turnover in the pericentromere. Pericentromeric histone dynamics are influenced by the chromatin-remodeling activities of STH1/NPS1 and ISW2. Sth1p is the ATPase component of the Remodels the Structure of Chromatin (RSC) complex, and Isw2p is an ATP-dependent DNA translocase member of the Imitation Switch (ISWI) subfamily of chromatin-remodeling factors. The balance between displacement and insertion of pericentromeric histones provides a mechanism to accommodate spindle-based tension while maintaining proper chromatin packaging during mitosis.

  1. ATP-dependent incorporation of 20S protease into the 26S complex that degrades proteins conjugated to ubiquitin.

    PubMed Central

    Eytan, E; Ganoth, D; Armon, T; Hershko, A

    1989-01-01

    Previous studies have indicated that the ATP-dependent 26S protease complex that degrades proteins conjugated to ubiquitin is formed by the assembly of three factors in an ATP-requiring process. We now identify one of the factors as the 20S "multicatalytic" protease, a complex of low molecular weight subunits widely distributed in eukaryotic cells. Comparison of the subunit compositions of purified 20S and 26S complexes indicates that the former is an integral part of the latter. By the use of detergent treatment to activate latent protease activity, we show that the 20S protease becomes incorporated into the 26S complex in the ATP-dependent assembly process. It thus seems that the 20S protease is the "catalytic core" of the 26S complex of the ubiquitin proteolytic pathway. Images PMID:2554287

  2. INO80 and gamma-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair.

    PubMed

    Morrison, Ashby J; Highland, Jessica; Krogan, Nevan J; Arbel-Eden, Ayelet; Greenblatt, Jack F; Haber, James E; Shen, Xuetong

    2004-12-17

    While the role of ATP-dependent chromatin remodeling in transcription is well established, a link between chromatin remodeling and DNA repair has remained elusive. We have found that the evolutionarily conserved INO80 chromatin remodeling complex directly participates in the repair of a double-strand break (DSB) in yeast. The INO80 complex is recruited to a HO endonuclease-induced DSB through a specific interaction with the DNA damage-induced phosphorylated histone H2A (gamma-H2AX). This interaction requires Nhp10, an HMG-like subunit of the INO80 complex. The loss of Nhp10 or gamma-H2AX results in reduced INO80 recruitment to the DSB. Finally, components of the INO80 complex show synthetic genetic interactions with the RAD52 DNA repair pathway, the main pathway for DSB repair in yeast. Our findings reveal a new role of ATP-dependent chromatin remodeling in nuclear processes and suggest that an ATP-dependent chromatin remodeling complex can read a DNA repair histone code.

  3. Reciprocal translocations

    SciTech Connect

    1993-12-31

    Chapter 26, describes reciprocal translocations of chromosomes: their occurrence, breakpoints, and multiple rearrangements. In addition, phenotypes of balanced and unbalanced translocation carriers and fetal death are discussed. Examples of translocation families are given. Meiosis and genetic risk in translocation carriers is presented. Finally, sperm chromosomes in meiotic segregation analysis is mentioned. 39 refs., 3 figs., 1 tab.

  4. 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.

  5. 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.

  6. Regulation of nucleosome positioning by a CHD Type III chromatin remodeler and its relationship to developmental gene expression in Dictyostelium

    PubMed Central

    Platt, James L.; Kent, Nicholas A.; Kimmel, Alan R.

    2017-01-01

    Nucleosome placement and repositioning can direct transcription of individual genes; however, the precise interactions of these events are complex and largely unresolved at the whole-genome level. The Chromodomain-Helicase-DNA binding (CHD) Type III proteins are a subfamily of SWI2/SNF2 proteins that control nucleosome positioning and are associated with several complex human disorders, including CHARGE syndrome and autism. Type III CHDs are required for multicellular development of animals and Dictyostelium but are absent in plants and yeast. These CHDs can mediate nucleosome translocation in vitro, but their in vivo mechanism is unknown. Here, we use genome-wide analysis of nucleosome positioning and transcription profiling to investigate the in vivo relationship between nucleosome positioning and gene expression during development of wild-type (WT) Dictyostelium and mutant cells lacking ChdC, a Type III CHD protein ortholog. We demonstrate major nucleosome positional changes associated with developmental gene regulation in WT. Loss of chdC caused an increase of intragenic nucleosome spacing and misregulation of gene expression, affecting ∼50% of the genes that are repositioned during WT development. These analyses demonstrate active nucleosome repositioning during Dictyostelium multicellular development, establish an in vivo function of CHD Type III chromatin remodeling proteins in this process, and reveal the detailed relationship between nucleosome positioning and gene regulation, as cells transition between developmental states. PMID:28330902

  7. 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

  8. 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.

  9. Fast, long-range, reversible conformational fluctuations in nucleosomes revealed by single-pair fluorescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Tomschik, Miroslav; Zheng, Haocheng; van Holde, Ken; Zlatanova, Jordanka; Leuba, Sanford H.

    2005-03-01

    The nucleosome core particle, the basic repeated structure in chromatin fibers, consists of an octamer of eight core histone molecules, organized as dimers (H2A/H2B) and tetramers [(H3/H4)2] around which DNA wraps tightly in almost two left-handed turns. The nucleosome has to undergo certain conformational changes to allow processes that need access to the DNA template to occur. By single-pair fluorescence resonance energy transfer, we demonstrate fast, long-range, reversible conformational fluctuations in nucleosomes between two states: fully folded (closed), with the DNA wrapped around the histone core, or open, with the DNA significantly unraveled from the histone octamer. The brief excursions into an extended open state may create windows of opportunity for protein factors involved in DNA transactions to bind to or translocate along the DNA. conformational transitions | evanescent field fluorescence microscope | nucleosome dynamics | nucleosome opening

  10. ATP-dependent transport of leukotrienes B4 and C4 by the multidrug resistance protein ABCC4 (MRP4).

    PubMed

    Rius, Maria; Hummel-Eisenbeiss, Johanna; Keppler, Dietrich

    2008-01-01

    The proinflammatory mediators leukotriene (LT) B(4) and LTC(4) must be transported out of cells before they can interact with LT receptors. Previously, we identified the multidrug resistance protein ABCC1 (MRP1) as an efflux pump for LTC(4). However, the molecular basis for the efflux of LTB(4) was unknown. Here, we demonstrate that human ABCC4 mediates the ATP-dependent efflux of LTB(4) in the presence of reduced glutathione (GSH), whereby the latter can be replaced by S-methyl GSH. Transport studies were performed with inside-out membrane vesicles from V79 fibroblasts and Sf9 insect cells that contained recombinant ABCC4, with vesicles from human platelets and myelomonocytic U937 cells, which were rich in endogenous ABCC4, but ABCC1 was below detectability. Moreover, human polymorphonuclear leukocytes contained ABCC4. K(m) values for LTB(4) were 5.2 muM with vesicles from fibroblasts and 5.6 muM with vesicles from platelets. ABCC4, with its broad substrate specificity, also functioned as an ATP-dependent efflux pump for LTC(4) with a K(m) of 0.13 muM in vesicles from fibroblasts and 0.32 muM in vesicles from platelets. However, GSH was not required for the transport of this glutathionylated leukotriene. The transport of LTC(4) by ABCC4 explains its release from platelets during transcellular synthesis. ATP-dependent transport of LTB(4) and LTC(4) by ABCC4 was inhibited by several organic anions, including S-decyl GSH, sulindac sulfide, and by the LTD(4) receptor antagonists montelukast and 3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-((3-dimethyl-amino-3-oxopropyl)-thio)-methyl)thio)propanoic acid (MK571). Thus, as an efflux pump for the proinflammatory mediators LTB(4) and LTC(4), ABCC4 may represent a novel target for anti-inflammatory therapies.

  11. 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

  12. Loss of ATP-dependent transport activity in pseudoxanthoma elasticum-associated mutants of human ABCC6 (MRP6).

    PubMed

    Iliás, Attila; Urbán, Zsolt; Seidl, Thomas L; Le Saux, Olivier; Sinkó, Emese; Boyd, Charles D; Sarkadi, Balázs; Váradi, András

    2002-05-10

    Mutations in the ABCC6 (MRP6) gene cause pseudoxanthoma elasticum (PXE), a rare heritable disorder resulting in the calcification of elastic fibers. In the present study a cDNA encoding a full-length normal variant of ABCC6 was amplified from a human kidney cDNA library, and the protein was expressed in Sf9 insect cells. In isolated membranes ATP binding as well as ATP-dependent active transport by ABCC6 was demonstrated. We found that glutathione conjugates, including leukotriene C(4) and N-ethylmaleimide S-glutathione (NEM-GS), were actively transported by human ABCC6. Organic anions (probenecid, benzbromarone, indomethacin), known to interfere with glutathione conjugate transport of human ABCC1 and ABCC2, inhibited the ABCC6-mediated NEM-GS transport in a specific manner, indicating that ABCC6 has a unique substrate specificity. We have also expressed three missense mutant forms of ABCC6, which have recently been shown to cause PXE. MgATP binding was normal in these proteins; ATP-dependent NEM-GS or leukotriene C(4) transport, however, was abolished. Our data indicate that human ABCC6 is a primary active transporter for organic anions. In the three ABCC6 mutant forms examined, the loss of transport activity suggests that these mutations result in a PXE phenotype through a direct influence on the transport activity of this ABC transporter.

  13. C1-transport in gastric micorsomes. An ATP-dependent influx sensitive to membrane potential and to protein kinase inhibitor.

    PubMed

    Soumarmon, A; Abastado, M; Bonfils, S; Lewin, M J

    1980-12-25

    Uptakes of radioactive C1- or 1- by gastric microsomal vesicles were stimulated 2- to 8-fold by AtP. The sensitivity of those uptakes to a C1- in equilibrium OH- ionophore and to osmotic swelling suggested they were due to transport rather than to binding. The ATP effect was labile, but dithiothreitol and methanol improved its stability. The stimulation of anion transport required magnesium; GTP and UTP were less potent than ATP whereas ADP and AMP had no effect. The apparent Km for ATP was estimated to be 2 X 10(-4) M at 22 degrees C. The rate of the ATP-dependent transport showed saturation-type kinetics, with half-maximal uptake at 10 mM for I- and 15 mM for C1-. Nonradioactive C1-, I-, and SCN- competed with 125I- uptake while SO42- did not. K+ valinomycin increased the ATP-dependent C1- uptake. The thermostable inhibitor of cAMP-dependent protein kinases inhibited the effect of ATP. These results suggest the existence of an anion conductance, permeant to C1-, I-, and SCN- and nonpermeant to SO42-, which could be linked to a cAMP-dependent protein kinase.

  14. 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

  15. 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.

  16. CHD4 Is a Peripheral Component of the Nucleosome Remodeling and Deacetylase Complex.

    PubMed

    Low, Jason K K; Webb, Sarah R; Silva, Ana P G; Saathoff, Hinnerk; Ryan, Daniel P; Torrado, Mario; Brofelth, Mattias; Parker, Benjamin L; Shepherd, Nicholas E; Mackay, Joel P

    2016-07-22

    Chromatin remodeling enzymes act to dynamically regulate gene accessibility. In many cases, these enzymes function as large multicomponent complexes that in general comprise a central ATP-dependent Snf2 family helicase that is decorated with a variable number of regulatory subunits. The nucleosome remodeling and deacetylase (NuRD) complex, which is essential for normal development in higher organisms, is one such macromolecular machine. The NuRD complex comprises ∼10 subunits, including the histone deacetylases 1 and 2 (HDAC1 and HDAC2), and is defined by the presence of a CHD family remodeling enzyme, most commonly CHD4 (chromodomain helicase DNA-binding protein 4). The existing paradigm holds that CHD4 acts as the central hub upon which the complex is built. We show here that this paradigm does not, in fact, hold and that CHD4 is a peripheral component of the NuRD complex. A complex lacking CHD4 that has HDAC activity can exist as a stable species. The addition of recombinant CHD4 to this nucleosome deacetylase complex reconstitutes a NuRD complex with nucleosome remodeling activity. These data contribute to our understanding of the architecture of the NuRD complex.

  17. An rtt109-independent role for vps75 in transcription-associated nucleosome dynamics.

    PubMed

    Selth, Luke A; Lorch, Yahli; Ocampo-Hafalla, Maria T; Mitter, Richard; Shales, Michael; Krogan, Nevan J; Kornberg, Roger D; Svejstrup, Jesper Q

    2009-08-01

    The histone chaperone Vps75 forms a complex with, and stimulates the activity of, the histone acetyltransferase Rtt109. However, Vps75 can also be isolated on its own and might therefore possess Rtt109-independent functions. Analysis of epistatic miniarray profiles showed that VPS75 genetically interacts with factors involved in transcription regulation whereas RTT109 clusters with genes linked to DNA replication/repair. Additional genetic and biochemical experiments revealed a close relationship between Vps75 and RNA polymerase II. Furthermore, Vps75 is recruited to activated genes in an Rtt109-independent manner, and its genome-wide association with genes correlates with transcription rate. Expression microarray analysis identified a number of genes whose normal expression depends on VPS75. Interestingly, histone H2B dynamics at some of these genes are consistent with a role for Vps75 in histone H2A/H2B eviction/deposition during transcription. Indeed, reconstitution of nucleosome disassembly using the ATP-dependent chromatin remodeler Rsc and Vps75 revealed that these proteins can cooperate to remove H2A/H2B dimers from nucleosomes. These results indicate a role for Vps75 in nucleosome dynamics during transcription, and importantly, this function appears to be largely independent of Rtt109.

  18. 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.

  19. 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.

  20. Proton motive force-driven and ATP-dependent drug extrusion systems in multidrug-resistant Lactococcus lactis.

    PubMed Central

    Bolhuis, H; Molenaar, D; Poelarends, G; van Veen, H W; Poolman, B; Driessen, A J; Konings, W N

    1994-01-01

    Three mutants of Lactococcus lactis subsp. lactis MG1363, termed EthR, DauR, and RhoR, were selected for resistance to high concentrations of ethidium bromide, daunomycin, and rhodamine 6G, respectively. These mutants were found to be cross resistant to a number of structurally and functionally unrelated drugs, among which were typical substrates of the mammalian multidrug transporter (P-glycoprotein) such as daunomycin, quinine, actinomycin D, gramicidin D, and rhodamine 6G. The three multidrug-resistant strains showed an increased rate of energy-dependent ethidium and daunomycin efflux compared with that of the wild-type strain. This suggests that resistance to these toxic compounds is at least partly due to active efflux. Efflux of ethidium from the EthR strain could occur against a 37-fold inwardly directed concentration gradient. In all strains, ethidium efflux was inhibited by reserpine, a well-known inhibitor of P-glycoprotein. Ionophores which selectively dissipate the membrane potential or the pH gradient across the membrane inhibited ethidium and daunomycin efflux in the wild-type strain, corresponding with a proton motive force-driven efflux system. The ethidium efflux system in the EthR strain, on the other hand, was inhibited by ortho-vanadate and not upon dissipation of the proton motive force, which suggests the involvement of ATP in the energization of transport. The partial inhibition of ethidium efflux by ortho-vanadate and nigericin in the DauR and RhoR strains suggest that a proton motive force-dependent and an ATP-dependent system are expressed simultaneously. This is the first report of an ATP-dependent transport system in prokaryotes which confers multidrug resistance to the organism. PMID:7961458

  1. A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1.

    PubMed

    Chang, Xiu-bao

    2007-03-01

    Over a million new cases of cancers are diagnosed each year in the United States and over half of these patients die from these devastating diseases. Thus, cancers cause a major public health problem in the United States and worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Numerous mechanisms of MDR exist in cancer cells, such as intrinsic or acquired MDR. Overexpression of ATP-binding cassette (ABC) drug transporters, such as P-glycoprotein (P-gp or ABCB1), breast cancer resistance protein (BCRP or ABCG2) and/or multidrug resistance-associated protein (MRP1 or ABCC1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs. In addition to their roles in MDR, there is substantial evidence suggesting that these drug transporters have functions in tissue defense. Basically, these drug transporters are expressed in tissues important for absorption, such as in lung and gut, and for metabolism and elimination, such as in liver and kidney. In addition, these drug transporters play an important role in maintaining the barrier function of many tissues including blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier. Thus, these ATP-dependent drug transporters play an important role in the absorption, disposition and elimination of the structurally diverse array of the endobiotics and xenobiotics. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

  2. ATP-dependent regulation of actin monomer-filament equilibrium by cyclase-associated protein and ADF/cofilin.

    PubMed

    Nomura, Kazumi; Ono, Shoichiro

    2013-07-15

    CAP (cyclase-associated protein) is a conserved regulator of actin filament dynamics. In the nematode Caenorhabditis elegans, CAS-1 is an isoform of CAP that is expressed in striated muscle and regulates sarcomeric actin assembly. In the present study, we report that CAS-2, a second CAP isoform in C. elegans, attenuates the actin-monomer-sequestering effect of ADF (actin depolymerizing factor)/cofilin to increase the steady-state levels of actin filaments in an ATP-dependent manner. CAS-2 binds to actin monomers without a strong preference for either ATP- or ADP-actin. CAS-2 strongly enhances the exchange of actin-bound nucleotides even in the presence of UNC-60A, a C. elegans ADF/cofilin that inhibits nucleotide exchange. UNC-60A induces the depolymerization of actin filaments and sequesters actin monomers, whereas CAS-2 reverses the monomer-sequestering effect of UNC-60A in the presence of ATP, but not in the presence of only ADP or the absence of ATP or ADP. A 1:100 molar ratio of CAS-2 to UNC-60A is sufficient to increase actin filaments. CAS-2 has two independent actin-binding sites in its N- and C-terminal halves, and the C-terminal half is necessary and sufficient for the observed activities of the full-length CAS-2. These results suggest that CAS-2 (CAP) and UNC-60A (ADF/cofilin) are important in the ATP-dependent regulation of the actin monomer-filament equilibrium.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. ATP-dependent degradation of /sup 125/I-bovine serum albumin by rabbit reticulocytes does not need repression of an endogenous inhibitor

    SciTech Connect

    Saus, J.; Timoneda, J.

    1987-01-01

    The ubiquitin-dependent proteolysis of /sup 125/I-bovine serum albumin in rabbit reticulocytes has been investigated. Using various reticulocyte fractions (reticulocyte protease, inhibitor-free protease, ubiquitin and inhibitor) in the presence or absence of ATP, we found that the repression of an endogenous inhibitor, as suggested by others for alpha-casein proteolysis, is unlikely for bovine serum albumin. Therefore, differences exist in the ATP-dependent proteolytic pathway of rabbit reticulocytes depending on the substrate. Fractionation of the reticulocyte ATP-dependent proteolytic system revealed at least two proteolytic and two inhibitory fractions involved in the proteolysis of bovine serum albumin.

  8. 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.

  9. 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.

  10. 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

  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. Tetrahydroisoquinolines affect the whole-cell phenotype of Mycobacterium tuberculosis by inhibiting the ATP-dependent MurE ligase

    PubMed Central

    Guzman, Juan D.; Pesnot, Thomas; Barrera, Diana A.; Davies, Heledd M.; McMahon, Eleanor; Evangelopoulos, Dimitrios; Mortazavi, Parisa N.; Munshi, Tulika; Maitra, Arundhati; Lamming, Eleanor D.; Angell, Richard; Gershater, Markus C.; Redmond, Joanna M.; Needham, Deborah; Ward, John M.; Cuca, Luis E.; Hailes, Helen C.; Bhakta, Sanjib

    2015-01-01

    Objectives (S)-Leucoxine, isolated from the Colombian Lauraceae tree Rhodostemonodaphne crenaticupula Madriñan, was found to inhibit the growth of Mycobacterium tuberculosis H37Rv. A biomimetic approach for the chemical synthesis of a wide array of 1-substituted tetrahydroisoquinolines was undertaken with the aim of elucidating a common pharmacophore for these compounds with novel mode(s) of anti-TB action. Methods Biomimetic Pictet–Spengler or Bischler–Napieralski synthetic routes were employed followed by an evaluation of the biological activity of the synthesized compounds. Results In this work, the synthesized tetrahydroisoquinolines were found to inhibit the growth of M. tuberculosis H37Rv and affect its whole-cell phenotype as well as the activity of the ATP-dependent MurE ligase, a key enzyme involved in the early stage of cell wall peptidoglycan biosynthesis. Conclusions As the correlation between the MIC and the half-inhibitory enzymatic concentration was not particularly strong, there is a credible possibility that these compounds have pleiotropic mechanism(s) of action in M. tuberculosis. PMID:25656411

  13. EM single particle analysis of the ATP-dependent BchI complex of magnesium chelatase: an AAA+ hexamer.

    PubMed

    Willows, R D; Hansson, A; Birch, D; Al-Karadaghi, S; Hansson, M

    2004-01-01

    BchI, belonging to the AAA+ -protein family, forms the enzyme magnesium chelatase together with BchD and BchH. This enzyme catalyses the insertion of Mg2+ into protoporphyrin IX upon ATP hydrolysis. Previous studies have indicated that BchI forms ATP-dependent complexes and it is a member of the AAA+ -protein family (ATPases associated with various cellular activities) and it was suggested based on structural homology that the BchI formed hexameric complexes. AAA+ -proteins are Mg2+ -dependent ATPases that normally form oligomeric ring complexes in the presence of ATP. Single particle analysis of fully formed ring complexes of BchI observed by negative staining EM indicate that the BchI has strong 6- and 2-fold rotational symmetries and a weaker 4-fold rotational symmetry which are reminiscent of DNA helicase. A 2D average of the fully formed BchI-ATP ring complex is presented here from images of the complex obtained from negative staining EM. Other complexes are also observed in the EM micrographs and the class averages of these are indicative of the fragility and dynamic nature of the BchI complex which has been reported and they are suggestive of partially circular complexes with six or less protomers per particle. The resolution of the average circular complex is estimated at approximately 30A and it is similar in shape and size to an atomic resolution hexameric model of BchI rendered at 30A.

  14. ATP-dependent DNA ligase from Thermococcus sp. 1519 displays a new arrangement of the OB-fold domain.

    PubMed

    Petrova, T; Bezsudnova, E Y; Boyko, K M; Mardanov, A V; Polyakov, K M; Volkov, V V; Kozin, M; Ravin, N V; Shabalin, I G; Skryabin, K G; Stekhanova, T N; Kovalchuk, M V; Popov, V O

    2012-12-01

    DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini. Their function is essential for maintaining genome integrity in the replication, recombination and repair of DNA. High flexibility is important for the function of DNA ligase molecules. Two types of overall conformations of archaeal DNA ligase that depend on the relative position of the OB-fold domain have previously been revealed: closed and open extended conformations. The structure of ATP-dependent DNA ligase from Thermococcus sp. 1519 (LigTh1519) in the crystalline state determined at a resolution of 3.02 Å shows a new relative arrangement of the OB-fold domain which is intermediate between the positions of this domain in the closed and the open extended conformations of previously determined archaeal DNA ligases. However, small-angle X-ray scattering (SAXS) measurements indicate that in solution the LigTh1519 molecule adopts either an open extended conformation or both an intermediate and an open extended conformation with the open extended conformation being dominant.

  15. Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface.

    PubMed

    Williamson, Adele; Rothweiler, Ulli; Leiros, Hanna Kirsti Schrøder

    2014-11-01

    DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.

  16. Werner syndrome protein directly binds to the AAA ATPase p97/VCP in an ATP-dependent fashion.

    PubMed

    Indig, Fred Eliezer; Partridge, Juneth Joaquin; von Kobbe, Cayetano; Aladjem, Mirit I; Latterich, Martin; Bohr, Vilhelm A

    2004-01-01

    We have previously shown that the Werner syndrome helicase, WRNp, a member of the RecQ helicase family, forms a tight molecular complex with the p97/Valosin containing protein (VCP), a member of the AAA (ATPases associated with diverse cellular activities) family of proteins. This interaction is disrupted by chemical agents that confer DNA damage, suggesting that VCP plays an important role in the signal-dependent release of WRNp from its nucleolar sequestration site. Here, we characterized the structural requirements for interactions between WRNp and VCP and for the nuclear localization of VCP. We discovered that VCP directly binds to the RQC (RecQ conserved) domain of WRNp, which is a highly conserved motif common to the RecQ helicase family. This interaction is ATP-dependent, suggesting that VCP plays a mechanistic role in releasing WRNp from the nucleolus. Immunohistochemical analysis of various VCP domains and mutated proteins expressed in vitro demonstrated that VCP may contain several hierarchical cellular localization motifs within its domain structure.

  17. ATP-dependent acidification and tonoplast hyperpolarization in isolated vacuoles from green suspension cells of Chenopodium rubrum L

    PubMed Central

    Bentrup, F.-W.; Gogarten-Boekels, M.; Hoffmann, B.; Gogarten, J. P.; Baumann, C.

    1986-01-01

    The tonoplast of isolated vacuoles from photoautotrophic suspension cells of Chenopodium rubrum L. was studied by means of the patch-clamp technique. In a symmetrical K+ concentration of 46 mM, similar to in vivo conditions, the tonoplast displayed a membrane potential near zero and a linear current-voltage relationship with a mean slope of 1.0 S/m2. ATP at 2 mM hyperpolarized the tonoplast (vacuole positive) by 15-20 mV and, in a parallel experiment, acidified the vacuole (outside pH 7.0) to pH 5.0, as monitored by accumulation of acridine orange. Analysis of the voltage-clamp current indicates a 2-fold, ATP-dependent increase of the membrane capacitance, from 4 to 8 mF/m2, and an ATP-independent, unidentified ion channel having a mean opening time of about 5 msec and a conductivity of 0.5-1.0 pS. PMID:16593685

  18. The Copper Efflux Regulator CueR Is Subject to ATP-Dependent Proteolysis in Escherichia coli.

    PubMed

    Bittner, Lisa-Marie; Kraus, Alexander; Schäkermann, Sina; Narberhaus, Franz

    2017-01-01

    The trace element copper serves as cofactor for many enzymes but is toxic at elevated concentrations. In bacteria, the intracellular copper level is maintained by copper efflux systems including the Cue system controlled by the transcription factor CueR. CueR, a member of the MerR family, forms homodimers, and binds monovalent copper ions with high affinity. It activates transcription of the copper tolerance genes copA and cueO via a conserved DNA-distortion mechanism. The mechanism how CueR-induced transcription is turned off is not fully understood. Here, we report that Escherichia coli CueR is prone to proteolysis by the AAA(+) proteases Lon, ClpXP, and ClpAP. Using a set of CueR variants, we show that CueR degradation is not altered by mutations affecting copper binding, dimerization or DNA binding of CueR, but requires an accessible C terminus. Except for a twofold stabilization shortly after a copper pulse, proteolysis of CueR is largely copper-independent. Our results suggest that ATP-dependent proteolysis contributes to copper homeostasis in E. coli by turnover of CueR, probably to allow steady monitoring of changes of the intracellular copper level and shut-off of CueR-dependent transcription.

  19. Activation of ATP-dependent K+ channels by hypoxia in smooth muscle cells isolated from the pig coronary artery.

    PubMed Central

    Dart, C; Standen, N B

    1995-01-01

    1. The perforated patch technique with amphotericin B was used to record whole-cell currents activated by hypoxia in smooth muscle cells, isolated enzymatically from pig coronary arteries. 2. Superfusion with hypoxic solution (O2 partial pressure, 25-40 mmHg) activated an inward current at -60 mV in 143 mM extracellular K+. The reversal potential of the current induced by hypoxia shifted with extracellular [K+] as expected for a K+ current, while its current-voltage relation was consistent with the channels showing little voltage dependence. 3. The hypoxia-induced current was inhibited by glibenclamide (10 microM), but was unaffected by charybdotoxin (50 nM). 4. In whole-cell recordings at -60 mV in 143 mM K+ solution, openings of single channels passing a current close to -2 pA could sometimes be detected in normoxic solution. Openings became more frequent during the onset of the response to hypoxia, when several levels could be detected. Channels with a similar conductance were activated by hypoxia in cell-attached patches. 5. Our results suggest that hypoxia activates ATP-dependent K+ channels. We discuss possible mechanisms by which this activation may occur. PMID:7539841

  20. Role of ATP-dependent potassium channels in pulmonary vascular tone of fetal lambs with congenital diaphragmatic hernia.

    PubMed

    de Buys Roessingh, Anthony S; de Lagausie, Pascal; Barbet, Jacques-Patrick; Mercier, Jean-Christophe; Aigrain, Yves; Dinh-Xuan, Anh Tuan

    2006-11-01

    High mortality in newborn babies with congenital diaphragmatic hernia (CDH) is principally due to persistent pulmonary hypertension. ATP-dependent potassium (K(ATP)) channels might modulate pulmonary vascular tone. We have assessed the effects of Pinacidil, a K(ATP) channel opener, and glibenclamide (GLI), a K(ATP) channel blocker, in near full-term lambs with and without CDH. In vivo, pulmonary hemodynamics were assessed by means of pressure and blood flow catheters. In vitro, we used isolated pulmonary vessels and immunohistochemistry to detect the presence of K(ATP) channels in pulmonary tissue. In vivo, pinacidil (2 mg) significantly reduced pulmonary vascular resistance (PVR) in both controls and CDH animals. GLI (30 mg) significantly increased pulmonary arterial pressure (PAP) and PVR in control animals only. In vitro, pinacidil (10 microM) relaxed, precontracted arteries from lambs with and without CDH. GLI (10(-5) microM) did not raise the basal tone of vessels. We conclude that activation of K(ATP) channels could be of interest to reduce pulmonary vascular tone in fetal lambs with CDH, a condition often associated with persistent pulmonary hypertension of the newborn.

  1. Template Supercoiling during ATP-Dependent DNA Helix Tracking: Studies with Simian Virus 40 Large Tumor Antigen

    NASA Astrophysics Data System (ADS)

    Yang, Liu; Jessee, C. Bret; Lau, Kawai; Zhang, Hui; Liu, Leroy F.

    1989-08-01

    Incubation of topologically relaxed plasmid DNA with simian virus 40 (SV40) large tumor antigen (T antigen), ATP, and eubacterial DNA topoisomerase I resulted in the formation of highly positively supercoiled DNA. Eukaryotic DNA topoisomerase I could not substitute for eubacterial DNA topoisomerase I in this reaction. Furthermore, the addition of eukaryotic topoisomerase I to a preincubated reaction mixture containing both T antigen and eubacterial topoisomerase I caused rapid relaxation of the positively supercoiled DNA. These results suggest that SV40 T antigen can introduce topoisomerase-relaxable supercoils into DNA in a reaction coupled to ATP hydrolysis. We interpret the observed T antigen supercoiling reaction in terms of a recently proposed twin-supercoiled-domain model that describes the mechanics of DNA helix-tracking processes. According to this model, positive and negative supercoils are generated ahead of and behind the moving SV40 T antigen, respectively. The preferential relaxation of negative supercoils by eubacterial DNA topoisomerase I explains the accumulation of positive supercoils in the DNA template. The supercoiling assay using DNA conformation-specific eubacterial DNA topoisomerase I may be of general use for the detection of ATP-dependent DNA helix-tracking proteins.

  2. The kinesin-14 Klp2 organizes microtubules into parallel bundles by an ATP-dependent sorting mechanism.

    PubMed

    Braun, Marcus; Drummond, Douglas R; Cross, Robert A; McAinsh, Andrew D

    2009-06-01

    The dynamic organization of microtubules into parallel arrays allows interphase cells to set up multi-lane highways for intracellular transport and M-phase cells to build the mitotic and meiotic spindles. Here we show that a minimally reconstituted system composed of Klp2, a kinesin-14 from the fission yeast Schizosaccharomyces pombe, together with microtubules assembled from purified S. pombe tubulin, autonomously assembles bundles of parallel microtubules. Bundles form by an ATP-dependent sorting mechanism that requires the full-length Klp2 motor. By this mechanism, antiparallel-overlapped microtubules slide over one another until they dissociate from the bundles, whereas parallel-overlapped microtubules are selectively trapped by an energy-dissipating force-balance mechanism. Klp2-driven microtubule sorting provides a robust pathway for the organization of microtubules into parallel arrays. In vivo evidence indicates that Klp2 is required for the proper organization of S. pombe interphase microtubules into bipolar arrays of parallel-overlapped microtubules, suggesting that kinesin-14-dependent microtubule sorting may have wide biological importance.

  3. Identification of human and rat FAD-AMP lyase (cyclic FMN forming) as ATP-dependent dihydroxyacetone kinases.

    PubMed

    Cabezas, Alicia; Costas, María Jesús; Pinto, Rosa María; Couto, Ana; Cameselle, José Carlos

    2005-12-30

    Rat liver FAD-AMP lyase or FMN cyclase is the only known enzymatic source of the unusual flavin nucleotide riboflavin 4',5'-cyclic phosphate. To determine its molecular identity, a peptide-mass fingerprint of the purified rat enzyme was obtained. It pointed to highly related, mammalian hypothetical proteins putatively classified as dihydroxyacetone (Dha) kinases due to weaker homologies to biochemically proven Dha kinases of plants, yeasts, and bacteria. The human protein LOC26007 cDNA was used to design PCR primers. The product amplified from human brain cDNA was cloned, sequenced (GenBank Accession No. ), and found to differ from protein LOC26007 cDNA by three SNPs. Its heterologous expression yielded a protein active both as FMN cyclase and ATP-dependent Dha kinase, each activity being inhibited by the substrate(s) of the other. Cyclase and kinase activities copurified from rat liver extracts. Evidence supports that a single protein sustains both activities, probably in a single active center. Putative Dha kinases from other mammals are likely to be FMN cyclases too. Future work will profit from the availability of the structure of Citrobacter freundii Dha kinase, which contains substrate-interacting residues conserved in human Dha kinase/FMN cyclase.

  4. The Copper Efflux Regulator CueR Is Subject to ATP-Dependent Proteolysis in Escherichia coli

    PubMed Central

    Bittner, Lisa-Marie; Kraus, Alexander; Schäkermann, Sina; Narberhaus, Franz

    2017-01-01

    The trace element copper serves as cofactor for many enzymes but is toxic at elevated concentrations. In bacteria, the intracellular copper level is maintained by copper efflux systems including the Cue system controlled by the transcription factor CueR. CueR, a member of the MerR family, forms homodimers, and binds monovalent copper ions with high affinity. It activates transcription of the copper tolerance genes copA and cueO via a conserved DNA-distortion mechanism. The mechanism how CueR-induced transcription is turned off is not fully understood. Here, we report that Escherichia coli CueR is prone to proteolysis by the AAA+ proteases Lon, ClpXP, and ClpAP. Using a set of CueR variants, we show that CueR degradation is not altered by mutations affecting copper binding, dimerization or DNA binding of CueR, but requires an accessible C terminus. Except for a twofold stabilization shortly after a copper pulse, proteolysis of CueR is largely copper-independent. Our results suggest that ATP-dependent proteolysis contributes to copper homeostasis in E. coli by turnover of CueR, probably to allow steady monitoring of changes of the intracellular copper level and shut-off of CueR-dependent transcription. PMID:28293558

  5. ATP-Dependent C–F Bond Cleavage Allows the Complete Degradation of 4-Fluoroaromatics without Oxygen

    PubMed Central

    Tiedt, Oliver; Mergelsberg, Mario; Boll, Kerstin; Müller, Michael; Adrian, Lorenz; Jehmlich, Nico; von Bergen, Martin

    2016-01-01

    ABSTRACT Complete biodegradation of the abundant and persistent fluoroaromatics requires enzymatic cleavage of an arylic C–F bond, probably the most stable single bond of a biodegradable organic molecule. While in aerobic microorganisms defluorination of fluoroaromatics is initiated by oxygenases, arylic C–F bond cleavage has never been observed in the absence of oxygen. Here, an oxygen-independent enzymatic aryl fluoride bond cleavage is described during the complete degradation of 4-fluorobenzoate or 4-fluorotoluene to CO2 and HF in the denitrifying Thauera aromatica: the ATP-dependent defluorination of 4-fluorobenzoyl-coenzyme A (4-F-BzCoA) to benzoyl-coenzyme A (BzCoA) and HF, catalyzed by class I BzCoA reductase (BCR). Adaptation to growth with the fluoroaromatics was accomplished by the downregulation of a promiscuous benzoate-CoA ligase and the concomitant upregulation of 4-F-BzCoA-defluorinating/dearomatizing BCR on the transcriptional level. We propose an unprecedented mechanism for reductive arylic C–F bond cleavage via a Birch reduction-like mechanism resulting in a formal nucleophilic aromatic substitution. In the proposed anionic 4-fluorodienoyl-CoA transition state, fluoride elimination to BzCoA is favored over protonation to a fluorinated cyclic dienoyl-CoA. PMID:27507824

  6. Structure of the acetophenone carboxylase core complex: prototype of a new class of ATP-dependent carboxylases/hydrolases

    PubMed Central

    Weidenweber, Sina; Schühle, Karola; Demmer, Ulrike; Warkentin, Eberhard; Ermler, Ulrich; Heider, Johann

    2017-01-01

    Degradation of the aromatic ketone acetophenone is initiated by its carboxylation to benzoylacetate catalyzed by acetophenone carboxylase (Apc) in a reaction dependent on the hydrolysis of two ATP to ADP and Pi. Apc is a large protein complex which dissociates during purification into a heterooctameric Apc(αα′βγ)2 core complex of 482 kDa and Apcε of 34 kDa. In this report, we present the X-ray structure of the Apc(αα′βγ)2 core complex from Aromatoleum aromaticum at ca. 3 Å resolution which reveals a unique modular architecture and serves as model of a new enzyme family. Apcβ contains a novel domain fold composed of two β-sheets in a barrel-like arrangement running into a bundle of eight short polyproline (type II)-like helical segments. Apcα and Apcα′ possess ATP binding modules of the ASKHA superfamily integrated into their multidomain structures and presumably operate as ATP-dependent kinases for acetophenone and bicarbonate, respectively. Mechanistic aspects of the novel carboxylation reaction requiring massive structural rearrangements are discussed and criteria for specifically annotating the family members Apc, acetone carboxylase and hydantoinase are defined. PMID:28054554

  7. Inhibitory effect of pyridoxal 5'-phosphate on the DNA binding site of ATP-dependent deoxyribonuclease from Bacillus laterosporus.

    PubMed

    Fujiyoshi, T; Nakayama, J; Anai, M

    1981-04-01

    Bacillus laterosporus ATP-dependent deoxyribonuclease has been found to be inhibited by pyridoxal 5'-phosphate. The inhibition is specific for pyridoxal 5'-phosphate and pyridoxal which are required in relatively high concentrations. Pyridoxamine 5'-phosphate, pyridoxamine, and pyridoxine are ineffective. The inhibition is reversed by dilution or dialysis but can be changed to an irreversible inactivation by reduction of the enzyme . pyridoxal 5'-phosphate complex with sodium borohydride. The compound is a competitive inhibitor with respect to DNA but not ATP. Moreover, the presence of DNA substrate protects the enzyme against this inactivation but the presence of ATP shows no effect. The reduced enzyme . pyridoxal 5'-phosphate complex displays a new absorption maximum at 325 nm and a fluorescence emission at 390-400 nm when excited at 325 nm which are characteristic for epsilon-N-(phosphopyridoxyl)lysine. Thus, B. laterosporus DNase appears to have an essential lysine residue at the DNA binding site of the enzyme, and the enzyme possess two different active sites, a DNA binding site and an ATP binding site.

  8. ATP-dependent interaction of propranolol and local anaesthetic with sarcoplasmic reticulum. Stimulation of Ca2+ efflux.

    PubMed Central

    Shoshan-Barmatz, V

    1988-01-01

    Preincubation of sarcoplasmic reticulum (SR) with propranolol or tetracaine inhibits Ca2+ accumulation and stimulates ATPase activity by more than 2-fold. This effect is obtained only when the preincubation is carried out in the presence of ATP or other nucleoside triphosphates. The (ATP + drug)-induced inhibition of Ca2+ accumulation is pH-dependent, increasing as the pH rises above 7.5. The presence of micromolar concentrations of Ca2+ or Mg2+ during the preincubation prevents the inhibitory effect of ATP plus drug on Ca2+ accumulation or ATPase activity. The (ATP + drug) modification of SR vesicles resulted in stimulation of a rapid Ca2+ efflux from passively loaded vesicles. The ATP-dependent inhibition of Ca2+ accumulation by the drug is obtained with other local anaesthetics. The drug concentration required for 50% inhibition was 0.15 mM for dibucaine and 0.4 mM for both propranolol and tetracaine, whereas it was 5 mM, 8 mM and greater than 10 mM for lidocaine, benzocaine and procaine respectively. The heavy SR vesicles were only slightly affected by the incubation with propranolol or tetracaine in the presence of ATP, but their sensitivity increased markedly after storage at 0 degrees C for 24-48 h. These results suggest that propranolol and some local anaesthetics, in the presence of ATP, stimulate Ca2+ efflux by modifying a protein factor(s) rather than the phospholipid bilayer. PMID:2975944

  9. An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon iosAsp formation but is restored by Protein Isoaspartyl Methltransferase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arabidopsis thaliana PLANT RNA HELICASE75 (AtPRH75) demonstrated an ATP-dependent, RNA duplex unwinding capacity and an ATP-independent, RNA duplex reforming ability. It is known to accumulate isoAsp, but the consequences of isoAsp formation in AtPRH75 are unknown. Duplex unwinding was abolished by ...

  10. 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.

  11. 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

  12. 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

  13. Redox-dependent complex formation by an ATP-dependent activator of the corrinoid/iron-sulfur protein

    PubMed Central

    Hennig, Sandra E.; Jeoung, Jae-Hun; Goetzl, Sebastian; Dobbek, Holger

    2012-01-01

    Movement, cell division, protein biosynthesis, electron transfer against an electrochemical gradient, and many more processes depend on energy conversions coupled to the hydrolysis of ATP. The reduction of metal sites with low reduction potentials (E0′ < -500 mV) is possible by connecting an energetical uphill electron transfer with the hydrolysis of ATP. The corrinoid-iron/sulfur protein (CoFeSP) operates within the reductive acetyl-CoA pathway by transferring a methyl group from methyltetrahydrofolate bound to a methyltransferase to the [Ni-Ni-Fe4S4] cluster of acetyl-CoA synthase. Methylation of CoFeSP only occurs in the low-potential Co(I) state, which can be sporadically oxidized to the inactive Co(II) state, making its reductive reactivation necessary. Here we show that an open-reading frame proximal to the structural genes of CoFeSP encodes an ATP-dependent reductive activator of CoFeSP. Our biochemical and structural analysis uncovers a unique type of reductive activator distinct from the electron-transferring ATPases found to reduce the MoFe-nitrogenase and 2-hydroxyacyl-CoA dehydratases. The CoFeSP activator contains an ASKHA domain (acetate and sugar kinases, Hsp70, and actin) harboring the ATP-binding site, which is also present in the activator of 2-hydroxyacyl-CoA dehydratases and a ferredoxin-like [2Fe-2S] cluster domain acting as electron donor. Complex formation between CoFeSP and its activator depends on the oxidation state of CoFeSP, which provides evidence for a unique strategy to achieve unidirectional electron transfer between two redox proteins. PMID:22431597

  14. ATP-dependent phosphorylation of serine-46 in the phosphocarrier protein HPr regulates lactose/H+ symport in Lactobacillus brevis.

    PubMed Central

    Ye, J J; Reizer, J; Cui, X; Saier, M H

    1994-01-01

    Lactobacillus brevis takes up lactose and the nonmetabolizable lactose analogue thiomethyl beta-galactoside (TMG) by a permease-catalyzed lactose/H+ symport mechanism. Earlier studies have shown that TMG, previously accumulated in L. brevis cells, rapidly effluxes from the cells upon addition of glucose, and that glucose inhibits further uptake of TMG. We have developed a vesicular system to analyze this regulatory mechanism and have used electroporation to shock proteins and membrane-impermeant metabolites into the vesicles. Uptake of TMG was dependent on an energy source, effectively provided by intravesicular ATP or extravesicular arginine. TMG uptake into these vesicles was not inhibited, and preaccumulated TMG did not efflux from them upon addition of glucose. Intravesicular but not extravesicular wild-type phosphocarrier protein HPr of Bacillus subtilis restored regulation. Glucose could be replaced by intravesicular (but not extravesicular) fructose 1,6-bisphosphate, gluconate 6-phosphate, or 2-phosphoglycerate, but not by other phosphorylated metabolites, in agreement with the allosteric activating effects of these compounds on HPr(Ser) kinase measured in vitro. Intravesicular serine-46-->alanine mutant HPr cold not promote regulation of lactose permease activity when electroporated into the vesicles with or without glucose or the various phosphorylated metabolites, but the serine-46-->aspartate mutant HPr promoted regulation, even in the absence of glucose or a metabolite. HPr(Ser-P) appears to convert the lactose/H+ symporter into a sugar uniporter. These results establish that HPr serine phosphorylation by the ATP-dependent metabolite-activated HPr kinase regulates lactose permease activity in L. brevis. A direct allosteric mechanism is proposed. Images PMID:8159711

  15. Mitochondria present in excised patches from pancreatic B-cells may form microcompartments with ATP-dependent potassium channels.

    PubMed

    Rustenbeck, I; Dickel, C; Herrmann, C; Grimmsmann, T

    1999-04-01

    Experiments with inside-out patches excised from pancreatic B-cells have yielded evidence that mitochondria are often contained in the cytoplasmic plug protruding into the tip of patch pipette. When intact B-cells were loaded with the fluorescent mitochondrial stain, rhodamine 123, and membrane patches excised from these cells, a green fluorescence could be observed in the lumen at the tip of the patch pipette. The same result was obtained with the mitochondrial stain, MitoTracker Green FM, which is only fluorescent in a membrane-bound state. Furthermore, the open probability of ATP-dependent potassium (K(ATP)) channels in inside-out patches was influenced by mitochondrial fuels and inhibitors. Respiratory substrates like tetramethyl phenylene diamine (2 mM) plus ascorbate (5 mM) or alpha-ketoisocaproic acid (10 mM) reduced the open probability of K(ATP) channels in inside-out patches significantly (down to 57% or 65% of control, respectively). This effect was antagonized by the inhibitor of cytochrome oxidase, sodium azide (5 mM). Likewise, the inhibitor of succinate dehydrogenase, malonate (5 mM), increased the open probability of K(ATP) channels in the presence of succinate (1 mM). However, oligomycin in combination with antimycin and rotenone did not increase open probability. Although it cannot be excluded that these effects result from a direct interaction with the K(ATP) channels, the presence of mitochondria in the close vicinity permits the hypothesis that changes in mitochondrial metabolism are involved, mitochondria and K(ATP) channels thus forming functional microcompartments.

  16. Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

    SciTech Connect

    Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

    1986-05-01

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca/sup 2 +/ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting /sup 32/P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated /sup 32/P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor.

  17. period-1 encodes an ATP-dependent RNA helicase that influences nutritional compensation of the Neurospora circadian clock

    SciTech Connect

    Emerson, Jillian M.; Bartholomai, Bradley M.; Ringelberg, Carol; Baker, Scott E.; Loros, Jennifer J.; Dunlap, Jay C.

    2015-12-22

    Mutants in the period-1 (prd-1) gene, characterized by a recessive allele, display a reduced growth rate and period lengthening of the developmental cycle controlled by the circadian clock. We refined the genetic location of prd-1 and used whole genome sequencing to find the mutation defining it, confirming the identity of prd-1 by rescuing the mutant circadian phenotype via transformation. PRD-1 is an RNA helicase whose orthologs, DDX5 and DDX17 in humans and Dbp2p in yeast, are implicated in various processes including transcriptional regulation, elongation, and termination, 23 ribosome biogenesis, and RNA decay. Although prdi-1smutantssiois an ATP-dependent RNA helicase, member of a sub-family display a long period (~25 hrs) circadian developmental cycle, they interestingly display a wild type period when the core circadian oscillator is tracked using a frq-luciferase transcriptional fusion under conditions of limiting nutritional carbon; the core oscillator runs with a long period under glucose-sufficient conditions. Thus PRD-1 clearly impacts the circadian oscillator and is not only part of a metabolic oscillator ancillary to the core clock. PRD-1 is an essential protein and its expression is neither light-regulated nor clock-regulated. However, it is transiently induced by glucose; in the presence of sufficient glucose PRD-1 is in the nucleus until glucose runs out which elicits its disappearance from the nucleus. Because circadian period length is carbon concentration-dependent, prd­-1 may be formally viewed as clock mutant with defective nutritional compensation of circadian period length.

  18. A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels.

    PubMed

    Zhang, Roger S; Wright, Jordan D; Pless, Stephan A; Nunez, John-Jose; Kim, Robin Y; Li, Jenny B W; Yang, Runying; Ahern, Christopher A; Kurata, Harley T

    2015-06-19

    ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Regulation of the glucose:H+ symporter by metabolite-activated ATP-dependent phosphorylation of HPr in Lactobacillus brevis.

    PubMed Central

    Ye, J J; Neal, J W; Cui, X; Reizer, J; Saier, M H

    1994-01-01

    phosphorylation by the ATP-dependent, metabolite-activated HPr kinase regulates glucose and lactose permease activities in L. brevis and suggest that other permeases may also be subject to this mode of regulation. PMID:8206825

  7. ATP-dependent roles of the DEAD-box protein Mss116p in group II intron splicing in vitro and in vivo

    PubMed Central

    Potratz, Jeffrey P.; Campo, Mark Del; Wolf, Rachel Z.; Lambowitz, Alan M.; Russell, Rick

    2011-01-01

    The yeast DEAD-box protein Mss116p functions as a general RNA chaperone in splicing mitochondrial group I and group II introns. For most of its functions, Mss116p is thought to use ATP-dependent RNA unwinding to facilitate RNA structural transitions, but it has been suggested to assist folding of one group II intron (aI5γ) primarily by stabilizing a folding intermediate. Here we compare three aI5γ constructs: one with long exons, one with short exons, and a ribozyme construct lacking exons. The long exons result in slower splicing, suggesting that they misfold and/or stabilize non-native intronic structure. Nevertheless, Mss116p acceleration of all three constructs depends upon ATP and is inhibited by mutations that compromise RNA unwinding, suggesting similar mechanisms. Results of splicing assays and a new two-stage assay that separates ribozyme folding and catalysis indicate that maximal folding of all three constructs by Mss116p requires ATP-dependent RNA unwinding. ATP-independent activation is appreciable for only a subpopulation of the minimal ribozyme construct and not for constructs containing exons. As expected for a general RNA chaperone, Mss116p can also disrupt the native ribozyme, which can refold after Mss116p removal. Finally, using yeast strains with mtDNA containing only the single intron aI5γ, we show that Mss116p mutants promote splicing in vivo to degrees that correlate with their residual ATP-dependent RNA-unwinding activities. Together, our results indicate that, although DEAD-box proteins play multiple roles in RNA folding, the physiological function of Mss116p in aI5γ splicing includes a requirement for ATP-dependent local unfolding, allowing conversion of non-functional to functional RNA structure. PMID:21679717

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Robertsonian translocations

    SciTech Connect

    1993-12-31

    Chapter 27, describes the occurrence of Robertsonian translocations (RTs), which refer to the recombination of whole chromosome arms, in both monocentric and dicentric chromosomes. The nonrandom participation of acrocentric chromosomes in RTs is documented by various methods, including unbiased ascertainment and ascertainment through trisomy, infertility, unspecified mental retardation, and Prader-Willi syndrome. Causes of nonrandom participation of chromosomes in RTs is presented, as are the following topics: segregation in carriers of RTs and segregation in sperm cells of RT carriers, interchromosomal effects and conclusions. 48 refs., 3 figs., 2 tabs.

  15. 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.

  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. Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein.

    PubMed

    Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

    2015-12-29

    The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

  18. 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.

  19. Nucleosome remodeling by the SWI/SNF complex is enhanced by yeast High Mobility Group Box (HMGB) proteins

    PubMed Central

    Hepp, Matias I.; Alarcon, Valentina; Dutta, Arnob; Workman, Jerry L.; Gutiérrez, José L.

    2014-01-01

    Regulation of gene expression at the level of transcription involves the concerted action of several proteins and protein complexes committed to dynamically alter the surrounding chromatin environment of a gene being activated or repressed. ATP-dependent chromatin remodeling complexes are key actors in chromatin remodeling, and the SWI/SNF complex is the founding member. While many studies have linked the action of these complexes to specific transcriptional regulation of a large number of genes and much is known about their catalytic activity, less is known about the nuclear elements that can enhance or modulate their activity. A number of studies have found that certain High Mobility Group (HMG) proteins are able to stimulate ATP-dependent chromatin remodeling activity, but their influence on the different biochemical outcomes of this activity is still unknown. In this work we studied the influence of the yeast Nhp6A, Nhp6B and Hmo1 proteins (HMGB family members) on different biochemical outcomes of yeast SWI/SNF remodeling activity. We found that all these HMG proteins stimulate the sliding activity of ySWI/SNF, while transient exposure of nucleosomal DNA and octamer transfer catalyzed by this complex are only stimulated by Hmo1. Consistently, only Hmo1 stimulates SWI/SNF binding to the nucleosome. Additionally, the sliding activity of another chromatin remodeling complex, ISW1a, is only stimulated by Hmo1. Further analyses show that these differential stimulatory effects of Hmo1 are dependent on the presence of its C-terminal tail, which contains a stretch of acidic and basic residues. PMID:24972368

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. Conformational transition of the lid helix covering the protease active site is essential for the ATP-dependent protease activity of FtsH.

    PubMed

    Suno, Ryoji; Shimoyama, Masakazu; Abe, Akiko; Shimamura, Tatsuro; Shimodate, Natsuka; Watanabe, Yo-hei; Akiyama, Yoshinori; Yoshida, Masasuke

    2012-09-21

    When bound to ADP, ATP-dependent protease FtsH subunits adopt either an "open" or "closed" conformation. In the open state, the protease catalytic site is located in a narrow space covered by a lidlike helix. This space disappears in the closed form because the lid helix bends at Gly448. Here, we replaced Gly448 with various residues that stabilize helices. Most mutants retained low ATPase activity and bound to the substrate protein, but lost protease activity. However, a mutant proline substitution lost both activities. Our study shows that the conformational transition of the lid helix is essential for the function of FtsH.

  7. Genome-Wide Mapping Targets of the Metazoan Chromatin Remodeling Factor NURF Reveals Nucleosome Remodeling at Enhancers, Core Promoters and Gene Insulators

    PubMed Central

    Kwon, So Yeon; Grisan, Valentina; Jang, Boyun; Herbert, John; Badenhorst, Paul

    2016-01-01

    NURF is a conserved higher eukaryotic ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF is able to alter chromatin dynamics to control transcription and genome organization. Previous biochemical and genetic analysis of the specificity-subunit of Drosophila NURF (Nurf301/Enhancer of Bithorax (E(bx)) has defined NURF as a critical regulator of homeotic, heat-shock and steroid-responsive gene transcription. It has been speculated that NURF controls pathway specific transcription by co-operating with sequence-specific transcription factors to remodel chromatin at dedicated enhancers. However, conclusive in vivo demonstration of this is lacking and precise regulatory elements targeted by NURF are poorly defined. To address this, we have generated a comprehensive map of in vivo NURF activity, using MNase-sequencing to determine at base pair resolution NURF target nucleosomes, and ChIP-sequencing to define sites of NURF recruitment. Our data show that, besides anticipated roles at enhancers, NURF interacts physically and functionally with the TRF2/DREF basal transcription factor to organize nucleosomes downstream of active promoters. Moreover, we detect NURF remodeling and recruitment at distal insulator sites, where NURF functionally interacts with and co-localizes with DREF and insulator proteins including CP190 to establish nucleosome-depleted domains. This insulator function of NURF is most apparent at subclasses of insulators that mark the boundaries of chromatin domains, where multiple insulator proteins co-associate. By visualizing the complete repertoire of in vivo NURF chromatin targets, our data provide new insights into how chromatin remodeling can control genome organization and regulatory interactions. PMID:27046080

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Dbp7p, a putative ATP-dependent RNA helicase from Saccharomyces cerevisiae, is required for 60S ribosomal subunit assembly.

    PubMed Central

    Daugeron, M C; Linder, P

    1998-01-01

    Putative ATP-dependent RNA helicases are ubiquitous, highly conserved proteins that are found in most organisms and they are implicated in all aspects of cellular RNA metabolism. Here we present the functional characterization of the Dbp7 protein, a putative ATP-dependent RNA helicase of the DEAD-box protein family from Saccharomyces cerevisiae. The complete deletion of the DBP7 ORF causes a severe slow-growth phenotype. In addition, the absence of Dbp7p results in a reduced amount of 60S ribosomal subunits and an accumulation of halfmer polysomes. Subsequent analysis of pre-rRNA processing indicates that this 60S ribosomal subunit deficit is due to a strong decrease in the production of 27S and 7S precursor rRNAs, which leads to reduced levels of the mature 25S and 5.8S rRNAs. Noticeably, the overall decrease of the 27S pre-rRNA species is neither associated with the accumulation of preceding precursors nor with the emergence of abnormal processing intermediates, suggesting that these 27S pre-rRNA species are degraded rapidly in the absence of Dbp7p. Finally, an HA epitope-tagged Dbp7 protein is localized in the nucleolus. We propose that Dbp7p is involved in the assembly of the pre-ribosomal particle during the biogenesis of the 60S ribosomal subunit. PMID:9582098

  13. The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes.

    PubMed Central

    Wagner, J D; Jankowsky, E; Company, M; Pyle, A M; Abelson, J N

    1998-01-01

    Of the proteins required for pre-mRNA splicing, at least four, the DEAH-box proteins, are closely related due to the presence of a central 'RNA helicase-like' region, and extended homology through a large portion of the protein. A major unresolved question is the function of these proteins. Indirect evidence suggests that several of these proteins are catalysts for important structural rearrangements in the spliceosome. However, the mechanism for the proposed alterations is presently unknown. We present evidence that PRP22, a DEAH-box protein required for mRNA release from the spliceosome, unwinds RNA duplexes in a concentration- and ATP-dependent manner. This demonstrates that PRP22 can modify RNA structure directly. We also show that the PRP22-dependent release of mRNA from the spliceosome is an ATP-dependent process and that recombinant PRP22 is an ATPase. Non-hydrolyzable ATP analogs did not substitute for ATP in the RNA-unwinding reaction, suggesting that ATP hydrolysis is required for this reaction. Specific mutation of a putative ATP phosphate-binding motif in the recombinant protein eliminated the ATPase and RNA-unwinding capacity. Significantly, these data suggest that the DEAH-box proteins act directly on RNA substrates within the spliceosome. PMID:9582286

  14. The nucleotide exchange factor MGE exerts a key function in the ATP-dependent cycle of mt-Hsp70-Tim44 interaction driving mitochondrial protein import.

    PubMed Central

    Schneider, H C; Westermann, B; Neupert, W; Brunner, M

    1996-01-01

    Import of preproteins into the mitochondrial matrix is driven by the ATP-dependent interaction of mt-Hsp70 with the peripheral inner membrane import protein Tim44 and the preprotein in transit. We show that Mge1p, a co-chaperone of mt-Hsp70, plays a key role in the ATP-dependent import reaction cycle in yeast. Our data suggest a cycle in which the mt-Hsp70-Tim44 complex forms with ATP: Mge1p promotes assembly of the complex in the presence of ATP. Hydrolysis of ATP by mt-Hsp70 occurs in complex with Tim44. Mge1p is then required for the dissociation of the ADP form of mt-Hsp70 from Tim44 after release of inorganic phosphate but before release of ADP. ATP hydrolysis and complex dissociation are accompanied by tight binding of mt-Hsp70 to the preprotein in transit. Subsequently, the release of mt-Hsp70 from the polypeptide chain is triggered by Mge1p which promotes release of ADP from mt-Hsp70. Rebinding of ATP to mt-Hsp70 completes the reaction cycle. Images PMID:8918457

  15. 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.

  16. The chromatin remodeling complex NuRD establishes the poised state of rRNA genes characterized by bivalent histone modifications and altered nucleosome positions.

    PubMed

    Xie, Wenbing; Ling, Te; Zhou, Yonggang; Feng, Weijun; Zhu, Qiaoyun; Stunnenberg, Henk G; Grummt, Ingrid; Tao, Wei

    2012-05-22

    rRNA genes (rDNA) exist in two distinct epigenetic states, active promoters being unmethylated and marked by euchromatic histone modifications, whereas silent ones are methylated and exhibit heterochromatic features. Here we show that the nucleosome remodeling and deacetylation (NuRD) complex establishes a specific chromatin structure at rRNA genes that are poised for transcription activation. The promoter of poised rRNA genes is unmethylated, associated with components of the preinitiation complex, marked by bivalent histone modifications and covered by a nucleosome in the "off" position, which is refractory to transcription initiation. Repression of rDNA transcription in growth-arrested and differentiated cells correlates with elevated association of NuRD and increased levels of poised rRNA genes. Reactivation of transcription requires resetting the promoter-bound nucleosome into the "on" position by the DNA-dependent ATPase CSB (Cockayne syndrome protein B). The results uncover a unique mechanism by which ATP-dependent chromatin remodeling complexes with opposing activities establish a specific chromatin state and regulate transcription.

  17. 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.

  18. 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).

  19. 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.

  20. 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

  1. Monitoring Conformational Dynamics with Single-Molecule Fluorescence Energy Transfer: Applications in Nucleosome Remodeling

    PubMed Central

    Deindl, Sebastian; Zhuang, Xiaowei

    2016-01-01

    Due to its ability to track distance changes within individual molecules or molecular complexes on the nanometer scale and in real time, single-molecule fluorescence resonance energy transfer (single-molecule FRET) is a powerful tool to tackle a wide range of important biological questions. Using our recently developed single-molecule FRET assay to monitor nucleosome translocation as an illustrative example, we describe here in detail how to set up, carry out, and analyze single-molecule FRET experiments that provide time-dependent information on biomolecular processes. PMID:22929765

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. Overexpression of the Gene Encoding the Multidrug Resistance-Associated Protein Results in Increased ATP-Dependent Glutathione S-Conjugate Transport

    NASA Astrophysics Data System (ADS)

    Muller, Michael; Meijer, Coby; Zaman, Guido J. R.; Borst, Piet; Scheper, Rik J.; Mulder, Nanno H.; de Vries, Elisabeth G. E.; Jansen, Peter L. M.

    1994-12-01

    The multidrug resistance-associated protein (MRP) is a 180- to 195-kDa glycoprotein associated with multidrug resistance of human tumor cells. MRP is mainly located in the plasma membrane and it confers resistance by exporting natural product drugs out of the cell. Here we demonstrate that overexpression of the MRP gene in human cancer cells increases the ATP-dependent glutathione S-conjugate carrier activity in plasma membrane vesicles isolated from these cells. The glutathione S-conjugate export carrier is known to mediate excretion of bivalent anionic conjugates from mammalian cells and is thought to play a role in the elimination of conjugated xenobiotics. Our results suggest that MRP can cause multidrug resistance by promoting the export of drug modification products from cells and they shed light on the reported link between drug resistance and cellular glutathione and glutathione S-transferase levels.

  7. Distinct roles for the RSC and Swi/Snf ATP-dependent chromatin remodelers in DNA double-strand break repair.

    PubMed

    Chai, Bob; Huang, Jian; Cairns, Bradley R; Laurent, Brehon C

    2005-07-15

    The failure of cells to repair damaged DNA can result in genomic instability and cancer. To efficiently repair chromosomal DNA lesions, the repair machinery must gain access to the damaged DNA in the context of chromatin. Here we report that both the RSC and Swi/Snf ATP-dependent chromatin-remodeling complexes play key roles in double-strand break (DSB) repair, specifically by homologous recombination (HR). RSC and Swi/Snf are each recruited to an in vivo DSB site but with distinct kinetics. We show that Swi/Snf is required earlier, at or preceding the strand invasion step of HR, while RSC is required following synapsis for completion of the recombinational repair event.

  8. Overexpression of DEAD box protein pMSS116 promotes ATP-dependent splicing of a yeast group II intron in vitro.

    PubMed Central

    Niemer, I; Schmelzer, C; Börner, G V

    1995-01-01

    The group II intron bI1, the first intron of the mitochondrial cytochrome b gene in yeast is self-splicing in vitro. Genetic evidence suggests that trans-acting factors are required for in vivo splicing of this intron. In accordance with these findings, we present in vitro data showing that splicing of bI1 under physiological conditions depends upon the presence of proteins of a mitochondrial lysate. ATP is an essential component is this reaction. Overexpression of the nuclear-encoded DEAD box protein pMSS-116 results in a marked increase in the ATP-dependent splicing activity of the extract, suggesting that pMSS116 may play an important role in splicing of bI1. Images PMID:7659519

  9. The inhibitory effect of some chlorinated hydrocarbon pesticides on the ATP-dependent Ca2+ binding of the particulate fraction of the eggshell gland mucosa cells.

    PubMed

    Lundholm, C E; Mathson, K

    1983-05-01

    The pesticide p-p'-DDT and its persistent metabolite p-p'-DDE cause thinning of the eggshells in several species of birds. In earlier investigations on ducks this thinning was found to be associated with a reduction of the ATP-dependent Ca2+ binding to a homogenate of the shell gland mucosal cells by DDE. The activity of a Ca2+-Mg2+-activated ATPase in the homogenate was also decreased on administration of DDE in vivo. We have therefore investigated the in vitro effects of some other chlorinated hydrocarbon pesticides of ecotoxicological interest on the ATP-dependent Ca2+ binding and the Ca2+-Mg2+-activated ATPase activity in a homogenate of the eggshell gland mucosa of the hen and determined the molar concentrations that produced 50% inhibition (=IC50). Several of the investigated compounds, namely toxaphene, chlordane, p-p'-DDD, o-p'-DDE, p-p'-DDT, methoxychlor and PCB (Arochlor 1242), had a similar IC50 to inhibit the Ca2+ binding as p-p'-DDE. Lindane, p-p'-DDA and biphenyl had an IC50 3.3-4 times higher and that of 2.4 D was 13.5 times higher than that of p-p'-DDE. When the IC50 of some of the compounds (p-p'-DDE, PCB, toxaphene, Lindane) was determined that decreased the Ca2+-Mg2+-activated ATPase of the homogenate it was found to be only 18 to 29 per cent of that needed to inhibit the Ca2+ binding by the homogenate. It is therefore probable that some other effect than inhibition of this enzyme is also involved in the Ca2+-binding process and affected by the compounds.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Assembly of Drosophila Centromeric Nucleosomes Requires CID Dimerization

    PubMed Central

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

    2012-01-01

    SUMMARY Centromeres are essential chromosomal regions required for kinetochore assembly and chromosome segregation. The composition and organization of centromeric nucleosomes containing the essential histone H3 variant CENP-A (CID in Drosophila) is a fundamental, unresolved issue. Using immunoprecipitation of CID mononucleosomes and cysteine crosslinking, we demonstrate that centromeric nucleosomes contain CID dimers in vivo. Furthermore, CID dimerization and centromeric targeting require a residue implicated in formation of the four helix bundle, which mediates intra-nucleosomal H3 dimerization and nucleosome integrity. Taken together, our findings suggest that CID nucleosomes are octameric in vivo and that CID dimerization is essential for correct centromere assembly. PMID:22209075

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. Dephosphorylation of phosphoproteins and synthetic phosphopeptides. Study of the specificity of the polycation-stimulated and MgATP-dependent phosphorylase phosphatases.

    PubMed

    Agostinis, P; Goris, J; Waelkens, E; Pinna, L A; Marchiori, F; Merlevede, W

    1987-01-25

    The substrate specificity of different forms of polycation-stimulated (PCSH, PCSL, and PCSC) phosphorylase phosphatases and of the catalytic subunit of the MgATP-dependent protein phosphatase from rabbit skeletal muscle was investigated. This was done, with phosphorylase a as the reference substrate, using the synthetic phosphopeptides patterned after the phosphorylated sites of pyruvate kinase (type L) (Arg2-Ala-Ser(32P)-Val-Ala (S2), and its Thr(32P) substitute (T4)), inhibitor-1 (Arg4-Pro-Thr(32P)-Pro-Ala (T5), Arg2-Pro-Thr(32P)-Pro-Ala (T1), and its Ser(32P) substitute (S1)), and some modified phosphopeptides (Arg2-Ala-Thr(32P)-Pro-Ala (T2) and Arg2-Pro-Thr(32P)-Val-Ala (T3)), all phosphorylated by cyclic AMP-dependent protein kinase. In addition, casein(Thr-32P), phosphorylated by casein kinase-2, was also tested. The PCS phosphatases show a striking preference for the T4 configuration, PCSC being the least efficient. The catalytic subunit of the MgATP-dependent phosphatase was almost completely inactive toward all these substrates. As shown for the PCSH phosphatase, and comparing with T4, the two proline residues flanking the Thr(P) in T1 and T5, just as in inhibitor-1, drastically imparied the dephosphorylation by lowering the Vmax and not by affecting the apparent Km. The C-terminal proline (as in T2) by itself represents a highly unfavorable factor in the dephosphorylation. The critical effect of the sequence X-Thr(P)-Pro or Pro-Thr(P)-Pro (T1, T2, T5, and inhibitor-1) can be overcome by manganese ions. The additional finding that this is not the case with the Pro-Ser(P)-Pro sequence (S1) suggests that the effect of Mn2+ is highly substrate specific. These observations show the considerable importance of the primary structure of the substrate in determining the specificity of the protein phosphatases.

  7. 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.

  8. 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.

  9. 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

  10. A Conserved Phenylalanine of Motif IV in Superfamily 2 Helicases Is Required for Cooperative, ATP-Dependent Binding of RNA Substrates in DEAD-Box Proteins▿ †

    PubMed Central

    Banroques, Josette; Cordin, Olivier; Doère, Monique; Linder, Patrick; Tanner, N. Kyle

    2008-01-01

    We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be an anchor that maintains the rigidity of the RecA-like domain. For DEAD-box proteins, the phenylalanine also aligns a highly conserved arginine of motif VI through van der Waals and cation-π interactions, thereby helping to maintain the network of interactions that exist between the different motifs involved in ATP and RNA binding. PMID:18332124

  11. Evidence for the presence of heat-stable protein (HPr) and ATP-dependent HPr kinase in heterofermentative lactobacilli lacking phosphoenolpyruvate:glycose phosphotransferase activity.

    PubMed Central

    Reizer, J; Peterkofsky, A; Romano, A H

    1988-01-01

    An analysis of the biochemical basis for the lack of phosphoenolpyruvate:glycose phosphotransferase activity in heterofermentative lactobacilli was carried out. Extracts of Lactobacillus brevis and Lactobacillus buchneri failed to reconstitute phosphotransferase activity of extracts of Staphylococcus aureus mutants impaired in the phosphotransferase system due to the absence of enzyme I, enzyme IILac, or enzyme IIILac activity, suggesting that these lactobacilli lack those phosphotransferase system components. In contrast, complementation tests with an extract of a S. aureus mutant deficient in heat-stable protein (HPr) indicated the presence of HPr activity in heterofermentative lactobacilli. The HPr of L. brevis was purified and shown to have properties similar to those of a typical HPr. In addition, L. brevis possesses an ATP-dependent protein kinase that phosphorylates a serine residue of the endogenous HPr as well as other HPrs of Gram-positive origin. The kinase activity is markedly stimulated by phosphorylated compounds related to sugar metabolism and is negatively modulated by orthophosphate, pyrophosphate, or arsenate and by a low molecular weight endogenous factor. In keeping with the idea of a regulatory role for the phosphorylation of HPr in lactobacilli, a HPr[Ser(P)] phosphatase activity in L. brevis was also demonstrated. On the basis of the finding of HPr and a system for its reversible covalent modification in an organism devoid of a functional phosphotransferase system we propose that, in lactobacilli, HPr has a role in the regulation of pathways other than the phosphotransferase system. Images PMID:2832843

  12. The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATP-dependent ubiquitin-proteasome pathway.

    PubMed Central

    Bailey, J L; Wang, X; England, B K; Price, S R; Ding, X; Mitch, W E

    1996-01-01

    Chronic renal failure (CRF) is associated with negative nitrogen balance and loss of lean body mass. To identify specific proteolytic pathways activated by CRF, protein degradation was measured in incubated epitrochlearis muscles from CRF and sham-operated, pair-fed rats. CRF stimulated muscle proteolysis, and inhibition of lysosomal and calcium-activated proteases did not eliminate this increase. When ATP production was blocked, proteolysis in CRF muscles fell to the same level as that in control muscles. Increased proteolysis was also prevented by feeding CRF rats sodium bicarbonate, suggesting that activation depends on acidification. Evidence that the ATP-dependent ubiquitin-proteasome pathway is stimulated by the acidemia of CRF includes the following findings: (a) An inhibitor of the proteasome eliminated the increase in muscle proteolysis; and (b) there was an increase in mRNAs encoding ubiquitin (324%) and proteasome subunits C3 (137%) and C9 (251%) in muscle. This response involved gene activation since transcription of mRNAs for ubiquitin and the C3 subunit were selectively increased in muscle of CRF rats. We conclude that CRF stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent pathway. The mechanism depends on acidification and increased expression of genes encoding components of the system. These responses could contribute to the loss of muscle mass associated with CRF. PMID:8617877

  13. Dbp9p, a putative ATP-dependent RNA helicase involved in 60S-ribosomal-subunit biogenesis, functionally interacts with Dbp6p.

    PubMed Central

    Daugeron, M C; Kressler, D; Linder, P

    2001-01-01

    Ribosome synthesis is a highly complex process and constitutes a major cellular activity. The biogenesis of this ribonucleoprotein assembly requires a multitude of protein trans-acting factors including several putative ATP-dependent RNA helicases of the DEAD-box and related protein families. Here we show that the previously uncharacterized Saccharomyces cerevisiae open reading frame YLR276C, hereafter named DBP9 (DEAD-box protein 9), encodes an essential nucleolar protein involved in 60S-ribosomal-subunit biogenesis. Genetic depletion of Dbp9p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This terminal phenotype is likely due to the instability of early pre-ribosomal particles, as evidenced by the low steady-state levels and the decreased synthesis of the 27S precursors to mature 25S and 5.8S rRNAs. In agreement with a role of Dbp9p in 60S subunit synthesis, we find that increased Dbp9p dosage efficiently suppresses certain dbp6 alleles and that dbp6/dbp9 double mutants show synthetic lethality. Furthermore, Dbp6p and Dbp9p weakly interact in a yeast two-hybrid assay. Altogether, our findings indicate an intimate functional interaction between Dbp6p and Dbp9p during the process of 60S-ribosomal-subunit assembly. PMID:11565753

  14. Toll-Like Receptors 2, -3 and -4 Prime Microglia but not Astrocytes Across Central Nervous System Regions for ATP-Dependent Interleukin-1β Release

    PubMed Central

    Facci, Laura; Barbierato, Massimo; Marinelli, Carla; Argentini, Carla; Skaper, Stephen D.; Giusti, Pietro

    2014-01-01

    Interleukin-1β (IL-1β) is a crucial mediator in the pathogenesis of inflammatory diseases at the periphery and in the central nervous system (CNS). Produced as an unprocessed and inactive pro-form which accumulates intracellularly, release of the processed cytokine is strongly promoted by ATP acting at the purinergic P2X7 receptor (P2X7R) in cells primed with lipopolysaccharide (LPS), a Toll-like receptor (TLR) 4 ligand. Microglia are central to the inflammatory process and a major source of IL-1β when activated. Here we show that purified (>99%) microglia cultured from rat cortex, spinal cord and cerebellum respond robustly to ATP-dependent IL-1β release, upon priming with a number of TLR isoform ligands (zymosan and Pam3CSK4 for TLR2, poly(I:C) for TLR3). Cytokine release was prevented by a P2X7R antagonist and inhibitors of stress-activated protein kinases. Enriched astrocytes (≤5% microglia) from these CNS regions displayed responses qualitatively similar to microglia but became unresponsive upon eradication of residual microglia with the lysosomotropic agent Leu-Leu-OMe. Activation of multiple TLR isoforms in nervous system pathology, coupled with elevated extracellular ATP levels and subsequent P2X7R activation may represent an important route for microglia-derived IL-1β. This phenomenon may have important consequences for neuroinflammation and its position to the common pathology of CNS diseases. PMID:25351234

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. "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.

  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. 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.

  6. 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.

  7. Assembly of Drosophila centromeric nucleosomes requires CID dimerization.

    PubMed

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

    2012-01-27

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

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. The Amyloid Precursor Protein of Alzheimer’s Disease Clusters at the Organelle/Microtubule Interface on Organelles that Bind Microtubules in an ATP Dependent Manner

    PubMed Central

    Stevenson, James W.; Conaty, Eliza A.; Walsh, Rylie B.; Poidomani, Paul J.; Samoriski, Colin M.; Scollins, Brianne J.; DeGiorgis, Joseph A.

    2016-01-01

    The amyloid precursor protein (APP) is a causal agent in the pathogenesis of Alzheimer’s disease and is a transmembrane protein that associates with membrane-limited organelles. APP has been shown to co-purify through immunoprecipitation with a kinesin light chain suggesting that APP may act as a trailer hitch linking kinesin to its intercellular cargo, however this hypothesis has been challenged. Previously, we identified an mRNA transcript that encodes a squid homolog of human APP770. The human and squid isoforms share 60% sequence identity and 76% sequence similarity within the cytoplasmic domain and share 15 of the final 19 amino acids at the C-terminus establishing this highly conserved domain as a functionally import segment of the APP molecule. Here, we study the distribution of squid APP in extruded axoplasm as well as in a well-characterized reconstituted organelle/microtubule preparation from the squid giant axon in which organelles bind microtubules and move towards the microtubule plus-ends. We find that APP associates with microtubules by confocal microscopy and co-purifies with KI-washed axoplasmic organelles by sucrose density gradient fractionation. By electron microscopy, APP clusters at a single focal point on the surfaces of organelles and localizes to the organelle/microtubule interface. In addition, the association of APP-organelles with microtubules is an ATP dependent process suggesting that the APP-organelles contain a microtubule-based motor protein. Although a direct kinesin/APP association remains controversial, the distribution of APP at the organelle/microtubule interface strongly suggests that APP-organelles have an orientation and that APP like the Alzheimer’s protein tau has a microtubule-based function. PMID:26814888

  13. ATP-dependent transport of glutathione conjugate of 7beta, 8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in murine hepatic canalicular plasma membrane vesicles.

    PubMed Central

    Srivastava, S K; Hu, X; Xia, H; Bleicher, R J; Zaren, H A; Orchard, J L; Awasthi, S; Singh, S V

    1998-01-01

    Glutathione (GSH) S-transferases (GSTs) have an important role in the detoxification of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9, 10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogen of benzo[a]pyrene. However, the fate and/or biological activity of the GSH conjugate of (+)-anti-BPDE [(-)-anti-BPD-SG] is not known. We now report that (-)-anti-BPD-SG is a competitive inhibitor (Ki 19 microM) of Pi-class isoenzyme mGSTP1-1, which among murine hepatic GSTs is most efficient in the GSH conjugation of (+)-anti-BPDE. Thus the inhibition of mGSTP1-1 activity by (-)-anti-BPD-SG might interfere with the GST-catalysed GSH conjugation of (+)-anti-BPDE unless one or more mechanisms exist for the removal of the conjugate. The results of the present study indicate that (-)-anti-BPD-SG is transported across canalicular liver plasma membrane (cLPM) in an ATP-dependent manner. The ATP-dependent transport of (-)-anti-[3H]BPD-SG followed Michaelis-Menten kinetics (Km 46 microM). The ATP dependence of the (-)-anti-BPD-SG transport was confirmed by measuring the stimulation of ATP hydrolysis (ATPase activity) by the conjugate in the presence of cLPM protein, which also followed Michaelis-Menten kinetics. In contrast, a kinetic analysis of ATP-dependent uptake of the model conjugate S-[3H](2,4-dinitrophenyl)-glutathione ([3H]DNP-SG) revealed the presence of a high-affinity and a low-affinity transport system in mouse cLPM, with apparent Km values of 18 and 500 microM respectively. The ATP-dependent transport of (-)-anti-BPD-SG was inhibited competitively by DNP-SG (Ki 1.65 microM). Likewise, (-)-anti-BPD-SG was found to be a potent competitive inhibitor of the high-affinity component of DNP-SG transport (Ki 6.3 microM). Our results suggest that GST-catalysed conjugation of (+)-anti-BPDE with GSH, coupled with ATP-dependent transport of the resultant conjugate across cLPM, might be the ultimate detoxification pathway for this carcinogen. PMID:9620885

  14. Physiology in conservation translocations

    PubMed Central

    Tarszisz, Esther; Dickman, Christopher R.; Munn, Adam J.

    2014-01-01

    Conservation translocations aim to restore species to their indigenous ranges, protect populations from threats and/or reinstate ecosystem functions. They are particularly important for the conservation and management of rare and threatened species. Despite tremendous efforts and advancement in recent years, animal conservation translocations generally have variable success, and the reasons for this are often uncertain. We suggest that when little is known about the physiology and wellbeing of individuals either before or after release, it will be difficult to determine their likelihood of survival, and this could limit advancements in the science of translocations for conservation. In this regard, we argue that physiology offers novel approaches that could substantially improve translocations and associated practices. As a discipline, it is apparent that physiology may be undervalued, perhaps because of the invasive nature of some physiological measurement techniques (e.g. sampling body fluids, surgical implantation). We examined 232 publications that dealt with translocations of terrestrial vertebrates and aquatic mammals and, defining ‘success’ as high or low, determined how many of these studies explicitly incorporated physiological aspects into their protocols and monitoring. From this review, it is apparent that physiological evaluation before and after animal releases could progress and improve translocation/reintroduction successes. We propose a suite of physiological measures, in addition to animal health indices, for assisting conservation translocations over the short term and also for longer term post-release monitoring. Perhaps most importantly, we argue that the incorporation of physiological assessments of animals at all stages of translocation can have important welfare implications by helping to reduce the total number of animals used. Physiological indicators can also help to refine conservation translocation methods. These approaches fall

  15. 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.

  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. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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.

  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. 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.

  6. 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

  7. 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.

  8. 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

  9. Kinetic and thermodynamic aspects of lipid translocation in biological membranes.

    PubMed Central

    Frickenhaus, S; Heinrich, R

    1999-01-01

    A theoretical analysis of the lipid translocation in cellular bilayer membranes is presented. We focus on an integrative model of active and passive transport processes determining the asymmetrical distribution of the major lipid components between the monolayers. The active translocation of the aminophospholipids phosphatidylserine and phosphatidylethanolamine is mathematically described by kinetic equations resulting from a realistic ATP-dependent transport mechanism. Concerning the passive transport of the aminophospholipids as well as of phosphatidylcholine, sphingomyelin, and cholesterol, two different approaches are used. The first treatment makes use of thermodynamic flux-force relationships. Relevant forces are transversal concentration differences of the lipids as well as differences in the mechanical states of the monolayers due to lateral compressions. Both forces, originating primarily from the operation of an aminophospholipid translocase, are expressed as functions of the lipid compositions of the two monolayers. In the case of mechanical forces, lipid-specific parameters such as different molecular surface areas and compression force constants are taken into account. Using invariance principles, it is shown how the phenomenological coefficients depend on the total lipid amounts. In a second approach, passive transport is analyzed in terms of kinetic mechanisms of carrier-mediated translocation, where mechanical effects are incorporated into the translocation rate constants. The thermodynamic as well as the kinetic approach are applied to simulate the time-dependent redistribution of the lipid components in human red blood cells. In the thermodynamic model the steady-state asymmetrical lipid distribution of erythrocyte membranes is simulated well under certain parameter restrictions: 1) the time scales of uncoupled passive transbilayer movement must be different among the lipid species; 2) positive cross-couplings of the passive lipid fluxes are

  10. Transcriptional and genomic mayhem due to aging-induced nucleosome loss in budding yeast

    PubMed Central

    Hu, Zheng; Chen, Kaifu; Li, Wei; Tyler, Jessica K.

    2014-01-01

    All eukaryotic genomes are assembled into a nucleoprotein structure termed chromatin, which is comprised of regular arrays of nucleosomes. Each nucleosome consists of eight core histone protein molecules around which the DNA is wrapped 1.75 times. The ultimate consequence of packaging the genome into chromatin is that the DNA sequences are relatively inaccessible. This allows the cell to use a comprehensive toolbox of chromatin-altering machineries to reveal access to the DNA sequence at the right time and right place in order to allow genomic processes, such as DNA repair, transcription and replication, to occur in a tightly-regulated manner. In other words, chromatin provides the framework that allows the regulation of all genomic processes, because the machineries that mediate transcription, repair and DNA replication themselves are relatively non-sequence specific and if the genome were naked, they would presumably perform their tasks in a random and unregulated manner. We recently provided support for this prediction in Zheng et al., [Genes and Development (2014) 28: 396-408] by investigating a physiologically relevant scenario in which we had found that cells lose half of the core histone proteins, that is, during the mitotic aging (also called replicative aging) of budding yeast. Using new spike-in normalization techniques, we found that the occupancy of nucleosomes at most DNA sequences is reduced by 50%, leading to transcriptional induction of every single gene. This loss of histones during aging was also accompanied by a significantly-increased frequency of genomic instability including DNA breaks, chromosomal translocations, retrotransposition, and transfer of mitochondrial DNA into the nuclear genome (Figure 1).

  11. Role of Nhp6 and Hmo1 in SWI/SNF occupancy and nucleosome landscape at gene regulatory regions.

    PubMed

    Hepp, Matias I; Smolle, Michaela; Gidi, Cristian; Amigo, Roberto; Valenzuela, Nicole; Arriagada, Axel; Maureira, Alejandro; Gogol, Madelaine M; Torrejón, Marcela; Workman, Jerry L; Gutiérrez, José L

    2017-03-01

    Diverse chromatin modifiers are involved in regulation of gene expression at the level of transcriptional regulation. Among these modifiers are ATP-dependent chromatin remodelers, where the SWI/SNF complex is the founding member. It has been observed that High Mobility Group (HMG) proteins can influence the activity of a number of these chromatin remodelers. In this context, we have previously demonstrated that the yeast HMG proteins Nhp6 and Hmo1 can stimulate SWI/SNF activity. Here, we studied the genome-wide binding patterns of Nhp6, Hmo1 and the SWI/SNF complex, finding that most of gene promoters presenting high occupancy of this complex also display high enrichment of these HMG proteins. Using deletion mutant strains we demonstrate that binding of SWI/SNF is significantly reduced at numerous genomic locations by deletion of NHP6 and/or deletion of HMO1. Moreover, alterations in the nucleosome landscape take place at gene promoters undergoing reduced SWI/SNF binding. Additional analyses show that these effects also correlate with alterations in transcriptional activity. Our results suggest that, besides the ability to stimulate SWI/SNF activity, these HMG proteins are able to assist the loading of this complex onto gene regulatory regions.

  12. Epigenetic regulation of stem cell maintenance in the Drosophila testis via the nucleosome-remodeling factor NURF.

    PubMed

    Cherry, Christopher M; Matunis, Erika L

    2010-06-04

    Regulation of stem cells depends on both tissue-specific transcriptional regulators and changes in chromatin organization, yet the coordination of these events in endogenous niches is poorly understood. In the Drosophila testis, local JAK-STAT signaling maintains germline and somatic stem cells (GSCs and cyst progenitor cells, or CPCs) in a single niche. Here we show that epigenetic regulation via the nucleosome-remodeling factor (NURF) complex ensures GSC and CPC maintenance by positively regulating JAK-STAT signaling, thereby preventing premature differentiation. Conversely, NURF is not required in early differentiating daughter cells of either lineage. Because three additional ATP-dependent chromatin remodelers (ACF, CHRAC, and dMi-2/NuRD) are dispensable for stem cell maintenance in the testis, epigenetic regulation of stem cells within this niche may rely primarily on NURF. Thus, local signals cooperate with specific chromatin-remodeling complexes in intact niches to coordinately regulate a common set of target genes to prevent premature stem cell differentiation.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. Impact of the F508del mutation on ovine CFTR, a Cl− channel with enhanced conductance and ATP-dependent gating

    PubMed Central

    Cai, Zhiwei; Palmai-Pallag, Timea; Khuituan, Pissared; Mutolo, Michael J; Boinot, Clément; Liu, Beihui; Scott-Ward, Toby S; Callebaut, Isabelle; Harris, Ann; Sheppard, David N

    2015-01-01

    Cross-species comparative studies are a powerful approach to understanding the epithelial Cl− channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl− channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl− currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl− channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del. Key points Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life-shortening genetic disease cystic fibrosis (CF). Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR. We found that

  18. ATP-Dependent Binding Cassette Transporter G Family Member 16 Increases Plant Tolerance to Abscisic Acid and Assists in Basal Resistance against Pseudomonas syringae DC30001[W][OPEN

    PubMed Central

    Ji, Hao; Peng, Yanhui; Meckes, Nicole; Allen, Sara; Stewart, C. Neal; Traw, M. Brian

    2014-01-01

    Plants have been shown previously to perceive bacteria on the leaf surface and respond by closing their stomata. The virulent bacterial pathogen Pseudomonas syringae pv tomato DC3000 (PstDC3000) responds by secreting a virulence factor, coronatine, which blocks the functioning of guard cells and forces stomata to reopen. After it is inside the leaf, PstDC3000 has been shown to up-regulate abscisic acid (ABA) signaling and thereby suppress salicylic acid-dependent resistance. Some wild plants exhibit resistance to PstDC3000, but the mechanisms by which they achieve this resistance remain unknown. Here, we used genome-wide association mapping to identify an ATP-dependent binding cassette transporter gene (ATP-dependent binding cassette transporter G family member16) in Arabidopsis (Arabidopsis thaliana) that contributes to wild plant resistance to PstDC3000. Through microarray analysis and β-glucuronidase reporter lines, we showed that the gene is up-regulated by ABA, bacterial infection, and coronatine. We also used a green fluorescent protein fusion protein and found that transporter is more likely to localize on plasma membranes than in cell walls. Transferred DNA insertion lines exhibited consistent defective tolerance of exogenous ABA and reduced resistance to infection by PstDC3000. Our conclusion is that ATP-dependent binding cassette transporter G family member16 is involved in ABA tolerance and contributes to plant resistance against PstDC3000. This is one of the first examples, to our knowledge, of ATP-dependent binding cassette transporter involvement in plant resistance to infection by a bacterial pathogen. It also suggests a possible mechanism by which plants reduce the deleterious effects of ABA hijacking during pathogen attack. Collectively, these results improve our understanding of basal resistance in Arabidopsis and offer unique ABA-related targets for improving the innate resistance of plants to bacterial infection. PMID:25146567

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein

    PubMed Central

    Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

    2015-01-01

    The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins. DOI: http://dx.doi.org/10.7554/eLife.11897.001 PMID:26714107

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. Hormone activation induces nucleosome positioning in vivo

    PubMed Central

    Belikov, Sergey; Gelius, Birgitta; Almouzni, Geneviève; Wrange, Örjan

    2000-01-01

    The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone. A robust hormone- and receptor-dependent activation could be reproduced in Xenopus laevis oocytes. The homogeneous response in this system allowed a detailed analysis of the transition in chromatin structure following hormone activation. This revealed two novel findings: hormone activation led to the establishment of specific translational positioning of nucleosomes despite the lack of significant positioning in the inactive state; and, in the active promoter, a subnucleosomal particle encompassing the glucocorticoid receptor (GR)-binding region was detected. The presence of only a single GR-binding site was sufficient for the structural transition to occur. Both basal promoter elements and ongoing transcription were dispensable. These data reveal a stepwise process in the transcriptional activation by glucocorticoid hormone. PMID:10698943

  3. Translocated effectors of Yersinia

    PubMed Central

    Matsumoto, Hiroyuki; Young, Glenn M.

    2009-01-01

    Summary Currently, all known translocated effectors of Yersinia are delivered into host cells by type III secretion systems (T3SSs). Pathogenic Yersinia maintain the plasmid-encoded Ysc T3SS for the specific delivery of the well-studied Yop effectors. New horizons for effector biology have opened with the discovery of the Ysps of Y. enterocolitica Biovar 1B, which are translocated into host cells by the chromosome-endoded Ysa T3SS. The reported arsenal of effectors is likely to expand since genomic analysis has revealed gene-clusters in some Yersinia that code for other T3SSs. These efforts also revealed possible type VI secretion (T6S) systems, which may indicate translocation of effectors occurs by multiple mechanisms. PMID:19185531

  4. 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+.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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)

  13. 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

  14. Simulations of Polymer Translocation

    NASA Astrophysics Data System (ADS)

    Vocks, H.

    2008-07-01

    Transport of molecules across membranes is an essential mechanism for life processes. These molecules are often long, and the pores in the membranes are too narrow for the molecules to pass through as a single unit. In such circumstances, the molecules have to squeeze -- i.e., translocate -- themselves through the pores. DNA, RNA and proteins are such naturally occuring long molecules in a variety of biological processes. Understandably, the process of translocation has been an active topic of current research: not only because it is a cornerstone of many biological processes, but also due to its relevance for practical applications. Translocation is a complicated process in living organisms -- the presence of chaperone molecules, pH, chemical potential gradients, and assisting molecular motors strongly influence its dynamics. Consequently, the translocation process has been empirically studied in great variety in biological literature. Study of translocation as a biophysical process is more recent. Herein, the polymer is simplified to a sequentially connected string of N monomers as it passes through a narrow pore on a membrane. The quantities of interest are the typical time scale for the polymer to leave a confining cell (the ``escape of a polymer from a vesicle'' time scale), and the typical time scale the polymer spends in the pore (the ``dwell'' time scale) as a function of N and other parameters like membrane thickness, membrane adsorption, electrochemical potential gradient, etc. Our research is focused on computer simulations of translocation. Since our main interest is in the scaling properties, we use a highly simplified description of the translocation process. The polymer is described as a self-avoiding walk on a lattice, and its dynamics consists of single-monomer jumps from one lattice site to another neighboring one. Since we have a very efficient program to simulate such polymer dynamics, which we decribe in Chapter 2, we can perform long

  15. 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.

  16. Problem-Elephant Translocation: Translocating the Problem and the Elephant?

    PubMed Central

    Fernando, Prithiviraj; Leimgruber, Peter; Prasad, Tharaka; Pastorini, Jennifer

    2012-01-01

    Human-elephant conflict (HEC) threatens the survival of endangered Asian elephants (Elephas maximus). Translocating “problem-elephants” is an important HEC mitigation and elephant conservation strategy across elephant range, with hundreds translocated annually. In the first comprehensive assessment of elephant translocation, we monitored 16 translocations in Sri Lanka with GPS collars. All translocated elephants were released into national parks. Two were killed within the parks where they were released, while all the others left those parks. Translocated elephants showed variable responses: “homers” returned to the capture site, “wanderers” ranged widely, and “settlers” established home ranges in new areas soon after release. Translocation caused wider propagation and intensification of HEC, and increased elephant mortality. We conclude that translocation defeats both HEC mitigation and elephant conservation goals. PMID:23236404

  17. Oncogene Translocations and NHL

    Cancer.gov

    A colloboration with several large population-based cohorts to determine whether the prevalence or level of t14;18 is associated with risk of NHL and to investigate the clonal relationship between translocation-bearing cells and subsequent tumors

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  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. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Hofmeister effect of anions on calcium translocation by sarcoplasmic reticulum Ca2+-ATPase

    PubMed Central

    Tadini-Buoninsegni, Francesco; Moncelli, Maria Rosa; Peruzzi, Niccolò; Ninham, Barry W.; Dei, Luigi; Nostro, Pierandrea Lo

    2015-01-01

    The occurrence of Hofmeister (specific ion) effects in various membrane-related physiological processes is well documented. For example the effect of anions on the transport activity of the ion pump Na+, K+-ATPase has been investigated. Here we report on specific anion effects on the ATP-dependent Ca2+ translocation by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Current measurements following ATP concentration jumps on SERCA-containing vesicles adsorbed on solid supported membranes were carried out in the presence of different potassium salts. We found that monovalent anions strongly interfere with ATP-induced Ca2+ translocation by SERCA, according to their increasing chaotropicity in the Hofmeister series. On the contrary, a significant increase in Ca2+ translocation was observed in the presence of sulphate. We suggest that the anions can affect the conformational transition between the phosphorylated intermediates E1P and E2P of the SERCA cycle. In particular, the stabilization of the E1P conformation by chaotropic anions seems to be related to their adsorption at the enzyme/water and/or at the membrane/water interface, while the more kosmotropic species affect SERCA conformation and functionality by modifying the hydration layers of the enzyme. PMID:26435197

  18. 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.

  19. 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.

  20. Interaction of nucleosome assembly proteins abolishes nuclear localization of DGK{zeta} by attenuating its association with importins

    SciTech Connect

    Okada, Masashi; Hozumi, Yasukazu; Ichimura, Tohru; Tanaka, Toshiaki; Hasegawa, Hiroshi; Yamamoto, Masakazu; Takahashi, Nobuya; Iseki, Ken; Yagisawa, Hitoshi; Shinkawa, Takashi; Isobe, Toshiaki; Goto, Kaoru

    2011-12-10

    Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGK{zeta}, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGK{zeta}. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGK{zeta} binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGK{zeta} and NAP1Ls prohibits nuclear import of DGK{zeta} because binding of NAP1Ls to DGK{zeta} blocks import carrier proteins, Qip1 and NPI1, to interact with DGK{zeta}, leading to cytoplasmic tethering of DGK{zeta}. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGK{zeta} and provide a clue to examine functional significance of its translocation under pathological conditions.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. [Peptide hydrolases with catalytic dyad Ser-Lys. Similarity and distinctions of the active centers of ATP-dependent Lon proteases, LexA repressors, signal peptidases and C-terminal processing proteases].

    PubMed

    Rotanova, T V

    2002-01-01

    It is established that ATP-dependent protease Lon family belongs to the serine-lysine peptide hydrolases clan. Significant similarity of amino acid sequences of proteases Lon and repressors LexA in the regions including the catalytic serine and lysine residues is revealed by comparing primary structures of different families of the enzymes with Ser-Lys catalytic dyad. The both Lon and LexA families are shown to be divided into two subfamilies in accordance with the nature of amino acids in the catalytically active serine environment. Putative DNA binding sites are revealed in proteolytic domains of Lon A subfamily. Similarities and distinctions of the all families peptide hydrolases of the clan in the regions of their active centers are discussed.

  6. 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.

  7. Genetic and epigenetic determinants mediate proneness of oncogene breakpoint sites for involvement in TCR translocations.

    PubMed

    Larmonie, N S D; van der Spek, A; Bogers, A J J C; van Dongen, J J M; Langerak, A W

    2014-03-01

    T-cell receptor (TCR) translocations are a genetic hallmark of T-cell acute lymphoblastic leukemia and lead to juxtaposition of oncogene and TCR loci. Oncogene loci become involved in translocations because they are accessible to the V(D)J recombination machinery. Such accessibility is predicted at cryptic recombination signal sequence (cRSS) sites ('Type 1') as well as other sites that are subject to DNA double-strand breaks (DSBs) ('Type 2') during early stages of thymocyte development. As chromatin accessibility markers have not been analyzed in the context of TCR-associated translocations, various genetic and epigenetic determinants of LMO2, TAL1 and TLX1 translocation breakpoint (BP) sites and BP cluster regions (BCRs) were examined in human thymocytes to establish DSB proneness and heterogeneity of BP site involvement in TCR translocations. Our data show that DSBs in BCRs are primarily induced in the presence of a genetic element of sequence vulnerability (cRSSs, transposable elements), whereas breaks at single BP sites lacking such elements are more likely induced by chance or perhaps because of patient-specific genetic vulnerability. Vulnerability to obtain DSBs is increased by features that determine chromatin organization, such as methylation status and nucleosome occupancy, although at different levels at different BP sites.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. Lupus nephritis: the central role of nucleosomes revealed.

    PubMed

    Mortensen, Elin S; Fenton, Kristin A; Rekvig, Ole P

    2008-02-01

    Systemic lupus erythematosus (SLE) is an autoimmune syndrome characterized by autoantibodies to nuclear constituents. Some of these antibodies are diagnostically important, whereas others act as disease-modifying factors. One clinically important factor is autoantibodies against dsDNA and nucleosomes, which have overlapping diagnostic and nephritogenic impact in SLE. Although a scientific focus for 5 decades, the molecular and cellular origin of these antibodies, and why they are associated with lupus nephritis, is still not fully understood. A consensus has, however, evolved that antibodies to dsDNA and nucleosomes are central pathogenic factors in the development of lupus nephritis. In contrast, no agreement has been reached as to which glomerular structures are bound by nephritogenic anti-nucleosome antibodies in vivo. Mutually contradictory paradigms and models have evolved simply because we still lack precise and conclusive data to provide definitive insight into how autoantibodies induce lupus nephritis and which specificity is critical in the nephritic process(es). In this review, data demonstrating the central role of nucleosomes in inducing and binding potentially nephritogenic antibodies to DNA and nucleosomes are presented and discussed. These autoimmune-inducing processes are discussed in the context of Matzinger's danger model (Matzinger P: Friendly and dangerous signals: is the tissue in control? Nat Immunol 2007, 8:11-13; Matzinger P: The danger model: a renewed sense of self. Science 2002, 296:301-305; Matzinger P: Tolerance, danger, and the extended family. Annu Rev Immunol 1994, 12:991-1045) and Medzhitov's and Janeway's (Medzhitov R, Janeway CA Jr: Decoding the patterns of self and nonself by the innate immune system. Science 2002, 296:298-300; Medzhitov R, Janeway CA Jr: How does the immune system distinguish self from nonself? Semin Immunol 2000, 12:185-188; Janeway CA Jr, Medzhitov R: Innate immune recognition. Annu Rev Immunol 2002, 20

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed.

    PubMed Central

    Agueli, C; Geraci, F; Giudice, G; Chimenti, L; Cascino, D; Sconzo, G

    2001-01-01

    In the present study, double immunofluorescence and immunoblot analysis have been used to show that centrosomes, isolated from Paracentrotus lividus sea urchin embryos at the first mitotic metaphase, contain the constitutive chaperone, heat-shock protein (HSP) 70. More specifically, we demonstrate that centrosomes contain only the HSP70-d isoform, which is one of the four isoforms identified in P. lividus. We also provide evidence that p34(cell division control kinase-2) and t complex polypeptide-1 (TCP-1) alpha, a subunit of the TCP-1 complex, are localized on the centrosomes. Furthermore, inhibition of TCP-1 in vivo, via microinjecting an anti-(TCP-1 alpha) antibody into P. lividus eggs before fertilization, either impaired mitosis or induced severe malformations in more than 50% of embryos. In addition, we have isolated the whole mitotic apparatus and shown that HSP70 localizes on the fibres of spindles and asters, and binds them in an ATP-dependent manner. These observations suggest that HSP70 has a chaperone role in assisting the TCP-1 complex in tubulin folding, when localized on centrosomes, and during the assembling and disassembling of the mitotic apparatus, when localized on the fibres of spindles and asters. PMID:11716770

  20. Interconversion of inositol (1,4,5)-trisphosphate to inositol (1,3,4,5)-tetrakisphosphate and (1,3,4)-trisphosphate in permeabilized adrenal glomerulosa cells is calcium-sensitive and ATP-dependent

    SciTech Connect

    Rossier, M.F.; Dentand, I.A.; Lew, P.D.; Capponi, A.M.; Vallotton, M.B.

    1986-08-29

    The metabolism of (/sub 3/H)inositol (1,4,5)-trisphosphate was followed in permeabilized bovine adrenal glomerulosa cells. At low Ca++ concentration (pCa = 7.2), more than 90% of (/sub 3/H)inositol (1,4,5)-trisphosphate had disappeared within 2 min, while two other metabolites, (/sub 3/H)inositol (1,3,4)-trisphosphate and (/sub 3/H)inositol (1,3,4,5)-tetrakisphosphate appeared progressively. At higher Ca++ concentrations (pCa = 5.7 and 4.8), the formation of these two metabolites was markedly increased, but completely abolished if the medium was ATP-depleted. The peak levels for the generation of (/sub 3/H)inositol (1,3,4,5)-tetrakisphosphate (1 min) preceded those of (3H)inositol (1,3,4)-trisphosphate and were closely correlated. These results suggest that, in adrenal glomerulosa cells, the isomer inositol (1,3,4)-trisphosphate is generated from inositol (1,4,5)-trisphosphate via a calcium-sensitive and ATP-dependent phosphorylation/dephosphorylation pathway involving the formation of inositol (1,3,4,5)-tetrakisphosphate.

  1. Identification of a MAP 2-like ATP-binding protein associated with axoplasmic vesicles that translocate on isolated microtubules

    PubMed Central

    1986-01-01

    Axoplasmic vesicles were purified and observed to translocate on isolated microtubules in an ATP-dependent, trypsin-sensitive manner, implying that ATP-binding polypeptides essential for force generation were present on the vesicle surface. To identify these proteins [alpha 32P]8-azidoadenosine 5'-triphosphate ([alpha 32P]8-N3ATP), a photoaffinity analogue of ATP, was used. The results presented here identify and characterize a vesicle-associated polypeptide having a relative molecular mass of 292 kD that bound [alpha 32P]8-N3ATP. The incorporation of label is ultraviolet light-dependent and ATP- sensitive. Moreover, the 292-kD polypeptide could be isolated in association with vesicles or microtubules, depending on the conditions used, and the data indicate that the 292-kD polypeptide is similar to mammalian brain microtubule-associated protein 2 (MAP 2) for the following reasons: The 292-kD polypeptide isolated from either squid axoplasm or optic lobe cross-reacts with antiserum to porcine brain MAP 2. Furthermore, it purifies with taxol-stabilized microtubules and is released with salt. Based on these characteristics, the 292-kD polypeptide is distinct from the known force-generating molecules myosin and flagellar dynein, as well as the 110-130-kD kinesin-like polypeptides that have recently been described (Brady, S. T., 1985, Nature (Lond.), 317:73-75; Vale, R. D., T. S. Reese, and M. P. Sheetz, 1985b, Cell, 42:39-50; Scholey, J. M., M. E. Porter, P. M. Grissom, and J. R. McIntosh, 1985, Nature (Lond.), 318:483-486). Because the 292-kD polypeptide binds ATP and is associated with vesicles that translocate on purified MAP-free microtubules in an ATP-dependent fashion, it is therefore believed to be involved in vesicle-microtubule interactions that promote organelle motility. PMID:3091608

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  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

    2017-01-24

    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. Abdominal radiation causes bacterial translocation

    SciTech Connect

    Guzman-Stein, G.; Bonsack, M.; Liberty, J.; Delaney, J.P.

    1989-02-01

    The purpose of this study was to determine if a single dose of radiation to the rat abdomen leads to bacterial translocation into the mesenteric lymph nodes (MLN). A second issue addressed was whether translocation correlates with anatomic damage to the mucosa. The radiated group (1100 cGy) which received anesthesia also was compared with a control group and a third group which received anesthesia alone but no abdominal radiation. Abdominal radiation lead to 100% positive cultures of MLN between 12 hr and 4 days postradiation. Bacterial translocation was almost nonexistent in the control and anesthesia group. Signs of inflammation and ulceration of the intestinal mucosa were not seen until Day 3 postradiation. Mucosal damage was maximal by Day 4. Bacterial translocation onto the MLN after a single dose of abdominal radiation was not apparently dependent on anatomical, histologic damage of the mucosa.

  19. 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.

  20. Structural insights into ribosome translocation

    PubMed Central

    Ling, Clarence

    2016-01-01

    During protein synthesis, tRNA and mRNA are translocated from the A to P to E sites of the ribosome thus enabling the ribosome to translate one codon of mRNA after the other. Ribosome translocation along mRNA is induced by the universally conserved ribosome GTPase, elongation factor G (EF‐G) in bacteria and elongation factor 2 (EF‐2) in eukaryotes. Recent structural and single‐molecule studies revealed that tRNA and mRNA translocation within the ribosome is accompanied by cyclic forward and reverse rotations between the large and small ribosomal subunits parallel to the plane of the intersubunit interface. In addition, during ribosome translocation, the ‘head’ domain of small ribosomal subunit undergoes forward‐ and back‐swiveling motions relative to the rest of the small ribosomal subunit around the axis that is orthogonal to the axis of intersubunit rotation. tRNA/mRNA translocation is also coupled to the docking of domain IV of EF‐G into the A site of the small ribosomal subunit that converts the thermally driven motions of the ribosome and tRNA into the forward translocation of tRNA/mRNA inside the ribosome. Despite recent and enormous progress made in the understanding of the molecular mechanism of ribosome translocation, the sequence of structural rearrangements of the ribosome, EF‐G and tRNA during translocation is still not fully established and awaits further investigation. WIREs RNA 2016, 7:620–636. doi: 10.1002/wrna.1354 For further resources related to this article, please visit the WIREs website. PMID:27117863

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative.

    PubMed Central

    Adams, C C; Workman, J L

    1995-01-01

    To investigate mechanisms by which multiple transcription factors access complex promoters and enhancers within cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores. We used a purified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kappa B (KBF1), to reconstituted nucleosome cores containing different combinations of binding sites. Here we show that binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative. Thus, the binuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kappa B all participated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particular DNA-binding domain. Simultaneous binding by two factors increased the affinity of individual factors for nucleosomal DNA by up to 2 orders of magnitude. Importantly, cooperative binding resulted in efficient nucleosome binding by factors (USF and NF-kappa B) which independently possess little nucleosome-binding ability. The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-binding and dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was responsible for the increased affinity of additional factors. Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of the orientation of the binding sites on the nucleosome. These results indicate that cooperative nucleosome binding is a general mechanism that may play a significant role in loading complex enhancer and promoter elements with multiple diverse factors in chromatin and contribute to the generation of threshold responses and transcriptional synergy by multiple activator sites in vivo. PMID:7862134

  10. TALEN-Induced Translocations in Human Cells.

    PubMed

    Piganeau, Marion; Renouf, Benjamin; Ghezraoui, Hind; Brunet, Erika

    2016-01-01

    Induction of chromosomal translocations in human cells is of a great interest to study tumorigenesis and genome instability. Here, we explain in detail a method to induce translocations using the transcription activator-like effector nucleases (TALENs). We describe how to detect translocation formation by PCR, calculate translocation frequency by 96-well PCR screen, and analyze breakpoint junctions. When inducing cancer translocations, it is also possible to detect the fusion gene by FISH analysis or western blot.

  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. Downside risk of wildlife translocation.

    PubMed

    Chipman, R; Slate, D; Rupprecht, C; Mendoza, M

    2008-01-01

    Translocation has been used successfully by wildlife professionals to enhance or reintroduce populations of rare or extirpated wildlife, provide hunting or wildlife viewing opportunities, farm wild game, and reduce local human-wildlife conflicts. However, accidental and intentional translocations may have multiple unintended negative consequences, including increased stress and mortality of relocated animals, negative impacts on resident animals at release sites, increased conflicts with human interests, and the spread of diseases. Many wildlife professionals now question the practice of translocation, particularly in light of the need to contain or eliminate high profile, economically important wildlife diseases and because using this technique may jeopardize international wildlife disease management initiatives to control rabies in raccoons, coyotes, and foxes in North America. Incidents have been documented where specific rabies variants (Texas gray fox, canine variant in coyotes, and raccoon) have been moved well beyond their current range as a result of translocation, including the emergence of raccoon rabies in the eastern United States. Here, we review and discuss the substantial challenges of curtailing translocation in the USA, focusing on movement of animals by the public, nuisance wildlife control operators, and wildlife rehabilitators.

  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. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. The elongation factor Spt4/5 regulates RNA polymerase II transcription through the nucleosome.

    PubMed

    Crickard, John B; Lee, Jaehyoun; Lee, Tae-Hee; Reese, Joseph C

    2017-04-03

    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.

  6. 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

  7. 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

  8. 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.

  9. The Arabidopsis Adh gene exhibits diverse nucleosome arrangements within a small DNase I-sensitive domain.

    PubMed Central

    Vega-Palas, M A; Ferl, R J

    1995-01-01

    The alcohol dehydrogenase (Adh) gene from Arabidopsis shows enhanced sensitivity to DNase I in cells that express the gene. This generalized sensitivity to DNase I is demarcated by position -500 on the 5' side and the end of the mRNA on the 3' side. Thus, the gene defined as the promoter and mRNA coding region corresponds very closely in size with the gene defined as a nuclease-sensitive domain. This is a remarkably close correspondence between a sensitive domain and a eukaryotic transcriptional unit, because previously reported DNase I-sensitive domains include large regions of DNA that are not transcribed. Nucleosomes are present in the coding region of the Adh gene when it is expressed, indicating that the transcriptional elongation process causes nucleosome disruption rather than release of nucleosomes from the coding region. In addition, the regulatory region contains a loosely positioned nucleosome that is separated from adjacent nucleosomes by internucleosomic DNA segments longer than the average linker DNA in bulk chromatin. This specific array of nucleosomes coexists with bound transcription factors that could contribute to the organization of the nucleosome arrangement. These results enhance our understanding of the complex interactions among DNA, nucleosomes, and transcription factors during gene expression in plants. PMID:8535143

  10. A molecule that detects the length of DNA by using chain fluctuations

    NASA Astrophysics Data System (ADS)

    Iwasa, Kuni H.; Florescu, Ana Maria

    2016-05-01

    A class of nucleosome remodelling motors translocates the nucleosomes, to which they are attached, towards the middle of the DNA chain in the presence of ATP during in vitro experiments. This biological activity is likely based on a physical mechanism for detecting and comparing the lengths of the flanking polymer chains. Here we propose that a pivoting mode of DNA fluctuations near the surface of the nucleosome coupled with a binding reaction with a DNA binding site of the motor provides a physical basis for length detection. Since the mean frequency of the fluctuations is higher for a shorter chain than a longer one due to its lower drag coefficient, a shorter chain has a higher rate of receptor binding, which triggers the ATP-dependent activity of the remodelling motor. The dimerisation of these units allows the motor to compare the length of the flanking DNA chains, enabling the translocation of the nucleosome towards the centre of the DNA.

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

    PubMed

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

    2015-05-08

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

  12. Structure and dynamics of DNA loops on nucleosomes studied with atomistic, microsecond-scale molecular dynamics

    PubMed Central

    Pasi, Marco; Lavery, Richard

    2016-01-01

    DNA loop formation on nucleosomes is strongly implicated in chromatin remodeling and occurs spontaneously in nucleosomes subjected to superhelical stress. The nature of such loops depends crucially on the balance between DNA deformation and DNA interaction with the nucleosome core. Currently, no high-resolution structural data on these loops exist. Although uniform rod models have been used to study loop size and shape, these models make assumptions concerning DNA mechanics and DNA–core binding. We present here atomic-scale molecular dynamics simulations for two different loop sizes. The results point to the key role of localized DNA kinking within the loops. Kinks enable the relaxation of DNA bending strain to be coupled with improved DNA–core interactions. Kinks lead to small, irregularly shaped loops that are asymmetrically positioned with respect to the nucleosome core. We also find that loop position can influence the dynamics of the DNA segments at the extremities of the nucleosome. PMID:27098037

  13. The elongation rate of RNA polymerase determines the fate of transcribed nucleosomes

    PubMed Central

    Bintu, Lacramioara; Kopaczynska, Marta; Hodges, Courtney; Lubkowska, Lucyna; Kashlev, Mikhail; Bustamante, Carlos

    2011-01-01

    Upon transcription, histones can either detach from DNA or transfer behind the polymerase through a process believed to involve template looping. The details governing nucleosomal fate during transcription are not well understood. Our atomic force microscopy images of RNA polymerase II-nucleosome complexes confirm the presence of looped transcriptional intermediates and provide mechanistic insight into the histone-transfer process via the distribution of transcribed nucleosome positions. Significantly, we find that a fraction of the transcribed nucleosomes are remodeled to hexasomes, and that this fraction depends on the transcription elongation rate. A simple model involving the kinetic competition between transcription elongation, histone transfer, and histone-histone dissociation quantitatively rationalizes our observations and unifies results obtained with other polymerases. Factors affecting the relative magnitude of these processes provide the physical basis for nucleosomal fate during transcription and, therefore, for the regulation of gene expression. PMID:22081017

  14. Electrostatic effect of H1-histone protein binding on nucleosome repeat length

    NASA Astrophysics Data System (ADS)

    Cherstvy, Andrey G.; Teif, Vladimir B.

    2014-08-01

    Within a simple biophysical model we describe the effect of electrostatic binding of H1 histone proteins on the nucleosome repeat length in chromatin. The length of wrapped DNA optimizes its binding energy to the histone core and the elastic energy penalty of DNA wrapping. The magnitude of the effect predicted from our model is in agreement with the systematic experimental data on the linear variation of nucleosome repeat lengths with H1/nucleosome ratio (Woodcock C L et al 2006 Chromos. Res. 14 17-25). We compare our model to the data for different cell types and organisms, with a widely varying ratio of bound H1 histones per nucleosome. We underline the importance of this non-specific histone-DNA charge-balance mechanism in regulating the positioning of nucleosomes and the degree of compaction of chromatin fibers in eukaryotic cells.

  15. The effects of transcription on the nucleosome structure of four Dictyostelium genes.

    PubMed Central

    Pavlovic, J; Banz, E; Parish, R W

    1989-01-01

    Micrococcal nuclease digestion of Dictyostelium discoideum nuclei from various developmental stages was used to investigate transcription-related changes in the chromatin structure of the coding region of four genes. Gene activity was determined by Northern blotting and nuclear run on experiments. During strong transcription of the developmentally regulated cysteine proteinase I gene, a smear superimposed on a nucleosomal ladder was observed, indicating perturbation of nucleosomal structure was occurring. However, two other developmentally regulated genes, discoidin I and pSC253, showed only slight nucleosome disruption during high levels of transcription. The chromatin structure of a fourth gene (pCZ22) was disrupted throughout development, even at those stages where transcription was greatly reduced. We suggest that although nucleosome structure can be transiently perturbed by the passage of the transcription complex in vivo, the degree of perturbation and the speed with which nucleosomes reassemble is also influenced by the DNA sequence. Images PMID:2704621

  16. Nuclear magnetic resonance and biochemical studies of histone mobility in nucleosomes

    SciTech Connect

    Smith, R.M. II

    1985-01-01

    /sup 13/C NMR spectra were recorded for nucleosome cores and nucleosome cores with specific segments removed in an effort to identify regions of histone mobility in the nucleosome core. Three types of particles were generated by limited proteolysis of nucleosome cores. Brief ..cap alpha..-chmyotrypsin treatment yielded a particle in which H3 residues 1-20 had been removed. Further ..cap alpha..-chymotrypsin treatment of this particle yielded a particle in which H3 residues 1-20, H2B residues 1-18, and H4 residues 1022 were absent. Very brief digestion of this particle with trypsin produced a particle in which H3 residues 1-20, H2B residues 1-18, H4 residues 1-22, and H2A residues 1-11 and 119-128 were removed. Comparisons of /sup 13/C NMR spectra of proteolysed nucleosome core particles with /sup 13/ C NMR data of native nucleosome cores and the compositions of different peptide regions with potential mobility showed that the random coil regions observed in the core particles were located in H3 residues 1-23 and H2A residues 1-16 and 116-128. Particles with the N-terminal tails removed still retained sedimentation and circular dichroism characteristics similar to intact core particles. Random coil resonances were also seen in preparations of nucleosome oligomers, nucleosome oligomers depleted of H1 and H5, nucleosome oligomers in a condensed form, and in intact nuclei. These resonances appeared to be very similar to those appearing in the nucleosome core spectrum.

  17. A compendium of nucleosome and transcript profiles reveals determinants of chromatin architecture and transcription.

    PubMed

    van Bakel, Harm; Tsui, Kyle; Gebbia, Marinella; Mnaimneh, Sanie; Hughes, Timothy R; Nislow, Corey

    2013-05-01

    Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies.

  18. A Compendium of Nucleosome and Transcript Profiles Reveals Determinants of Chromatin Architecture and Transcription

    PubMed Central

    van Bakel, Harm; Tsui, Kyle; Gebbia, Marinella; Mnaimneh, Sanie; Hughes, Timothy R.; Nislow, Corey

    2013-01-01

    Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies. PMID:23658529

  19. 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

  20. Chiral discotic columnar germs of nucleosome core particles.

    PubMed Central

    Livolant, F; Leforestier, A

    2000-01-01

    In concentrated solution and in the presence of high concentrations of monovalent cations, nucleosome core particles order into a discotic columnar mesophase. This phase is limited to finite-sized hexagonal germs that further divide into six coiled branches, following an iterative process. We show how the structure of the germs comes from the competition between hexagonal packing and chirality with a combination of dendritic facetting and double-twist configurations. Geometrical considerations lead us to suspect that the chirality of the eukaryotic chromosomes may originate from the same competition. PMID:10777768

  1. Structure-based analysis of DNA sequence patterns guiding nucleosome positioning in vitro.

    PubMed

    Cui, Feng; Zhurkin, Victor B

    2010-06-01

    Recent studies of genome-wide nucleosomal organization suggest that the DNA sequence is one of the major determinants of nucleosome positioning. Although the search for underlying patterns encoded in nucleosomal DNA has been going on for about 30 years, our knowledge of these patterns still remains limited. Based on our evaluations of DNA deformation energy, we developed new scoring functions to predict nucleosome positioning. There are three principal differences between our approach and earlier studies: (i) we assume that the length of nucleosomal DNA varies from 146 to 147 bp; (ii) we consider the anisotropic flexibility of pyrimidine-purine (YR) dimeric steps in the context of their neighbors (e.g., YYRR versus RYRY); (iii) we postulate that alternating AT-rich and GC-rich motifs reflect sequence-dependent interactions between histone arginines and DNA in the minor groove. Using these functions, we analyzed 20 nucleosome positions mapped in vitro at single nucleotide resolution (including clones 601, 603, 605, the pGUB plasmid, chicken beta-globin and three 5S rDNA genes). We predicted 15 of the 20 positions with 1-bp precision, and two positions with 2-bp precision. The predicted position of the '601' nucleosome (i.e., the optimum of the computed score) deviates from the experimentally determined unique position by no more than 1 bp - an accuracy exceeding that of earlier predictions. Our analysis reveals a clear heterogeneity of the nucleosomal sequences which can be divided into two groups based on the positioning 'rules' they follow. The sequences of one group are enriched by highly deformable YR/YYRR motifs at the minor-groove bending sites SHL+/- 3.5 and +/- 5.5, which is similar to the alpha-satellite sequence used in most crystallized nucleosomes. Apparently, the positioning of these nucleosomes is determined by the interactions between histones H2A/H2B and the terminal parts of nucleosomal DNA. In the other group (that includes the '601' clone) the

  2. Structure-based Analysis of DNA Sequence Patterns Guiding Nucleosome Positioning in vitro

    PubMed Central

    Cui, Feng; Zhurkin, Victor B.

    2010-01-01

    Recent studies of genome-wide nucleosomal organization suggest that the DNA sequence is one of the major determinants of nucleosome positioning. Although the search for underlying patterns encoded in nucleosomal DNA has been going on for about 30 years, our knowledge of these patterns still remains limited. Based on our evaluations of DNA deformation energy, we developed new scoring functions to predict nucleosome positioning. There are three principal differences between our approach and earlier studies: (i) we assume that the length of nucleosomal DNA varies from 146 to 147 bp; (ii) we consider the anisotropic flexibility of pyrimidine-purine (YR) dimeric steps in the context of their neighbors (e.g., YYRR versus RYRY); (iii) we postulate that alternating AT-rich and GC-rich motifs reflect sequence-dependent interactions between histone arginines and DNA in the minor groove. Using these functions, we analyzed 20 nucleosome positions mapped in vitro at single nucleotide resolution (including clones 601, 603, 605, the pGUB plasmid, chicken β-globin and three 5S rDNA genes). We predicted 15 of the 20 positions with 1-bp precision, and two positions with 2-bp precision. The predicted position of the ‘601’ nucleosome (i.e., the optimum of the computed score) deviates from the experimentally determined unique position by no more than 1 bp — an accuracy exceeding that of earlier predictions. Our analysis reveals a clear heterogeneity of the nucleosomal sequences which can be divided into two groups based on the positioning ‘rules’ they follow. The sequences of one group are enriched by highly deformable YR/YYRR motifs at the minor-groove bending sites SHL ±3.5 and ±5.5, which is similar to the α-satellite sequence used in most crystallized nucleosomes. Apparently, the positioning of these nucleosomes is determined by the interactions between histones H2A/H2B and the terminal parts of nucleosomal DNA. In the other group (that includes the ‘601’ clone

  3. CENP-C directs a structural transition of CENP-A nucleosomes mainly through sliding of DNA gyres.

    PubMed

    Falk, Samantha J; Lee, Jaehyoun; Sekulic, Nikolina; Sennett, Michael A; Lee, Tae-Hee; Black, Ben E

    2016-03-01

    The histone H3 variant CENP-A is incorporated into nucleosomes that mark centromere location. We have recently reported that CENP-A nucleosomes, compared with their H3 counterparts, confer an altered nucleosome shape. Here, using a single-molecule fluorescence resonance energy transfer (FRET) approach with recombinant human histones and centromere DNA, we found that the nucleosome shape change directed by CENP-A is dominated by lateral passing of two DNA gyres (gyre sliding). A nonhistone centromere protein, CENP-C, binds and reshapes the nucleosome, sliding the DNA gyres back to positions similar to those in canonical nucleosomes containing conventional histone H3. The model that we generated to explain the CENP-A-nucleosome transition provides an example of a shape change imposed by external binding proteins and has notable implications for understanding of the epigenetic basis of the faithful inheritance of centromere location on chromosomes.

  4. Dissecting relative contributions of cis- and trans-determinants to nucleosome distribution by comparing Tetrahymena macronuclear and micronuclear chromatin

    PubMed Central

    Xiong, Jie; Gao, Shan; Dui, Wen; Yang, Wentao; Chen, Xiao; Taverna, Sean D.; Pearlman, Ronald E.; Ashlock, Wendy; Miao, Wei; Liu, Yifan

    2016-01-01

    The ciliate protozoan Tetrahymena thermophila contains two types of structurally and functionally differentiated nuclei: the transcriptionally active somatic macronucleus (MAC) and the transcriptionally silent germ-line micronucleus (MIC). Here, we demonstrate that MAC features well-positioned nucleosomes downstream of transcription start sites and flanking splice sites. Transcription-associated trans-determinants promote nucleosome positioning in MAC. By contrast, nucleosomes in MIC are dramatically delocalized. Nucleosome occupancy in MAC and MIC are nonetheless highly correlated with each other, as well as with in vitro reconstitution and predictions based upon DNA sequence features, revealing unexpectedly strong contributions from cis-determinants. In particular, well-positioned nucleosomes are often matched with GC content oscillations. As many nucleosomes are coordinately accommodated by both cis- and trans-determinants, we propose that their distribution is shaped by the impact of these nucleosomes on the mutational and transcriptional landscape, and driven by evolutionary selection. PMID:27488188

  5. Partially Assembled Nucleosome Structures at Atomic Detail.

    PubMed

    Rychkov, Georgy N; Ilatovskiy, Andrey V; Nazarov, Igor B; Shvetsov, Alexey V; Lebedev, Dmitry V; Konev, Alexander Y; Isaev-Ivanov, Vladimir V; Onufriev, Alexey V

    2017-02-07

    The evidence is now overwhelming that partially assembled nucleosome states (PANS) are as important as the canonical nucleosome structure for the understanding of how accessibility to genomic DNA is regulated in cells. We use a combination of molecular dynamics simulation and atomic force microscopy to deliver, in atomic detail, structural models of three key PANS: the hexasome (H2A·H2B)·(H3·H4)2, the tetrasome (H3·H4)2, and the disome (H3·H4). Despite fluctuations of the conformation of the free DNA in these structures, regions of protected DNA in close contact with the histone core remain stable, thus establishing the basis for the understanding of the role of PANS in DNA accessibility regulation. On average, the length of protected DNA in each structure is roughly 18 basepairs per histone protein. Atomistically detailed PANS are used to explain experimental observations; specifically, we discuss interpretation of atomic force microscopy, Förster resonance energy transfer, and small-angle x-ray scattering data obtained under conditions when PANS are expected to exist. Further, we suggest an alternative interpretation of a recent genome-wide study of DNA protection in active chromatin of fruit fly, leading to a conclusion that the three PANS are present in actively transcribing regions in a substantial amount. The presence of PANS may not only be a consequence, but also a prerequisite for fast transcription in vivo.

  6. Electrostatic origin of salt-induced nucleosome array compaction.

    PubMed

    Korolev, Nikolay; Allahverdi, Abdollah; Yang, Ye; Fan, Yanping; Lyubartsev, Alexander P; Nordenskiöld, Lars

    2010-09-22

    The physical mechanism of the folding and unfolding of chromatin is fundamentally related to transcription but is incompletely characterized and not fully understood. We experimentally and theoretically studied chromatin compaction by investigating the salt-mediated folding of an array made of 12 positioning nucleosomes with 177 bp repeat length. Sedimentation velocity measurements were performed to monitor the folding provoked by addition of cations Na(+), K(+), Mg(2+), Ca(2+), spermidine(3+), Co(NH(3))(6)(3+), and spermine(4+). We found typical polyelectrolyte behavior, with the critical concentration of cation needed to bring about maximal folding covering a range of almost five orders of magnitude (from 2 μM for spermine(4+) to 100 mM for Na(+)). A coarse-grained model of the nucleosome array based on a continuum dielectric description and including the explicit presence of mobile ions and charged flexible histone tails was used in computer simulations to investigate the cation-mediated compaction. The results of the simulations with explicit ions are in general agreement with the experimental data, whereas simple Debye-Hückel models are intrinsically incapable of describing chromatin array folding by multivalent cations. We conclude that the theoretical description of the salt-induced chromatin folding must incorporate explicit mobile ions that include ion correlation and ion competition effects.

  7. Nucleosome Free Regions in Yeast Promoters Result from Competitive Binding of Transcription Factors That Interact with Chromatin Modifiers

    PubMed Central

    Ozonov, Evgeniy A.; van Nimwegen, Erik

    2013-01-01

    Because DNA packaging in nucleosomes modulates its accessibility to transcription factors (TFs), unraveling the causal determinants of nucleosome positioning is of great importance to understanding gene regulation. Although there is evidence that intrinsic sequence specificity contributes to nucleosome positioning, the extent to which other factors contribute to nucleosome positioning is currently highly debated. Here we obtained both in vivo and in vitro reference maps of positions that are either consistently covered or free of nucleosomes across multiple experimental data-sets in Saccharomyces cerevisiae. We then systematically quantified the contribution of TF binding to nucleosome positiong using a rigorous statistical mechanics model in which TFs compete with nucleosomes for binding DNA. Our results reconcile previous seemingly conflicting results on the determinants of nucleosome positioning and provide a quantitative explanation for the difference between in vivo and in vitro positioning. On a genome-wide scale, nucleosome positioning is dominated by the phasing of nucleosome arrays over gene bodies, and their positioning is mainly determined by the intrinsic sequence preferences of nucleosomes. In contrast, larger nucleosome free regions in promoters, which likely have a much more significant impact on gene expression, are determined mainly by TF binding. Interestingly, of the 158 yeast TFs included in our modeling, we find that only 10–20 significantly contribute to inducing nucleosome-free regions, and these TFs are highly enriched for having direct interations with chromatin remodelers. Together our results imply that nucleosome free regions in yeast promoters results from the binding of a specific class of TFs that recruit chromatin remodelers. PMID:23990766

  8. Nucleosomes Are Essential for Proper Regulation of a Multigated Promoter in Saccharomyces cerevisiae.

    PubMed

    Yarrington, Robert M; Goodrum, Jenna M; Stillman, David J

    2016-02-01

    Nucleosome-depleted regions (NDRs) are present immediately adjacent to the transcription start site in most eukaryotic promoters. Here we show that NDRs in the upstream promoter region can profoundly affect gene regulation. Chromatin at the yeast HO promoter is highly repressive and numerous coactivators are required for expression. We modified the HO promoter with segments from the well-studied CLN2 NDR, creating chimeric promoters differing in nucleosome occupancy but with binding sites for the same activator, SBF. Nucleosome depletion resulted in substantial increases in both factor binding and gene expression and allowed activation from a much longer distance, probably by allowing recruited coactivators to act further downstream. Nucleosome depletion also affected sequential activation of the HO promoter; HO activation typically requires the ordered recruitment of activators first to URS1, second to the left-half of URS2 (URS2-L), and finally to the right-half of URS2 (URS2-R), with each region representing distinct gates that must be unlocked to achieve activation. The absence of nucleosomes at URS2-L resulted in promoters no longer requiring both the URS1 and URS2-L gates, as either gate alone is now sufficient to promote binding of the SBF factor to URS2-R. Furthermore, nucleosome depletion at URS2 altered the timing of HO expression and bypassed the regulation that restricts expression to mother cells. Our results reveal insight into how nucleosomes can create a requirement for ordered recruitment of factors to facilitate complex transcriptional regulation.

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

    PubMed

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

    2015-11-25

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

  10. Evaluation of the protective capabilities of nucleosome STRs obtained by large-scale sequencing.

    PubMed

    Dong, Chunnan; Yang, Yadong; Yan, Jiangwei; Fu, Lihong; Zhang, Xiaojing; Cong, Bin; Li, Shujin

    2015-07-01

    Partial DNA profiles are often obtained from degraded forensic samples and are hard to analyze and interpret. With in-depth studies on degraded DNA, an increasing number of forensic scientists have focused on the intrinsic structural properties of DNA. In theory, nucleosomes offer protection to the bound DNA by limiting access to enzymes. In our study, we performed large-scale DNA sequencing on nucleosome core DNA of human leucocytes. Five nucleosome short tandem repeats (STRs) were selected (including three forensic common STRs (i.e. TPOX, TH01, and D10S1248) and two unpublished STRs (i.e. AC012568.7 and AC007160.3)). We performed a population genetic investigation and forensic genetic statistical analysis of these two unpublished loci on 108 healthy unrelated individuals of the HeBei Han population in China. We estimated the protective capabilities of five selected nucleosome loci and MiniFiler™ loci with artificial degraded DNA and case samples. We also analyzed differences between sequencing results and software predicted results. Our findings showed that nucleosome STRs were more likely to be detected than MiniFiler™ loci. They were well protected from degradation by nucleosomes and could be candidates for further nucleosome multiplex construction, which would increase the chances of obtaining a better balanced profile with fewer allelic drop-outs.

  11. FSAP-mediated nucleosome release from late apoptotic cells is inhibited by autoantibodies present in SLE.

    PubMed

    Marsman, Gerben; Stephan, Femke; de Leeuw, Karina; Bulder, Ingrid; Ruinard, Jessica T; de Jong, Jan; Westra, Johanna; Bultink, Irene E M; Voskuyl, Alexandre E; Aarden, Lucien A; Luken, Brenda M; Kallenberg, Cees G M; Zeerleder, Sacha

    2016-03-01

    Inefficient clearance of apoptotic cells and the subsequent exposure of the immune system to nuclear contents are crucially involved in the pathogenesis of systemic lupus erythematosus (SLE). Factor VII-activating protease (FSAP) is activated in serum upon contact with dead cells, and releases nucleosomes from late apoptotic cells into the extracellular environment. We investigated whether FSAP-mediated nucleosome release from late apoptotic cells is affected in SLE patients. Nucleosome release in sera of 27 SLE patients and 30 healthy controls was investigated by incubating late apoptotic Jurkat cells with serum and analyzing the remaining DNA content by flow cytometry. We found that nucleosome release in sera of SLE patients with high disease activity was significantly decreased when compared with that in SLE sera obtained during low disease activity or from healthy individuals. Upon removal of IgG/IgM antibodies from SLE sera, nucleosome release was restored. Similarly, monoclonal antinuclear antibodies inhibited nucleosome release in healthy donor serum or by plasma-purified FSAP. This inhibition was lost when Fab fragments were used, suggesting that antigen cross-linking is involved. In conclusion, FSAP-mediated nucleosome release from late apoptotic cells is greatly impaired in SLE patient sera, possibly hampering the clearance of these cells and thereby propagating inflammation.

  12. 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.

  13. The role of the nucleosome acidic patch in modulating higher order chromatin structure.

    PubMed

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

    2013-05-06

    Higher order folding of chromatin fibre is mediated by interactions of the histone H4 N-terminal tail domains with neighbouring nucleosomes. Mechanistically, the H4 tails of one nucleosome bind to the acidic patch region on the surface of adjacent nucleosomes, causing fibre compaction. The functionality of the chromatin fibre can be modified by proteins that interact with the nucleosome. The co-structures of five different proteins with the nucleosome (LANA, IL-33, RCC1, Sir3 and HMGN2) recently have been examined by experimental and computational studies. Interestingly, each of these proteins displays steric, ionic and hydrogen bond complementarity with the acidic patch, and therefore will compete with each other for binding to the nucleosome. We first review the molecular details of each interface, focusing on the key non-covalent interactions that stabilize the protein-acidic patch interactions. We then propose a model in which binding of proteins to the nucleosome disrupts interaction of the H4 tail domains with the acidic patch, preventing the intrinsic chromatin folding pathway and leading to assembly of alternative higher order chromatin structures with unique biological functions.

  14. A nucleosome assembly factor is a constituent of simian virus 40 minichromosomes.

    PubMed Central

    Krude, T; de Maddalena, C; Knippers, R

    1993-01-01

    Using in vitro replication assays, we compared native with salt-treated simian virus 40 minichromosomes isolated from infected cell nuclei. Minichromosomes from both preparations contain the full complement of nucleosomes, but salt treatment removes histone H1 and a fraction of nonhistone chromatin proteins. Both types of minichromosomes served well as templates for in vitro replication, but the structures of the replication products were strikingly different. Replicated salt-treated minichromosomes contained, on average, about half the normal number of nucleosomes as previously shown (T. Krude and R. Knippers, Mol. Cell. Biol. 11:6257-6267, 1991). In contrast, the replicated untreated minichromosomes were found to be densely packed with nucleosomes, indicating that an assembly of new nucleosomes occurred during in vitro replication. Biochemical and immunological data showed that the fraction of nonhistone chromatin proteins associated with native minichromosomes includes a nucleosome assembly activity that appears to be closely related to chromatin assembly factor I (S. Smith and B. W. Stillman, Cell 58:15-25, 1989). Furthermore, this minichromosome-bound nucleosome assembly factor is able to exert its activity in trans to replicating protein-free competitor DNA. Thus, native chromatin itself contains the activities required for an ordered assembly of nucleosomes during the replication process. Images PMID:8380890

  15. RSC remodeling of oligo-nucleosomes: an atomic force microscopy study

    PubMed Central

    Montel, Fabien; Castelnovo, Martin; Menoni, Hervé; Angelov, Dimitar; Dimitrov, Stefan; Faivre-Moskalenko, Cendrine

    2011-01-01

    The ‘remodels structure of chromatin’ (RSC) complex is an essential chromatin remodeling factor that is required for the control of several processes including transcription, repair and replication. The ability of RSC to relocate centrally positioned mononucleosomes at the end of nucleosomal DNA is firmly established, but the data on RSC action on oligo-nucleosomal templates remains still scarce. By using atomic force microscopy (AFM) imaging, we have quantitatively studied the RSC-induced mobilization of positioned di- and trinucleosomes as well as the directionality of mobilization on mononucleosomal template labeled at one end with streptavidin. AFM imaging showed only a limited set of distinct configurational states for the remodeling products. No stepwise or preferred directionality of the nucleosome motion was observed. Analysis of the corresponding reaction pathways allows deciphering the mechanistic features of RSC-induced nucleosome relocation. The final outcome of RSC remodeling of oligosome templates is the packing of the nucleosomes at the edge of the template, providing large stretches of DNA depleted of nucleosomes. This feature of RSC may be used by the cell to overcome the barrier imposed by the presence of nucleosomes. PMID:21138962

  16. Statistical Mechanics of Nucleosomes Constrained by Higher-Order Chromatin Structure

    NASA Astrophysics Data System (ADS)

    Chereji, Răzvan V.; Morozov, Alexandre V.

    2011-07-01

    Eukaryotic DNA is packaged into chromatin: one-dimensional arrays of nucleosomes separated by stretches of linker DNA are folded into 30-nm chromatin fibers which in turn form higher-order structures (Felsenfeld and Groudine in Nature 421:448, 2003). Each nucleosome, the fundamental unit of chromatin, has 147 base pairs (bp) of DNA wrapped around a histone octamer (Richmond and Davey in Nature 423:145, 2003). In order to describe how chromatin fiber formation affects nucleosome positioning and energetics, we have developed a thermodynamic model of finite-size particles with effective nearest-neighbor interactions and arbitrary DNA-binding energies. We show that both one- and two-body interactions can be extracted from one-particle density profiles based on high-throughput maps of in vitro or in vivo nucleosome positions. Although a simpler approach that neglects two-body interactions (even if they are in fact present in the system) can be used to predict sequence determinants of nucleosome positions, the full theory is required to disentangle one- and two-body effects. Finally, we construct a minimal model in which nucleosomes are positioned primarily by steric exclusion and two-body interactions rather than intrinsic histone-DNA sequence preferences. The model reproduces nucleosome occupancy patterns observed over transcribed regions in living cells.

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  18. CENP-A and H3 Nucleosomes Display a Similar Stability to Force-Mediated Disassembly

    PubMed Central

    Kim, Sung Hyun; Vlijm, Rifka; van der Torre, Jaco; Dalal, Yamini; Dekker, Cees

    2016-01-01

    Centromere-specific nucleosomes are a central feature of the kinetochore complex during mitosis, in which microtubules exert pulling and pushing forces upon the centromere. CENP-A nucleosomes have been assumed to be structurally unique, thereby providing resilience under tension relative to their H3 canonical counterparts. Here, we directly test this hypothesis by subjecting CENP-A and H3 octameric nucleosomes, assembled on random or on centromeric DNA sequences, to varying amounts of applied force by using single-molecule magnetic tweezers. We monitor individual disassembly events of CENP-A and H3 nucleosomes. Regardless of the DNA sequence, the force-mediated disassembly experiments for CENP-A and H3 nucleosomes demonstrate similar rupture forces, life time residency and disassembly steps. From these experiments, we conclude that CENP-A does not, by itself, contribute unique structural features to the nucleosome that lead to a significant resistance against force-mediated disruption. The data present insights into the mechanistic basis for how CENP-A nucleosomes might contribute to the structural foundation of the centromere in vivo. PMID:27820823

  19. The FACT Histone Chaperone Guides Histone H4 Into Its Nucleosomal Conformation in Saccharomyces cerevisiae

    PubMed Central

    McCullough, Laura; Poe, Bryan; Connell, Zaily; Xin, Hua; Formosa, Tim

    2013-01-01

    The pob3-Q308K mutation alters the small subunit of the Saccharomyces cerevisiae histone/nucleosome chaperone Facilitates Chromatin Transactions (FACT), causing defects in both transcription and DNA replication. We describe histone mutations that suppress some of these defects, providing new insight into the mechanism of FACT activity in vivo. FACT is primarily known for its ability to promote reorganization of nucleosomes into a more open form, but neither the pob3-Q308K mutation nor the compensating histone mutations affect this activity. Instead, purified mutant FACT complexes fail to release from nucleosomes efficiently, and the histone mutations correct this flaw. We confirm that pob3-T252E also suppresses pob3-Q308K and show that combining two suppressor mutations can be detrimental, further demonstrating the importance of balance between association and dissociation for efficient FACT:nucleosome interactions. To explain our results, we propose that histone H4 can adopt multiple conformations, most of which are incompatible with nucleosome assembly. FACT guides H4 to adopt appropriate conformations, and this activity can be enhanced or diminished by mutations in Pob3 or histones. FACT can therefore destabilize nucleosomes by favoring the reorganized state, but it can also promote assembly by tethering histones and DNA together and maintaining them in conformations that promote canonical nucleosome formation. PMID:23833181

  20. 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

  1. Investigation of the interaction between berberine and nucleosomes in solution: Spectroscopic and equilibrium dialysis approach

    NASA Astrophysics Data System (ADS)

    Rabbani-Chadegani, Azra; Mollaei, Hossein; Sargolzaei, Javad

    2017-02-01

    Berberine is a natural plant alkaloid with high pharmacological potential. Although its interaction with free DNA has been the subject of several reports, to date there is no work concerning the effect of berberine on nucleoprotein structure of DNA, the nucleosomes. The present study focuses on the binding affinity of berberine to nucleosomes and histone H1 employing various spectroscopic techniques, fluorescence, circular dichroism, thermal denaturation as well as equilibrium dialysis. The results showed that the binding of berberine to nucleosomes is positive cooperative with Ka = 5.57 × 103 M- 1. Berberine quenched with the chromophores of protein moiety of nucleosomes and reduced fluorescence emission intensity at 335 nm with Ksv value of 0.135. Binding of berberine to nucleosomes decreased the absorbance at 210 and 260 nm, produced hypochromicity in thermal denaturation profiles and its affinity to nucleoprotein structure of nucleosomes was much higher than to free DNA. Berberine also exhibited high affinity to histone H1 in solution and the binding was positive cooperative with. Ka = 3.61 × 103 M- 1. Moreover berberine decreased fluorescence emission intensity of H1 by quenching with tyrosine residue in its globular core domain. The circular dichroism profiles demonstrated that the binding of drug induced secondary structural changes in both DNA stacking and histone H1. It is concluded that berberine is genotoxic drug, interacts with nucleosomes and in this process histone H1 is involved to exert its anticancer activity.

  2. Genome-Wide Chromatin Remodeling Identified at GC-Rich Long Nucleosome-Free Regions

    PubMed Central

    Hochreiter, Sepp

    2012-01-01

    To gain deeper insights into principles of cell biology, it is essential to understand how cells reorganize their genomes by chromatin remodeling. We analyzed chromatin remodeling on next generation sequencing data from resting and activated T cells to determine a whole-genome chromatin remodeling landscape. We consider chromatin remodeling in terms of nucleosome repositioning which can be observed most robustly in long nucleosome-free regions (LNFRs) that are occupied by nucleosomes in another cell state. We found that LNFR sequences are either AT-rich or GC-rich, where nucleosome repositioning was observed much more prominently in GC-rich LNFRs — a considerable proportion of them outside promoter regions. Using support vector machines with string kernels, we identified a GC-rich DNA sequence pattern indicating loci of nucleosome repositioning in resting T cells. This pattern appears to be also typical for CpG islands. We found out that nucleosome repositioning in GC-rich LNFRs is indeed associated with CpG islands and with binding sites of the CpG-island-binding ZF-CXXC proteins KDM2A and CFP1. That this association occurs prominently inside and also prominently outside of promoter regions hints at a mechanism governing nucleosome repositioning that acts on a whole-genome scale. PMID:23144837

  3. DNA repair of a single UV photoproduct in a designed nucleosome

    SciTech Connect

    Kosmoskil, Joseph V.; Ackerman, Eric J. ); Smerdon, Michael J.

    2001-08-28

    Eukaryotic DNA repair enzymes must interact with the architectural hierarchy of chromatin. The challenge of finding damaged DNA complexed with histone proteins in nucleosomes is complicated by the need to maintain local chromatin structures involved in regulating other DNA processing events. The heterogeneity of lesions induced by DNA-damaging agents has led us to design homogeneously damaged substrates to directly compare repair of naked DNA with that of nucleosomes. Here we report that nucleotide excision repair in Xenopus nuclear extracts can effectively repair a single UV radiation photoproduct located 5 bases from the dyad center of a positioned nucleosome, although the nucleosome is repaired at about half the rate at which the naked DNA fragment is. Extract repair within the nucleosome is > 50-fold more rapid than either enzymatic photoreversal or endonuclease cleavage of the lesion in vitro. Furthermore, nucleosome formation occurs (after repair) only on damaged naked DNA ( 165-bp fragments) during a 1-h incubation in these extracts, even in the presence of a large excess of undamaged DNA. This is an example of selective nucleosome assembly by Xenopus nuclear extracts on a short linear DNA fragment containing a DNA lesion.

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

    PubMed Central

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

    2015-01-01

    The centromeric nucleosome is a key epigenetic determinant of centromere identity and function. Consequently, deciphering how CENP-A containing nucleosomes contribute structurally to centromere function is a fundamental question in chromosome biology. Here, we performed microsecond timescale all-atom molecular dynamics (MD) simulations of CENP-A and H3 nucleosomes, and report that the octameric CENP-A core particles and nucleosomes display different dynamics from their canonical H3-containing counterparts. The most significant motion observed is within key interactions at the heart of the CENP-A octameric core, wherein shearing of contacts within the CENP-A:CENP-A’ dimerization interface results in a weaker four helix bundle, and an extrusion of 10–30 bp of DNA near the pseudo-dyad. Coupled to other local and global fluctuations, the CENP-A nucleosome occupies a more rugged free energy landscape than the canonical H3 nucleosome. Taken together, our data suggest that CENP-A encodes enhanced distortability to the octameric nucleosome, which may allow for enhanced flexing of the histone core in vivo. PMID:26602160

  5. High nucleosome occupancy is encoded at X-linked gene promoters in C. elegans

    PubMed Central

    Ercan, Sevinç; Lubling, Yaniv; Segal, Eran; Lieb, Jason D.

    2011-01-01

    We mapped nucleosome occupancy by paired-end Illumina sequencing in C. elegans embryonic cells, adult somatic cells, and a mix of adult somatic and germ cells. In all three samples, the nucleosome occupancy of gene promoters on the X chromosome differed from autosomal promoters. While both X and autosomal promoters exhibit a typical nucleosome-depleted region upstream of transcript start sites and a well-positioned +1 nucleosome, X-linked gene promoters on average exhibit higher nucleosome occupancy relative to autosomal promoters. We show that the difference between X and autosomes does not depend on the somatic dosage compensation machinery. Instead, the chromatin difference at promoters is partly encoded by DNA sequence, because a model trained on nucleosome sequence preferences from S. cerevisiae in vitro data recapitulate nearly completely the experimentally observed difference between X and autosomal promoters. The model predictions also correlate very well with experimentally determined occupancy values genome-wide. The nucleosome occupancy differences observed on X promoters may bear on mechanisms of X chromosome dosage compensation in the soma, and chromosome-wide repression of X in the germline. PMID:21177966

  6. Linker histones H1 and H5 prevent the mobility of positioned nucleosomes

    SciTech Connect

    Pennings, S.; Meersseman, G. ); Bradbury, E.M. Los Alamos National Lab., NM )

    1994-10-25

    We have previously identified a generally occurring short-range mobility of nucleosome cores on DNA in relatively low ionic strength conditions. Here we report that this mobility of histone octamers positioned on constructs of 5S rDNA is suppressed by the binding of histone H1 or H5 to the nucleosome. Histone H5 is the more potent inhibitor of nucleosome mobility, in accordance with its higher affinity for chromatin. We propose that this reversible restraint on chromatin dynamics may play a role in local regulation of processes that require access to the DNA. 42 refs., 2 figs.

  7. DNA stretching in the nucleosome facilitates alkylation by an intercalating antitumour agent

    PubMed Central

    Dong, Yuancai; Surana, Uttam; Davey, Curt A.

    2010-01-01

    DNA stretching in the nucleosome core can cause dramatic structural distortions, which may influence compaction and factor recognition in chromatin. We find that the base pair unstacking arising from stretching-induced extreme minor groove kinking near the nucleosome centre creates a hot spot for intercalation and alkylation by a novel anticancer compound. This may have far reaching implications for how chromatin structure can influence binding of intercalator species and indicates potential for the development of site selective DNA-binding agents that target unique conformational features of the nucleosome. PMID:20026584

  8. MutS mediates heteroduplex loop formation by a translocation mechanism.

    PubMed Central

    Allen, D J; Makhov, A; Grilley, M; Taylor, J; Thresher, R; Modrich, P; Griffith, J D

    1997-01-01

    Interaction of Escherichia coli MutS and MutL with heteroduplex DNA has been visualized by electron microscopy. In a reaction dependent on ATP hydrolysis, complexes between a MutS dimer and a DNA heteroduplex are converted to protein-stabilized, alpha-shaped loop structures with the mismatch in most cases located within the DNA loop. Loop formation depends on ATP hydrolysis and loop size increases linearly with time at a rate of 370 base pairs/min in phosphate buffer and about 10,000 base pairs/min in the HEPES buffer used for repair assay. These observations suggest a translocation mechanism in which a MutS dimer bound to a mismatch subsequently leaves this site by ATP-dependent tracking or unidimensional movement that is in most cases bidirectional from the mispair. In view of the bidirectional capability of the methyl-directed pathway, this reaction may play a role in determination of heteroduplex orientation. The rate of MutS-mediated DNA loop growth is enhanced by MutL, and when both proteins are present, both are found at the base of alpha-loop structures, and both can remain associated with excision intermediates produced in later stages of the reaction. PMID:9250691

  9. Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase.

    PubMed

    Pai, Chien-Hua; Chiang, Bing-Yu; Ko, Tzu-Ping; Chou, Chia-Cheng; Chong, Cheong-Meng; Yen, Fang-Jiun; Chen, Shoujun; Coward, James K; Wang, Andrew H-J; Lin, Chun-Hung

    2006-12-13

    Most organisms use glutathione to regulate intracellular thiol redox balance and protect against oxidative stress; protozoa, however, utilize trypanothione for this purpose. Trypanothione biosynthesis requires ATP-dependent conjugation of glutathione (GSH) to the two terminal amino groups of spermidine by glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), which are considered as drug targets. GspS catalyzes the penultimate step of the biosynthesis-amide bond formation between spermidine and the glycine carboxylate of GSH. We report herein five crystal structures of Escherichia coli GspS in complex with substrate, product or inhibitor. The C-terminal of GspS belongs to the ATP-grasp superfamily with a similar fold to the human glutathione synthetase. GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine. We also identify essential amino acids involved in the catalysis. Our results constitute the first structural information on the biochemical features of parasite homologs (including TryS) that underlie their broad specificity for polyamines.

  10. The photomorphogenesis regulator DET1 binds the amino-terminal tail of histone H2B in a nucleosome context.

    PubMed

    Benvenuto, Giovanna; Formiggini, Fabio; Laflamme, Pierre; Malakhov, Mikhail; Bowler, Chris

    2002-09-03

    Light provides a major source of information from the environment during plant growth and development. Recent results suggest that the key events controlling light-regulated gene expression in plants are translocation of the phytochrome photoreceptors into the nucleus, followed by their binding to transcription factors such as PIF3. Coupled with this, the degradation of positively acting intermediates such as the transcription factor HY5 by COP1 and the COP9 signalosome appears to be an important process whereby photomorphogenesis is repressed in darkness (e.g., ). Genetic analyses in Arabidopsis and tomato have revealed that the nuclear protein DET1 also plays a key role in the repression of photomorphogenesis. However, the function of this protein has remained a mystery. In a series of in vitro experiments, we provide persuasive evidence that DET1 binds to nonacetylated amino-terminal tails of the core histone H2B in the context of the nucleosome. Furthermore, we have utilized FRET (fluorescence resonance energy transfer) imaging with GFP variants to demonstrate this interaction within the nucleus of living plant cells. Given the dramatic photomorphogenic phenotypes of det1 mutants, we propose that chromatin remodeling plays a heretofore unsuspected role in regulating gene expression during photomorphogenesis.

  11. Suitability of amphibians and reptiles for translocation.

    PubMed

    Germano, Jennifer M; Bishop, Phillip J

    2009-02-01

    Translocations are important tools in the field of conservation. Despite increased use over the last few decades, the appropriateness of translocations for amphibians and reptiles has been debated widely over the past 20 years. To provide a comprehensive evaluation of the suitability of amphibians and reptiles for translocation, we reviewed the results of amphibian and reptile translocation projects published between 1991 and 2006. The success rate of amphibian and reptile translocations reported over this period was twice that reported in an earlier review in 1991. Success and failure rates were independent of the taxonomic class (Amphibia or Reptilia) released. Reptile translocations driven by human-wildlife conflict mitigation had a higher failure rate than those motivated by conservation, and more recent projects of reptile translocations had unknown outcomes. The outcomes of amphibian translocations were significantly related to the number of animals released, with projects releasing over 1000 individuals being most successful. The most common reported causes of translocation failure were homing and migration of introduced individuals out of release sites and poor habitat. The increased success of amphibian and reptile translocations reviewed in this study compared with the 1991 review is encouraging for future conservation projects. Nevertheless, more preparation, monitoring, reporting of results, and experimental testing of techniques and reintroduction questions need to occur to improve translocations of amphibians and reptiles as a whole.

  12. Yeast Isw1a and Isw1b exhibit similar nucleosome mobilization capacities for mononucleosomes, but differently mobilize dinucleosome templates.

    PubMed

    Krajewski, Wladyslaw A

    2014-03-15

    Nucleosome remodeling studies in vitro have primarily focused on the use of mononucleosome templates, which, however, can provide only limited information on how nucleosome mobilization occurs in the context of chromatin, in which internucleosome interactions might influence remodeling. We tried to evaluate whether nucleosome mobilization by yeast Isw1a, Isw1b and Isw2 could be affected by neighboring nucleosomes. We compared mono- and dinucleosomes positioned by the '601' sequence, the studied constructs contain variation in linker length between nucleosomes and variation in the length of flanking sequences. The data characterizing the remodeling were based on gel retardation of the mono and dinucleosomes, keeping in mind the observation that the relative position of the nucleosome will change the mobility of the complex in well defined ways. We found that Isw1a, Isw1b and Isw2 process nucleosomes differently whether they exist as mononucleosomes or dinucleosomes, such as, the Isw1a and Isw1b nucleosome repositioning patterns, which were very similar for mononucleosomes, appeared to be profoundly different in case of dinucleosome templates. We also examined the DNase I protection patterns of remodeled mono- and dinucleosomes. The data suggest that nucleosome mobilizing activity of Isw1a, Isw1b and Isw2 complexes could be significantly influenced by neighboring nucleosomes.

  13. Activity of FEN1 endonuclease on nucleosome substrates is dependent upon DNA sequence but not flap orientation.

    PubMed

    Jagannathan, Indu; Pepenella, Sharon; Hayes, Jeffrey J

    2011-05-20

    We demonstrated previously that human FEN1 endonuclease, an enzyme involved in excising single-stranded DNA flaps that arise during Okazaki fragment processing and base excision repair, cleaves model flap substrates assembled into nucleosomes. Here we explore the effect of flap orientation with respect to the surface of the histone octamer on nucleosome structure and FEN1 activity in vitro. We find that orienting the flap substrate toward the histone octamer does not significantly alter the rotational orientation of two different nucleosome positioning sequences on the surface of the histone octamer but does cause minor perturbation of nucleosome structure. Surprisingly, flaps oriented toward the nucleosome surface are accessible to FEN1 cleavage in nucleosomes containing the Xenopus 5S positioning sequence. In contrast, neither flaps oriented toward nor away from the nucleosome surface are cleaved by the enzyme in nucleosomes containing the high-affinity 601 nucleosome positioning sequence. The data are consistent with a model in which sequence-dependent motility of DNA on the nucleosome is a major determinant of FEN1 activity. The implications of these findings for the activity of FEN1 in vivo are discussed.

  14. Dnmt1-independent CG methylation contributes to nucleosome positioning in diverse eukaryotes.

    PubMed

    Huff, Jason T; Zilberman, Daniel

    2014-03-13

    Dnmt1 epigenetically propagates symmetrical CG methylation in many eukaryotes. Their genomes are typically depleted of CG dinucleotides because of imperfect repair of deaminated methylcytosines. Here, we extensively survey diverse species lacking Dnmt1 and show that, surprisingly, symmetrical CG methylation is nonetheless frequently present and catalyzed by a different DNA methyltransferase family, Dnmt5. Numerous Dnmt5-containing organisms that diverged more than a billion years ago exhibit clustered methylation, specifically in nucleosome linkers. Clustered methylation occurs at unprecedented densities and directly disfavors nucleosomes, contributing to nucleosome positioning between clusters. Dense methylation is enabled by a regime of genomic sequence evolution that enriches CG dinucleotides and drives the highest CG frequencies known. Species with linker methylation have small, transcriptionally active nuclei that approach the physical limits of chromatin compaction. These features constitute a previously unappreciated genome architecture, in which dense methylation influences nucleosome positions, likely facilitating nuclear processes under extreme spatial constraints.

  15. Rearrangement of the histone H2A C-terminal domain in the nucleosome

    SciTech Connect

    Usachenko, S.I.; Bavykin, S.G.; Gavin, I.M.; Bradbury, M. |

    1994-07-19

    Using zero-length covalent protein-DNA crosslinking, the authors have mapped the histone-DNA contacts in nucleosome core particles from which the C- and N-terminal domains of histone H2A were selectively trimmed by trypsin or clostripain. They found that the flexible trypsin-sensitive C-terminal domain of histone H2A contacts the dyad axis, whereas its globular domain contacts the end of DNA in the nucleosome core particle. The appearance of the histone H2A contact at the dyad axis occurs only in the absence of linker DNA and does not depend on the absence of linker histones. The results show the ability of the histone H2A C-terminal domain to rearrange. This rearrangement might play a biological role in nucleosome disassembly and reassembly and the retention of the H2A-H2B dimer (or the whole octamer) during the passing of polymerases through the nucleosome.

  16. 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.

  17. Transcription-dependent and transcription-independent nucleosome disruption induced by dioxin

    SciTech Connect

    Morgan, J.E.; Whitlock, J.P. Jr. )

    1992-12-01

    In mouse hepatoma cells, both the regulatory and the transcribed regions of the cyp1a1 gene assume a nucleosomal configuration when the gene is silent; two nucleosomes occupy specific sites at the transcriptional promoter. Activation of transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin is accompanied by changes in chromatin structure, which depend upon a functional aromatic hydrocarbon (Ah) receptor. In the transcribed region of the gene, nucleosome disruption occurs as a consequence of RNA elongation. In contrast, at the promoter, loss of positioned nucleosomes is independent of transcription and represents an event in the mechanism by which the liganded Ah receptor enhances transcriptional initiation. 38 refs., 5 figs.

  18. Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA.

    PubMed

    Osakabe, Akihisa; Adachi, Fumiya; Arimura, Yasuhiro; Maehara, Kazumitsu; Ohkawa, Yasuyuki; Kurumizaka, Hitoshi

    2015-10-01

    DNA methylation occurs on CpG sites and is important to form pericentric heterochromatin domains. The satellite 2 sequence, containing seven CpG sites, is located in the pericentric region of human chromosome 1 and is highly methylated in normal cells. In contrast, the satellite 2 region is reportedly hypomethylated in cancer cells, suggesting that the methylation status may affect the chromatin structure around the pericentric regions in tumours. In this study, we mapped the nucleosome positioning on the satellite 2 sequence in vitro and found that DNA methylation modestly affects the distribution of the nucleosome positioning. The micrococcal nuclease assay revealed that the DNA end flexibility of the nucleosomes changes, depending on the DNA methylation status. However, the structures and thermal stabilities of the nucleosomes are unaffected by DNA methylation. These findings provide new information to understand how DNA methylation functions in regulating pericentric heterochromatin formation and maintenance in normal and malignant cells.

  19. Atomic Force Microscopy Imaging of SWI/SNF Action: Mapping the Nucleosome Remodeling and Sliding

    PubMed Central

    Montel, Fabien; Fontaine, Emeline; St-Jean, Philippe; Castelnovo, Martin; Faivre-Moskalenko, Cendrine

    2007-01-01

    We propose a combined experimental (atomic force microscopy) and theoretical study of the structural and dynamical properties of nucleosomes. In contrast to biochemical approaches, this method allows us to determine simultaneously the DNA-complexed length distribution and nucleosome position in various contexts. First, we show that differences in the nucleoproteic structure observed between conventional H2A and H2A.Bbd variant nucleosomes induce quantitative changes in the length distribution of DNA-complexed with histones. Then, the sliding action of remodeling complex SWI/SNF is characterized through the evolution of the nucleosome position and wrapped DNA length mapping. Using a linear energetic model for the distribution of DNA-complexed length, we extract the net-wrapping energy of DNA onto the histone octamer and compare it to previous studies. PMID:17468167

  20. Synthetic Nucleosomes Reveal that GlcNAcylation Modulates Direct Interaction with the FACT Complex

    PubMed Central

    Raj, Ritu; Lercher, Lukas; Mohammed, Shabaz

    2016-01-01

    Abstract Transcriptional regulation can be established by various post‐translational modifications (PTMs) on histone proteins in the nucleosome and by nucleobase modifications on chromosomal DNA. Functional consequences of histone O‐GlcNAcylation (O‐GlcNAc=O‐linked β‐N‐acetylglucosamine) are largely unexplored. Herein, we generate homogeneously GlcNAcylated histones and nucleosomes by chemical post‐translational modification. Mass‐spectrometry‐based quantitative interaction proteomics reveals a direct interaction between GlcNAcylated nucleosomes and the “facilitates chromatin transcription” (FACT) complex. Preferential binding of FACT to GlcNAcylated nucleosomes may point towards O‐GlcNAcylation as one of the triggers for FACT‐driven transcriptional control. PMID:27272618

  1. Atomic force microscopy imaging of SWI/SNF action: mapping the nucleosome remodeling and sliding.

    PubMed

    Montel, Fabien; Fontaine, Emeline; St-Jean, Philippe; Castelnovo, Martin; Faivre-Moskalenko, Cendrine

    2007-07-15

    We propose a combined experimental (atomic force microscopy) and theoretical study of the structural and dynamical properties of nucleosomes. In contrast to biochemical approaches, this method allows us to determine simultaneously the DNA-complexed length distribution and nucleosome position in various contexts. First, we show that differences in the nucleoproteic structure observed between conventional H2A and H2A.Bbd variant nucleosomes induce quantitative changes in the length distribution of DNA-complexed with histones. Then, the sliding action of remodeling complex SWI/SNF is characterized through the evolution of the nucleosome position and wrapped DNA length mapping. Using a linear energetic model for the distribution of DNA-complexed length, we extract the net-wrapping energy of DNA onto the histone octamer and compare it to previous studies.

  2. Nucleosome arrangement in alpha-satellite chromatin of African green monkey cells.

    PubMed Central

    Smith, M R; Lieberman, M W

    1984-01-01

    By analyzing the accessibility of restriction endonuclease sites in African green monkey alpha-satellite chromatin, we demonstrate the absence of a unique phase relationship between nucleosomes and alpha-satellite DNA. The data indicate a minimum of three different positions for nucleosome cores relative to the alpha-satellite sequence and suggest a random distribution in at least some regions. In addition, while we confirm published reports that staphylococcal nuclease cuts the alpha-satellite sequence in chromatin at a highly preferred site, two-dimensional gel electrophoresis of nuclear digests demonstrates that this site is preferentially cut by staphylococcal nuclease even when it is within the nucleosome core. These data indicate that staphylococcal nuclease is not useful for determining nucleosome positions on alpha-satellite DNA, and perhaps on other specific DNA sequences as well. Images PMID:6089117

  3. Histone chaperones: assisting histone traffic and nucleosome dynamics.

    PubMed

    Gurard-Levin, Zachary A; Quivy, Jean-Pierre; Almouzni, Geneviève

    2014-01-01

    The functional organization of eukaryotic DNA into chromatin uses histones as components of its building block, the nucleosome. Histone chaperones, which are proteins that escort histones throughout their cellular life, are key actors in all facets of histone metabolism; they regulate the supply and dynamics of histones at chromatin for its assembly and disassembly. Histone chaperones can also participate in the distribution of histone variants, thereby defining distinct chromatin landscapes of importance for genome function, stability, and cell identity. Here, we discuss our current knowledge of the known histone chaperones and their histone partners, focusing on histone H3 and its variants. We then place them into an escort network that distributes these histones in various deposition pathways. Through their distinct interfaces, we show how they affect dynamics during DNA replication, DNA damage, and transcription, and how they maintain genome integrity. Finally, we discuss the importance of histone chaperones during development and describe how misregulation of the histone flow can link to disease.

  4. Polymorphism of apyrimidinic DNA structures in the nucleosome

    PubMed Central

    Osakabe, Akihisa; Arimura, Yasuhiro; Matsumoto, Syota; Horikoshi, Naoki; Sugasawa, Kaoru; Kurumizaka, Hitoshi

    2017-01-01

    Huge amounts (>10,000/day) of apurinic/apyrimidinic (AP) sites are produced in genomes, but their structures in chromatin remain undetermined. We determined the crystal structure of the nucleosome containing AP-site analogs at two symmetric sites, which revealed structural polymorphism: one forms an inchworm configuration without an empty space at the AP site, and the other forms a B-form-like structure with an empty space and the orphan base. This unexpected inchworm configuration of the AP site is important to understand the AP DNA repair mechanism, because it may not be recognized by the major AP-binding protein, APE1, during the base excision repair process. PMID:28139742

  5. Reconstitution and electron spin resonance spin labeling studies of nucleosomes

    SciTech Connect

    Chan, D.C.F.; Grover, T.A.; Piette, L.H.

    1980-10-01

    The spin label, N-(2,2,5,5-tetramethyl-3-carbonylpyrrolidine-1-oxyl)-imidazole, was used to study the mode of reconstitution of nucleosome core particles. The histone cores in 2 M NaCl were first reacted with the imidazole spin label. After the removal of unreacted label, the histone cores were mixed with purified core DNA (145 base pairs) in 2 M NaCl. The mixture was then reconstituted by salt step-gradient dialysis according to Tatchell and Van Holde. At each step of the dialysis, an electron spin resonance (ESR) spectrum of the labeled tyrosyls was recorded and the correlation time of the label determined. As the ionic strength was gradually decreased, the correlation time of the spin label increased. This is in contrast to what we observed previously for the histone core alone, in which a decrease in the ionic strength caused the histone core (in the absence of DNA) to dissociate, freeing up the label and decreasing its correlation tie. Judging from the change in rotational correlation times for the spin label, we concluded that the histone core binds progressively to the DNA in the range of 2 M-0.3 M NaCl. When the ionic strength is <0.3 M, full association between the histone core and DNA takes place. These reconstituted spin labelled nucleosome core complexes, purified by isokinetic sucrose gradient, were found to have identical physical properties (histone content, sedimentation coefficient, thermal melting profile, and ciruclar dichroism) as the native particle.

  6. Crystal structure of the human centromeric nucleosome containing CENP-A.

    PubMed

    Tachiwana, Hiroaki; Kagawa, Wataru; Shiga, Tatsuya; Osakabe, Akihisa; Miya, Yuta; Saito, Kengo; Hayashi-Takanaka, Yoko; Oda, Takashi; Sato, Mamoru; Park, Sam-Yong; Kimura, Hiroshi; Kurumizaka, Hitoshi

    2011-07-10

    In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate α-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg 80 and Gly 81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.

  7. A region of the nucleosome required for multiple types of transcriptional silencing in Saccharomyces cerevisiae.

    PubMed

    Prescott, Eugenia T; Safi, Alexias; Rusche, Laura N

    2011-07-01

    Extended heterochromatin domains, which are repressive to transcription and help define centromeres and telomeres, are formed through specific interactions between silencing proteins and nucleosomes. This study reveals that in Saccharomyces cerevisiae, the same nucleosomal surface is critical for the formation of multiple types of heterochromatin, but not for local repression mediated by a related transcriptional repressor. Thus, this region of the nucleosome may be generally important to long-range silencing. In S. cerevisiae, the Sir proteins perform long-range silencing, whereas the Sum1 complex acts locally to repress specific genes. A mutant form of Sum1p, Sum1-1p, achieves silencing in the absence of Sir proteins. A genetic screen identified mutations