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Sample records for atp-dependent nucleosome translocation

  1. Multiple aspects of ATP-dependent nucleosome translocation by RSC and Mi-2 are directed by the underlying DNA sequence.

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    Joke J F A van Vugt

    Full Text Available BACKGROUND: Chromosome structure, DNA metabolic processes and cell type identity can all be affected by changing the positions of nucleosomes along chromosomal DNA, a reaction that is catalysed by SNF2-type ATP-driven chromatin remodelers. Recently it was suggested that in vivo, more than 50% of the nucleosome positions can be predicted simply by DNA sequence, especially within promoter regions. This seemingly contrasts with remodeler induced nucleosome mobility. The ability of remodeling enzymes to mobilise nucleosomes over short DNA distances is well documented. However, the nucleosome translocation processivity along DNA remains elusive. Furthermore, it is unknown what determines the initial direction of movement and how new nucleosome positions are adopted. METHODOLOGY/PRINCIPAL FINDINGS: We have used AFM imaging and high resolution PAGE of mononucleosomes on 600 and 2500 bp DNA molecules to analyze ATP-dependent nucleosome repositioning by native and recombinant SNF2-type enzymes. We report that the underlying DNA sequence can control the initial direction of translocation, translocation distance, as well as the new positions adopted by nucleosomes upon enzymatic mobilization. Within a strong nucleosomal positioning sequence both recombinant Drosophila Mi-2 (CHD-type and native RSC from yeast (SWI/SNF-type repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalyzed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps. CONCLUSIONS/SIGNIFICANCE: Nucleosome repositioning thus appears to be influenced by both remodeler intrinsic and DNA sequence specific properties that interplay to define ATPase

  2. Multiple aspects of atp-dependent nucleosome translocation by rsc and mi-2 are directed by the underlying DNA sequence

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    Vugt, J. van; Jager, M. de; Murawska, M.; Brehm, A.; Noort, J. van; Logie, C.

    2009-01-01

    Background Chromosome structure, DNA metabolic processes and cell type identity can all be affected by changing the positions of nucleosomes along chromosomal DNA, a reaction that is catalysed by SNF2-type ATP-driven chromatin remodelers. Recently it was suggested that in vivo, more than 50% of the

  3. ATP-Dependent Chromatin Remodeling Factors and Their Roles in Affecting Nucleosome Fiber Composition

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    Alexandra Lusser

    2011-10-01

    Full Text Available ATP-dependent chromatin remodeling factors of the SNF2 family are key components of the cellular machineries that shape and regulate chromatin structure and function. Members of this group of proteins have broad and heterogeneous functions ranging from controlling gene activity, facilitating DNA damage repair, promoting homologous recombination to maintaining genomic stability. Several chromatin remodeling factors are critical components of nucleosome assembly processes, and recent reports have identified specific functions of distinct chromatin remodeling factors in the assembly of variant histones into chromatin. In this review we will discuss the specific roles of ATP-dependent chromatin remodeling factors in determining nucleosome composition and, thus, chromatin fiber properties.

  4. Visualizing ATP-dependent RNA translocation by the NS3 helicase from HCV.

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    Appleby, Todd C; Anderson, Robert; Fedorova, Olga; Pyle, Anna M; Wang, Ruth; Liu, Xiaohong; Brendza, Katherine M; Somoza, John R

    2011-02-04

    The structural mechanism by which nonstructural protein 3 (NS3) from the hepatitis C virus (HCV) translocates along RNA is currently unknown. HCV NS3 is an ATP-dependent motor protein essential for viral replication and a member of the superfamily 2 helicases. Crystallographic analysis using a labeled RNA oligonucleotide allowed us to unambiguously track the positional changes of RNA bound to full-length HCV NS3 during two discrete steps of the ATP hydrolytic cycle. The crystal structures of HCV NS3, NS3 bound to bromine-labeled RNA, and a tertiary complex of NS3 bound to labeled RNA and a non-hydrolyzable ATP analog provide a direct view of how large domain movements resulting from ATP binding and hydrolysis allow the enzyme to translocate along the phosphodiester backbone. While directional translocation of HCV NS3 by a single base pair per ATP hydrolyzed is observed, the 3' end of the RNA does not shift register with respect to a conserved tryptophan residue, supporting a "spring-loading" mechanism that leads to larger steps by the enzyme as it moves along a nucleic acid substrate.

  5. Stepwise nucleosome translocation by RSC remodeling complexes.

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    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.

  6. Selective and ATP-dependent translocation of peptides by the homodimeric ATP binding cassette transporter TAP-like (ABCB9)

    NARCIS (Netherlands)

    Wolters, Justina Clarinda; Abele, Rupert; Tampé, Robert

    2005-01-01

    The transporter associated with antigen processing (TAP)-like (TAPL, ABCB9) belongs to the ATP-binding cassette transporter family, which translocates a vast variety of solutes across membranes. The function of this half-size transporter has not yet been determined. Here, we show that TAPL forms a h

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

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    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.

  8. Regulation of DNA Translocation Efficiency within the Chromatin Remodeler RSC/Sth1 Potentiates Nucleosome Sliding and Ejection.

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    Clapier, Cedric R; Kasten, Margaret M; Parnell, Timothy J; Viswanathan, Ramya; Szerlong, Heather; Sirinakis, George; Zhang, Yongli; Cairns, Bradley R

    2016-05-01

    The RSC chromatin remodeler slides and ejects nucleosomes, utilizing a catalytic subunit (Sth1) with DNA translocation activity, which can pump DNA around the nucleosome. A central question is whether and how DNA translocation is regulated to achieve sliding versus ejection. Here, we report the regulation of DNA translocation efficiency by two domains residing on Sth1 (Post-HSA and Protrusion 1) and by actin-related proteins (ARPs) that bind Sth1. ARPs facilitated sliding and ejection by improving "coupling"-the amount of DNA translocation by Sth1 relative to ATP hydrolysis. We also identified and characterized Protrusion 1 mutations that promote "coupling," and Post-HSA mutations that improve ATP hydrolysis; notably, the strongest mutations conferred efficient nucleosome ejection without ARPs. Taken together, sliding-to-ejection involves a continuum of DNA translocation efficiency, consistent with higher magnitudes of ATPase and coupling activities (involving ARPs and Sth1 domains), enabling the simultaneous rupture of multiple histone-DNA contacts facilitating ejection.

  9. Functional delineation of three groups of the ATP-dependent family of chromatin remodeling enzymes.

    NARCIS (Netherlands)

    Boyer, L.A.; Logie, C.; Bonte, E; Becker, P.B.; Wade, P.A.; Wolff, A.P.; Wu, C.; Imbalzano, A.N.; Peterson, C.L.

    2000-01-01

    ATP-dependent chromatin remodeling enzymes antagonize the inhibitory effects of chromatin. We compare six different remodeling complexes: ySWI/SNF, yRSC, hSWI/SNF, xMi-2, dCHRAC, and dNURF. We find that each complex uses similar amounts of ATP to remodel nucleosomal arrays at nearly identical rates.

  10. Kinetic mechanism of DNA translocation by the RSC molecular motor.

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

    2013-04-15

    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.

  11. Baculoviruses and nucleosome management.

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    Volkman, Loy E

    2015-02-01

    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.

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

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    Wioletta Czaja

    2012-09-01

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

  13. Functional roles of nucleosome stability and dynamics.

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    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.

  14. Baculoviruses and nucleosome management

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    Volkman, Loy E., E-mail: lvolkman@berkeley.edu

    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.

  15. ATP-dependent chromatin remodeling in the DNA-damage response

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    Lans Hannes

    2012-01-01

    Full Text Available Abstract The integrity of DNA is continuously challenged by metabolism-derived and environmental genotoxic agents that cause a variety of DNA lesions, including base alterations and breaks. DNA damage interferes with vital processes such as transcription and replication, and if not repaired properly, can ultimately lead to premature aging and cancer. Multiple DNA pathways signaling for DNA repair and DNA damage collectively safeguard the integrity of DNA. Chromatin plays a pivotal role in regulating DNA-associated processes, and is itself subject to regulation by the DNA-damage response. Chromatin influences access to DNA, and often serves as a docking or signaling site for repair and signaling proteins. Its structure can be adapted by post-translational histone modifications and nucleosome remodeling, catalyzed by the activity of ATP-dependent chromatin-remodeling complexes. In recent years, accumulating evidence has suggested that ATP-dependent chromatin-remodeling complexes play important, although poorly characterized, roles in facilitating the effectiveness of the DNA-damage response. In this review, we summarize the current knowledge on the involvement of ATP-dependent chromatin remodeling in three major DNA repair pathways: nucleotide excision repair, homologous recombination, and non-homologous end-joining. This shows that a surprisingly large number of different remodeling complexes display pleiotropic functions during different stages of the DNA-damage response. Moreover, several complexes seem to have multiple functions, and are implicated in various mechanistically distinct repair pathways.

  16. Nucleosome repositioning underlies dynamic gene expression.

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    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.

  17. Nucleosome switches.

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    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.

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

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    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.

  19. The RSC chromatin remodelling ATPase translocates DNA with high force and small step size.

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    Sirinakis, George; Clapier, Cedric R; Gao, Ying; Viswanathan, Ramya; Cairns, Bradley R; Zhang, Yongli

    2011-06-15

    ATP-dependent chromatin remodelling complexes use the energy of ATP hydrolysis to reposition and reconfigure nucleosomes. Despite their diverse functions, all remodellers share highly conserved ATPase domains, many shown to translocate DNA. Understanding remodelling requires biophysical knowledge of the DNA translocation process: how the ATPase moves DNA and generates force, and how translocation and force generation are coupled on nucleosomes. Here, we characterize the real-time activity of a minimal RSC translocase 'motor' on bare DNA, using high-resolution optical tweezers and a 'tethered' translocase system. We observe on dsDNA a processivity of ∼35 bp, a speed of ∼25 bp/s, and a step size of 2.0 (±0.4, s.e.m.) bp. Surprisingly, the motor is capable of moving against high force, up to 30 pN, making it one of the most force-resistant motors known. We also provide evidence for DNA 'buckling' at initiation. These observations reveal the ATPase as a powerful DNA translocating motor capable of disrupting DNA-histone interactions by mechanical force.

  20. Roles of histones and nucleosomes in gene transcription

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    This article reviews the latest research developments in the field of eukaryotic gene regulation by the structural alterations of chromatin and nucleosomes. The following issues are briefly addressed: (ⅰ) nucleosome and histone modifications by both the ATP-dependent remodel- ing com-plexes and the histone acetyltransferases and their roles in gene activation; (ⅱ) competitive binding of histones and transcription factors on gene promoters, and transcription repression by nucleosomes; and (ⅲ) influences of linker histone H1 on gene regulation. Meanwhile, the significance and impact of these new research progresses, as well as issues worthwhile for further study are commented.

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

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    Steven E Weicksel

    Full Text Available 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.

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

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    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.

  3. Structural studies on Helicobacter pylori ATP-dependent protease, FtsH

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    Kim, Sung Hyun; Kang, Gil Bu; Song, Hye-Eun; Park, Sang Jin; Bea, Man-Ho; Eom, Soo Hyun, E-mail: eom@gist.ac.kr [Department of Life Science, Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712 (Korea, Republic of)

    2008-05-01

    The crystal structures of the Helicobacter pylori FtsH ATPase domain in the nucleotide-free state and complexed with ADP have been determined. The ATP-dependent protease, FtsH, degrades misassembled membrane proteins for quality control like SecY, subunit a of FoF1-ATPase, and YccA, and digests short-lived soluble proteins in order to control their cellular regulation, including σ32, LpxC and λcII. The FtsH protein has an N-terminal transmembrane segment and a large cytosolic region that consists of two domains, an ATPase and a protease domain. To provide a structural basis for the nucleotide-dependent domain motions and a better understanding of substrate translocation, the crystal structures of the Helicobacter pylori (Hp) FtsH ATPase domain in the nucleotide-free state and complexed with ADP, were determined. Two different structures of HpFtsH ATPase were observed, with the nucleotide-free state in an asymmetric unit, and these structures reveal the new forms and show other conformational differences between the nucleotide-free and ADP-bound state compared with previous structures. In particular, one HpFtsH Apo structure has a considerable rotation difference compared with the HpFtsH ADP complex, and this large conformational change reveals that FtsH may have the mechanical force needed for substrate translocation.

  4. ATP-independent cooperative binding of yeast Isw1a to bare and nucleosomal DNA.

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    Anne De Cian

    Full Text Available Among chromatin remodeling factors, the ISWI family displays a nucleosome-enhanced ATPase activity coupled to DNA translocation. While these enzymes are known to bind to DNA, their activity has not been fully characterized. Here we use TEM imaging and single molecule manipulation to investigate the interaction between DNA and yeast Isw1a. We show that Isw1a displays a highly cooperative ATP-independent binding to and bridging between DNA segments. Under appropriate tension, rare single nucleation events can sometimes be observed and loop DNA with a regular step. These nucleation events are often followed by binding of successive complexes bridging between nearby DNA segments in a zipper-like fashion, as confirmed by TEM observations. On nucleosomal substrates, we show that the specific ATP-dependent remodeling activity occurs in the context of cooperative Isw1a complexes bridging extranucleosomal DNA. Our results are interpreted in the context of the recently published partial structure of Isw1a and support its acting as a "protein ruler" (with possibly more than one tick.

  5. The nucleosome-remodeling ATPase ISWI is regulated by poly-ADP-ribosylation.

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    Anna Sala

    2008-10-01

    Full Text Available ATP-dependent nucleosome-remodeling enzymes and covalent modifiers of chromatin set the functional state of chromatin. However, how these enzymatic activities are coordinated in the nucleus is largely unknown. We found that the evolutionary conserved nucleosome-remodeling ATPase ISWI and the poly-ADP-ribose polymerase PARP genetically interact. We present evidence showing that ISWI is target of poly-ADP-ribosylation. Poly-ADP-ribosylation counteracts ISWI function in vitro and in vivo. Our work suggests that ISWI is a physiological target of PARP and that poly-ADP-ribosylation can be a new, important post-translational modification regulating the activity of ATP-dependent nucleosome remodelers.

  6. Role of nucleosome remodeling in neurodevelopmental and intellectual disability disorders

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    Alberto J Lopez

    2015-04-01

    Full Text Available It is becoming increasingly important to understand how epigenetic mechanisms control gene expression during neurodevelopment. Two epigenetic mechanisms that have received considerable attention are DNA methylation and histone acetylation. Human exome sequencing and genome-wide association studies have linked several neurobiological disorders to genes whose products actively regulate DNA methylation and histone acetylation. More recently, a third major epigenetic mechanism, nucleosome remodeling, has been implicated in human developmental and intellectual disability disorders. Nucleosome remodeling is driven primarily through nucleosome remodeling complexes with specialized ATP-dependent enzymes. These enzymes directly interact with DNA or chromatin structure, as well as histone subunits, to restructure the shape and organization of nucleosome positioning to ultimately regulate gene expression. Of particular interest is the neuron-specific Brg1/hBrm Associated Factor (nBAF complex. Mutations in nBAF subunit genes have so far been linked to Coffin-Siris syndrome, Nicolaides-Baraitser syndrome, schizophrenia, and Autism Spectrum Disorder. Together, these human developmental and intellectual disability disorders are powerful examples of the impact of epigenetic modulation on gene expression. This review focuses on the new and emerging role of nucleosome remodeling in neurodevelopmental and intellectual disability disorders and whether nucleosome remodeling affects gene expression required for cognition independently of its role in regulating gene expression required for development.

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

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    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.

  8. CLONING, SEQUENCING, AND EXPRESSION OF BACILLUS-SUBTILIS GENES INVOLVED IN ATP-DEPENDENT NUCLEASE SYNTHESIS

    NARCIS (Netherlands)

    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-

  9. Phosphorylation of linker histones regulates ATP-dependent chromatin remodeling enzymes.

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    Horn, P.J.; Carruthers, L.M.; Logie, C.; Hill, D.A.; Solomon, M.J.; Wade, P.A.; Imbalzano, A.N.; Hansen, J.; Peterson, C.L.

    2002-01-01

    Members of the ATP-dependent family of chromatin remodeling enzymes play key roles in the regulation of transcription, development, DNA repair and cell cycle control. We find that the remodeling activities of the ySWI/SNF, hSWI/SNF, xMi-2 and xACF complexes are nearly abolished by incorporation of l

  10. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells.

    Science.gov (United States)

    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

    2016-02-01

    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.

  11. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells

    Science.gov (United States)

    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

  12. Structural Analysis of the Key Intermediate Formed during Transcription through a Nucleosome.

    Science.gov (United States)

    Chang, H-W; Shaytan, A K; Hsieh, F-K; Kulaeva, O I; Kirpichnikov, M P; Studitsky, V M

    2013-01-01

    Transcription through chromatin by different RNA polymerases produces different biological outcomes and is accompanied by either nucleosome survival at the original location (Pol II-type mechanism) or backward nucleosome translocation along DNA (Pol III-type mechanism). It has been proposed that differences in the structure of the key intermediates formed during transcription dictate the fate of the nucleosomes. To evaluate this possibility, structure of the key intermediate formed during transcription by Pol III-type mechanism was studied by DNase I footprinting and molecular modeling. The Pol III-type mechanism is characterized by less efficient formation of the key intermediate required for nucleosome survival (Ø-loop, Pol II-type mechanism), most likely due to steric interference between the RNA polymerase and DNA in the Ø-loop. The data suggest that the lower efficiency of Ø-loop formation induces formation of a lower nucleosomal barrier and nucleosome translocation during transcription by Pol III-type mechanism.

  13. What controls nucleosome positions?

    Science.gov (United States)

    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.

  14. Transfer RNA is an essential component of the ubiquitin- and ATP-dependent proteolytic system

    Energy Technology Data Exchange (ETDEWEB)

    Ciechanover, A.; Wolin, S.L.. Steitz, J.A.; Lodish, H.F.

    1985-03-01

    Protein degradation via the nonlysosomal ATP-dependent pathway in rabbit reticulocytes involves a number of components. In the initial event, ubiquitin, an abundant 76-residue polypeptide, becomes covalently linked to the protein substrate in an ATP-requiring reaction. Once marked in this way, the conjugated protein is proteolyzed in a reaction that also requires ATP. Here the authors show that tRNA is another essential component of the system. Ribonucleases strongly inhibit the ubiquitin- and ATP-dependent degradation of /sup 125/I-labeled bovine serum albumin in the reticulocyte system in vitro. RNAs extracted from fractions of the reticulocyte extract or from mouse cells restore proteolytic activity. When the RNA is fractionated by gel electrophoresis, only the tRNA fraction is active in restoring proteolysis. Furthermore, pure mouse tRNA/sup His/, isolated by immunoprecipitation with patient autoimmune sera, restores the proteolytic activity. The possibility that the level of uncharged tRNA in mammalian cells regulates the ubiquitin- and ATP-dependent proteolytic system is discussed.

  15. Comparative Genomics Reveals Chd1 as a Determinant of Nucleosome Spacing in Vivo.

    Science.gov (United States)

    Hughes, Amanda L; Rando, Oliver J

    2015-07-14

    Packaging of genomic DNA into nucleosomes is nearly universally conserved in eukaryotes, and many features of the nucleosome landscape are quite conserved. Nonetheless, quantitative aspects of nucleosome packaging differ between species because, for example, the average length of linker DNA between nucleosomes can differ significantly even between closely related species. We recently showed that the difference in nucleosome spacing between two Hemiascomycete species-Saccharomyces cerevisiae and Kluyveromyces lactis-is established by trans-acting factors rather than being encoded in cis in the DNA sequence. Here, we generated several S. cerevisiae strains in which endogenous copies of candidate nucleosome spacing factors are deleted and replaced with the orthologous factors from K. lactis. We find no change in nucleosome spacing in such strains in which H1 or Isw1 complexes are swapped. In contrast, the K. lactis gene encoding the ATP-dependent remodeler Chd1 was found to direct longer internucleosomal spacing in S. cerevisiae, establishing that this remodeler is partially responsible for the relatively long internucleosomal spacing observed in K. lactis. By analyzing several chimeric proteins, we find that sequence differences that contribute to the spacing activity of this remodeler are dispersed throughout the coding sequence, but that the strongest spacing effect is linked to the understudied N-terminal end of Chd1. Taken together, our data find a role for sequence evolution of a chromatin remodeler in establishing quantitative aspects of the chromatin landscape in a species-specific manner.

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

    Science.gov (United States)

    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.

  17. ATP-dependent mitochondrial porphyrin importer ABCB6 protects against phenylhydrazine toxicity.

    Science.gov (United States)

    Ulrich, Dagny L; Lynch, John; Wang, Yao; Fukuda, Yu; Nachagari, Deepa; Du, Guoqing; Sun, Daxi; Fan, Yiping; Tsurkan, Lyudmila; Potter, Philip M; Rehg, Jerold E; Schuetz, John D

    2012-04-13

    Abcb6 is a mammalian mitochondrial ATP-binding cassette (ABC) transporter that regulates de novo porphyrin synthesis. In previous studies, haploinsufficient (Abcb6(+/-)) embryonic stem cells showed impaired porphyrin synthesis. Unexpectedly, Abcb6(-/-) mice derived from these stem cells appeared phenotypically normal. We hypothesized that other ATP-dependent and/or -independent mechanisms conserve porphyrins. Here, we demonstrate that Abcb6(-/-) mice lack mitochondrial ATP-driven import of coproporphyrin III. Gene expression analysis revealed that loss of Abcb6 results in up-regulation of compensatory porphyrin and iron pathways, associated with elevated protoporphyrin IX (PPIX). Phenylhydrazine-induced stress caused higher mortality in Abcb6(-/-) mice, possibly because of sustained elevation of PPIX and an inability to convert PPIX to heme despite elevated ferrochelatase levels. Therefore, Abcb6 is the sole ATP-dependent porphyrin importer, and loss of Abcb6 produces up-regulation of heme and iron pathways necessary for normal development. However, under extreme demand for porphyrins (e.g. phenylhydrazine stress), these adaptations appear inadequate, which suggests that under these conditions Abcb6 is important for optimal survival.

  18. Nucleosome Positioning and Epigenetics

    Science.gov (United States)

    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.

  19. RSC-dependent constructive and destructive interference between opposing arrays of phased nucleosomes in yeast.

    Science.gov (United States)

    Ganguli, Dwaipayan; Chereji, Răzvan V; Iben, James R; Cole, Hope A; Clark, David J

    2014-10-01

    RSC and SWI/SNF are related ATP-dependent chromatin remodeling machines that move nucleosomes, regulating access to DNA. We addressed their roles in nucleosome phasing relative to transcription start sites in yeast. SWI/SNF has no effect on phasing at the global level. In contrast, RSC depletion results in global nucleosome repositioning: Both upstream and downstream nucleosomal arrays shift toward the nucleosome-depleted region (NDR), with no change in spacing, resulting in a narrower and partly filled NDR. The global picture of RSC-depleted chromatin represents the average of a range of chromatin structures, with most genes showing a shift of the +1 or the -1 nucleosome into the NDR. Using RSC ChIP data reported by others, we show that RSC occupancy is highest on the coding regions of heavily transcribed genes, though not at their NDRs. We propose that RSC has a role in restoring chromatin structure after transcription. Analysis of gene pairs in different orientations demonstrates that phasing patterns reflect competition between phasing signals emanating from neighboring NDRs. These signals may be in phase, resulting in constructive interference and a regular array, or out of phase, resulting in destructive interference and fuzzy positioning. We propose a modified barrier model, in which a stable complex located at the NDR acts as a bidirectional phasing barrier. In RSC-depleted cells, this barrier has a smaller footprint, resulting in narrower NDRs. Thus, RSC plays a critical role in organizing yeast chromatin.

  20. ATP-dependent DNA binding, unwinding, and resection by the Mre11/Rad50 complex.

    Science.gov (United States)

    Liu, Yaqi; Sung, Sihyun; Kim, Youngran; Li, Fuyang; Gwon, Gwanghyun; Jo, Aera; Kim, Ae-Kyoung; Kim, Taeyoon; Song, Ok-Kyu; Lee, Sang Eun; Cho, Yunje

    2016-04-01

    ATP-dependent DNA end recognition and nucleolytic processing are central functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair. However, it is still unclear how ATP binding and hydrolysis primes the MR function and regulates repair pathway choice in cells. Here,Methanococcus jannaschii MR-ATPγS-DNA structure reveals that the partly deformed DNA runs symmetrically across central groove between two ATPγS-bound Rad50 nucleotide-binding domains. Duplex DNA cannot access the Mre11 active site in the ATP-free full-length MR complex. ATP hydrolysis drives rotation of the nucleotide-binding domain and induces the DNA melting so that the substrate DNA can access Mre11. Our findings suggest that the ATP hydrolysis-driven conformational changes in both DNA and the MR complex coordinate the melting and endonuclease activity.

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

    Science.gov (United States)

    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.

  2. SecA supports a constant rate of preprotein translocation

    NARCIS (Netherlands)

    Tomkiewicz, D; Nouwen, N; van Leeuwen, R; Tans, S; Driessen, AJM

    2006-01-01

    In Escherichia coli, secretory proteins (preproteins) are translocated across the cytoplasmic membrane by the Sec system composed of a protein-conducting channel, SecYEG, and an ATP-dependent motor protein, SecA. After binding of the preprotein to SecYEG-bound SecA, cycles of ATP binding and hydroly

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

    Science.gov (United States)

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

    2016-09-01

    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.

  4. The size of the nucleosome

    DEFF Research Database (Denmark)

    Bohr, Jakob; Olsen, Kasper

    2011-01-01

    The structural origin of the size of the 11 nm nucleosomal disc is addressed. On the nanometer length-scale the organization of DNA as chromatin in the chromosomes involves a coiling of DNA around the histone core of the nucleosome. We suggest that the size of the nucleosome core particle is dict......-pairs of the linker-DNA is included the estimate of the size of an ideal nucleosome is in close agreement with the experimental numbers. Interestingly, the size of the nucleosome is shown to be a consequence of intrinsic properties of the DNA double helix....

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

    Directory of Open Access Journals (Sweden)

    Nicola Wiechens

    2016-03-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2016-03-01

    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.

  7. Antispasmodic activity of Symplocos paniculata is mediated through opening of ATP-dependent K+ channel

    Directory of Open Access Journals (Sweden)

    Khalid Hussain Janbaz

    2016-06-01

    Full Text Available Symplocos paniculata is a medicinal plant used by native healers to manage gastrointestinal ailments. The crude methanolic extract of S. paniculata was screened pharmacologically both in vitro and in vivo for the validation of its therapeutic potential. It suppressed the spontaneous activity of isolated rabbit jejunum preparations and also caused inhibition of the low K+ (20 mM- induced spastic contractions in isolated rabbit jejunum preparations in a manner comparable to cromakalim. The relaxant effect was found to be blocked following glibenclamide exposure of the isolated tissue preparations similar to cromakalim, suggesting that observed response was likely to be mediated through opening of ATP dependent K+ channels. Following oral administration to mice provided protection against castor oil-induced diarrhea in a manner similar to loperamide. The plant material was found safe in toxicity study up to oral dose of 8 g/kg in mice. Hence, present study provides a scientific basis for the vernacular use of S. paniculata in gastro-intestinal system.

  8. Properties of mutants of haemophilus influenzae deficient in ATP-dependent deoxyribonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, J.K.

    1976-01-01

    Eight isogenic Haemophilus influenzae strains whose extracts lack ATP-dependent deoxyribonuclease activity (Add/sup -/ mutants) form three complementation and genetic linkage groups. Since there are known to be three subunits of the enzyme, these data suggest that each of the three genes specifies a different subunit. Gel electrophoresis of partially purified mutant extracts indicates that the smallest subunit is missing in one of the groups but is present in all the other mutants. The mutants are more sensitive to a variety of chemical agents than the wild type. The most sensitive mutants lack the ATPase activity associated with the enzyme. These strains exhibit aberrant incorporation of tritiated thymidine, which starts up more rapidly and shuts off sooner than in the wild type. An extracellular compound is responsible for most of this effect, in that wild type cells put into medium in which Add/sup -/ cells have been growing show a similar aberrant incorporation. The effect of these media can be mimicked by cyclic AMP and cyclic GMP, although millimolar concentrations are required. It is postulated that the Add/sup -/ mutants are more permeable to many substances than the wild type, partly because of the extracellular compound usually surrounding them, and the increased permeability might be responsible for the mutants' nonviability.

  9. Selective removal of promoter nucleosomes by the RSC chromatin-remodeling complex.

    Science.gov (United States)

    Lorch, Yahli; Griesenbeck, Joachim; Boeger, Hinrich; Maier-Davis, Barbara; Kornberg, Roger D

    2011-08-01

    Purified chromatin rings, excised from the PHO5 locus of Saccharomyces cerevisiae in transcriptionally repressed and activated states, were remodeled with RSC and ATP. Nucleosomes were translocated, and those originating on the promoter of repressed rings were removed, whereas those originating on the open reading frame (ORF) were retained. Treatment of the repressed rings with histone deacetylase diminished the removal of promoter nucleosomes. These findings point to a principle of promoter chromatin remodeling for transcription, namely that promoter specificity resides primarily in the nucleosomes rather than in the remodeling complex that acts upon them.

  10. Sequence-dependent nucleosome positioning.

    Science.gov (United States)

    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.

  11. Nucleosomes undergo slow spontaneous gaping.

    Science.gov (United States)

    Ngo, Thuy T M; Ha, Taekjip

    2015-04-30

    In eukaryotes, DNA is packaged into a basic unit, the nucleosome which consists of 147 bp of DNA wrapped around a histone octamer composed of two copies each of the histones H2A, H2B, H3 and H4. Nucleosome structures are diverse not only by histone variants, histone modifications, histone composition but also through accommodating different conformational states such as DNA breathing and dimer splitting. Variation in nucleosome structures allows it to perform a variety of cellular functions. Here, we identified a novel spontaneous conformational switching of nucleosomes under physiological conditions using single-molecule FRET. Using FRET probes placed at various positions on the nucleosomal DNA to monitor conformation of the nucleosome over a long period of time (30-60 min) at various ionic conditions, we identified conformational changes we refer to as nucleosome gaping. Gaping transitions are distinct from nucleosome breathing, sliding or tightening. Gaping modes switch along the direction normal to the DNA plane through about 5-10 angstroms and at minutes (1-10 min) time scale. This conformational transition, which has not been observed previously, may be potentially important for enzymatic reactions/transactions on nucleosomal substrate and the formation of multiple compression forms of chromatin fibers.

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

    Science.gov (United States)

    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.

  13. A split personality for nucleosomes.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  15. Circulating Nucleosomes and Nucleosome Modifications as Biomarkers in Cancer

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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. The ATP-dependent remodeler RSC transfers histone dimers and octamers through the rapid formation of an unstable encounter intermediate.

    Science.gov (United States)

    Rowe, Claire E; Narlikar, Geeta J

    2010-11-16

    RSC, an essential chromatin remodeling complex in budding yeast, is involved in a variety of biological processes including transcription, recombination, repair, and replication. How RSC participates in such diverse processes is not fully understood. In vitro, RSC uses ATP to carry out several seemingly distinct reactions: it repositions nucleosomes, transfers H2A/H2B dimers between nucleosomes, and transfers histone octamers between pieces of DNA. This raises the intriguing mechanistic question of how this molecular machine can use a single ATPase subunit to create these varied products. Here, we use a FRET-based approach to kinetically order the products of the RSC reaction. Surprisingly, transfer of H2A/H2B dimers and histone octamers is initiated on a time scale of seconds when assayed by FRET, but formation of stable nucleosomal products occurs on a time scale of minutes when assayed by native gel. These results suggest a model in which RSC action rapidly generates an unstable encounter intermediate that contains the two exchange substrates in close proximity. This intermediate then collapses more slowly to form the stable transfer products seen on native gels. The rapid, biologically relevant time scale on which the transfer products are generated implies that such products can play key roles in vivo.

  18. A conserved residue cluster that governs kinetics of ATP-dependent gating of Kir6.2 potassium channels

    DEFF Research Database (Denmark)

    Zhang, Roger S; Wright, Jordan; Pless, Stephan Alexander;

    2015-01-01

    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, potentially necessary to localize the ε-amine of Lys......ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly-rectifying Kir channel (Kir6.x) and sulfonylurea receptors (SUR). 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 approximately 60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly...

  19. Conditions for positioning of nucleosomes on DNA.

    Science.gov (United States)

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

    Science.gov (United States)

    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.

  1. Plant-like phosphofructokinase from Plasmodium falciparum belongs to a novel class of ATP-dependent enzymes.

    Science.gov (United States)

    Mony, Binny M; Mehta, Monika; Jarori, Gotam K; Sharma, Shobhona

    2009-11-01

    Malaria parasite-infected erythrocytes exhibit enhanced glucose utilisation and 6-phospho-1-fructokinase (PFK) is a key enzyme in glycolysis. Here we present the characterisation of PFK from the human malaria parasite Plasmodium falciparum. Of the two putative PFK genes on chromosome 9 (PfPFK9) and 11 (PfPFK11), only the PfPFK9 gene appeared to possess all the catalytic features appropriate for PFK activity. The deduced PfPFK proteins contain domains homologous to the plant-like pyrophosphate (PPi)-dependent PFK beta and alpha subunits, which are quite different from the human erythrocyte PFK protein. The PfPFK9 gene beta and alpha regions were cloned and expressed as His(6)- and GST-tagged proteins in Escherichia coli. Complementation of PFK-deficient E. coli and activity analysis of purified recombinant proteins confirmed that PfPFK9beta possessed catalytic activity. Monoclonal antibodies against the recombinant beta protein confirmed that the PfPFK9 protein has beta and alpha domains fused into a 200 kDa protein, as opposed to the independent subunits found in plants. Despite an overall structural similarity to plant PPi-PFKs, the recombinant protein and the parasite extract exhibited only ATP-dependent enzyme activity, and none with PPi. Unlike host PFK, the Plasmodium PFK was insensitive to fructose-2,6-bisphosphate (F-2,6-bP), phosphoenolpyruvate (PEP) and citrate. A comparison of the deduced PFK proteins from several protozoan PFK genome databases implicates a unique class of ATP-dependent PFK present amongst the apicomplexan protozoans.

  2. Nucleosome Organization in Human Embryonic Stem Cells.

    Science.gov (United States)

    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

  3. Nucleosome Organization in Human Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Puya G Yazdi

    Full Text Available 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

  4. The size of the nucleosome

    CERN Document Server

    Bohr, Jakob

    2011-01-01

    The structural origin of the size of the 11 nm nucleosomal disc is addressed. On the nanometer length-scale the organization of DNA as chromatin in the chromosomes involves a coiling of DNA around the histone core of the nucleosome. We suggest that the size of the nucleosome core particle is dictated by the fulfillment of two criteria: One is optimizing the volume fraction of the DNA double helix; this requirement for close-packing has its root in optimizing atomic and molecular interactions. The other criterion being that of having a zero strain-twist coupling; being a zero-twist structure is a necessity when allowing for transient tensile stresses during the reorganization of DNA, e.g., during the reposition, or sliding, of a nucleosome along the DNA double helix. The mathematical model we apply is based on a tubular description of double helices assuming hard walls. When the base-pairs of the linker-DNA is included the estimate of the size of an ideal nucleosome is in close agreement with the experimental ...

  5. Visible periodicity of strong nucleosome DNA sequences.

    Science.gov (United States)

    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. Nucleosome repositioning via loop formation

    CERN Document Server

    Kulic, M L

    2002-01-01

    Active (catalysed) and passive (intrinsic) nucleosome repositioning is known to be a crucial event during the transcriptional activation of certain eucaryotic genes. Here we consider theoretically the intrinsic mechanism and study in detail the energetics and dynamics of DNA-loop-mediated nucleosome repositioning, as previously proposed by Schiessel et al. (H. Schiessel, J. Widom, R. F. Bruinsma, and W. M. Gelbart. 2001. {\\it Phys. Rev. Lett.} 86:4414-4417). The surprising outcome of the present study is the inherent nonlocality of nucleosome motion within this model -- being a direct physical consequence of the loop mechanism. On long enough DNA templates the longer jumps dominate over the previously predicted local motion, a fact that contrasts simple diffusive mechanisms considered before. The possible experimental outcome resulting from the considered mechanism is predicted, discussed and compared to existing experimental findings.

  7. Review fifteen years of search for strong nucleosomes.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. Relationship between nucleosome positioning and DNA methylation

    Science.gov (United States)

    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

  10. Conditions for positioning of nucleosomes on DNA

    CERN Document Server

    Sheinman, Michael

    2015-01-01

    Positioning of nucleosomes along eukaryotic genomes plays an important role in their 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 analyzed how well nucleosomes are positioned along the DNA as a function of strength of the preferential binding, correlation length of the binding energy landscape, interactions between neighboring nucleosomes and others relevant system properties. We analyze different scenarios: designed energy landscapes and generically disordered ones and derive conditions for good positioning. Using analytic and numerical approaches we find that, even if the binding preferences are very weak, synergistic interplay between the interactions and the binding preferences is essential for a good positioning of nucleosomes, especially on correlated energy landscapes. Ana...

  11. ATP-dependent recruitment of export factor Aly/REF onto intronless mRNAs by RNA helicase UAP56.

    Science.gov (United States)

    Taniguchi, Ichiro; Ohno, Mutsuhito

    2008-01-01

    Loading of export factors onto mRNAs is a key step in gene expression. In vertebrates, splicing plays a role in this process. Specific protein complexes, exon junction complex and transcription/export complex, are loaded onto mRNAs in a splicing-dependent manner, and adaptor proteins such as Aly/REF in the complexes in turn recruit mRNA exporter TAP-p15 onto the RNA. By contrast, how export factors are recruited onto intronless mRNAs is largely unknown. We previously showed that Aly/REF is preferentially associated with intronless mRNAs in the nucleus. Here we show that Aly/REF could preferentially bind intronless mRNAs in vitro and that this binding was stimulated by RNA helicase UAP56 in an ATP-dependent manner. Consistently, an ATP binding-deficient UAP56 mutant specifically inhibited mRNA export in Xenopus oocytes. Interestingly, ATP activated the RNA binding activity of UAP56 itself. ATP-bound UAP56 therefore bound to both RNA and Aly/REF, and as a result ATPase activity of UAP56 was cooperatively stimulated. These results are consistent with a model in which ATP-bound UAP56 chaperones Aly/REF onto RNA, ATP is then hydrolyzed, and UAP56 dissociates from RNA for the next round of Aly/REF recruitment. Our finding provides a mechanistic insight into how export factors are recruited onto mRNAs.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  14. Identification of ATP-Dependent Phosphofructokinase as a Regulatory Step in the Glycolytic Pathway of the Actinomycete Streptomyces coelicolor A3(2)

    NARCIS (Netherlands)

    Alves, A.M.C.R.; Euverink, G.J.W.; Bibb, M.J.; Dijkhuizen, L.

    1997-01-01

    The ATP-dependent phosphofructokinase (ATP-PFK) of Streptomyces coelicolor A3(2) was purified to homogeneity (1,600-fold) and characterized (110 kDa, with a single type of subunit of 40 kDa); it is allosterically inhibited by phosphoenolpyruvate. Cloning of the pfk gene of S. coelicolor A3(2) and an

  15. Nucleosome architecture throughout the cell cycle.

    Science.gov (United States)

    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.

  16. Insights into DNA signals for nucleosome positioning

    Institute of Scientific and Technical Information of China (English)

    Zhiming DAI; Xianhua DAI; Jihua FENG; Qian XIANG; Yangyang DENG; Jiang WANG

    2008-01-01

    The nucleosome is the fundamental unit of eukaryotic genomes. Its positioning in the promoter region plays a central role in regulating gene transcription. Experimental evidence suggests that the genomic DNA sequence is one important determinant of nucleosome positioning. Several approaches have been developed to predict nucleosome positions based on DNA sequence features, but the results indicate that there is room for improvement. This paper presents a new computational approach to predict genome-wide nucleosome locations in promoter regions. Importantly, the proposed approach outperforms existing approaches in yeast. Further anal-ysis demonstrates that DNA signals for nucleosome posi-tioning vary with species and composition of histones. Analysis of individual genes reveals that the role of the underlying DNA sequence in nucleosome positioning var-ies with genes.

  17. Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Tulika Munshi

    Full Text Available ATP-dependent Mur ligases (Mur synthetases play essential roles in the biosynthesis of cell wall peptidoglycan (PG as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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. spFRET studies of nucleosome dynamics modulated by histone modifications, histone variants and neighboring nucleosomes

    NARCIS (Netherlands)

    Buning, Ruth

    2015-01-01

    At the basis of the regulation of the genetic code (DNA) in eukaryotes is its organization into nucleosomes. Nucleosomes modulate DNA accessibility through conformational dynamics like DNA breathing - the transient unwrapping of DNA from the nucleosome. Single-pair Fluorescence Resonance Energy Tran

  1. Reading sequence-directed computational nucleosome maps.

    Science.gov (United States)

    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.

  2. A Positive Twist to the Centromeric Nucleosome

    Directory of Open Access Journals (Sweden)

    Josefina Ocampo

    2015-10-01

    Full Text Available Centromeric nucleosomes are critical for chromosome attachment to the mitotic spindle. In this issue of Cell Reports, Diaz-Ingelmo et al. (2015 propose that the yeast centromeric nucleosome is stabilized by a positively supercoiled loop formed by the sequence-specific CBF3 complex.

  3. Fragile Nucleosomes Influence Pol II Promoter Function.

    Science.gov (United States)

    Pradhan, Suman K; Xue, Yong; Carey, Michael F

    2015-11-05

    In this issue of Molecular Cell, Kubik et al. (2015) describe how the RSC chromatin remodeling complex collaborates with two DNA sequence motifs and sequence-specific general regulatory factors to assemble fragile nucleosomes at highly transcribed yeast Pol II promoters, and they distinguish these from promoters bearing stable nucleosomes.

  4. Structural analysis of nucleosomal barrier to transcription.

    Science.gov (United States)

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

    2015-10-27

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

  5. A brief review of nucleosome structure.

    Science.gov (United States)

    Cutter, Amber R; Hayes, Jeffrey J

    2015-10-01

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

  6. Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays.

    Science.gov (United States)

    Schrader, Anna; Gross, Thomas; Thalhammer, Verena; Längst, Gernot

    2015-01-01

    The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.

  7. Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays.

    Directory of Open Access Journals (Sweden)

    Anna Schrader

    Full Text Available The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.

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

    Science.gov (United States)

    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.

  9. Structural basis for retroviral integration into nucleosomes.

    Science.gov (United States)

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

    2015-07-16

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

  10. Histone chaperone-mediated nucleosome assembly process.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    So Yeon Kwon

    2016-04-01

    Full Text Available 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.

  13. Dynamics of nucleosome invasion by DNA binding proteins.

    Science.gov (United States)

    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.

  14. Analysis of the mechanism of nucleosome survival during transcription.

    Science.gov (United States)

    Chang, Han-Wen; Kulaeva, Olga I; Shaytan, Alexey K; Kibanov, Mikhail; Kuznedelov, Konstantin; Severinov, Konstantin V; Kirpichnikov, Mikhail P; Clark, David J; Studitsky, Vasily M

    2014-02-01

    Maintenance of nucleosomal structure in the cell nuclei is essential for cell viability, regulation of gene expression and normal aging. Our previous data identified a key intermediate (a small intranucleosomal DNA loop, Ø-loop) that is likely required for nucleosome survival during transcription by RNA polymerase II (Pol II) through chromatin, and suggested that strong nucleosomal pausing guarantees efficient nucleosome survival. To evaluate these predictions, we analysed transcription through a nucleosome by different, structurally related RNA polymerases and mutant yeast Pol II having different histone-interacting surfaces that presumably stabilize the Ø-loop. The height of the nucleosomal barrier to transcription and efficiency of nucleosome survival correlate with the net negative charges of the histone-interacting surfaces. Molecular modeling and analysis of Pol II-nucleosome intermediates by DNase I footprinting suggest that efficient Ø-loop formation and nucleosome survival are mediated by electrostatic interactions between the largest subunit of Pol II and core histones.

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

    Science.gov (United States)

    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. Insights into distinct regulatory modes of nucleosome positioning

    Directory of Open Access Journals (Sweden)

    Feng Jihua

    2009-12-01

    Full Text Available Abstract Background The nucleosome is the fundamental unit of eukaryotic genomes. Experimental evidence suggests that the genomic DNA sequence and a variety of protein factors contribute to nucleosome positioning in vivo. However, how nucleosome positioning is determined locally is still largely unknown. Results We found that transcription factor binding sites (TFBSs with particular nucleosomal contexts show a preference to reside on specific chromosomes. We identified four typical gene classes associated with distinct regulatory modes of nucleosome positioning, and further showed that they are distinguished by transcriptional regulation patterns. The first mode involves the cooperativity between chromatin remodeling and stable transcription factor (TF-DNA binding that is linked to high intrinsic DNA binding affinities, evicting nucleosomes from favorable DNA sequences. The second is the DNA-encoded low nucleosome occupancy that is associated with high gene activity. The third is through chromatin remodeling and histone acetylation, sliding nucleosomes along DNA. This mode is linked to more cryptic sites for TF binding. The last consists of the nucleosome-enriched organization driven by other factors that overrides nucleosome sequence preferences. In addition, we showed that high polymerase II (Pol II occupancy is associated with high nucleosome occupancy around the transcription start site (TSS. Conclusions We identified four different regulatory modes of nucleosome positioning and gave insights into mechanisms that specify promoter nucleosome location. We suggest two distinct modes of recruitment of Pol II, which are selectively employed by different genes.

  17. Nucleosome positioning, nucleotide excision repair and photoreactivation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Guintini, Laetitia; Charton, Romain; Peyresaubes, François; Thoma, Fritz; Conconi, Antonio

    2015-12-01

    The position of nucleosomes on DNA participates in gene regulation and DNA replication. Nucleosomes can be repressors by limiting access of factors to regulatory sequences, or activators by facilitating binding of factors to exposed DNA sequences on the surface of the core histones. The formation of UV induced DNA lesions, like cyclobutane pyrimidine dimers (CPDs), is modulated by DNA bending around the core histones. Since CPDs are removed by nucleotide excision repair (NER) and photolyase repair, it is of paramount importance to understand how DNA damage and repair are tempered by the position of nucleosomes. In vitro, nucleosomes inhibit NER and photolyase repair. In vivo, nucleosomes slow down NER and considerably obstruct photoreactivation of CPDs. However, over-expression of photolyase allows repair of nucleosomal DNA in a second time scale. It is proposed that the intrinsic abilities of nucleosomes to move and transiently unwrap could facilitate damage recognition and repair in nucleosomal DNA.

  18. Featuring the nucleosome surface as a therapeutic target.

    Science.gov (United States)

    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. From nucleosome to chromosome: a dynamic organization of genetic information

    NARCIS (Netherlands)

    P. Fransz; H. de Jong

    2011-01-01

    Gene activity is controlled at different levels of chromatin organization, which involve genomic sequences, nucleosome structure, chromatin folding and chromosome arrangement. These levels are interconnected and influence each other. At the basic level nucleosomes generally occlude the DNA sequence

  20. Dynamic regulation of transcription factors by nucleosome remodeling.

    Science.gov (United States)

    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.

  1. Insights into distinct regulatory modes of nucleosome positioning

    OpenAIRE

    Feng Jihua; Xiang Qian; Dai Xianhua; Dai Zhiming; Deng Yangyang; Wang Jiang

    2009-01-01

    Abstract Background The nucleosome is the fundamental unit of eukaryotic genomes. Experimental evidence suggests that the genomic DNA sequence and a variety of protein factors contribute to nucleosome positioning in vivo. However, how nucleosome positioning is determined locally is still largely unknown. Results We found that transcription factor binding sites (TFBSs) with particular nucleosomal contexts show a preference to reside on specific chromosomes. We identified four typical gene clas...

  2. The Effect of Micrococcal Nuclease Digestion on Nucleosome Positioning Data

    OpenAIRE

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

    2010-01-01

    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 mod...

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

    Science.gov (United States)

    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.

  4. Traceless Synthesis of Asymmetrically Modified Bivalent Nucleosomes.

    Science.gov (United States)

    Lechner, Carolin C; Agashe, Ninad D; Fierz, Beat

    2016-02-18

    Nucleosomes carry extensive post-translational modifications (PTMs), which results in complex modification patterns that are involved in epigenetic signaling. Although two copies of each histone coexist in a nucleosome, they may not carry the same PTMs and are often differently modified (asymmetric). In bivalent domains, a chromatin signature prevalent in embryonic stem cells (ESCs), namely H3 methylated at lysine 4 (H3K4me3), coexists with H3K27me3 in asymmetric nucleosomes. We report a general, modular, and traceless method for producing asymmetrically modified nucleosomes. We further show that in bivalent nucleosomes, H3K4me3 inhibits the activity of the H3K27-specific lysine methyltransferase (KMT) polycomb repressive complex 2 (PRC2) solely on the same histone tail, whereas H3K27me3 stimulates PRC2 activity across tails, thereby partially overriding the H3K4me3-mediated repressive effect. To maintain bivalent domains in ESCs, PRC2 activity must thus be locally restricted or reversed.

  5. Dynamics and function of compact nucleosome arrays.

    Science.gov (United States)

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

    2009-09-01

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

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

    OpenAIRE

    Zhang, Y; Moqtaderi, Z.; Rattner, BP; Euskirchen, G.; Snyder, M; Kadonaga, JT; Liu, XS; Struhl, K

    2009-01-01

    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 regi...

  7. Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro.

    Science.gov (United States)

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

    2015-12-01

    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.

  8. Probing Nucleosome Stability with a DNA Origami Nanocaliper.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-07-27

    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.

  10. Tension-dependent Free Energies of Nucleosome Unwrapping

    CERN Document Server

    Lequieu, Joshua; Schwartz, David C; de Pablo, Juan J

    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 are crucial f...

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

    Directory of Open Access Journals (Sweden)

    Ho-Ryun Chung

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  13. Human nucleosomes: special role of CG dinucleotides and Alu-nucleosomes

    Directory of Open Access Journals (Sweden)

    Trifonov Edward N

    2011-05-01

    Full Text Available Abstract Background The periodical occurrence of dinucleotides with a period of 10.4 bases now is undeniably a hallmark of nucleosome positioning. Whereas many eukaryotic genomes contain visible and even strong signals for periodic distribution of dinucleotides, the human genome is rather featureless in this respect. The exact sequence features in the human genome that govern the nucleosome positioning remain largely unknown. Results When analyzing the human genome sequence with the positional autocorrelation method, we found that only the dinucleotide CG shows the 10.4 base periodicity, which is indicative of the presence of nucleosomes. There is a high occurrence of CG dinucleotides that are either 31 (10.4 × 3 or 62 (10.4 × 6 base pairs apart from one another - a sequence bias known to be characteristic of Alu-sequences. In a similar analysis with repetitive sequences removed, peaks of repeating CG motifs can be seen at positions 10, 21 and 31, the nearest integers of multiples of 10.4. Conclusions Although the CG dinucleotides are dominant, other elements of the standard nucleosome positioning pattern are present in the human genome as well. The positional autocorrelation analysis of the human genome demonstrates that the CG dinucleotide is, indeed, one visible element of the human nucleosome positioning pattern, which appears both in Alu sequences and in sequences without repeats. The dominant role that CG dinucleotides play in organizing human chromatin is to indicate the involvement of human nucleosomes in tuning the regulation of gene expression and chromatin structure, which is very likely due to cytosine-methylation/-demethylation in CG dinucleotides contained in the human nucleosomes. This is further confirmed by the positions of CG-periodical nucleosomes on Alu sequences. Alu repeats appear as monomers, dimers and trimers, harboring two to six nucleosomes in a run. Considering the exceptional role CG dinucleotides play in the

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

    Science.gov (United States)

    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

  15. Acetylation Mimics Within a Single Nucleosome Alter Local DNA Accessibility In Compacted Nucleosome Arrays

    Science.gov (United States)

    Mishra, Laxmi N.; Pepenella, Sharon; Rogge, Ryan; Hansen, Jeffrey C.; Hayes, Jeffrey J.

    2016-01-01

    The activation of a silent gene locus is thought to involve pioneering transcription factors that initiate changes in the local chromatin structure to increase promoter accessibility and binding of downstream effectors. To better understand the molecular requirements for the first steps of locus activation, we investigated whether acetylation of a single nucleosome is sufficient to alter DNA accessibility within a condensed 25-nucleosome array. We found that acetylation mimics within the histone H4 tail domain increased accessibility of the surrounding linker DNA, with the increased accessibility localized to the immediate vicinity of the modified nucleosome. In contrast, acetylation mimics within the H3 tail had little effect, but were able to synergize with H4 tail acetylation mimics to further increase accessibility. Moreover, replacement of the central nucleosome with a nucleosome free region also resulted in increased local, but not global DNA accessibility. Our results indicate that modification or disruption of only a single target nucleosome results in significant changes in local chromatin architecture and suggest that very localized chromatin modifications imparted by pioneer transcription factors are sufficient to initiate a cascade of events leading to promoter activation. PMID:27708426

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  18. A futile cycle, formed between two ATP-dependant -glutamyl cycle enzymes, -glutamyl cysteine synthetase and 5-oxoprolinase: the cause of cellular ATP depletion in nephrotic cystinosis?

    Indian Academy of Sciences (India)

    Akhilesh Kumar; Anand Kumar Bachhawat

    2010-03-01

    Cystinosis, an inherited disease caused by a defect in the lysosomal cystine transporter (CTNS), is characterized by renal proximal tubular dysfunction. Adenosine triphosphate (ATP) depletion appears to be a key event in the pathophysiology of the disease, even though the manner in which ATP depletion occurs is still a puzzle. We present a model that explains how a futile cycle that is generated between two ATP-utilizing enzymes of the -glutamyl cycle leads to ATP depletion. The enzyme -glutamyl cysteine synthetase (-GCS), in the absence of cysteine, forms 5-oxoproline (instead of the normal substrate, -glutamyl cysteine) and the 5-oxoproline is converted into glutamate by the ATP-dependant enzyme, 5-oxoprolinase. Thus, in cysteine-limiting conditions, glutamate is cycled back into glutamate via 5-oxoproline at the cost of two ATP molecules without production of glutathione and is the cause of the decreased levels of glutathione synthesis, as well as the ATP depletion observed in these cells. The model is also compatible with the differences seen in the human patients and the mouse model of cystinosis, where renal failure is not observed.

  19. Conserved substitution patterns around nucleosome footprints in eukaryotes and Archaea derive from frequent nucleosome repositioning through evolution.

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    Tobias Warnecke

    Full Text Available Nucleosomes, the basic repeat units of eukaryotic chromatin, have been suggested to influence the evolution of eukaryotic genomes, both by altering the propensity of DNA to mutate and by selection acting to maintain or exclude nucleosomes in particular locations. Contrary to the popular idea that nucleosomes are unique to eukaryotes, histone proteins have also been discovered in some archaeal genomes. Archaeal nucleosomes, however, are quite unlike their eukaryotic counterparts in many respects, including their assembly into tetramers (rather than octamers from histone proteins that lack N- and C-terminal tails. Here, we show that despite these fundamental differences the association between nucleosome footprints and sequence evolution is strikingly conserved between humans and the model archaeon Haloferax volcanii. In light of this finding we examine whether selection or mutation can explain concordant substitution patterns in the two kingdoms. Unexpectedly, we find that neither the mutation nor the selection model are sufficient to explain the observed association between nucleosomes and sequence divergence. Instead, we demonstrate that nucleosome-associated substitution patterns are more consistent with a third model where sequence divergence results in frequent repositioning of nucleosomes during evolution. Indeed, we show that nucleosome repositioning is both necessary and largely sufficient to explain the association between current nucleosome positions and biased substitution patterns. This finding highlights the importance of considering the direction of causality between genetic and epigenetic change.

  20. Universal full-length nucleosome mapping sequence probe.

    Science.gov (United States)

    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.

  1. High nucleosome occupancy is encoded at human regulatory sequences.

    Directory of Open Access Journals (Sweden)

    Desiree Tillo

    Full Text Available Active eukaryotic regulatory sites are characterized by open chromatin, and yeast promoters and transcription factor binding sites (TFBSs typically have low intrinsic nucleosome occupancy. Here, we show that in contrast to yeast, DNA at human promoters, enhancers, and TFBSs generally encodes high intrinsic nucleosome occupancy. In most cases we examined, these elements also have high experimentally measured nucleosome occupancy in vivo. These regions typically have high G+C content, which correlates positively with intrinsic nucleosome occupancy, and are depleted for nucleosome-excluding poly-A sequences. We propose that high nucleosome preference is directly encoded at regulatory sequences in the human genome to restrict access to regulatory information that will ultimately be utilized in only a subset of differentiated cells.

  2. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function.

    Science.gov (United States)

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

    2016-04-28

    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.

  3. Prediction of nucleosome positioning based on transcription factor binding sites.

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    Xianfu Yi

    Full Text Available BACKGROUND: The DNA of all eukaryotic organisms is packaged into nucleosomes, the basic repeating units of chromatin. The nucleosome consists of a histone octamer around which a DNA core is wrapped and the linker histone H1, which is associated with linker DNA. By altering the accessibility of DNA sequences, the nucleosome has profound effects on all DNA-dependent processes. Understanding the factors that influence nucleosome positioning is of great importance for the study of genomic control mechanisms. Transcription factors (TFs have been suggested to play a role in nucleosome positioning in vivo. PRINCIPAL FINDINGS: Here, the minimum redundancy maximum relevance (mRMR feature selection algorithm, the nearest neighbor algorithm (NNA, and the incremental feature selection (IFS method were used to identify the most important TFs that either favor or inhibit nucleosome positioning by analyzing the numbers of transcription factor binding sites (TFBSs in 53,021 nucleosomal DNA sequences and 50,299 linker DNA sequences. A total of nine important families of TFs were extracted from 35 families, and the overall prediction accuracy was 87.4% as evaluated by the jackknife cross-validation test. CONCLUSIONS: Our results are consistent with the notion that TFs are more likely to bind linker DNA sequences than the sequences in the nucleosomes. In addition, our results imply that there may be some TFs that are important for nucleosome positioning but that play an insignificant role in discriminating nucleosome-forming DNA sequences from nucleosome-inhibiting DNA sequences. The hypothesis that TFs play a role in nucleosome positioning is, thus, confirmed by the results of this study.

  4. Controls of nucleosome positioning in the human genome.

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    Daniel J Gaffney

    Full Text Available Nucleosomes are important for gene regulation because their arrangement on the genome can control which proteins bind to DNA. Currently, few human nucleosomes are thought to be consistently positioned across cells; however, this has been difficult to assess due to the limited resolution of existing data. We performed paired-end sequencing of micrococcal nuclease-digested chromatin (MNase-seq from seven lymphoblastoid cell lines and mapped over 3.6 billion MNase-seq fragments to the human genome to create the highest-resolution map of nucleosome occupancy to date in a human cell type. In contrast to previous results, we find that most nucleosomes have more consistent positioning than expected by chance and a substantial fraction (8.7% of nucleosomes have moderate to strong positioning. In aggregate, nucleosome sequences have 10 bp periodic patterns in dinucleotide frequency and DNase I sensitivity; and, across cells, nucleosomes frequently have translational offsets that are multiples of 10 bp. We estimate that almost half of the genome contains regularly spaced arrays of nucleosomes, which are enriched in active chromatin domains. Single nucleotide polymorphisms that reduce DNase I sensitivity can disrupt the phasing of nucleosome arrays, which indicates that they often result from positioning against a barrier formed by other proteins. However, nucleosome arrays can also be created by DNA sequence alone. The most striking example is an array of over 400 nucleosomes on chromosome 12 that is created by tandem repetition of sequences with strong positioning properties. In summary, a large fraction of nucleosomes are consistently positioned--in some regions because they adopt favored sequence positions, and in other regions because they are forced into specific arrangements by chromatin remodeling or DNA binding proteins.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  8. Nucleosome conformational flexibility in experiments with single chromatin fibers

    Directory of Open Access Journals (Sweden)

    Sivolob A. V.

    2010-09-01

    Full Text Available Studies on the chromatin nucleosome organization play an ever increasing role in our comprehension of mechanisms of the gene activity regulation. This minireview describes the results on the nucleosome conformational flexibility, which were obtained using magnetic tweezers to apply torsion to oligonucleosome fibers reconstituted on single DNA molecules. Such an approach revealed a new structural form of the nucleosome, the reversome, in which DNA is wrapped in a right-handed superhelix around a distorted histone octamer. Molecular mechanisms of the nucleosome structural flexibility and its biological relevance are discussed.

  9. Predicting nucleosome positioning using a duration Hidden Markov Model

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    Widom Jonathan

    2010-06-01

    Full Text Available Abstract Background The nucleosome is the fundamental packing unit of DNAs in eukaryotic cells. Its detailed positioning on the genome is closely related to chromosome functions. Increasing evidence has shown that genomic DNA sequence itself is highly predictive of nucleosome positioning genome-wide. Therefore a fast software tool for predicting nucleosome positioning can help understanding how a genome's nucleosome organization may facilitate genome function. Results We present a duration Hidden Markov model for nucleosome positioning prediction by explicitly modeling the linker DNA length. The nucleosome and linker models trained from yeast data are re-scaled when making predictions for other species to adjust for differences in base composition. A software tool named NuPoP is developed in three formats for free download. Conclusions Simulation studies show that modeling the linker length distribution and utilizing a base composition re-scaling method both improve the prediction of nucleosome positioning regarding sensitivity and false discovery rate. NuPoP provides a user-friendly software tool for predicting the nucleosome occupancy and the most probable nucleosome positioning map for genomic sequences of any size. When compared with two existing methods, NuPoP shows improved performance in sensitivity.

  10. Assembly of Drosophila centromeric nucleosomes requires CID dimerization.

    Science.gov (United States)

    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.

  11. Nucleosome DNA sequence structure of isochores

    Directory of Open Access Journals (Sweden)

    Trifonov Edward N

    2011-04-01

    Full Text Available Abstract Background Significant differences in G+C content between different isochore types suggest that the nucleosome positioning patterns in DNA of the isochores should be different as well. Results Extraction of the patterns from the isochore DNA sequences by Shannon N-gram extension reveals that while the general motif YRRRRRYYYYYR is characteristic for all isochore types, the dominant positioning patterns of the isochores vary between TAAAAATTTTTA and CGGGGGCCCCCG due to the large differences in G+C composition. This is observed in human, mouse and chicken isochores, demonstrating that the variations of the positioning patterns are largely G+C dependent rather than species-specific. The species-specificity of nucleosome positioning patterns is revealed by dinucleotide periodicity analyses in isochore sequences. While human sequences are showing CG periodicity, chicken isochores display AG (CT periodicity. Mouse isochores show very weak CG periodicity only. Conclusions Nucleosome positioning pattern as revealed by Shannon N-gram extension is strongly dependent on G+C content and different in different isochores. Species-specificity of the pattern is subtle. It is reflected in the choice of preferentially periodical dinucleotides.

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

    Science.gov (United States)

    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.

  13. Uncovering the forces between nucleosomes using DNA origami.

    Science.gov (United States)

    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.

  14. Training-free atomistic prediction of nucleosome occupancy.

    Science.gov (United States)

    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.

  15. The split personality of CENP-A nucleosomes.

    Science.gov (United States)

    Westhorpe, Frederick G; Straight, Aaron F

    2012-07-20

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

  16. Tetrameric structure of centromeric nucleosomes in interphase Drosophila cells.

    Directory of Open Access Journals (Sweden)

    Yamini Dalal

    2007-08-01

    Full Text Available Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3. However, the mechanistic basis for centromere maintenance is unknown. We investigated biochemical properties of CenH3 nucleosomes from Drosophila melanogaster cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical "beads-on-a-string" appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity.

  17. Uncovering the forces between nucleosomes using DNA origami

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2015-12-01

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  2. Touch, act and go: landing and operating on nucleosomes.

    Science.gov (United States)

    Speranzini, Valentina; Pilotto, Simona; Sixma, Titia K; Mattevi, Andrea

    2016-02-15

    Chromatin-associated enzymes are responsible for the installation, removal and reading of precise post-translation modifications on DNA and histone proteins. They are specifically recruited to the target gene by associated factors, and as a result of their activity, they contribute in modulating cell identity and differentiation. Structural and biophysical approaches are broadening our knowledge on these processes, demonstrating that DNA, histone tails and histone surfaces can each function as distinct yet functionally interconnected anchoring points promoting nucleosome binding and modification. The mechanisms underlying nucleosome recognition have been described for many histone modifiers and related readers. Here, we review the recent literature on the structural organization of these nucleosome-associated proteins, the binding properties that drive nucleosome modification and the methodological advances in their analysis. The overarching conclusion is that besides acting on the same substrate (the nucleosome), each system functions through characteristic modes of action, which bring about specific biological functions in gene expression regulation.

  3. Functioning of the mitochondrial ATP-dependent potassium channel in rats varying in their resistance to hypoxia. Involvement of the channel in the process of animal's adaptation to hypoxia.

    Science.gov (United States)

    Mironova, Galina D; Shigaeva, Maria I; Gritsenko, Elena N; Murzaeva, Svetlana V; Gorbacheva, Olga S; Germanova, Elena L; Lukyanova, Ludmila D

    2010-12-01

    The mechanism of tissue protection from ischemic damage by activation of the mitochondrial ATP-dependent K(+) channel (mitoK(ATP)) remains unexplored. In this work, we have measured, using various approaches, the ATP-dependent mitochondrial K(+) transport in rats that differed in their resistance to hypoxia. The transport was found to be faster in the hypoxia-resistant rats as compared to that in the hypoxia-sensitive animals. Adaptation of animals to the intermittent normobaric hypoxia increased the rate of transport. At the same time, the intramitochondrial concentration of K(+) in the hypoxia-sensitive rats was higher than that in the resistant and adapted animals. This indicates that adaptation to hypoxia stimulates not only the influx of potassium into mitochondria, but also K(+)/H(+) exchange. When mitoK(ATP) was blocked, the rate of the mitochondrial H(2)O(2) production was found to be significantly higher in the hypoxia-resistant rats than that in the hypoxia-sensitive animals. The natural flavonoid-containing adaptogen Extralife, which has an evident antihypoxic effect, increased the rate of the mitochondrial ATP-dependent K(+) transport in vitro and increased the in vivo tolerance of hypoxia-sensitive rats to acute hypoxia 5-fold. The involvement of the mitochondrial K(+) transport in the mechanism of cell adaptation to hypoxia is discussed.

  4. Ionizing Radiation Induces HMGB1 Cytoplasmic Translocation and Extracellular Release

    Institute of Scientific and Technical Information of China (English)

    Lili Wang; Li He; Guoqiang Bao; Xin He; Saijun Fan; Haichao Wang

    2016-01-01

    Objective A nucleosomal protein,HMGBI,can be secreted by activated immune cells or passively released by dying cells,thereby amplifying rigorous inflammatory responses.In this study we aimed to test the possibility that radiation similarly induces cytoplasmic HMGB1 translocation and release.Methods Human skin fibroblast (GM0639) and bronchial epithelial (16HBE) cells and rats were exposed to X-ray radiation,and HMGB1 translocation and release were then assessed by immunocytochemistry and immunoassay,respectively.Results At a wide dose range(4.0-12.0 Gy),X-ray radiation induced a dramatic cytoplasmic HMGB1 translocation,and triggered a time-and dose-dependent HMGB1 release both in vitro and in vivo.The radiation-mediated HMGB1 release was also associated with noticeable chromosomal DNA damage and loss of cell viability.Conclusions Radiation induces HMGB1 cytoplasmic translocation and extracellular release through active secretion and passive leakage processes.

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

    Science.gov (United States)

    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.

  6. Nucleosome Stability Distinguishes Two Different Promoter Types at All Protein-Coding Genes in Yeast.

    Science.gov (United States)

    Kubik, Slawomir; Bruzzone, Maria Jessica; Jacquet, Philippe; Falcone, Jean-Luc; Rougemont, Jacques; Shore, David

    2015-11-01

    Previous studies indicate that eukaryotic promoters display a stereotypical chromatin landscape characterized by a well-positioned +1 nucleosome near the transcription start site and an upstream -1 nucleosome that together demarcate a nucleosome-free (or -depleted) region. Here we present evidence that there are two distinct types of promoters distinguished by the resistance of the -1 nucleosome to micrococcal nuclease digestion. These different architectures are characterized by two sequence motifs that are broadly deployed at one set of promoters where a nuclease-sensitive ("fragile") nucleosome forms, but concentrated in a narrower, nucleosome-free region at all other promoters. The RSC nucleosome remodeler acts through the motifs to establish stable +1 and -1 nucleosome positions, while binding of a small set of general regulatory (pioneer) factors at fragile nucleosome promoters plays a key role in their destabilization. We propose that the fragile nucleosome promoter architecture is adapted for regulation of highly expressed, growth-related genes.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  10. Dynamics of Nucleosome Positioning Maturation following Genomic Replication

    Directory of Open Access Journals (Sweden)

    Pauline Vasseur

    2016-09-01

    Full Text Available Chromatin is thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture occurring during genomic replication. Here, we measure a key aspect of chromatin structure dynamics during replication—how rapidly nucleosome positions are established on the newly replicated daughter genomes. By isolating newly synthesized DNA marked with 5-ethynyl-2′-deoxyuridine (EdU, we characterize nucleosome positions on both daughter genomes of S. cerevisiae during chromatin maturation. We find that nucleosomes rapidly adopt their mid-log positions at highly transcribed genes, which is consistent with a role for transcription in positioning nucleosomes in vivo. Additionally, experiments in hir1Δ mutants reveal a role for HIR in nucleosome spacing. We also characterized nucleosome positions on the leading and lagging strands, uncovering differences in chromatin maturation dynamics at hundreds of genes. Our data define the maturation dynamics of newly replicated chromatin and support a role for transcription in sculpting the chromatin template.

  11. Dynamics of forced biopolymer translocation

    CERN Document Server

    Lehtola, V V; Kaski, K; 10.1209/0295-5075/85/58006

    2009-01-01

    We present results from our simulations of biopolymer translocation in a solvent which explain the main experimental findings. The forced translocation can be described by simple force balance arguments for the relevant range of pore potentials in experiments and biological systems. Scaling of translocation time with polymer length varies with pore force and friction. Hydrodynamics affects this scaling and significantly reduces translocation times.

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

    Bohr, Jakob; Olsen, Kasper

    2012-01-01

    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...

  14. Recycling of protein subunits during DNA translocation and cleavage by Type I restriction-modification enzymes.

    Science.gov (United States)

    Simons, Michelle; Szczelkun, Mark D

    2011-09-01

    The Type I restriction-modification enzymes comprise three protein subunits; HsdS and HsdM that form a methyltransferase (MTase) and HsdR that associates with the MTase and catalyses Adenosine-5'-triphosphate (ATP)-dependent DNA translocation and cleavage. Here, we examine whether the MTase and HsdR components can 'turnover' in vitro, i.e. whether they can catalyse translocation and cleavage events on one DNA molecule, dissociate and then re-bind a second DNA molecule. Translocation termination by both EcoKI and EcoR124I leads to HsdR dissociation from linear DNA but not from circular DNA. Following DNA cleavage, the HsdR subunits appear unable to dissociate even though the DNA is linear, suggesting a tight interaction with the cleaved product. The MTases of EcoKI and EcoAI can dissociate from DNA following either translocation or cleavage and can initiate reactions on new DNA molecules as long as free HsdR molecules are available. In contrast, the MTase of EcoR124I does not turnover and additional cleavage of circular DNA is not observed by inclusion of RecBCD, a helicase-nuclease that degrades the linear DNA product resulting from Type I cleavage. Roles for Type I restriction endonuclease subunit dynamics in restriction alleviation in the cell are discussed.

  15. Asymmetric unwrapping of nucleosomes under tension directed by DNA local flexibility.

    Science.gov (United States)

    Ngo, Thuy T M; Zhang, Qiucen; Zhou, Ruobo; Yodh, Jaya G; Ha, Taekjip

    2015-03-12

    Dynamics of the nucleosome and exposure of nucleosomal DNA play key roles in many nuclear processes, but local dynamics of the nucleosome and its modulation by DNA sequence are poorly understood. Using single-molecule assays, we observed that the nucleosome can unwrap asymmetrically and directionally under force. The relative DNA flexibility of the inner quarters of nucleosomal DNA controls the unwrapping direction such that the nucleosome unwraps from the stiffer side. If the DNA flexibility is similar on two sides, it stochastically unwraps from either side. The two ends of the nucleosome are orchestrated such that the opening of one end helps to stabilize the other end, providing a mechanism to amplify even small differences in flexibility to a large asymmetry in nucleosome stability. Our discovery of DNA flexibility as a critical factor for nucleosome dynamics and mechanical stability suggests a novel mechanism of gene regulation by DNA sequence and modifications.

  16. DNA Shape Dominates Sequence Affinity in Nucleosome Formation

    Science.gov (United States)

    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.

  17. Local Nucleosome Dynamics Facilitate Chromatin Accessibility in Living Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Saera Hihara

    2012-12-01

    Full Text Available Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (∼50 nm movement/30 ms, which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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)

  1. Single-nucleosome mapping of histone modifications in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Chih Long Liu

    2005-10-01

    Full Text Available Covalent modification of histone proteins plays a role in virtually every process on eukaryotic DNA, from transcription to DNA repair. Many different residues can be covalently modified, and it has been suggested that these modifications occur in a great number of independent, meaningful combinations. Published low-resolution microarray studies on the combinatorial complexity of histone modification patterns suffer from confounding effects caused by the averaging of modification levels over multiple nucleosomes. To overcome this problem, we used a high-resolution tiled microarray with single-nucleosome resolution to investigate the occurrence of combinations of 12 histone modifications on thousands of nucleosomes in actively growing S. cerevisiae. We found that histone modifications do not occur independently; there are roughly two groups of co-occurring modifications. One group of lysine acetylations shows a sharply defined domain of two hypo-acetylated nucleosomes, adjacent to the transcriptional start site, whose occurrence does not correlate with transcription levels. The other group consists of modifications occurring in gradients through the coding regions of genes in a pattern associated with transcription. We found no evidence for a deterministic code of many discrete states, but instead we saw blended, continuous patterns that distinguish nucleosomes at one location (e.g., promoter nucleosomes from those at another location (e.g., over the 3' ends of coding regions. These results are consistent with the idea of a simple, redundant histone code, in which multiple modifications share the same role.

  2. Problem-elephant translocation: translocating the problem and the elephant?

    Directory of Open Access Journals (Sweden)

    Prithiviraj Fernando

    Full Text Available 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.

  3. Problem-elephant translocation: translocating the problem and the elephant?

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Imtiaz Nisar Lone

    Full Text Available 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.

  5. Multiple distinct stimuli increase measured nucleosome occupancy around human promoters.

    Directory of Open Access Journals (Sweden)

    Chuong D Pham

    Full Text Available Nucleosomes can block access to transcription factors. Thus the precise localization of nucleosomes relative to transcription start sites and other factor binding sites is expected to be a critical component of transcriptional regulation. Recently developed microarray approaches have allowed the rapid mapping of nucleosome positions over hundreds of kilobases (kb of human genomic DNA, although these approaches have not yet been widely used to measure chromatin changes associated with changes in transcription. Here, we use custom tiling microarrays to reveal changes in nucleosome positions and abundance that occur when hormone-bound glucocorticoid receptor (GR binds to sites near target gene promoters in human osteosarcoma cells. The most striking change is an increase in measured nucleosome occupancy at sites spanning ∼1 kb upstream and downstream of transcription start sites, which occurs one hour after addition of hormone, but is lost at 4 hours. Unexpectedly, this increase was seen both on GR-regulated and GR-non-regulated genes. In addition, the human SWI/SNF chromatin remodeling factor (a GR co-activator was found to be important for increased occupancy upon hormone treatment and also for low nucleosome occupancy without hormone. Most surprisingly, similar increases in nucleosome occupancy were also seen on both regulated and non-regulated promoters during differentiation of human myeloid leukemia cells and upon activation of human CD4+ T-cells. These results indicate that dramatic changes in chromatin structure over ∼2 kb of human promoters may occur genomewide and in response to a variety of stimuli, and suggest novel models for transcriptional regulation.

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

    Science.gov (United States)

    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.

  7. Nucleosome Assembly Dynamics Involve Spontaneous Fluctuations in the Handedness of Tetrasomes

    NARCIS (Netherlands)

    Vlijm, R.; Lee, M.; Lipfert, J.; Lusser, A.; Dekker, C.; Dekker, N.H.

    2015-01-01

    DNA wrapping around histone octamers generates nucleosomes, the basic compaction unit of eukaryotic chromatin. Nucleosome stability is carefully tuned to maintain DNA accessibility in transcription, replication, and repair. Using freely orbiting magnetic tweezers, which measure the twist and length

  8. The RSC chromatin remodelling enzyme has a unique role in directing the accurate positioning of nucleosomes.

    Science.gov (United States)

    Wippo, Christian J; Israel, Lars; Watanabe, Shinya; Hochheimer, Andreas; Peterson, Craig L; Korber, Philipp

    2011-04-01

    Nucleosomes impede access to DNA. Therefore, nucleosome positioning is fundamental to genome regulation. Nevertheless, the molecular nucleosome positioning mechanisms are poorly understood. This is partly because in vitro reconstitution of in vivo-like nucleosome positions from purified components is mostly lacking, barring biochemical studies. Using a yeast extract in vitro reconstitution system that generates in vivo-like nucleosome patterns at S. cerevisiae loci, we find that the RSC chromatin remodelling enzyme is necessary for nucleosome positioning. This was previously suggested by genome-wide in vivo studies and is confirmed here in vivo for individual loci. Beyond the limitations of conditional mutants, we show biochemically that RSC functions directly, can be sufficient, but mostly relies on other factors to properly position nucleosomes. Strikingly, RSC could not be replaced by either the closely related SWI/SNF or the Isw2 remodelling enzyme. Thus, we pinpoint that nucleosome positioning specifically depends on the unique properties of the RSC complex.

  9. Statistical mechanics of chromatin: Inferring free energies of nucleosome formation from high-throughput data sets

    Science.gov (United States)

    Morozov, Alexandre

    2009-03-01

    Formation of nucleosome core particles is a first step towards packaging genomic DNA into chromosomes in living cells. Nucleosomes are formed by wrapping 147 base pairs of DNA around a spool of eight histone proteins. It is reasonable to assume that formation of single nucleosomes in vitro is determined by DNA sequence alone: it costs less elastic energy to wrap a flexible DNA polymer around the histone octamer, and more if the polymer is rigid. However, it is unclear to which extent this effect is important in living cells. Cells have evolved chromatin remodeling enzymes that expend ATP to actively reposition nucleosomes. In addition, nucleosome positioning on long DNA sequences is affected by steric exclusion - many nucleosomes have to form simultaneously without overlap. Currently available bioinformatics methods for predicting nucleosome positions are trained on in vivo data sets and are thus unable to distinguish between extrinsic and intrinsic nucleosome positioning signals. In order to see the relative importance of such signals for nucleosome positioning in vivo, we have developed a model based on a large collection of DNA sequences from nucleosomes reconstituted in vitro by salt dialysis. We have used these data to infer the free energy of nucleosome formation at each position along the genome. The method uses an exact result from the statistical mechanics of classical 1D fluids to infer the free energy landscape from nucleosome occupancy. We will discuss the degree to which in vitro nucleosome occupancy profiles are predictive of in vivo nucleosome positions, and will estimate how many nucleosomes are sequence-specific and how many are positioned purely by steric exclusion. Our approach to nucleosome energetics should be applicable across multiple organisms and genomic regions.

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

    Science.gov (United States)

    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.

  11. Lysine Acetylation Facilitates Spontaneous DNA Dynamics in the Nucleosome.

    Science.gov (United States)

    Kim, Jongseong; Lee, Jaehyoun; Lee, Tae-Hee

    2015-12-01

    The nucleosome, comprising a histone protein core wrapped around by DNA, is the fundamental packing unit of DNA in cells. Lysine acetylation at the histone core elevates DNA accessibility in the nucleosome, the mechanism of which remains largely unknown. By employing our recently developed hybrid single molecule approach, here we report how the structural dynamics of DNA in the nucleosome is altered upon acetylation at histone H3 lysine 56 (H3K56) that is critical for elevated DNA accessibility. Our results indicate that H3K56 acetylation facilitates the structural dynamics of the DNA at the nucleosome termini that spontaneously and repeatedly open and close on a ms time scale. The results support a molecular mechanism of histone acetylation in catalyzing DNA unpacking whose efficiency is ultimately limited by the spontaneous DNA dynamics at the nucleosome temini. This study provides the first and unique experimental evidence revealing a role of protein chemical modification in directly regulating the kinetic stability of the DNA packing unit.

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

    Science.gov (United States)

    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.

  13. Reactivity in ELISA with DNA-loaded nucleosomes in patients with proliferative lupus nephritis

    NARCIS (Netherlands)

    Dieker, J.W.; Schlumberger, W.; McHugh, N.; Hamann, P.; Vlag, J. van der; Berden, J.H.M.

    2015-01-01

    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. Fi

  14. Specificity of monoclonal anti-nucleosome auto-antibodies derived from lupus mice

    NARCIS (Netherlands)

    Kramers, K; Stemmer, C; Monestier, M; vanBruggen, MCJ; RijkeSchilder, TPM; Hylkema, MN; Smeenk, RJT; Muller, S; Berden, JHM

    1996-01-01

    Recently, anti-nucleosome antibodies, which do not bind to DNA or to individual histones, have been identified in longitudinal studies in lupus mice. These anti-nucleosome antibodies occur early in spontaneous SLE and are formed prior to other anti-nuclear specificities. However, nucleosomal epitope

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

    Science.gov (United States)

    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.

  16. Distinct modes of regulation by chromatin encoded through nucleosome positioning signals.

    Directory of Open Access Journals (Sweden)

    Yair Field

    2008-11-01

    Full Text Available The detailed positions of nucleosomes profoundly impact gene regulation and are partly encoded by the genomic DNA sequence. However, less is known about the functional consequences of this encoding. Here, we address this question using a genome-wide map of approximately 380,000 yeast nucleosomes that we sequenced in their entirety. Utilizing the high resolution of our map, we refine our understanding of how nucleosome organizations are encoded by the DNA sequence and demonstrate that the genomic sequence is highly predictive of the in vivo nucleosome organization, even across new nucleosome-bound sequences that we isolated from fly and human. We find that Poly(dA:dT tracts are an important component of these nucleosome positioning signals and that their nucleosome-disfavoring action results in large nucleosome depletion over them and over their flanking regions and enhances the accessibility of transcription factors to their cognate sites. Our results suggest that the yeast genome may utilize these nucleosome positioning signals to regulate gene expression with different transcriptional noise and activation kinetics and DNA replication with different origin efficiency. These distinct functions may be achieved by encoding both relatively closed (nucleosome-covered chromatin organizations over some factor binding sites, where factors must compete with nucleosomes for DNA access, and relatively open (nucleosome-depleted organizations over other factor sites, where factors bind without competition.

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

    Science.gov (United States)

    Weng, Liwei; Greenberg, Marc M

    2015-09-01

    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.

  18. Naturally occuring nucleosome positioning signals in human exons and introns

    DEFF Research Database (Denmark)

    Baldi, Pierre; Brunak, Søren; Chauvin, Yves;

    1996-01-01

    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...... 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...... 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 nucleosome. Analysis of mRNA sequences encoding proteins with known tertiary structure exclude the possibility that the pattern is a consequence...

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

    Energy Technology Data Exchange (ETDEWEB)

    Okada, Masashi; Hozumi, Yasukazu [Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585 (Japan); Ichimura, Tohru [Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Hachioji 192-0397 (Japan); Tanaka, Toshiaki; Hasegawa, Hiroshi; Yamamoto, Masakazu; Takahashi, Nobuya [Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585 (Japan); Iseki, Ken [Department of Emergency and Critical Care Medicine, Yamagata University School of Medicine, Yamagata 990-9585 (Japan); Yagisawa, Hitoshi [Laboratory of Biological Signaling, Graduate School of Life Science, University of Hyogo, Hyogo 678-1297 (Japan); Shinkawa, Takashi; Isobe, Toshiaki [Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Hachioji 192-0397 (Japan); Goto, Kaoru, E-mail: kgoto@med.id.yamagata-u.ac.jp [Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585 (Japan)

    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.

  20. NAP1-Assisted Nucleosome Assembly on DNA Measured in Real Time by Single-Molecule Magnetic Tweezers

    NARCIS (Netherlands)

    Vlijm, R.; Smitshuijzen, J.S.J.; Lusser, A.; Dekker, C.

    2012-01-01

    While many proteins are involved in the assembly and (re)positioning of nucleosomes, the dynamics of protein-assisted nucleosome formation are not well understood. We study NAP1 (nucleosome assembly protein 1) assisted nucleosome formation at the single-molecule level using magnetic tweezers. This m

  1. A map of nucleosome positions in yeast at base-pair resolution.

    Science.gov (United States)

    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.

  2. Coupling between Histone Conformations and DNA Geometry in Nucleosomes on a Microsecond Timescale: Atomistic Insights into Nucleosome Functions.

    Science.gov (United States)

    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.

  3. The dynamics of individual nucleosomes controls the chromatin condensation pathway: direct AFM visualization of variant chromatin

    CERN Document Server

    Montel, Fabien; Castelnovo, Martin; Bednar, Jan; Dimitrov, Stefan; Angelov, Dimitar; Faivre-Moskalenko, Cendrine

    2009-01-01

    Chromatin organization and dynamics is studied in this work at scales ranging from single nucleosome to nucleosomal array by using a unique combination of biochemical assays, single molecule imaging technique and numerical modeling. We demonstrate that a subtle modification in the nucleosome structure induced by the histone variant H2A.Bbd drastically modifies the higher order organization of the nucleosomal arrays. Importantly, as directly visualized by AFM, conventional H2A nucleosomal arrays exhibit specific local organization, in contrast to H2A.Bbd arrays, which show ?beads on a string? structure. The combination of systematic image analysis and theoretical modeling allows a quantitative description relating the observed gross structural changes of the arrays to their local organization. Our results strongly suggest that higher-order organization of H1-free nucleosomal arrays is mainly determined by the fluctuation properties of individual nucleosomes. Moreover, numerical simulations suggest the existenc...

  4. Large-scale ATP-independent nucleosome unfolding by a histone chaperone.

    Science.gov (United States)

    Valieva, Maria E; Armeev, Grigoriy A; Kudryashova, Kseniya S; Gerasimova, Nadezhda S; Shaytan, Alexey K; Kulaeva, Olga I; McCullough, Laura L; Formosa, Tim; Georgiev, Pavel G; Kirpichnikov, Mikhail P; Studitsky, Vasily M; Feofanov, Alexey V

    2016-12-01

    DNA accessibility to regulatory proteins is substantially influenced by nucleosome structure and dynamics. The facilitates chromatin transcription (FACT) complex increases the accessibility of nucleosomal DNA, but the mechanism and extent of its nucleosome reorganization activity are unknown. Here we determined the effects of FACT from the yeast Saccharomyces cerevisiae on single nucleosomes by using single-particle Förster resonance energy transfer (spFRET) microscopy. FACT binding results in dramatic ATP-independent, symmetrical and reversible DNA uncoiling that affects at least 70% of the DNA within a nucleosome, occurs without apparent loss of histones and proceeds via an 'all-or-none' mechanism. A mutated version of FACT is defective in uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus, FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this activity is an important function of FACT in vivo.

  5. Nucleosome Presence at AML-1 Binding Sites Inversely Correlates with Ly49 Expression: Revelations from an Informatics Analysis of Nucleosomes and Immune Cell Transcription Factors.

    Science.gov (United States)

    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.

  6. Enhancement of the nucleosomal pattern in sequences of lower complexity

    DEFF Research Database (Denmark)

    Bolshoy, Alexander; Shapiro, Kevin; Trifonov, Edward N.;

    1997-01-01

    in those of higher linguistic complexity. The nucleosome DNA positioning pattern is one of the weakest (highly degenerate) sequence patterns. It has been extracted recently by specially designed multiple alignment procedures. We applied the most sensitive of these procedures to nearly equal subsets...

  7. Dynamics of nucleosome assembly and effects of DNA methylation.

    Science.gov (United States)

    Lee, Ju Yeon; Lee, Jaehyoun; Yue, Hongjun; Lee, Tae-Hee

    2015-02-13

    The nucleosome is the fundamental packing unit of the eukaryotic genome, and CpG methylation is an epigenetic modification associated with gene repression and silencing. We investigated nucleosome assembly mediated by histone chaperone Nap1 and the effects of CpG methylation based on three-color single molecule FRET measurements, which enabled direct monitoring of histone binding in the context of DNA wrapping. According to our observation, (H3-H4)2 tetramer incorporation must precede H2A-H2B dimer binding, which is independent of DNA termini wrapping. Upon CpG methylation, (H3-H4)2 tetramer incorporation and DNA termini wrapping are facilitated, whereas proper incorporation of H2A-H2B dimers is inhibited. We suggest that these changes are due to rigidified DNA and increased random binding of histones to DNA. According to the results, CpG methylation expedites nucleosome assembly in the presence of abundant DNA and histones, which may help facilitate gene packaging in chromatin. The results also indicate that the slowest steps in nucleosome assembly are DNA termini wrapping and tetramer positioning, both of which are affected heavily by changes in the physical properties of DNA.

  8. Twin-Arginine Protein Translocation

    NARCIS (Netherlands)

    Goosens, Vivianne J; van Dijl, Jan Maarten

    2016-01-01

    Twin-arginine protein translocation systems (Tat) translocate fully folded and co-factor-containing proteins across biological membranes. In this review, we focus on the Tat pathway of Gram-positive bacteria. The minimal Tat pathway is composed of two components, namely a TatA and TatC pair, which a

  9. Nucleosomal DNA binding drives the recognition of H3K36-methylated nucleosomes by the PSIP1-PWWP domain

    NARCIS (Netherlands)

    van Nuland, R.; van Schaik, F.M.; Simonis, M.; van Heesch, S.; Cuppen, E.; Boelens, R.; Timmers, H.M.; van Ingen, H.

    2013-01-01

    BACKGROUND: Recognition of histone modifications by specialized protein domains is a key step in the regulation of DNA-mediated processes like gene transcription. The structural basis of these interactions is usually studied using histone peptide models, neglecting the nucleosomal context. Here, we

  10. Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions.

    Science.gov (United States)

    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.

  11. High mobility group protein 1: A collaborator in nucleosome dynamics and estrogen-responsive gene expression

    Institute of Scientific and Technical Information of China (English)

    William M Scovell

    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 siR NA 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.

  12. An in vitro-identified high-affinity nucleosome-positioning signal is capable of transiently positioning a nucleosome in vivo

    Directory of Open Access Journals (Sweden)

    Gracey Lia E

    2010-07-01

    Full Text Available Abstract Background The physiological function of eukaryotic DNA occurs in the context of nucleosomal arrays that can expose or obscure defined segments of the genome. Certain DNA sequences are capable of strongly positioning a nucleosome in vitro, suggesting the possibility that favorable intrinsic signals might reproducibly structure chromatin segments. As high-throughput sequencing analyses of nucleosome coverage in vitro and in vivo have become possible, a vigorous debate has arisen over the degree to which intrinsic DNA:nucleosome affinities orchestrate the in vivo positions of nucleosomes, thereby controlling physical accessibility of specific sequences in DNA. Results We describe here the in vivo consequences of placing a synthetic high-affinity nucleosome-positioning signal, the 601 sequence, into a DNA plasmid vector in mice. Strikingly, the 601 sequence was sufficient to position nucleosomes during an early phase after introduction of the DNA into the mice (when the plasmid vector transgene was active. This positioning capability was transient, with a loss of strong positioning at a later time point when the transgenes had become silent. Conclusions These results demonstrate an ability of DNA sequences selected solely for nucleosome affinity to organize chromatin in vivo, and the ability of other mechanisms to overcome these interactions in a dynamic nuclear environment.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  15. Crystal structure of the nucleosome containing ultraviolet light-induced cyclobutane pyrimidine dimer.

    Science.gov (United States)

    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. Genomic Comparison of Translocating and Non-Translocating Escherichia coli.

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    Nathan L Bachmann

    Full Text Available Translocation of E. coli across the gut epithelium can result in fatal sepsis in post-surgical patients. In vitro and in vivo experiments have identified the existence of a novel pathotype of translocating E. coli (TEC that employs an unknown mechanism for translocating across epithelial cells to the mesenteric lymph nodes and the blood stream in both humans and animal models. In this study the genomes of four TEC strains isolated from the mesenteric lymph nodes of a fatal case of hospitalised patient (HMLN-1, blood of pigs after experimental shock (PC-1 and after non-lethal haemorrhage in rats (KIC-1 and KIC-2 were sequenced in order to identify the genes associated with their adhesion and/or translocation. To facilitate the comparison, the genomes of a non-adhering, non-translocating E. coli (46-4 and adhering but non-translocating E. coli (73-89 were also sequenced and compared. Whole genome comparison revealed that three (HMLN-1, PC-1 and KIC-2 of the four TEC strains carried a genomic island that encodes a Type 6 Secretion System that may contribute to adhesion of the bacteria to gut epithelial cells. The human TEC strain HMLN-1 also carried the invasion ibeA gene, which was absent in the animal TEC strains and is likely to be associated with host-specific translocation. Phylogenetic analysis revealed that the four TEC strains were distributed amongst three distinct E. coli phylogroups, which was supported by the presence of phylogroup specific fimbriae gene clusters. The genomic comparison has identified potential genes that can be targeted with knock-out experiments to further characterise the mechanisms of E. coli translocation.

  17. An advanced coarse-grained nucleosome core particle model for computer simulations of nucleosome-nucleosome interactions under varying ionic conditions.

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    Yanping Fan

    Full Text Available In the eukaryotic cell nucleus, DNA exists as chromatin, a compact but dynamic complex with histone proteins. The first level of DNA organization is the linear array of nucleosome core particles (NCPs. The NCP is a well-defined complex of 147 bp DNA with an octamer of histones. Interactions between NCPs are of paramount importance for higher levels of chromatin compaction. The polyelectrolyte nature of the NCP implies that nucleosome-nucleosome interactions must exhibit a great influence from both the ionic environment as well as the positively charged and highly flexible N-terminal histone tails, protruding out from the NCP. The large size of the system precludes a modelling analysis of chromatin at an all-atom level and calls for coarse-grained approximations. Here, a model of the NCP that include the globular histone core and the flexible histone tails described by one particle per each amino acid and taking into account their net charge is proposed. DNA wrapped around the histone core was approximated at the level of two base pairs represented by one bead (bases and sugar plus four beads of charged phosphate groups. Computer simulations, using a Langevin thermostat, in a dielectric continuum with explicit monovalent (K(+, divalent (Mg(2+ or trivalent (Co(NH(3(6 (3+ cations were performed for systems with one or ten NCPs. Increase of the counterion charge results in a switch from repulsive NCP-NCP interaction in the presence of K(+, to partial aggregation with Mg(2+ and to strong mutual attraction of all 10 NCPs in the presence of CoHex(3+. The new model reproduced experimental results and the structure of the NCP-NCP contacts is in agreement with available data. Cation screening, ion-ion correlations and tail bridging contribute to the NCP-NCP attraction and the new NCP model accounts for these interactions.

  18. An advanced coarse-grained nucleosome core particle model for computer simulations of nucleosome-nucleosome interactions under varying ionic conditions.

    Science.gov (United States)

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

    2013-01-01

    In the eukaryotic cell nucleus, DNA exists as chromatin, a compact but dynamic complex with histone proteins. The first level of DNA organization is the linear array of nucleosome core particles (NCPs). The NCP is a well-defined complex of 147 bp DNA with an octamer of histones. Interactions between NCPs are of paramount importance for higher levels of chromatin compaction. The polyelectrolyte nature of the NCP implies that nucleosome-nucleosome interactions must exhibit a great influence from both the ionic environment as well as the positively charged and highly flexible N-terminal histone tails, protruding out from the NCP. The large size of the system precludes a modelling analysis of chromatin at an all-atom level and calls for coarse-grained approximations. Here, a model of the NCP that include the globular histone core and the flexible histone tails described by one particle per each amino acid and taking into account their net charge is proposed. DNA wrapped around the histone core was approximated at the level of two base pairs represented by one bead (bases and sugar) plus four beads of charged phosphate groups. Computer simulations, using a Langevin thermostat, in a dielectric continuum with explicit monovalent (K(+)), divalent (Mg(2+)) or trivalent (Co(NH(3))(6) (3+)) cations were performed for systems with one or ten NCPs. Increase of the counterion charge results in a switch from repulsive NCP-NCP interaction in the presence of K(+), to partial aggregation with Mg(2+) and to strong mutual attraction of all 10 NCPs in the presence of CoHex(3+). The new model reproduced experimental results and the structure of the NCP-NCP contacts is in agreement with available data. Cation screening, ion-ion correlations and tail bridging contribute to the NCP-NCP attraction and the new NCP model accounts for these interactions.

  19. Crystal structures of heterotypic nucleosomes containing histones H2A.Z and H2A.

    Science.gov (United States)

    Horikoshi, Naoki; Arimura, Yasuhiro; Taguchi, Hiroyuki; Kurumizaka, Hitoshi

    2016-06-01

    H2A.Z is incorporated into nucleosomes located around transcription start sites and functions as an epigenetic regulator for the transcription of certain genes. During transcriptional regulation, the heterotypic H2A.Z/H2A nucleosome containing one each of H2A.Z and H2A is formed. However, previous homotypic H2A.Z nucleosome structures suggested that the L1 loop region of H2A.Z would sterically clash with the corresponding region of canonical H2A in the heterotypic nucleosome. To resolve this issue, we determined the crystal structures of heterotypic H2A.Z/H2A nucleosomes. In the H2A.Z/H2A nucleosome structure, the H2A.Z L1 loop structure was drastically altered without any structural changes of the canonical H2A L1 loop, thus avoiding the steric clash. Unexpectedly, the heterotypic H2A.Z/H2A nucleosome is more stable than the homotypic H2A.Z nucleosome. These data suggested that the flexible character of the H2A.Z L1 loop plays an essential role in forming the stable heterotypic H2A.Z/H2A nucleosome.

  20. Structure of human nucleosome containing the testis-specific histone variant TSH2B

    Energy Technology Data Exchange (ETDEWEB)

    Urahama, Takashi; Horikoshi, Naoki; Osakabe, Akihisa; Tachiwana, Hiroaki; Kurumizaka, Hitoshi, E-mail: kurumizaka@waseda.jp [Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480 (Japan)

    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.

  1. The role of histone H4 biotinylation in the structure of nucleosomes.

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    Nina A Filenko

    Full Text Available BACKGROUND: Post-translational modifications of histones play important roles in regulating nucleosome structure and gene transcription. It has been shown that biotinylation of histone H4 at lysine-12 in histone H4 (K12Bio-H4 is associated with repression of a number of genes. We hypothesized that biotinylation modifies the physical structure of nucleosomes, and that biotin-induced conformational changes contribute to gene silencing associated with histone biotinylation. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis we used atomic force microscopy to directly analyze structures of nucleosomes formed with biotin-modified and non-modified H4. The analysis of the AFM images revealed a 13% increase in the length of DNA wrapped around the histone core in nucleosomes with biotinylated H4. This statistically significant (p<0.001 difference between native and biotinylated nucleosomes corresponds to adding approximately 20 bp to the classical 147 bp length of nucleosomal DNA. CONCLUSIONS/SIGNIFICANCE: The increase in nucleosomal DNA length is predicted to stabilize the association of DNA with histones and therefore to prevent nucleosomes from unwrapping. This provides a mechanistic explanation for the gene silencing associated with K12Bio-H4. The proposed single-molecule AFM approach will be instrumental for studying the effects of various epigenetic modifications of nucleosomes, in addition to biotinylation.

  2. The nucleosome landscape of Plasmodium falciparum reveals chromatin architecture and dynamics of regulatory sequences.

    Science.gov (United States)

    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.

  3. DNA-guided establishment of nucleosome patterns within coding regions of a eukaryotic genome.

    Science.gov (United States)

    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.

  4. Preferential Nucleosome Assembly at DNA Triplet Repeats from the Myotonic Dystrophy Gene

    Science.gov (United States)

    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.

  5. Stabilization of Nucleosomes by Histone Tails and by FACT Revealed by spFRET Microscopy.

    Science.gov (United States)

    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

    2017-01-06

    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.

  6. Stabilization of Nucleosomes by Histone Tails and by FACT Revealed by spFRET Microscopy

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    Maria E. Valieva

    2017-01-01

    Full Text Available 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.

  7. Electochemical detection of chromosome translocation

    DEFF Research Database (Denmark)

    Kwasny, Dorota; Dimaki, Maria; Silahtaroglu, Asli;

    2014-01-01

    Cytogenetics is a study of the cell structure with a main focus on chromosomes content and their structure. Chromosome abnormalities, such as translocations may cause various genetic disorders and heametological malignancies. Chromosome translocations are structural rearrangements of two...... chromosomes that results in formation of derivative chromosomes with a mixed DNA sequence. The method currently used for their detection is Fluorescent In Situ Hybridization, which requires a use of expensive, fluorescently labeled probes that target the derivative chromosomes. We present here a double...... hybridization approach developed for label-free detection of the chromosome translocations. For specific translocation detection it is necessary to determine that the two DNA sequences forming a derivative chromosome are connected, which is achieved by two subsequent hybridization steps. The electrochemical...

  8. Cracking the chromatin code: Precise rule of nucleosome positioning

    Science.gov (United States)

    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.

  9. Determinants of nucleosome positioning and their influence on plant gene expression.

    Science.gov (United States)

    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.

  10. Stable complex formation of CENP-B with the CENP-A nucleosome.

    Science.gov (United States)

    Fujita, Risa; Otake, Koichiro; Arimura, Yasuhiro; Horikoshi, Naoki; Miya, Yuta; Shiga, Tatsuya; Osakabe, Akihisa; Tachiwana, Hiroaki; Ohzeki, Jun-ichirou; Larionov, Vladimir; Masumoto, Hiroshi; Kurumizaka, Hitoshi

    2015-05-26

    CENP-A and CENP-B are major components of centromeric chromatin. CENP-A is the histone H3 variant, which forms the centromere-specific nucleosome. CENP-B specifically binds to the CENP-B box DNA sequence on the centromere-specific repetitive DNA. In the present study, we found that the CENP-A nucleosome more stably retains human CENP-B than the H3.1 nucleosome in vitro. Specifically, CENP-B forms a stable complex with the CENP-A nucleosome, when the CENP-B box sequence is located at the proximal edge of the nucleosome. Surprisingly, the CENP-B binding was weaker when the CENP-B box sequence was located in the distal linker region of the nucleosome. This difference in CENP-B binding, depending on the CENP-B box location, was not observed with the H3.1 nucleosome. Consistently, we found that the DNA-binding domain of CENP-B specifically interacted with the CENP-A-H4 complex, but not with the H3.1-H4 complex, in vitro. These results suggested that CENP-B forms a more stable complex with the CENP-A nucleosome through specific interactions with CENP-A, if the CENP-B box is located proximal to the CENP-A nucleosome. Our in vivo assay also revealed that CENP-B binding in the vicinity of the CENP-A nucleosome substantially stabilizes the CENP-A nucleosome on alphoid DNA in human cells.

  11. Reactivity in ELISA with DNA-loaded nucleosomes in patients with proliferative lupus nephritis.

    Science.gov (United States)

    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.

  12. TALEN-Induced Translocations in Human Cells.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  14. Effects of Alu elements on global nucleosome positioning in the human genome

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    Yamashita Riu

    2010-05-01

    Full Text Available Abstract Background Understanding the genome sequence-specific positioning of nucleosomes is essential to understand various cellular processes, such as transcriptional regulation and replication. As a typical example, the 10-bp periodicity of AA/TT and GC dinucleotides has been reported in several species, but it is still unclear whether this feature can be observed in the whole genomes of all eukaryotes. Results With Fourier analysis, we found that this is not the case: 84-bp and 167-bp periodicities are prevalent in primates. The 167-bp periodicity is intriguing because it is almost equal to the sum of the lengths of a nucleosomal unit and its linker region. After masking Alu elements, these periodicities were greatly diminished. Next, using two independent large-scale sets of nucleosome mapping data, we analyzed the distribution of nucleosomes in the vicinity of Alu elements and showed that (1 there are one or two fixed slot(s for nucleosome positioning within the Alu element and (2 the positioning of neighboring nucleosomes seems to be in phase, more or less, with the presence of Alu elements. Furthermore, (3 these effects of Alu elements on nucleosome positioning are consistent with inactivation of promoter activity in Alu elements. Conclusions Our discoveries suggest that the principle governing nucleosome positioning differs greatly across species and that the Alu family is an important factor in primate genomes.

  15. Dynamic Nucleosome Movement Provides Structural Information of Topological Chromatin Domains in Living Human Cells

    Science.gov (United States)

    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

  16. Nanopores suggest a negligible influence of CpG methylation on nucleosome packaging and stability.

    Science.gov (United States)

    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.

    Science.gov (United States)

    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. Multivalent Interactions by the Set8 Histone Methyltransferase With Its Nucleosome Substrate.

    Science.gov (United States)

    Girish, Taverekere S; McGinty, Robert K; Tan, Song

    2016-04-24

    Set8 is the only mammalian monomethyltransferase responsible for H4K20me1, a methyl mark critical for genomic integrity of eukaryotic cells. We present here a structural model for how Set8 uses multivalent interactions to bind and methylate the nucleosome based on crystallographic and solution studies of the Set8/nucleosome complex. Our studies indicate that Set8 employs its i-SET and c-SET domains to engage nucleosomal DNA 1 to 1.5 turns from the nucleosomal dyad and in doing so, it positions the SET domain for catalysis with H4 Lys20. Surprisingly, we find that a basic N-terminal extension to the SET domain plays an even more prominent role in nucleosome binding, possibly by making an arginine anchor interaction with the nucleosome H2A/H2B acidic patch. We further show that proliferating cell nuclear antigen and the nucleosome compete for binding to Set8 through this basic extension, suggesting a mechanism for how nucleosome binding protects Set8 from proliferating cell nuclear antigen-dependent degradation during the cell cycle.

  19. Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells.

    Science.gov (United States)

    Sebeson, Amy; Xi, Liqun; Zhang, Quanwei; Sigmund, Audrey; Wang, Ji-Ping; Widom, Jonathan; Wang, Xiaozhong

    2015-01-01

    The binding sequence for any transcription factor can be found millions of times within a genome, yet only a small fraction of these sequences encode functional transcription factor binding sites. One of the reasons for this dichotomy is that many other factors, such as nucleosomes, compete for binding. To study how the competition between nucleosomes and transcription factors helps determine a functional transcription factor site from a predicted transcription factor site, we compared experimentally-generated in vitro nucleosome occupancy with in vivo nucleosome occupancy and transcription factor binding in murine embryonic stem cells. Using a solution hybridization enrichment technique, we generated a high-resolution nucleosome map from targeted regions of the genome containing predicted sites and functional sites of Oct4/Sox2 regulation. We found that at Pax6 and Nes, which are bivalently poised in stem cells, functional Oct4 and Sox2 sites show high amounts of in vivo nucleosome displacement compared to in vitro. Oct4 and Sox2, which are active, show no significant displacement of in vivo nucleosomes at functional sites, similar to nonfunctional Oct4/Sox2 binding. This study highlights a complex interplay between Oct4 and Sox2 transcription factors and nucleosomes among different target genes, which may result in distinct patterns of stem cell gene regulation.

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

    Science.gov (United States)

    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.

  1. Novel nucleosomal particles containing core histones and linker DNA but no histone H1.

    Science.gov (United States)

    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.

  2. Molecular determinants of nucleosome retention at CpG-rich sequences in mouse spermatozoa

    NARCIS (Netherlands)

    Erkek, S.; Hisano, M.; Liang, C.Y.; Gill, M.; Murr, R.; Dieker, J.W.C.; Schubeler, D.; Vlag, J. van der; Stadler, M.B.; Peters, A.H.

    2013-01-01

    In mammalian spermatozoa, most but not all of the genome is densely packaged by protamines. Here we reveal the molecular logic underlying the retention of nucleosomes in mouse spermatozoa, which contain only 1% residual histones. We observe high enrichment throughout the genome of nucleosomes at CpG

  3. Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Amy Sebeson

    Full Text Available The binding sequence for any transcription factor can be found millions of times within a genome, yet only a small fraction of these sequences encode functional transcription factor binding sites. One of the reasons for this dichotomy is that many other factors, such as nucleosomes, compete for binding. To study how the competition between nucleosomes and transcription factors helps determine a functional transcription factor site from a predicted transcription factor site, we compared experimentally-generated in vitro nucleosome occupancy with in vivo nucleosome occupancy and transcription factor binding in murine embryonic stem cells. Using a solution hybridization enrichment technique, we generated a high-resolution nucleosome map from targeted regions of the genome containing predicted sites and functional sites of Oct4/Sox2 regulation. We found that at Pax6 and Nes, which are bivalently poised in stem cells, functional Oct4 and Sox2 sites show high amounts of in vivo nucleosome displacement compared to in vitro. Oct4 and Sox2, which are active, show no significant displacement of in vivo nucleosomes at functional sites, similar to nonfunctional Oct4/Sox2 binding. This study highlights a complex interplay between Oct4 and Sox2 transcription factors and nucleosomes among different target genes, which may result in distinct patterns of stem cell gene regulation.

  4. A molecule that detects the length of DNA by using chain fluctuation

    CERN Document Server

    Iwasa, Kuni H

    2015-01-01

    A class of nucleosome remodeling motors translocate nucleosomes, to which they are attached, toward the middle of DNA chain in the presence of ATP during in vitro experiments. Such a biological activity is likely be 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 fluctuation near the surface of the nucleosome coupled with binding reaction with a DNA binding site of the motor provides a physical basis for length detection. Since the mean frequency of fluctuation 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 remodeling motor. Dimerization of such units allows the motor to compare the length of the flanking DNA chains, enabling the translocation of the nucleosome toward the center of the DNA.

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

    Science.gov (United States)

    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

    2015-05-01

    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.

  6. Stimulation of the Drosophila immune system alters genome-wide nucleosome occupancy

    Directory of Open Access Journals (Sweden)

    Yingxue Ren

    2015-03-01

    Full Text Available In eukaryotes, nucleosomes participate in all DNA-templated events by regulating access to the underlying DNA sequence. However, nucleosome dynamics during a genome response have not been well characterized [1,2]. We stimulated Drosophila S2 cells with heat-killed Gram-negative bacteria Salmonella typhimurium, and mapped genome-wide nucleosome occupancy at high temporal resolution by MNase-seq using Illumina HiSeq 2500. We show widespread nucleosome occupancy change in S2 cells during the immune response, with the significant nucleosomal loss occurring at 4 h after stimulation. Data have been deposited to the Gene Expression Omnibus (GEO database repository with the dataset identifier GSE64507.

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

    Science.gov (United States)

    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.

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

    CERN Document Server

    Montel, Fabien; Menoni, Hervé; Angelov, Dimitar; Dimitrov, Stéfan; Faivre-Moskalenko, Cendrine

    2010-01-01

    RSC 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 edg...

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

    Directory of Open Access Journals (Sweden)

    Harm van Bakel

    2013-05-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  11. New insights into two distinct nucleosome distributions: comparison of cross-platform positioning datasets in the yeast genome

    Directory of Open Access Journals (Sweden)

    Deng Yangyang

    2010-01-01

    Full Text Available Abstract Background Recently, a number of high-resolution genome-wide maps of nucleosome locations in S. cerevisiae have been derived experimentally. However, nucleosome positions are determined in vivo by the combined effects of numerous factors. Consequently, nucleosomes are not simple static units, which may explain the discrepancies in reported nucleosome positions as measured by different experiments. In order to more accurately depict the genome-wide nucleosome distribution, we integrated multiple nucleosomal positioning datasets using a multi-angle analysis strategy. Results To evaluate the contribution of chromatin structure to transcription, we used the vast amount of available nucleosome analyzed data. Analysis of this data allowed for the comprehensive identification of the connections between promoter nucleosome positioning patterns and various transcription-dependent properties. Further, we characterised the function of nucleosome destabilisation in the context of transcription regulation. Our results indicate that genes with similar nucleosome occupancy patterns share general transcription attributes. We identified the local regulatory correlation (LRC regions for two distinct types of nucleosomes and we assessed their regulatory properties. We also estimated the nucleosome reproducibility and measurement accuracy for high-confidence transcripts. We found that by maintaining a distance of ~13 bp between the upstream border of the +1 nucleosome and the transcription start sites (TSSs, the stable +1 nucleosome may form a barrier against the accessibility of the TSS and shape an optimum chromatin conformation for gene regulation. An in-depth analysis of nucleosome positioning in normally growing and heat shock cells suggested that the extent and patterns of nucleosome sliding are associated with gene activation. Conclusions Our results, which combine different types of data, suggest that cross-platform information, including

  12. The Pioneer Transcription Factor FoxA Maintains an Accessible Nucleosome Configuration at Enhancers for Tissue-Specific Gene Activation.

    Science.gov (United States)

    Iwafuchi-Doi, Makiko; Donahue, Greg; Kakumanu, Akshay; Watts, Jason A; Mahony, Shaun; Pugh, B Franklin; Lee, Dolim; Kaestner, Klaus H; Zaret, Kenneth S

    2016-04-01

    Nuclear DNA wraps around core histones to form nucleosomes, which restricts the binding of transcription factors to gene regulatory sequences. Pioneer transcription factors can bind DNA sites on nucleosomes and initiate gene regulatory events, often leading to the local opening of chromatin. However, the nucleosomal configuration of open chromatin and the basis for its regulation is unclear. We combined low and high levels of micrococcal nuclease (MNase) digestion along with core histone mapping to assess the nucleosomal configuration at enhancers and promoters in mouse liver. We find that MNase-accessible nucleosomes, bound by transcription factors, are retained more at liver-specific enhancers than at promoters and ubiquitous enhancers. The pioneer factor FoxA displaces linker histone H1, thereby keeping enhancer nucleosomes accessible in chromatin and allowing other liver-specific transcription factors to bind and stimulate transcription. Thus, nucleosomes are not exclusively repressive to gene regulation when they are retained with, and exposed by, pioneer factors.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2011-04-01

    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.

  20. Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome

    DEFF Research Database (Denmark)

    Pedersen, Jakob Skou; Valen, Eivind; Velazquez, Amhed Missael Vargas;

    2014-01-01

    data generated from hair shafts of a 4000-yr-old Paleo-Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery...... of the expected signals at promoter regions, exon/intron boundaries, and CTCF sites. The top-scoring nucleosome calls revealed distinct DNA positioning biases, attesting to nucleotide-level accuracy. The ancient methylation levels exhibited high conservation over time, clustering closely with modern hair tissues...

  1. Characterization and partial purification of a proton translocating ATPase from corn coleoptile tonoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Mandala, S.M.

    1985-01-01

    ATP-dependent proton translocating activity in microsomal membranes from corn coleoptiles was characterized. Proton pumping activity, detected by either /sup 14/C-methylamine uptake or quinacrine fluorescence quenching, had a broad optimum at pH 7.5, and was substrate specific for MgATP. N,N'-dicyclohexylcarbodiimide, diethylstilbestrol, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and protonophores were found to inhibit proton transport, while vanadate and oligomycin had little effect. Proton pumping activity was stimulated 10 fold with Cl/sup -/ but was almost completely inhibited by 50 mM, KNO/sub 3/. Uptake studies with /sup 36/Cl/sup -/ indicated the Cl/sup -/ was transported into the microsomal vesicles in response to the pH gradient. ATP-stimulated proton pumping activity was localized on linear density gradients. On sucrose gradients, the activity cosedimented with the marker for endoplasmic reticulum at 1.11 g/cc. Sucrose gradients prepared in the presence of MgCl/sub 2/ were used to shift the ER marker to a heavier density, away from proton pumping activity. Linear dextran gradients also resulted in a clear separation of ATP-stimulated methylamine, thiocyanate, and /sup 36/Cl/sup -/ uptake, from markers for ER, Golgi, mitochondria, and plasma membranes. The tonoplast ATPase was solubilized with octylglucoside and partially purified on linear sucrose gradients. The specific activity of the KNO/sub 3/-sensitive ATPase increased 30-fold during purification.

  2. DNA Translocation through Graphene Nanopores

    CERN Document Server

    Schneider, Grégory F; Calado, Victor E; Pandraud, Grégory; Zandbergen, Henny W; Vandersypen, Lieven M K; Dekker, Cees

    2010-01-01

    Nanopores -- nanosized holes that can transport ions and molecules -- are very promising devices for genomic screening, in particular DNA sequencing. Both solid-state and biological pores suffer from the drawback, however, that the channel constituting the pore is long, viz. 10-100 times the distance between two bases in a DNA molecule (0.5 nm for single-stranded DNA). Here, we demonstrate that it is possible to realize and use ultrathin nanopores fabricated in graphene monolayers for single-molecule DNA translocation. The pores are obtained by placing a graphene flake over a microsize hole in a silicon nitride membrane and drilling a nanosize hole in the graphene using an electron beam. As individual DNA molecules translocate through the pore, characteristic temporary conductance changes are observed in the ionic current through the nanopore, setting the stage for future genomic screening.

  3. The supercoiling state of DNA determines the handedness of both H3 and CENP-A nucleosomes.

    Science.gov (United States)

    Vlijm, R; Kim, S H; De Zwart, P L; Dalal, Y; Dekker, C

    2017-02-02

    Nucleosomes form the unit structure of the genome in eukaryotes, thereby constituting a fundamental tenet of chromatin biology. In canonical nucleosomes, DNA wraps around the histone octamer in a left-handed toroidal ramp. Here, in single-molecule magnetic tweezers studies of chaperone-assisted nucleosome assembly, we show that the handedness of the DNA wrapping around the nucleosome core is intrinsically ambidextrous, and depends on the pre-assembly supercoiling state of the DNA, i.e., it is not uniquely determined by the octameric histone core. Nucleosomes assembled onto negatively supercoiled DNA are found to exhibit a left-handed conformation, whereas assembly onto positively supercoiled DNA results in right-handed nucleosomes. This intrinsic flexibility to adopt both chiralities is observed both for canonical H3 nucleosomes, and for centromere-specific variant CENP-A nucleosomes. These data support recent advances suggesting an intrinsic adaptability of the nucleosome, and provide insights into how nucleosomes might rapidly re-assemble after cellular processes that generate positive supercoiling in vivo.

  4. Understanding the connection between epigenetic DNA methylation and nucleosome positioning from computer simulations.

    Directory of Open Access Journals (Sweden)

    Guillem Portella

    Full Text Available Cytosine methylation is one of the most important epigenetic marks that regulate the process of gene expression. Here, we have examined the effect of epigenetic DNA methylation on nucleosomal stability using molecular dynamics simulations and elastic deformation models. We found that methylation of CpG steps destabilizes nucleosomes, especially when these are placed in sites where the DNA minor groove faces the histone core. The larger stiffness of methylated CpG steps is a crucial factor behind the decrease in nucleosome stability. Methylation changes the positioning and phasing of the nucleosomal DNA, altering the accessibility of DNA to regulatory proteins, and accordingly gene functionality. Our theoretical calculations highlight a simple physical-based explanation on the foundations of epigenetic signaling.

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

    Science.gov (United States)

    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.

  6. Genome-wide nucleosome occupancy and DNA methylation profiling of four human cell lines

    Directory of Open Access Journals (Sweden)

    Aaron L. Statham

    2015-03-01

    Full Text Available DNA methylation and nucleosome positioning are two key mechanisms that contribute to the epigenetic control of gene expression. During carcinogenesis, the expression of many genes is altered alongside extensive changes in the epigenome, with repressed genes often being associated with local DNA hypermethylation and gain of nucleosomes at their promoters. However the spectrum of alterations that occur at distal regulatory regions has not been extensively studied. To address this we used Nucleosome Occupancy and Methylation sequencing (NOMe-seq to compare the genome-wide DNA methylation and nucleosome occupancy profiles between normal and cancer cell line models of the breast and prostate. Here we describe the bioinformatic pipeline and methods that we developed for the processing and analysis of the NOMe-seq data published by (Taberlay et al., 2014 [1] and deposited in the Gene Expression Omnibus with accession GSE57498.

  7. A RSC/nucleosome complex determines chromatin architecture and facilitates activator binding.

    Science.gov (United States)

    Floer, Monique; Wang, Xin; Prabhu, Vidya; Berrozpe, Georgina; Narayan, Santosh; Spagna, Dan; Alvarez, David; Kendall, Jude; Krasnitz, Alexander; Stepansky, Asya; Hicks, James; Bryant, Gene O; Ptashne, Mark

    2010-04-30

    How is chromatin architecture established and what role does it play in transcription? We show that the yeast regulatory locus UASg bears, in addition to binding sites for the activator Gal4, sites bound by the RSC complex. RSC positions a nucleosome, evidently partially unwound, in a structure that facilitates Gal4 binding to its sites. The complex comprises a barrier that imposes characteristic features of chromatin architecture. In the absence of RSC, ordinary nucleosomes encroach over the UASg and compete with Gal4 for binding. Taken with our previous work, the results show that both prior to and following induction, specific DNA-binding proteins are the predominant determinants of chromatin architecture at the GAL1/10 genes. RSC/nucleosome complexes are also found scattered around the yeast genome. Higher eukaryotic RSC lacks the specific DNA-binding determinants found on yeast RSC, and evidently Gal4 works in those organisms despite whatever obstacle broadly positioned nucleosomes present.

  8. AFM Imaging of SWI/SNF action: mapping the nucleosome remodeling and sliding

    CERN Document Server

    Montel, Fabien; Saint-Jean, Philippe; Castelnovo, Martin; Moskalenko-Faivre, 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 to determine simultaneously the DNA complexed length distribution and nucleosome position in various contexts. First, we show that differences in the nucleo-proteic structure observed between conventional H2A and H2A.Bbd variant nucleosomes induce quantitative changes in the 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.

  9. nuMap:A Web Platform for Accurate Prediction of Nucleosome Positioning

    Institute of Scientific and Technical Information of China (English)

    Bader A Alharbi; Thamir H Alshammari; Nathan L Felton; Victor B Zhurkin; Feng Cui

    2014-01-01

    Nucleosome positioning is critical for gene expression and of major biological interest. The high cost of experimentally mapping nucleosomal arrangement signifies the need for computational approaches to predict nucleosome positions at high resolution. Here, we present a web-based application to fulfill this need by implementing two models, YR and W/S schemes, for the translational and rotational positioning of nucleosomes, respectively. Our methods are based on sequence-dependent anisotropic bending that dictates how DNA is wrapped around a histone octamer. This application allows users to specify a number of options such as schemes and param-eters for threading calculation and provides multiple layout formats. The nuMap is implemented in Java/Perl/MySQL and is freely available for public use at http://numap.rit.edu. The user manual, implementation notes, description of the methodology and examples are available at the site.

  10. Reassembly of nucleosomes at the MLH1 promoter initiates resilencing following decitabine exposure.

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    Luke B Hesson

    Full Text Available Hypomethylating agents reactivate tumor suppressor genes that are epigenetically silenced in cancer. Inevitably these genes are resilenced, leading to drug resistance. Using the MLH1 tumor suppressor gene as a model, we showed that decitabine-induced re-expression was dependent upon demethylation and eviction of promoter nucleosomes. Following decitabine withdrawal, MLH1 was rapidly resilenced despite persistent promoter demethylation. Single molecule analysis at multiple time points showed that gene resilencing was initiated by nucleosome reassembly on demethylated DNA and only then was followed by remethylation and stable silencing. Taken together, these data establish the importance of nucleosome positioning in mediating resilencing of drug-induced gene reactivation and suggest a role for therapeutic targeting of nucleosome assembly as a mechanism to overcome drug resistance.

  11. Nucleosome structure of the yeast CHA1 promoter

    DEFF Research Database (Denmark)

    Moreira, José Manuel Alfonso; Holmberg, S

    1998-01-01

    conditions. Five yeast TBP mutants defective in different steps in activated transcription abolished CHA1 expression, but failed to affect induction-dependent chromatin rearrangement of the promoter region. Progressive truncations of the RNA polymerase II C-terminal domain caused a progressive reduction......The Saccharomyces cerevisiae CHA1 gene encodes the catabolic L-serine (L-threonine) dehydratase. We have previously shown that the transcriptional activator protein Cha4p mediates serine/threonine induction of CHA1 expression. We used accessibility to micrococcal nuclease and DNase I to determine...... the in vivo chromatin structure of the CHA1 chromosomal locus, both in the non-induced state and upon induction. Upon activation, a precisely positioned nucleosome (nuc-1) occluding the TATA box and the transcription start site is removed. A strain devoid of Cha4p showed no chromatin alteration under inducing...

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

    Science.gov (United States)

    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.

  13. Serum nucleosomes during neoadjuvant chemotherapy in patients with cervical cancer. Predictive and prognostic significance

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    Cetina Lucely

    2005-06-01

    Full Text Available Abstract Background It has been shown that free DNA circulates in serum plasma of patients with cancer and that at least part is present in the form of oligo- and monucleosomes, a marker of cell death. Preliminary data has shown a good correlation between decrease of nucleosomes with response and prognosis. Here, we performed pre- and post-chemotherapy determinations of serum nucleosomes with an enzyme-linked immunosorbent assay (ELISA method in a group of patients with cervical cancer receiving neoadjuvant chemotherapy. Methods From December 2000 to June 2001, 41 patients with cervical cancer staged as FIGO stages IB2-IIIB received three 21-day courses of carboplatin and paclitaxel, both administered at day 1; then, patients underwent radical hysterectomy. Nucleosomes were measured the day before (baseline, at day seven of the first course and day seven of the third course of chemotherapy. Values of nucleosomes were analyzed with regard to pathologic response and to time to progression-free and overall survival. Results All patients completed chemotherapy, were evaluable for pathologic response, and had nucleosome levels determined. At a mean follow-up of 23 months (range, 7–26 months, projected progression time and overall survival were 80.3 and 80.4%, respectively. Mean differential values of nucleosomes were lower in the third course as compared with the first course (p >0.001. The decrease in the third course correlated with pathologic response (p = 0.041. Survival analysis showed a statistically significant, better progression-free and survival time in patients who showed lower levels at the third course (p = 0.0243 and p = 0.0260, respectively. Cox regression analysis demonstrated that nucleosome increase in the third course increased risk of death to 6.86 (95% confidence interval [CI 95%], 0.84–56.0. Conclusion Serum nucleosomes may have a predictive role for response and prognostic significance in patients with cervical cancer

  14. A role for FACT in repopulation of nucleosomes at inducible genes.

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    Warren P Voth

    Full Text Available Xenobiotic drugs induce Pleiotropic Drug Resistance (PDR genes via the orthologous Pdr1/Pdr3 transcription activators. We previously identified the Mediator transcription co-activator complex as a key target of Pdr1 orthologs and demonstrated that Pdr1 interacts directly with the Gal11/Med15 subunit of the Mediator complex. Based on an interaction between Pdr1 and the FACT complex, we show that strains with spt16 or pob3 mutations are sensitive to xenobiotic drugs and display diminished PDR gene induction. Although FACT acts during the activation of some genes by assisting in the nucleosomes eviction at promoters, PDR promoters already contain nucleosome-depleted regions (NDRs before induction. To determine the function of FACT at PDR genes, we examined the kinetics of RNA accumulation and changes in nucleosome occupancy following exposure to a xenobiotic drug in wild type and FACT mutant yeast strains. In the presence of normal FACT, PDR genes are transcribed within 5 minutes of xenobiotic stimulation and transcription returns to basal levels by 30-40 min. Nucleosomes are constitutively depleted in the promoter regions, are lost from the open reading frames during transcription, and the ORFs are wholly repopulated with nucleosomes as transcription ceases. While FACT mutations cause minor delays in activation of PDR genes, much more pronounced and significant defects in nucleosome repopulation in the ORFs are observed in FACT mutants upon transcription termination. FACT therefore has a major role in nucleosome redeposition following cessation of transcription at the PDR genes, the opposite of its better-known function in nucleosome disassembly.

  15. Nucleosome Repositioning: A Novel Mechanism for Nicotine- and Cocaine-Induced Epigenetic Changes.

    Science.gov (United States)

    Brown, Amber N; Vied, Cynthia; Dennis, Jonathan H; Bhide, Pradeep G

    2015-01-01

    Drugs of abuse modify behavior by altering gene expression in the brain. Gene expression can be regulated by changes in DNA methylation as well as by histone modifications, which alter chromatin structure, DNA compaction and DNA accessibility. In order to better understand the molecular mechanisms directing drug-induced changes in chromatin structure, we examined DNA-nucleosome interactions within promoter regions of 858 genes in human neuroblastoma cells (SH-SY5Y) exposed to nicotine or cocaine. Widespread, drug- and time-resolved repositioning of nucleosomes was identified at the transcription start site and promoter region of multiple genes. Nicotine and cocaine produced unique and shared changes in terms of the numbers and types of genes affected, as well as repositioning of nucleosomes at sites which could increase or decrease the probability of gene expression based on DNA accessibility. Half of the drug-induced nucleosome positions approximated a theoretical model of nucleosome occupancy based on physical and chemical characteristics of the DNA sequence, whereas the basal or drug naïve positions were generally DNA sequence independent. Thus we suggest that nucleosome repositioning represents an initial dynamic genome-wide alteration of the transcriptional landscape preceding more selective downstream transcriptional reprogramming, which ultimately characterizes the cell- and tissue-specific responses to drugs of abuse.

  16. Nucleosome Repositioning: A Novel Mechanism for Nicotine- and Cocaine-Induced Epigenetic Changes.

    Directory of Open Access Journals (Sweden)

    Amber N Brown

    Full Text Available Drugs of abuse modify behavior by altering gene expression in the brain. Gene expression can be regulated by changes in DNA methylation as well as by histone modifications, which alter chromatin structure, DNA compaction and DNA accessibility. In order to better understand the molecular mechanisms directing drug-induced changes in chromatin structure, we examined DNA-nucleosome interactions within promoter regions of 858 genes in human neuroblastoma cells (SH-SY5Y exposed to nicotine or cocaine. Widespread, drug- and time-resolved repositioning of nucleosomes was identified at the transcription start site and promoter region of multiple genes. Nicotine and cocaine produced unique and shared changes in terms of the numbers and types of genes affected, as well as repositioning of nucleosomes at sites which could increase or decrease the probability of gene expression based on DNA accessibility. Half of the drug-induced nucleosome positions approximated a theoretical model of nucleosome occupancy based on physical and chemical characteristics of the DNA sequence, whereas the basal or drug naïve positions were generally DNA sequence independent. Thus we suggest that nucleosome repositioning represents an initial dynamic genome-wide alteration of the transcriptional landscape preceding more selective downstream transcriptional reprogramming, which ultimately characterizes the cell- and tissue-specific responses to drugs of abuse.

  17. A 1-dimensional statistical mechanics model for nucleosome positioning on genomic DNA

    CERN Document Server

    Tesoro, S; Morozov, A N; Sulaiman, N; Marenduzzo, D

    2015-01-01

    The first level of folding of DNA in eukaryotes is provided by the so called '10nm chromatin fibre', where DNA wraps around histone proteins (approx. 10 nm in size) to form nucleosomes, which go on to create a zig zagging 'bead on a string' structure. In this work we present a one dimensional statistical mechanics model to study nucleosome positioning within one such 10 nm fibre. We consider both the case of homogeneous DNA, where the problem can be mapped to a Tonks gas, and that of genomic sheep DNA, where our modelling is informed by high-resolution nucleosome positioning data. First, we consider the simple, analytically solvable, case where nucleosomes are assumed to be point like. Then, we perform numerical simulations to gauge the effect of their finite size on the nucleosomal distribution probabilities. Finally, we compare nucleosome distributions and simulated nuclease digestion patterns for the two cases (homogeneous and sheep DNA), thereby providing testable predictions of the effect of sequence on ...

  18. Apoptotic lymphocytes of H. sapiens lose nucleosomes in GC-rich promoters.

    Science.gov (United States)

    Hosid, Sergey; Ioshikhes, Ilya

    2014-07-01

    We analyzed two sets of human CD4+ nucleosomal DNA directly sequenced by Illumina (Solexa) high throughput sequencing method. The first set has ∼40 M sequences and was produced from the normal CD4+ T lymphocytes by micrococcal nuclease. The second set has ∼44 M sequences and was obtained from peripheral blood lymphocytes by apoptotic nucleases. The different nucleosome sets showed similar dinucleotide positioning AA/TT, GG/CC, and RR/YY (R is purine, Y--pyrimidine) patterns with periods of 10-10.4 bp. Peaks of GG/CC and AA/TT patterns were shifted by 5 bp from each other. Two types of promoters in H. sapiens: AT and GC-rich were identified. AT-rich promoters in apoptotic cell had +1 nucleosome shifts 50-60 bp downstream from those in normal lymphocytes. GC-rich promoters in apoptotic cells lost 80% of nucleosomes around transcription start sites as well as in total DNA. Nucleosome positioning was predicted by combination of {AA, TT}, {GG, CC}, {WW, SS} and {RR, YY} patterns. In our study we found that the combinations of {AA, TT} and {GG, CC} provide the best results and successfully mapped 33% of nucleosomes 147 bp long with precision ±15 bp (only 31/147 or 21% is expected).

  19. Single-Molecule Studies of the Linker Histone H1 Binding to DNA and the Nucleosome.

    Science.gov (United States)

    Yue, Hongjun; Fang, He; Wei, Sijie; Hayes, Jeffrey J; Lee, Tae-Hee

    2016-04-12

    Linker histone H1 regulates chromatin structure and gene expression. Investigating the dynamics and stoichiometry of binding of H1 to DNA and the nucleosome is crucial to elucidating its functions. Because of the abundant positive charges and the strong self-affinity of H1, quantitative in vitro studies of its binding to DNA and the nucleosome have generated results that vary widely and, therefore, should be interpreted in a system specific manner. We sought to overcome this limitation by developing a specially passivated microscope slide surface to monitor binding of H1 to DNA and the nucleosome at a single-molecule level. According to our measurements, the stoichiometry of binding of H1 to DNA and the nucleosome is very heterogeneous with a wide distribution whose averages are in reasonable agreement with previously published values. Our study also revealed that H1 does not dissociate from DNA or the nucleosome on a time scale of tens of minutes. We found that histone chaperone Nap1 readily dissociates H1 from DNA and superstoichiometrically bound H1 from the nucleosome, supporting a hypothesis whereby histone chaperones contribute to the regulation of the H1 profile in chromatin.

  20. GAA triplet-repeats cause nucleosome depletion in the human genome.

    Science.gov (United States)

    Zhao, Hongyu; Xing, Yongqiang; Liu, Guoqing; Chen, Ping; Zhao, Xiujuan; Li, Guohong; Cai, Lu

    2015-08-01

    Although there have been many investigations into how trinucleotide repeats affect nucleosome formation and local chromatin structure, the nucleosome positioning of GAA triplet-repeats in the human genome has remained elusive. In this work, the nucleosome occupancy around GAA triplet-repeats across the human genome was computed statistically. The results showed a nucleosome-depleted region in the vicinity of GAA triplet-repeats in activated and resting CD4(+) T cells. Furthermore, the A-tract was frequently adjacent to the upstream region of GAA triplet-repeats and could enhance the depletion surrounding GAA triplet-repeats. In vitro chromatin reconstitution assays with GAA-containing plasmids also demonstrated that the inserted GAA triplet-repeats destabilized the ability of recombinant plasmids to assemble nucleosomes. Our results suggested that GAA triplet-repeats have lower affinity to histones and can change local nucleosome positioning. These findings may be helpful for understanding the mechanism of Friedreich's ataxia, which is associated with GAA triplet-repeats at the chromatin level.

  1. Relationship between nucleosome positioning and progesterone-induced alternative splicing in breast cancer cells.

    Science.gov (United States)

    Iannone, Camilla; Pohl, Andy; Papasaikas, Panagiotis; Soronellas, Daniel; Vicent, Guillermo P; Beato, Miguel; ValcáRcel, Juan

    2015-03-01

    Splicing of mRNA precursors can occur cotranscriptionally and it has been proposed that chromatin structure influences splice site recognition and regulation. Here we have systematically explored potential links between nucleosome positioning and alternative splicing regulation upon progesterone stimulation of breast cancer cells. We confirm preferential nucleosome positioning in exons and report four distinct profiles of nucleosome density around alternatively spliced exons, with RNA polymerase II accumulation closely following nucleosome positioning. Hormone stimulation induces switches between profile classes, correlating with a subset of alternative splicing changes. Hormone-induced exon inclusion often correlates with higher nucleosome occupancy at the exon or the preceding intronic region and with higher RNA polymerase II accumulation. In contrast, exons skipped upon hormone stimulation display low nucleosome densities even before hormone treatment, suggesting that chromatin structure primes alternative splicing regulation. Skipped exons frequently harbor binding sites for hnRNP AB, a hormone-induced splicing regulator whose knock down prevents some hormone-induced skipping events. Collectively, our results argue that a variety of chromatin architecture mechanisms can influence alternative splicing decisions.

  2. AFM studies in diverse ionic environments of nucleosomes reconstituted on the 601 positioning sequence.

    Science.gov (United States)

    Nazarov, Igor; Chekliarova, Iana; Rychkov, Georgy; Ilatovskiy, Andrey V; Crane-Robinson, Colyn; Tomilin, Alexey

    2016-02-01

    Atomic force microscopy (AFM) was used to study mononucleosomes reconstituted from a DNA duplex of 353 bp containing the strong 601 octamer positioning sequence, together with recombinant human core histone octamers. Three parameters were measured: 1) the length of DNA wrapped around the core histones; 2) the number of superhelical turns, calculated from the total angle through which the DNA is bent, and 3) the volume of the DNA-histone core. This approach allowed us to define in detail the structural diversity of nucleosomes caused by disassembly of the octasome to form subnucleosomal structures containing hexasomes, tetrasomes and disomes. At low ionic strength (TE buffer) and in the presence of physiological concentrations of monovalent cations, the majority of the particles were subnucleosomal, but physiological concentrations of bivalent cations resulted in about half of the nucleosomes being canonical octasomes in which the exiting DNA duplexes cross orthogonally. The dominance of this last species explains why bivalent but not monovalent cations can induce the initial step towards compaction and convergence of neighboring nucleosomes in nucleosomal arrays to form the chromatin fiber in the absence of linker histone. The observed nucleosome structural diversity may reflect the functional plasticity of nucleosomes under physiological conditions.

  3. Inducible nucleosome depletion at OREBP-binding-sites by hypertonic stress.

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    Edith H Y Tong

    Full Text Available BACKGROUND: Osmotic Response Element-Binding Protein (OREBP, also known as TonEBP or NFAT5, is a unique transcription factor. It is hitherto the only known mammalian transcription factor that regulates hypertonic stress-induced gene transcription. In addition, unlike other monomeric members of the NFAT family, OREBP exists as a homodimer and it is the only transcription factor known to bind naked DNA targets by complete encirclement in vitro. Nevertheless, how OREBP interacts with target DNA, also known as ORE/TonE, and how it elicits gene transcription in vivo, remains unknown. METHODOLOGY: Using hypertonic induction of the aldose reductase (AR gene activation as a model, we showed that OREs contained dynamic nucleosomes. Hypertonic stress induced a rapid and reversible loss of nucleosome(s around the OREs. The loss of nucleosome(s was found to be initiated by an OREBP-independent mechanism, but was significantly potentiated in the presence of OREBP. Furthermore, hypertonic induction of AR gene was associated with an OREBP-dependent hyperacetylation of histones that spanned the 5' upstream sequences and at least some exons of the gene. Nevertheless, nucleosome loss was not regulated by the acetylation status of histone. SIGNIFICANCE: Our findings offer novel insights into the mechanism of OREBP-dependent transcriptional regulation and provide a basis for understanding how histone eviction and transcription factor recruitment are coupled.

  4. Theoretical estimates of exposure timescales of protein binding sites on DNA regulated by nucleosome kinetics.

    Science.gov (United States)

    Parmar, Jyotsana J; Das, Dibyendu; Padinhateeri, Ranjith

    2016-02-29

    It is being increasingly realized that nucleosome organization on DNA crucially regulates DNA-protein interactions and the resulting gene expression. While the spatial character of the nucleosome positioning on DNA has been experimentally and theoretically studied extensively, the temporal character is poorly understood. Accounting for ATPase activity and DNA-sequence effects on nucleosome kinetics, we develop a theoretical method to estimate the time of continuous exposure of binding sites of non-histone proteins (e.g. transcription factors and TATA binding proteins) along any genome. Applying the method to Saccharomyces cerevisiae, we show that the exposure timescales are determined by cooperative dynamics of multiple nucleosomes, and their behavior is often different from expectations based on static nucleosome occupancy. Examining exposure times in the promoters of GAL1 and PHO5, we show that our theoretical predictions are consistent with known experiments. We apply our method genome-wide and discover huge gene-to-gene variability of mean exposure times of TATA boxes and patches adjacent to TSS (+1 nucleosome region); the resulting timescale distributions have non-exponential tails.

  5. Increased Nucleosomes and Neutrophil Activation Link to Disease Progression in Patients with Scrub Typhus but Not Murine Typhus in Laos.

    Science.gov (United States)

    Paris, Daniel H; Stephan, Femke; Bulder, Ingrid; Wouters, Diana; van der Poll, Tom; Newton, Paul N; Day, Nicholas P J; Zeerleder, Sacha

    2015-01-01

    Cell-mediated immunity is essential in protection against rickettsial illnesses, but the role of neutrophils in these intracellular vasculotropic infections remains unclear. This study analyzed the plasma levels of nucleosomes, FSAP-activation (nucleosome-releasing factor), and neutrophil activation, as evidenced by neutrophil-elastase (ELA) complexes, in sympatric Lao patients with scrub typhus and murine typhus. In acute scrub typhus elevated nucleosome levels correlated with lower GCS scores, raised respiratory rate, jaundice and impaired liver function, whereas neutrophil activation correlated with fibrinolysis and high IL-8 plasma levels, a recently identified predictor of severe disease and mortality. Nucleosome and ELA complex levels were associated with a 4.8-fold and 4-fold increased risk of developing severe scrub typhus, beyond cut off values of 1,040 U/ml for nucleosomes and 275 U/ml for ELA complexes respectively. In murine typhus, nucleosome levels associated with pro-inflammatory cytokines and the duration of illness, while ELA complexes correlated strongly with inflammation markers, jaundice and increased respiratory rates. This study found strong correlations between circulating nucleosomes and neutrophil activation in patients with scrub typhus, but not murine typhus, providing indirect evidence that nucleosomes could originate from neutrophil extracellular trap (NET) degradation. High circulating plasma nucleosomes and ELA complexes represent independent risk factors for developing severe complications in scrub typhus. As nucleosomes and histones exposed on NETs are highly cytotoxic to endothelial cells and are strongly pro-coagulant, neutrophil-derived nucleosomes could contribute to vascular damage, the pro-coagulant state and exacerbation of disease in scrub typhus, thus indicating a detrimental role of neutrophil activation. The data suggest that increased neutrophil activation relates to disease progression and severe complications, and

  6. Increasing Nucleosome Occupancy Is Correlated with an Increasing Mutation Rate so Long as DNA Repair Machinery Is Intact.

    Science.gov (United States)

    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

    Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently, chromatin has been shown to play a part in this process. To elucidate this relationship further, we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed, that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally, cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp) and end (115 bp) of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts, and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed, our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer.

  7. Genome-Wide Nucleosome Occupancy and Positioning and Their Impact on Gene Expression and Evolution in Plants.

    Science.gov (United States)

    Zhang, Tao; Zhang, Wenli; Jiang, Jiming

    2015-08-01

    The fundamental unit of chromatin is the nucleosome that consists of a protein octamer composed of the four core histones (Hs; H3, H4, H2A, and H2B) wrapped by 147 bp of DNA. Nucleosome occupancy and positioning have proven to be dynamic and have a critical impact on expression, regulation, and evolution of eukaryotic genes. We developed nucleosome occupancy and positioning data sets using leaf tissue of rice (Oryza sativa) and both leaf and flower tissues of Arabidopsis (Arabidopsis thaliana). We show that model plant and animal species share the fundamental characteristics associated with nucleosome dynamics. Only 12% and 16% of the Arabidopsis and rice genomes, respectively, were occupied by well-positioned nucleosomes. The cores of positioned nucleosomes were enriched with G/C dinucleotides and showed a lower C→T mutation rate than the linker sequences. We discovered that nucleosomes associated with heterochromatic regions were more spaced with longer linkers than those in euchromatic regions in both plant species. Surprisingly, different nucleosome densities were found to be associated with chromatin in leaf and flower tissues in Arabidopsis. We show that deep MNase-seq data sets can be used to map nucleosome occupancy of specific genomic loci and reveal gene expression patterns correlated with chromatin dynamics in plant genomes.

  8. Mutation bias, rather than binding preference, underlies the nucleosome-associated G+C% variation in eukaryotes.

    Science.gov (United States)

    Xing, Ke; He, Xionglei

    2015-03-18

    The effects of genetic content on epigenetic status have been extensively studied, but how epigenetic status affects genetic content is not well understood. As a key epigenetic factor the nucleosome structure is highly correlated with local G+C% in eukaryotic genomes. The prevailing explanation to the pattern is that nucleosome occupancy favors higher G+C% sequences more than lower G+C% sequences. However, recent observation of a biased mutation spectrum caused by nucleosome occupancy suggests that the higher G+C% of nucleosomal DNA might be the evolutionary consequence of nucleosome occupancy. To distinguish the two explanations, we examined data from an in vitro nucleosome reconstitution experiment in which histones are incubated with yeast Saccharomyces cerevisiae and Escherichia coli genomic DNA, the former has been shaped by nucleosome structure while the latter has not. There is a strong positive correlation between nucleosome density and G+C% for the yeast DNA, an observation consistent with in vivo data, and such a pattern nearly vanishes for E. coli genomic DNA, suggesting that biased mutation, rather than biased occupancy, explains the most nucleosome-associated G+C% variation in eukaryotic genomes.

  9. Increasing Nucleosome Occupancy Is Correlated with an Increasing Mutation Rate so Long as DNA Repair Machinery Is Intact.

    Directory of Open Access Journals (Sweden)

    Puya G Yazdi

    Full Text Available Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently, chromatin has been shown to play a part in this process. To elucidate this relationship further, we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed, that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally, cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp and end (115 bp of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts, and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed, our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer.

  10. Translocations affecting human immunoglobulin heavy chain locus

    Directory of Open Access Journals (Sweden)

    Sklyar I. V.

    2014-03-01

    Full Text Available Translocations involving human immunoglobulin heavy chain (IGH locus are implicated in different leukaemias and lymphomas, including multiple myeloma, mantle cell lymphoma, Burkitt’s lymphoma and diffuse large B cell lymphoma. We have analysed published data and identified eleven breakpoint cluster regions (bcr related to these cancers within the IgH locus. These ~1 kbp bcrs are specific for one or several types of blood cancer. Our findings could help devise PCR-based assays to detect cancer-related translocations, to identify the mechanisms of translocations and to help in the research of potential translocation partners of the immunoglobulin locus at different stages of B-cell differentiation.

  11. Haloarchaeal Protein Translocation via the Twin Arginine Translocation Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Pohlschroder Mechthild

    2009-02-03

    Protein transport across hydrophobic membranes that partition cellular compartments is essential in all cells. The twin arginine translocation (Tat) pathway transports proteins across the prokaryotic cytoplasmic membranes. Distinct from the universally conserved Sec pathway, which secretes unfolded proteins, the Tat machinery is unique in that it secretes proteins in a folded conformation, making it an attractive pathway for the transport and secretion of heterologously expressed proteins that are Sec-incompatible. During the past 7 years, the DOE-supported project has focused on the characterization of the diversity of bacterial and archaeal Tat substrates as well as on the characterization of the Tat pathway of a model archaeon, Haloferax volcanii, a member of the haloarchaea. We have demonstrated that H. volcanii uses this pathway to transport most of its secretome.

  12. A one-dimensional statistical mechanics model for nucleosome positioning on genomic DNA.

    Science.gov (United States)

    Tesoro, S; Ali, I; Morozov, A N; Sulaiman, N; Marenduzzo, D

    2016-02-12

    The first level of folding of DNA in eukaryotes is provided by the so-called '10 nm chromatin fibre', where DNA wraps around histone proteins (∼10 nm in size) to form nucleosomes, which go on to create a zig-zagging bead-on-a-string structure. In this work we present a one-dimensional statistical mechanics model to study nucleosome positioning within one such 10 nm fibre. We focus on the case of genomic sheep DNA, and we start from effective potentials valid at infinite dilution and determined from high-resolution in vitro salt dialysis experiments. We study positioning within a polynucleosome chain, and compare the results for genomic DNA to that obtained in the simplest case of homogeneous DNA, where the problem can be mapped to a Tonks gas. First, we consider the simple, analytically solvable, case where nucleosomes are assumed to be point-like. Then, we perform numerical simulations to gauge the effect of their finite size on the nucleosomal distribution probabilities. Finally we compare nucleosome distributions and simulated nuclease digestion patterns for the two cases (homogeneous and sheep DNA), thereby providing testable predictions of the effect of sequence on experimentally observable quantities in experiments on polynucleosome chromatin fibres reconstituted in vitro.

  13. Nucleosome Positions and Differential Methylation Status of Various Regions within MLH1 CpG Island

    Institute of Scientific and Technical Information of China (English)

    BAI Hua; ZHOU Jing; DENG Da-jun

    2008-01-01

    Objective:To determine the relationship between nucleosome positions and formation of differential methylation of the reported region A,B,C,and D within the MLH1 CpG island. Methods:Methylation of the MLH1 promoter was analyzed by combined of bisulfite restriction assay.Chromatin of RKO and MGC803 cells were extracted and digested by Mnase.Mononucleosomal DNA fragment was isolated and used as templates for detection of nucleosomal distribution by a battery of quantitative PCRs covering the full MLH1 promoter region. Results:The MLH1 was methylated in RKO and unmethylated in MGC803.At the region B,where methylation of CpG sites did not correlated with transcription of this gene well,qPCR product of the M-3(-599nt~-475nt)fragment was amplified in both RKO and MGC803 cells.However,at the region C and D within the core promoter,where methylation of CpG sites correlated with loss of MLH1 transcription well,the M-7(-257nt~-153nt)and M-8(-189nt~-71nt)fragments were amplified remarkably only in RKO cells. Conclusion:Nucleosome may be the basic unit for both CpG methylation and methylation-related regulation of gene transcription.Methylation status of CpG sites within the same nucleosome may be homogeneous;between different nucleosomes,homogeneous or heterogeneous.

  14. Nucleosome assembly dynamics involve spontaneous fluctuations in the handedness of tetrasomes.

    Science.gov (United States)

    Vlijm, Rifka; Lee, Mina; Lipfert, Jan; Lusser, Alexandra; Dekker, Cees; Dekker, Nynke H

    2015-01-13

    DNA wrapping around histone octamers generates nucleosomes, the basic compaction unit of eukaryotic chromatin. Nucleosome stability is carefully tuned to maintain DNA accessibility in transcription, replication, and repair. Using freely orbiting magnetic tweezers, which measure the twist and length of single DNA molecules, we monitor the real-time loading of tetramers or complete histone octamers onto DNA by Nucleosome Assembly Protein-1 (NAP1). Remarkably, we find that tetrasomes exhibit spontaneous flipping between a preferentially occupied left-handed state (ΔLk = -0.73) and a right-handed state (ΔLk = +1.0), separated by a free energy difference of 2.3 kBT (1.5 kcal/mol). This flipping occurs without concomitant changes in DNA end-to-end length. The application of weak positive torque converts left-handed tetrasomes into right-handed tetrasomes, whereas nucleosomes display more gradual conformational changes. Our findings reveal unexpected dynamical rearrangements of the nucleosomal structure, suggesting that chromatin can serve as a "twist reservoir," offering a mechanistic explanation for the regulation of DNA supercoiling in chromatin.

  15. Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation.

    Science.gov (United States)

    Sun, Yujia; Nien, Chung-Yi; Chen, Kai; Liu, Hsiao-Yun; Johnston, Jeff; Zeitlinger, Julia; Rushlow, Christine

    2015-11-01

    The Drosophila genome activator Vielfaltig (Vfl), also known as Zelda (Zld), is thought to prime enhancers for activation by patterning transcription factors (TFs). Such priming is accompanied by increased chromatin accessibility, but the mechanisms by which this occurs are poorly understood. Here, we analyze the effect of Zld on genome-wide nucleosome occupancy and binding of the patterning TF Dorsal (Dl). Our results show that early enhancers are characterized by an intrinsically high nucleosome barrier. Zld tackles this nucleosome barrier through local depletion of nucleosomes with the effect being dependent on the number and position of Zld motifs. Without Zld, Dl binding decreases at enhancers and redistributes to open regions devoid of enhancer activity. We propose that Zld primes enhancers by lowering the high nucleosome barrier just enough to assist TFs in accessing their binding motifs and promoting spatially controlled enhancer activation if the right patterning TFs are present. We envision that genome activators in general will utilize this mechanism to activate the zygotic genome in a robust and precise manner.

  16. Abo1, a conserved bromodomain AAA-ATPase, maintains global nucleosome occupancy and organisation.

    Science.gov (United States)

    Gal, Csenge; Murton, Heather E; Subramanian, Lakxmi; Whale, Alex J; Moore, Karen M; Paszkiewicz, Konrad; Codlin, Sandra; Bähler, Jürg; Creamer, Kevin M; Partridge, Janet F; Allshire, Robin C; Kent, Nicholas A; Whitehall, Simon K

    2016-01-01

    Maintenance of the correct level and organisation of nucleosomes is crucial for genome function. Here, we uncover a role for a conserved bromodomain AAA-ATPase, Abo1, in the maintenance of nucleosome architecture in fission yeast. Cells lacking abo1(+) experience both a reduction and mis-positioning of nucleosomes at transcribed sequences in addition to increased intragenic transcription, phenotypes that are hallmarks of defective chromatin re-establishment behind RNA polymerase II. Abo1 is recruited to gene sequences and associates with histone H3 and the histone chaperone FACT. Furthermore, the distribution of Abo1 on chromatin is disturbed by impaired FACT function. The role of Abo1 extends to some promoters and also to silent heterochromatin. Abo1 is recruited to pericentromeric heterochromatin independently of the HP1 ortholog, Swi6, where it enforces proper nucleosome occupancy. Consequently, loss of Abo1 alleviates silencing and causes elevated chromosome mis-segregation. We suggest that Abo1 provides a histone chaperone function that maintains nucleosome architecture genome-wide.

  17. Promoter-proximal transcription factor binding is transcriptionally active when coupled with nucleosome repositioning in immediate vicinity

    Science.gov (United States)

    Yadav, Vinod Kumar; Thakur, Ram Krishna; Eckloff, Bruce; Baral, Aradhita; Singh, Ankita; Halder, Rashi; Kumar, Akinchan; Alam, Mohammad Parwez; Kundu, Tapas K.; Pandita, Raj; Pandita, Tej K.; Wieben, Eric D.; Chowdhury, Shantanu

    2014-01-01

    Previous studies have analyzed patterns of transcription, transcription factor (TF) binding or mapped nucleosome occupancy across the genome. These suggest that the three aspects are genetically connected but the cause and effect relationships are still unknown. For example, physiologic TF binding studies involve many TFs, consequently, it is difficult to assign nucleosome reorganization to the binding site occupancy of any particular TF. Therefore, several aspects remain unclear: does TF binding influence nucleosome (re)organizations locally or impact the chromatin landscape at a more global level; are all or only a fraction of TF binding a result of reorganization in nucleosome occupancy and do all TF binding and associated changes in nucleosome occupancy result in altered gene expression? With these in mind, following characterization of two states (before and after induction of a single TF of choice) we determined: (i) genomic binding sites of the TF, (ii) promoter nucleosome occupancy and (iii) transcriptome profiles. Results demonstrated that promoter-proximal TF binding influenced expression of the target gene when it was coupled to nucleosome repositioning at or close to its binding site in most cases. In contrast, only in few cases change in target gene expression was found when TF binding occurred without local nucleosome reorganization. PMID:25081206

  18. Nucleosome-induced neutrophil activation occurs independently of TLR9 and endosomal acidification: implications for systemic lupus erythematosus.

    NARCIS (Netherlands)

    Lindau, D.S.U.; Ronnefarth, V.; Erbacher, A.; Rammensee, H.G.; Decker, P. de

    2011-01-01

    The nucleosome is a major autoantigen known to activate PMN in systemic lupus erythematosus (SLE). TLR9 recognizes bacterial and even mammalian DNA under certain circumstances. Nevertheless, the role of TLR9 in SLE development is still unclear. Since nucleosomes are composed of DNA, we investigated

  19. Physical properties of naked DNA influence nucleosome positioning and correlate with transcription start and termination sites in yeast

    Directory of Open Access Journals (Sweden)

    Soler-López Montserrat

    2011-10-01

    Full Text Available Abstract Background In eukaryotic organisms, DNA is packaged into chromatin structure, where most of DNA is wrapped into nucleosomes. DNA compaction and nucleosome positioning have clear functional implications, since they modulate the accessibility of genomic regions to regulatory proteins. Despite the intensive research effort focused in this area, the rules defining nucleosome positioning and the location of DNA regulatory regions still remain elusive. Results Naked (histone-free and nucleosomal DNA from yeast were digested by microccocal nuclease (MNase and sequenced genome-wide. MNase cutting preferences were determined for both naked and nucleosomal DNAs. Integration of their sequencing profiles with DNA conformational descriptors derived from atomistic molecular dynamic simulations enabled us to extract the physical properties of DNA on a genomic scale and to correlate them with chromatin structure and gene regulation. The local structure of DNA around regulatory regions was found to be unusually flexible and to display a unique pattern of nucleosome positioning. Ab initio physical descriptors derived from molecular dynamics were used to develop a computational method that accurately predicts nucleosome enriched and depleted regions. Conclusions Our experimental and computational analyses jointly demonstrate a clear correlation between sequence-dependent physical properties of naked DNA and regulatory signals in the chromatin structure. These results demonstrate that nucleosome positioning around TSS (Transcription Start Site and TTS (Transcription Termination Site (at least in yeast is strongly dependent on DNA physical properties, which can define a basal regulatory mechanism of gene expression.

  20. Transmission of modified nucleosomes from the mouse male germline to the zygote and subsequent remodeling of paternal chromatin.

    NARCIS (Netherlands)

    Heijden, G.W. van der; Derijck, A.H.A.; Ramos, L.; Giele, M.M.; Vlag, J. van der; Boer, P. de

    2006-01-01

    Rapidly after gamete fusion, the sperm nucleus loses its specific chromatin conformation and the DNA is repopulated with maternally derived nucleosomes. We evaluated the nature of paternally derived nucleosomes and the dynamics of sperm chromatin remodeling in the zygote directly after gamete fusion

  1. Habitat drives dispersal and survival of translocated juvenile desert tortoises

    Science.gov (United States)

    Nafus, Melia G.; Esque, Todd; Averill-Murray, Roy C.; Nussear, Kenneth E.; Swaisgood, Ronald R.

    2017-01-01

    1.In spite of growing reliance on translocations in wildlife conservation, translocation efficacy remains inconsistent. One factor that can contribute to failed translocations is releasing animals into poor quality or otherwise inadequate habitat.

  2. Structural Basis of Silencing: Sir3 BAH Domain in Complex with a Nucleosome at 3.0 Å Resolution

    Energy Technology Data Exchange (ETDEWEB)

    Armache, Karim-Jean; Garlick, Joseph D.; Canzio, Daniele; Narlikar, Geeta J.; Kingston, Robert E. (UCSF); (Mass. Gen. Hosp.)

    2011-11-30

    Gene silencing is essential for regulating cell fate in eukaryotes. Altered chromatin architectures contribute to maintaining the silenced state in a variety of species. The silent information regulator (Sir) proteins regulate mating type in Saccharomyces cerevisiae. One of these proteins, Sir3, interacts directly with the nucleosome to help generate silenced domains. We determined the crystal structure of a complex of the yeast Sir3 BAH (bromo-associated homology) domain and the nucleosome core particle at 3.0 angstrom resolution. We see multiple molecular interactions between the protein surfaces of the nucleosome and the BAH domain that explain numerous genetic mutations. These interactions are accompanied by structural rearrangements in both the nucleosome and the BAH domain. The structure explains how covalent modifications on H4K16 and H3K79 regulate formation of a silencing complex that contains the nucleosome as a central component.

  3. The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

    Directory of Open Access Journals (Sweden)

    Vitali Bialevich

    2017-01-01

    Full Text Available Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested.

  4. The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

    Science.gov (United States)

    Shamayeva, Katsiaryna; Guzanova, Alena; Řeha, David; Csefalvay, Eva; Carey, Jannette; Weiserova, Marie

    2017-01-01

    Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested. PMID:28133570

  5. Comparative studies of genome-wide maps of nucleosomes between deletion mutants of elp3 and hos2 genes of Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Takashi Matsumoto

    Full Text Available In order to elucidate the influence of histone acetylation upon nucleosomal DNA length and nucleosome position, we compared nucleosome maps of the following three yeast strains; strain BY4741 (control, the elp3 (one of histone acetyltransferase genes deletion mutant, and the hos2 (one of histone deactylase genes deletion mutant of Saccharomyces cerevisiae. We sequenced mononucleosomal DNA fragments after treatment with micrococcal nuclease. After mapping the DNA fragments to the genome, we identified the nucleosome positions. We showed that the distributions of the nucleosomal DNA lengths of the control and the hos2 disruptant were similar. On the other hand, the distribution of the nucleosomal DNA lengths of the elp3 disruptant shifted toward shorter than that of the control. It strongly suggests that inhibition of Elp3-induced histone acetylation causes the nucleosomal DNA length reduction. Next, we compared the profiles of nucleosome mapping numbers in gene promoter regions between the control and the disruptant. We detected 24 genes with low conservation level of nucleosome positions in promoters between the control and the elp3 disruptant as well as between the control and the hos2 disruptant. It indicates that both Elp3-induced acetylation and Hos2-induced deacetylation influence the nucleosome positions in the promoters of those 24 genes. Interestingly, in 19 of the 24 genes, the profiles of nucleosome mapping numbers were similar between the two disruptants.

  6. DNA nanopore translocation in glutamate solutions

    NARCIS (Netherlands)

    Plesa, C.; Van Loo, N.; Dekker, C.

    2015-01-01

    Nanopore experiments have traditionally been carried out with chloride-based solutions. Here we introduce silver/silver-glutamate-based electrochemistry as an alternative, and study the viscosity, conductivity, and nanopore translocation characteristics of potassium-, sodium-, and lithium-glutamate

  7. Dudleya Variegata Translocation - San Diego [ds654

    Data.gov (United States)

    California Department of Resources — At Mission Trails Regional Park, a translocation project of Dudleya variegata was conducted in efforts to save the population from a private property undergoing...

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

    Science.gov (United States)

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

    2015-11-26

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

  9. MeCP2 binds to nucleosome free (linker DNA) regions and to H3K9/H3K27 methylated nucleosomes in the brain

    Science.gov (United States)

    Thambirajah, Anita A.; Ng, Marlee K.; Frehlick, Lindsay J.; Li, Andra; Serpa, Jason J.; Petrotchenko, Evgeniy V.; Silva-Moreno, Begonia; Missiaen, Kristal K.; Borchers, Christoph H.; Adam Hall, J.; Mackie, Ryan; Lutz, Frank; Gowen, Brent E.; Hendzel, Michael; Georgel, Philippe T.; Ausió, Juan

    2012-01-01

    Methyl-CpG-binding protein 2 (MeCP2) is a chromatin-binding protein that mediates transcriptional regulation, and is highly abundant in brain. The nature of its binding to reconstituted templates has been well characterized in vitro. However, its interactions with native chromatin are less understood. Here we show that MeCP2 displays a distinct distribution within fractionated chromatin from various tissues and cell types. Artificially induced global changes in DNA methylation by 3-aminobenzamide or 5-aza-2′-deoxycytidine, do not significantly affect the distribution or amount of MeCP2 in HeLa S3 or 3T3 cells. Most MeCP2 in brain is chromatin-bound and localized within highly nuclease-accessible regions. We also show that, while in most tissues and cell lines, MeCP2 forms stable complexes with nucleosome, in brain, a fraction of it is loosely bound to chromatin, likely to nucleosome-depleted regions. Finally, we provide evidence for novel associations of MeCP2 with mononucleosomes containing histone H2A.X, H3K9me2 and H3K27me3 in different chromatin fractions from brain cortex and in vitro. We postulate that the functional compartmentalization and tissue-specific distribution of MeCP2 within different chromatin types may be directed by its association with nucleosomes containing specific histone variants, and post-translational modifications. PMID:22144686

  10. Chromatin modification by PSC occurs at one PSC per nucleosome and does not require the acidic patch of histone H2A.

    Science.gov (United States)

    Lo, Stanley M; McElroy, Kyle A; Francis, Nicole J

    2012-01-01

    Chromatin architecture is regulated through both enzymatic and non-enzymatic activities. For example, the Polycomb Group (PcG) proteins maintain developmental gene silencing using an array of chromatin-based mechanisms. The essential Drosophila PcG protein, Posterior Sex Combs (PSC), compacts chromatin and inhibits chromatin remodeling and transcription through a non-enzymatic mechanism involving nucleosome bridging. Nucleosome bridging is achieved through a combination of nucleosome binding and self-interaction. Precisely how PSC interacts with chromatin to bridge nucleosomes is not known and is the subject of this work. We determine the stoichiometry of PSC-chromatin interactions in compact chromatin (in which nucleosomes are bridged) using Scanning Transmission Electron Microscopy (STEM). We find that full compaction occurs with one PSC per nucleosome. In addition to compacting chromatin, we show that PSC oligomerizes nucleosome arrays. PSC-mediated oligomerization of chromatin occurs at similar stoichiometry as compaction suggesting it may also involve nucleosome bridging. Interactions between the tail of histone H4 and the acidic patch of histone H2A are important for chromatin folding and oligomerization, and several chromatin proteins bind the histone H2A acidic patch. However, mutation of the acidic patch of histone H2A does not affect PSC's ability to inhibit chromatin remodeling or bridge nucleosomes. In fact, PSC does not require nucleosomes for bridging activity but can bridge naked DNA segments. PSC clusters nucleosomes on sparsely assembled templates, suggesting it interacts preferentially with nucleosomes over bare DNA. This may be due to the ability of PSC to bind free histones. Our data are consistent with a model in which each PSC binds a nucleosome and at least one other PSC to directly bridge nucleosomes and compact chromatin, but also suggest that naked DNA can be included in compacted structures. We discuss how our data highlight the diversity

  11. Chromatin modification by PSC occurs at one PSC per nucleosome and does not require the acidic patch of histone H2A.

    Directory of Open Access Journals (Sweden)

    Stanley M Lo

    Full Text Available Chromatin architecture is regulated through both enzymatic and non-enzymatic activities. For example, the Polycomb Group (PcG proteins maintain developmental gene silencing using an array of chromatin-based mechanisms. The essential Drosophila PcG protein, Posterior Sex Combs (PSC, compacts chromatin and inhibits chromatin remodeling and transcription through a non-enzymatic mechanism involving nucleosome bridging. Nucleosome bridging is achieved through a combination of nucleosome binding and self-interaction. Precisely how PSC interacts with chromatin to bridge nucleosomes is not known and is the subject of this work. We determine the stoichiometry of PSC-chromatin interactions in compact chromatin (in which nucleosomes are bridged using Scanning Transmission Electron Microscopy (STEM. We find that full compaction occurs with one PSC per nucleosome. In addition to compacting chromatin, we show that PSC oligomerizes nucleosome arrays. PSC-mediated oligomerization of chromatin occurs at similar stoichiometry as compaction suggesting it may also involve nucleosome bridging. Interactions between the tail of histone H4 and the acidic patch of histone H2A are important for chromatin folding and oligomerization, and several chromatin proteins bind the histone H2A acidic patch. However, mutation of the acidic patch of histone H2A does not affect PSC's ability to inhibit chromatin remodeling or bridge nucleosomes. In fact, PSC does not require nucleosomes for bridging activity but can bridge naked DNA segments. PSC clusters nucleosomes on sparsely assembled templates, suggesting it interacts preferentially with nucleosomes over bare DNA. This may be due to the ability of PSC to bind free histones. Our data are consistent with a model in which each PSC binds a nucleosome and at least one other PSC to directly bridge nucleosomes and compact chromatin, but also suggest that naked DNA can be included in compacted structures. We discuss how our data

  12. The human IL-2 gene promoter can assemble a positioned nucleosome that becomes remodeled upon T cell activation.

    Science.gov (United States)

    Attema, Joanne L; Reeves, Raymond; Murray, Vincent; Levichkin, Ilya; Temple, Mark D; Tremethick, David J; Shannon, M Frances

    2002-09-01

    Controlled production of the cytokine IL-2 plays a key role in the mammalian immune system. Expression from the gene is tightly regulated with no detectable expression in resting T cells and a strong induction following T cell activation. The IL-2 proximal promoter (+1 to -300) contains many well-defined transcriptional activation elements that respond to T cell stimulation. To determine the role of chromatin structure in the regulation of interleukin-2 gene transcription, nucleosome assembly across the IL-2 promoter region was examined using in vitro chromatin reconstitution assays. The IL-2 promoter assembles a nucleosome that is both translationally and rotationally positioned, spanning some of the major functional control elements. The binding of transcription factors to these elements, with the exception of the architectural protein HMGA1, was occluded by the presence of the nucleosome. Analysis of the chromatin architecture of the IL-2 gene in Jurkat T cells provided evidence for the presence of a similarly positioned nucleosome in vivo. The region encompassed by this nucleosome becomes remodeled following activation of Jurkat T cells. These observations suggest that the presence of a positioned nucleosome across the IL-2 proximal promoter may play an important role in maintaining an inactive gene in resting T cells and that remodeling of this nucleosome is important for gene activation.

  13. The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling.

    Science.gov (United States)

    Shukla, Manu Shubhdarshan; Syed, Sajad Hussain; Goutte-Gattat, Damien; Richard, John Lalith Charles; Montel, Fabien; Hamiche, Ali; Travers, Andrew; Faivre-Moskalenko, Cendrine; Bednar, Jan; Hayes, Jeffrey J; Angelov, Dimitar; Dimitrov, Stefan

    2011-04-01

    Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of ∼10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a 'defective' docking domain may be a primary structural role of H2A.Bbd in chromatin.

  14. Changes in nucleosome position at transcriptional start sites of specific genes in Zea mays mediator of paramutation1 mutants.

    Science.gov (United States)

    Labonne, Jonathan D J; Dorweiler, Jane E; McGinnis, Karen M

    2013-04-01

    Nucleosomes facilitate compaction of DNA within the confines of the eukaryotic nucleus. This packaging of DNA and histone proteins must accommodate cellular processes, such as transcription and DNA replication. The repositioning of nucleosomes to facilitate cellular processes is likely regulated by several factors. In Zea mays, Mediator of paramutation1 (MOP1) has been demonstrated to be an epigenetic regulator of gene expression. Based on sequence orthology and mutant phenotypes, MOP1 is likely to function in an RNA-dependent pathway to mediate changes to chromatin. High-resolution microarrays were used to assay the distribution of nucleosomes across the transcription start sites (TSSs) of ~400 maize genes in wild type and mutant mop1-1 tissues. Analysis of nucleosome distribution in leaf, immature tassel and ear shoot tissues resulted in the identification of three genes showing consistent differences in nucleosome positioning and occupancy between wild type and mutant mop1-1. These specific changes in nucleosome distribution were located upstream as well as downstream of the TSS. No direct relationship between the specific changes in nucleosome distribution and transcription were observed through quantitative expression analysis in these tissues. In silico prediction suggests that nucleosome positioning is not dictated by intrinsic DNA sequence signals in the TSSs of two of the identified genes, suggesting a role for chromatin remodeling proteins in MOP1-mediated pathways. These results also indicate that MOP1 contributions to nucleosome position may be either separate from changes in gene expression, or cooperative with development and other levels of regulation in coordinating gene expression.

  15. Dynamic Conformations of Nucleosome Arrays in Solution from Small-Angle X-ray Scattering

    Energy Technology Data Exchange (ETDEWEB)

    Howell, Steven C. [George Washington Univ., Washington, DC (United States)

    2016-01-31

    We set out to determine quantitative information regarding the dynamic conformation of nucleosome arrays in solution using experimental SAXS. Toward this end, we developed a CG simulation algorithm for dsDNA which rapidly generates ensembles of structures through Metropolis MC sampling of a Markov chain.

  16. Pre-mRNA splicing is a determinant of nucleosome organization.

    Directory of Open Access Journals (Sweden)

    Hadas Keren-Shaul

    Full Text Available Chromatin organization affects alternative splicing and previous studies have shown that exons have increased nucleosome occupancy compared with their flanking introns. To determine whether alternative splicing affects chromatin organization we developed a system in which the alternative splicing pattern switched from inclusion to skipping as a function of time. Changes in nucleosome occupancy were correlated with the change in the splicing pattern. Surprisingly, strengthening of the 5' splice site or strengthening the base pairing of U1 snRNA with an internal exon abrogated the skipping of the internal exons and also affected chromatin organization. Over-expression of splicing regulatory proteins also affected the splicing pattern and changed nucleosome occupancy. A specific splicing inhibitor was used to show that splicing impacts nucleosome organization endogenously. The effect of splicing on the chromatin required a functional U1 snRNA base pairing with the 5' splice site, but U1 pairing was not essential for U1 snRNA enhancement of transcription. Overall, these results suggest that splicing can affect chromatin organization.

  17. Regulation of Nucleosome Architecture and Factor Binding Revealed by Nuclease Footprinting of the ESC Genome.

    Science.gov (United States)

    Hainer, Sarah J; Fazzio, Thomas G

    2015-10-06

    Functional interactions between gene regulatory factors and chromatin architecture have been difficult to directly assess. Here, we use micrococcal nuclease (MNase) footprinting to probe the functions of two chromatin-remodeling complexes. By simultaneously quantifying alterations in small MNase footprints over the binding sites of 30 regulatory factors in mouse embryonic stem cells (ESCs), we provide evidence that esBAF and Mbd3/NuRD modulate the binding of several regulatory proteins. In addition, we find that nucleosome occupancy is reduced at specific loci in favor of subnucleosomes upon depletion of esBAF, including sites of histone H2A.Z localization. Consistent with these data, we demonstrate that esBAF is required for normal H2A.Z localization in ESCs, suggesting esBAF either stabilizes H2A.Z-containing nucleosomes or promotes subnucleosome to nucleosome conversion by facilitating H2A.Z deposition. Therefore, integrative examination of MNase footprints reveals insights into nucleosome dynamics and functional interactions between chromatin structure and key gene-regulatory factors.

  18. Regulation of Nucleosome Architecture and Factor Binding Revealed by Nuclease Footprinting of the ESC Genome

    Directory of Open Access Journals (Sweden)

    Sarah J. Hainer

    2015-10-01

    Full Text Available Functional interactions between gene regulatory factors and chromatin architecture have been difficult to directly assess. Here, we use micrococcal nuclease (MNase footprinting to probe the functions of two chromatin-remodeling complexes. By simultaneously quantifying alterations in small MNase footprints over the binding sites of 30 regulatory factors in mouse embryonic stem cells (ESCs, we provide evidence that esBAF and Mbd3/NuRD modulate the binding of several regulatory proteins. In addition, we find that nucleosome occupancy is reduced at specific loci in favor of subnucleosomes upon depletion of esBAF, including sites of histone H2A.Z localization. Consistent with these data, we demonstrate that esBAF is required for normal H2A.Z localization in ESCs, suggesting esBAF either stabilizes H2A.Z-containing nucleosomes or promotes subnucleosome to nucleosome conversion by facilitating H2A.Z deposition. Therefore, integrative examination of MNase footprints reveals insights into nucleosome dynamics and functional interactions between chromatin structure and key gene-regulatory factors.

  19. Structure-based identification of new high-affinity nucleosome binding sequences.

    Science.gov (United States)

    Battistini, Federica; Hunter, Christopher A; Moore, Irene K; Widom, Jonathan

    2012-06-29

    The substrate for the proteins that express genetic information in the cell is not naked DNA but an assembly of nucleosomes, where the DNA is wrapped around histone proteins. The organization of these nucleosomes on genomic DNA is influenced by the DNA sequence. Here, we present a structure-based computational approach that translates sequence information into the energy required to bend DNA into a nucleosome-bound conformation. The calculations establish the relationship between DNA sequence and histone octamer binding affinity. In silico selection using this model identified several new DNA sequences, which were experimentally found to have histone octamer affinities comparable to the highest-affinity sequences known. The results provide insights into the molecular mechanism through which DNA sequence information encodes its organization. A quantitative appreciation of the thermodynamics of nucleosome positioning and rearrangement will be one of the key factors in understanding the regulation of transcription and in the design of new promoter architectures for the purposes of tuning gene expression dynamics.

  20. Human tNASP promotes in vitro nucleosome assembly with histone H3.3.

    Science.gov (United States)

    Kato, Daiki; Osakabe, Akihisa; Tachiwana, Hiroaki; Tanaka, Hiroki; Kurumizaka, Hitoshi

    2015-02-10

    Nuclear autoantigenic sperm proteins (NASPs) are members of the acidic histone chaperones, which promote nucleosome assembly. In humans, two splicing variants proposed for the somatic and testicular isoforms, sNASP and tNASP, respectively, have been found, and the shorter form, sNASP, reportedly promotes nucleosome assembly with the histone H3 isoforms, H3.1, H3.2, and H3.3. However, the biochemical properties of the longer form, tNASP, have not been reported. tNASP is considered to exist specifically in the testis. Our present results revealed that the tNASP protein is ubiquitously produced in various human tissues, in addition to testis. Unexpectedly, we found that the nucleosome assembly activity of purified tNASP was extremely low with the canonical histone H3.1 or H3.2, but was substantially detected with the replacement histone H3.3 variant. A mutational analysis revealed that the H3.3 Ile89 residue, corresponding to the H3.1 Val89 residue, is responsible for the tNASP-mediated nucleosome assembly with H3.3. A histone deposition assay showed that the H3.3-H4 complex is more efficiently deposited onto DNA by tNASP than the H3.1-H4 complex. These results provide evidence that tNASP is ubiquitously produced in various types of human tissues and promotes in vitro nucleosome assembly with H3 variant specificity.

  1. An ensemble of B-DNA dinucleotide geometries lead to characteristic nucleosomal DNA structure and provide plasticity required for gene expression

    Directory of Open Access Journals (Sweden)

    Bansal Manju

    2011-01-01

    Full Text Available Abstract Background A nucleosome is the fundamental repeating unit of the eukaryotic chromosome. It has been shown that the positioning of a majority of nucleosomes is primarily controlled by factors other than the intrinsic preference of the DNA sequence. One of the key questions in this context is the role, if any, that can be played by the variability of nucleosomal DNA structure. Results In this study, we have addressed this question by analysing the variability at the dinucleotide and trinucleotide as well as longer length scales in a dataset of nucleosome X-ray crystal structures. We observe that the nucleosome structure displays remarkable local level structural versatility within the B-DNA family. The nucleosomal DNA also incorporates a large number of kinks. Conclusions Based on our results, we propose that the local and global level versatility of B-DNA structure may be a significant factor modulating the formation of nucleosomes in the vicinity of high-plasticity genes, and in varying the probability of binding by regulatory proteins. Hence, these factors should be incorporated in the prediction algorithms and there may not be a unique 'template' for predicting putative nucleosome sequences. In addition, the multimodal distribution of dinucleotide parameters for some steps and the presence of a large number of kinks in the nucleosomal DNA structure indicate that the linear elastic model, used by several algorithms to predict the energetic cost of nucleosome formation, may lead to incorrect results.

  2. Stochastic resonance during a polymer translocation process.

    Science.gov (United States)

    Mondal, Debasish; Muthukumar, M

    2016-04-14

    We have studied the occurrence of stochastic resonance when a flexible polymer chain undergoes a single-file translocation through a nano-pore separating two spherical cavities, under a time-periodic external driving force. The translocation of the chain is controlled by a free energy barrier determined by chain length, pore length, pore-polymer interaction, and confinement inside the donor and receiver cavities. The external driving force is characterized by a frequency and amplitude. By combining the Fokker-Planck formalism for polymer translocation and a two-state model for stochastic resonance, we have derived analytical formulas for criteria for emergence of stochastic resonance during polymer translocation. We show that no stochastic resonance is possible if the free energy barrier for polymer translocation is purely entropic in nature. The polymer chain exhibits stochastic resonance only in the presence of an energy threshold in terms of polymer-pore interactions. Once stochastic resonance is feasible, the chain entropy controls the optimal synchronization conditions significantly.

  3. Translocation pathways for inhaled asbestos fibers

    Directory of Open Access Journals (Sweden)

    Mantegazza F

    2008-01-01

    Full Text Available Abstract We discuss the translocation of inhaled asbestos fibers based on pulmonary and pleuro-pulmonary interstitial fluid dynamics. Fibers can pass the alveolar barrier and reach the lung interstitium via the paracellular route down a mass water flow due to combined osmotic (active Na+ absorption and hydraulic (interstitial pressure is subatmospheric pressure gradient. Fibers can be dragged from the lung interstitium by pulmonary lymph flow (primary translocation wherefrom they can reach the blood stream and subsequently distribute to the whole body (secondary translocation. Primary translocation across the visceral pleura and towards pulmonary capillaries may also occur if the asbestos-induced lung inflammation increases pulmonary interstitial pressure so as to reverse the trans-mesothelial and trans-endothelial pressure gradients. Secondary translocation to the pleural space may occur via the physiological route of pleural fluid formation across the parietal pleura; fibers accumulation in parietal pleura stomata (black spots reflects the role of parietal lymphatics in draining pleural fluid. Asbestos fibers are found in all organs of subjects either occupationally exposed or not exposed to asbestos. Fibers concentration correlates with specific conditions of interstitial fluid dynamics, in line with the notion that in all organs microvascular filtration occurs from capillaries to the extravascular spaces. Concentration is high in the kidney (reflecting high perfusion pressure and flow and in the liver (reflecting high microvascular permeability while it is relatively low in the brain (due to low permeability of blood-brain barrier. Ultrafine fibers (length

  4. Liver Cirrhosis and Intestinal Bacterial Translocation

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    Intestinal barrier dysfunction, facilitating translocation of bacteria and bacterial products, plays an important role in the pathophysiology of liver cirrhosis and its complications. Intestinal defense system including microbial barrier, immunologic barrier, mechanical barrier, chemical barrier, plays an important role in the maintenance of intestinal function. Under normal circumstances, the intestinal barrier can prevent intestinal bacteria through the intestinal wall from spreading to the body. Severe infection, trauma, shock, cirrhosis, malnutrition, immune suppression conditions, intestinal bacteria and endotoxin translocation, can lead to multiple organ dysfunction. The intestinal microlfora is not only involved in the digestion of nutrients, but also in local immunity, forming a barrier against pathogenic microorganisms. The derangement of the gut microlfora may lead to microbial translocation, deifned as the passage of viable microorganisms or bacterial products from the intestinal lumen to the mesenteric lymph nodes and other extraintestinal sites. In patients with cirrhosis, primary and intestinal lfora imbalance, intestinal bacterial overgrowth, intestinal mucosal barrier dysfunction, endotoxemia is associated with weakened immunity.

  5. Nuclear translocation and retention of growth hormone

    DEFF Research Database (Denmark)

    Mertani, Hichem C; Raccurt, Mireille; Abbate, Aude

    2003-01-01

    We have previously demonstrated that GH is subject to rapid receptor-dependent nuclear translocation. Here, we examine the importance of ligand activation of the GH-receptor (GHR)-associated Janus kinase (JAK) 2 and receptor dimerization for hormone internalization and nuclear translocation by use...... of cells stably transfected with cDNA for the GHR. Staurosporine and herbimycin A treatment of cells did not affect the ability of GH to internalize but resulted in increased nuclear accumulation of hormone. Similarly, receptor mutations, which prevent the association and activation of JAK2, did not affect...... the ability of the hormone to internalize or translocate to the nucleus but resulted in increased nuclear accumulation of GH. These results were observed both by nuclear isolation and confocal laser scanning microscopy. Staurosporine treatment of cells in which human GH (hGH) was targeted to the cytoplasm...

  6. Rank Modulation for Translocation Error Correction

    CERN Document Server

    Farnoud, Farzad; Milenkovic, Olgica

    2012-01-01

    We consider rank modulation codes for flash memories that allow for handling arbitrary charge drop errors. Unlike classical rank modulation codes used for correcting errors that manifest themselves as swaps of two adjacently ranked elements, the proposed \\emph{translocation rank codes} account for more general forms of errors that arise in storage systems. Translocations represent a natural extension of the notion of adjacent transpositions and as such may be analyzed using related concepts in combinatorics and rank modulation coding. Our results include tight bounds on the capacity of translocation rank codes, construction techniques for asymptotically good codes, as well as simple decoding methods for one class of structured codes. As part of our exposition, we also highlight the close connections between the new code family and permutations with short common subsequences, deletion and insertion error-correcting codes for permutations and permutation arrays.

  7. DNA nanopore translocation in glutamate solutions

    Science.gov (United States)

    Plesa, C.; van Loo, N.; Dekker, C.

    2015-08-01

    Nanopore experiments have traditionally been carried out with chloride-based solutions. Here we introduce silver/silver-glutamate-based electrochemistry as an alternative, and study the viscosity, conductivity, and nanopore translocation characteristics of potassium-, sodium-, and lithium-glutamate solutions. We show that it has a linear response at typical voltages and can be used to detect DNA translocations through a nanopore. The glutamate anion also acts as a redox-capable thickening agent, with high-viscosity solutions capable of slowing down the DNA translocation process by up to 11 times, with a corresponding 7 time reduction in signal. These results demonstrate that glutamate can replace chloride as the primary anion in nanopore resistive pulse sensing.

  8. Polymer translocation through a nanopore: DPD study.

    Science.gov (United States)

    Yang, Kan; Vishnyakov, Aleksey; Neimark, Alexander V

    2013-04-04

    Translocation of a polymer chain through a narrow pore is explored using 3D explicit solvent dissipative particle dynamics simulation. We study the dependence of the translocation dynamics and translocation time τ on the chain length N, driving force magnitude E, and solvent quality. Two types of driving forces are considered: uniform hydrostatic force, which is applied equally to the chain and solvent particles, and uniform electrostatic force, which is applied selectively to the charged particles in the chain and oppositely charged counterions in the solvent. We concluded that the scaling correlations τ ~ E(-ξ) and τ ~ N(β) are valid only for coil-like chains. For globular chains, the exponents ξ and β could not be identified with a reasonable accuracy. While the found value of ξ agrees with published experimental results and does not depend on the driving force type, the exponent β depends on the driving force and solvent quality. This is explained by nonequilibrium effects, as in the systems considered, the time of translocation is comparable with the time of chain relaxation. These effects, manifested in the changes of chain conformation in the process of translocation, were analyzed on the basis of the variation of the gyration radii of cis and trans segments of the chain in normal and lateral directions. A prominent chain expansion was observed for coils and was insignificant for globules. This work demonstrates the feasibility of the 3D dissipative particle dynamics modeling of translocation phenomena and accounting for the electrostatic interactions with explicit counterions, as well as for the solvent quality, in a computationally efficient manner.

  9. The structural basis of modified nucleosome recognition by 53BP1.

    Science.gov (United States)

    Wilson, Marcus D; Benlekbir, Samir; Fradet-Turcotte, Amélie; Sherker, Alana; Julien, Jean-Philippe; McEwan, Andrea; Noordermeer, Sylvie M; Sicheri, Frank; Rubinstein, John L; Durocher, Daniel

    2016-08-01

    DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases RNF8 and RNF168, respectively. RNF168 ubiquitinates H2A on lysine 13 and lysine 15 (refs 7, 8) (yielding H2AK13ub and H2AK15ub, respectively), an event that triggers the recruitment of 53BP1 (also known as TP53BP1) to chromatin flanking DSBs. 53BP1 binds specifically to H2AK15ub-containing nucleosomes through a peptide segment termed the ubiquitination-dependent recruitment motif (UDR), which requires the simultaneous engagement of histone H4 lysine 20 dimethylation (H4K20me2) by its tandem Tudor domain. How 53BP1 interacts with these two histone marks in the nucleosomal context, how it recognizes ubiquitin, and how it discriminates between H2AK13ub and H2AK15ub is unknown. Here we present the electron cryomicroscopy (cryo-EM) structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle (NCP-ubme) at 4.5 Å resolution. The structure reveals that H4K20me2 and H2AK15ub recognition involves intimate contacts with multiple nucleosomal elements including the acidic patch. Ubiquitin recognition by 53BP1 is unusual and involves the sandwiching of the UDR segment between ubiquitin and the NCP surface. The selectivity for H2AK15ub is imparted by two arginine fingers in the H2A amino-terminal tail, which straddle the nucleosomal DNA and serve to position ubiquitin over the NCP-bound UDR segment. The structure of the complex between NCP-ubme and 53BP1 reveals the basis of 53BP1 recruitment to DSB sites and illuminates how combinations of histone marks and nucleosomal elements cooperate to produce highly specific chromatin responses, such as those elicited following chromosome breaks.

  10. Nucleosome adaptability conferred by sequence and structural variations in histone H2A-H2B dimers.

    Science.gov (United States)

    Shaytan, Alexey K; Landsman, David; Panchenko, Anna R

    2015-06-01

    Nucleosome variability is essential for their functions in compacting the chromatin structure and regulation of transcription, replication and cell reprogramming. The DNA molecule in nucleosomes is wrapped around an octamer composed of four types of core histones (H3, H4, H2A, H2B). Nucleosomes represent dynamic entities and may change their conformation, stability and binding properties by employing different sets of histone variants or by becoming post-translationally modified. There are many variants of histones H2A and H2B. Specific H2A and H2B variants may preferentially associate with each other resulting in different combinations of variants and leading to the increased combinatorial complexity of nucleosomes. In addition, the H2A-H2B dimer can be recognized and substituted by chaperones/remodelers as a distinct unit, can assemble independently and is stable during nucleosome unwinding. In this review we discuss how sequence and structural variations in H2A-H2B dimers may provide necessary complexity and confer the nucleosome functional variability.

  11. Nucleosome-specific, time-dependent changes in histone modifications during activation of the early growth response 1 (Egr1) gene.

    Science.gov (United States)

    Riffo-Campos, Ángela L; Castillo, Josefa; Tur, Gema; González-Figueroa, Paula; Georgieva, Elena I; Rodríguez, José L; López-Rodas, Gerardo; Rodrigo, M Isabel; Franco, Luis

    2015-01-01

    Histone post-translational modifications and nucleosome remodeling are coordinate events involved in eukaryotic transcriptional regulation. There are relatively few data on the time course with which these events occur in individual nucleosomes. As a contribution to fill this gap, we first describe the nature and time course of structural changes in the nucleosomes -2, -1, and +1 of the murine Egr1 gene upon induction. To initiate the transient activation of the gene, we used the stimulation of MLP29 cells with phorbol esters and the in vivo activation after partial hepatectomy. In both models, nucleosomes -1 and +1 are partially evicted, whereas nucleosomes +1 and -2 slide downstream during transcription. The sliding of the latter nucleosome allows the EGR1 protein to bind its site, resulting in the repression of the gene. To decide whether EGR1 is involved in the sliding of nucleosome -2, Egr1 was knocked down. In the absence of detectable EGR1, the nucleosome still slides and remains downstream longer than in control cells, suggesting that the product of the gene may be rather involved in the returning of the nucleosome to the basal position. Moreover, the presence of eight epigenetic histone marks has been determined at a mononucleosomal level in that chromatin region. H3S10phK14ac, H3K4me3, H3K9me3, and H3K27me3 are characteristic of nucleosome +1, and H3K9ac and H4K16ac are mainly found in nucleosome -1, and H3K27ac predominates in nucleosomes -2 and -1. The temporal changes in these marks suggest distinct functions for some of them, although changes in H3K4me3 may result from histone turnover.

  12. Tissue Nitrogen and Fructan Translocation in Bread Wheat

    Institute of Scientific and Technical Information of China (English)

    HOU You-liang; L.O'Brien; ZHONG Gai-rong

    2002-01-01

    Translocation of previously accumulated nitrogen and carbohydrates from vegetative tissue of the wheat plant is a major assimilate source for grain filling. This study was conducted to examine genotype differences in nitrogen and fructan translocation and their relationships to grain yield and protein content. Effects indicated that significant genotype differences existed for nitrogen accumulation at anthesis and fructan at milk stage and their translocation. Two high protein genotypes, Cunningham and PST90-19, accumulated more nitrogen before anthesis and had greater nitrogen translocation, but lower post-anthesis nitrogen uptake,than two low protein genotypes, SUN109A and TM56. Among plant parts, leaves were the major storage for tissue nitrogen and provided the overwhelming proportion of the total nitrogen translocation, whereas for fructan accumulation and translocation it was the stems. The two high protein genotypes had a higher percentage of their grain nitrogen derived from nitrogen translocation, while for the two low protein ones, it was from postanthesis nitrogen uptake and assimilation. Increasing nitrogen application increased nitrogen accumulation and translocation, but decreased fructan accumulation and translocation. High grain protein content was associated with high nitrogen translocation from leaves, stems and the total plant, while high grain yield was related to high fructan translocation from stems and the total plant. Fructan translocation was negatively correlated to grain protein content. Nitrogen and fructan translocation were not correlated with each other.

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

    Science.gov (United States)

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

    2016-05-08

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

  14. Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.

    Science.gov (United States)

    Knight, Britta; Kubik, Slawomir; Ghosh, Bhaswar; Bruzzone, Maria Jessica; Geertz, Marcel; Martin, Victoria; Dénervaud, Nicolas; Jacquet, Philippe; Ozkan, Burak; Rougemont, Jacques; Maerkl, Sebastian J; Naef, Félix; Shore, David

    2014-08-01

    In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and -1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these "fragile" nucleosomes play an important role in regulating RPG transcriptional output.

  15. Making copies of chromatin: the challenge of nucleosomal organization and epigenetic information.

    Science.gov (United States)

    Corpet, Armelle; Almouzni, Geneviève

    2009-01-01

    Understanding the basic mechanisms underlying chromatin dynamics during DNA replication in eukaryotic cells is of fundamental importance. Beyond DNA compaction, chromatin organization represents a means to regulate genome function. Thus, the inheritance and maintenance of the DNA sequence, along with its organization into chromatin, is central for eukaryotic life. To orchestrate DNA replication in the context of chromatin is a challenge, both in terms of accessibility to the compact structures and maintenance of chromatin organization. To meet the challenge of maintenance, cells have evolved efficient nucleosome dynamics involving assembly pathways and chromatin maturation mechanisms that restore chromatin organization in the wake of DNA replication. In this review, we describe our current knowledge concerning how these pathways operate at the nucleosomal level and highlight the key players, such as histone chaperones, chromatin remodelers or modifiers, involved in the process of chromatin duplication. Major advances have been made recently concerning de novo nucleosome assembly and our understanding of its coordination with recycling of parental histones is progressing. Insights into the transmission of chromatin-based information during replication have important implications in the field of epigenetics to fully comprehend how the epigenetic landscape might, or at times might not, be stably maintained in the face of dramatic changes in chromatin structure.

  16. Dynamic Conformations of Nucleosome Arrays in Solution from Small-Angle X-ray Scattering

    Science.gov (United States)

    Howell, Steven C.

    Chromatin conformation and dynamics remains unsolved despite the critical role of the chromatin in fundamental genetic functions such as transcription, replication, and repair. At the molecular level, chromatin can be viewed as a linear array of nucleosomes, each consisting of 147 base pairs (bp) of double-stranded DNA (dsDNA) wrapped around a protein core and connected by 10 to 90 bp of linker dsDNA. Using small-angle X-ray scattering (SAXS), we investigated how the conformations of model nucleosome arrays in solution are modulated by ionic condition as well as the effect of linker histone proteins. To facilitate ensemble modeling of these SAXS measurements, we developed a simulation method that treats coarse-grained DNA as a Markov chain, then explores possible DNA conformations using Metropolis Monte Carlo (MC) sampling. This algorithm extends the functionality of SASSIE, a program used to model intrinsically disordered biological molecules, adding to the previous methods for simulating protein, carbohydrates, and single-stranded DNA. Our SAXS measurements of various nucleosome arrays together with the MC generated models provide valuable solution structure information identifying specific differences from the structure of crystallized arrays.

  17. Influence of Rotational Nucleosome Positioning on Transcription Start Site Selection in Animal Promoters

    Science.gov (United States)

    Ambrosini, Giovanna; Bucher, Philipp

    2016-01-01

    The recruitment of RNA-Pol-II to the transcription start site (TSS) is an important step in gene regulation in all organisms. Core promoter elements (CPE) are conserved sequence motifs that guide Pol-II to the TSS by interacting with specific transcription factors (TFs). However, only a minority of animal promoters contains CPEs. It is still unknown how Pol-II selects the TSS in their absence. Here we present a comparative analysis of promoters’ sequence composition and chromatin architecture in five eukaryotic model organisms, which shows the presence of common and unique DNA-encoded features used to organize chromatin. Analysis of Pol-II initiation patterns uncovers that, in the absence of certain CPEs, there is a strong correlation between the spread of initiation and the intensity of the 10 bp periodic signal in the nearest downstream nucleosome. Moreover, promoters’ primary and secondary initiation sites show a characteristic 10 bp periodicity in the absence of CPEs. We also show that DNA natural variants in the region immediately downstream the TSS are able to affect both the nucleosome-DNA affinity and Pol-II initiation pattern. These findings support the notion that, in addition to CPEs mediated selection, sequence–induced nucleosome positioning could be a common and conserved mechanism of TSS selection in animals. PMID:27716823

  18. A Meier-Gorlin syndrome mutation impairs the ORC1-nucleosome association.

    Science.gov (United States)

    Zhang, Wei; Sankaran, Saumya; Gozani, Or; Song, Jikui

    2015-05-15

    Recent studies have identified several genetic mutations within the BAH domain of human Origin Recognition Complex subunit 1 (hORC1BAH), including the R105Q mutation, implicated in Meier-Gorlin Syndrome (MGS). However, the pathological role of the hORC1 R105Q mutation remains unclear. In this study, we have investigated the interactions of the hORC1BAH domain with histone H4K20me2, DNA, and the nucleosome core particle labeled with H4Kc20me2, a chemical analog of H4K20me2. Our study revealed a nucleosomal DNA binding site for hORC1BAH. The R105Q mutation reduces the hORC1BAH-DNA binding affinity, leading to impaired hORC1BAH-nucleosome interaction, which likely influences DNA replication initiation and MGS pathogenesis. This study provides an etiologic link between the hORC1 R105Q mutation and MGS.

  19. Nucleosome Density ChIP-Seq Identifies Distinct Chromatin Modification Signatures Associated with MNase Accessibility.

    Science.gov (United States)

    Lorzadeh, Alireza; Bilenky, Misha; Hammond, Colin; Knapp, David J H F; Li, Luolan; Miller, Paul H; Carles, Annaick; Heravi-Moussavi, Alireza; Gakkhar, Sitanshu; Moksa, Michelle; Eaves, Connie J; Hirst, Martin

    2016-11-15

    Nucleosome position, density, and post-translational modification are widely accepted components of mechanisms regulating DNA transcription but still incompletely understood. We present a modified native ChIP-seq method combined with an analytical framework that allows MNase accessibility to be integrated with histone modification profiles. Application of this methodology to the primitive (CD34+) subset of normal human cord blood cells enabled genomic regions enriched in one versus two nucleosomes marked by histone 3 lysine 4 trimethylation (H3K4me3) and/or histone 3 lysine 27 trimethylation (H3K27me3) to be associated with their transcriptional and DNA methylation states. From this analysis, we defined four classes of promoter-specific profiles and demonstrated that a majority of bivalent marked promoters are heterogeneously marked at a single-cell level in this primitive cell type. Interestingly, extension of this approach to human embryonic stem cells revealed an altered relationship between chromatin modification state and nucleosome content at promoters, suggesting developmental stage-specific organization of histone methylation states.

  20. Monitoring interactions at ATP-dependent drug efflux pumps

    NARCIS (Netherlands)

    Hendrikse, NH

    2000-01-01

    Chemotherapeutic treatment of cancer patients is often unsuccessful, due to the involvement of various mechanisms, leading to multidrug resistance (MDR). In this review, I describe the mechanisms involved in MDR. Furthermore, results obtained by imaging of P-glycoprotein (P-gp) and the multidrug res

  1. Clinical relevance of ATP-dependent potassium channels

    NARCIS (Netherlands)

    Ligtenberg, JJM; vanHaeften, TW; Links, TP; Smit, AJ

    1995-01-01

    Many cells are equipped with so-called potassium (K+) channels which have an important role in maintaining transmembrane potential. Closure of these channels leads to membrane depolarization, which can be followed by cell-specific activity such as contraction of vascular smooth muscle, or secretion

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

    Science.gov (United States)

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

    2015-01-01

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

  3. Promiscuous presentation and recognition of nucleosomal autoepitopes in lupus: role of autoimmune T cell receptor alpha chain.

    Science.gov (United States)

    Shi, Y; Kaliyaperumal, A; Lu, L; Southwood, S; Sette, A; Michaels, M A; Datta, S K

    1998-02-01

    T cells specific for nucleosomal autoepitopes are selectively expanded in lupus mice and these Th cells drive autoimmune B cells to produce pathogenic antinuclear antibodies. We transfected the TCR-alpha and -beta chain genes of a representative, pathogenic autoantibody-inducing Th clone specific for the nucleosomal core histone peptide H471-94 into TCR-negative recipient cells. Although the autoimmune TCRs were originally derived from SNF1 (I-Ad/q) mice, the transfectants could recognize the nucleosomal autoepitope presented by APC-bearing I-A molecules of all haplotypes tested, as well as human DR molecules. Competition assays indicated that the autoepitopes bound to the MHC class II groove. Most remarkably, MHC-unrestricted recognition of the nucleosomal peptide epitope was conferred by the lupus TCR-alpha chain even when it paired with a TCR-beta chain of irrelevant specificity. Several other disease-relevant Th clones and splenic T cells of lupus mice had similar properties. The TCR-alpha chains of these murine lupus Th clones shared related motifs and charged residues in their CDRs, and similar motifs were apparent even in TCR-alpha chains of human lupus Th clones. The lupus TCR-alpha chains probably contact the nucleosomal peptide complexed with MHC with relatively high affinity/avidity to sustain TCR signaling, because CD4 coreceptor was not required for promiscuous recognition. Indeed, pathogenic autoantibody-inducing, CD4-negative, TCR-alphabeta+ Th cells are expanded in systemic lupus erythematosus. These results have implications regarding thymic selection and peripheral expansion of nucleosome-specific T cells in lupus. They also suggest that universally tolerogenic epitopes could be designed for therapy of lupus patients with diverse HLA alleles. We propose to designate nucleosomes and other antigens bearing universal epitopes "Pantigens" (for promiscuous antigens).

  4. Quantized biopolymer translocation through nanopores: departure from simple scaling

    CERN Document Server

    Melchionna, Simone; Fyta, Maria; Kaxiras, Efthimios; Succi, Sauro

    2009-01-01

    We discuss multiscale simulations of long biopolymer translocation through wide nanopores that can accommodate multiple polymer strands. The simulations provide clear evidence of folding quantization, namely, the translocation proceeds through multi-folded configurations characterized by a well-defined integer number of folds. As a consequence, the translocation time acquires a dependence on the average folding number, which results in a deviation from the single-exponent power-law characterizing single-file translocation through narrow pores. The mechanism of folding quantization allows polymers above a threshold length (approximately $1,000$ persistence lengths for double-stranded DNA) to exhibit cooperative behavior and as a result to translocate noticeably faster.

  5. Genes and translocations involved in POF.

    Science.gov (United States)

    Schlessinger, David; Herrera, Luisa; Crisponi, Laura; Mumm, Steven; Percesepe, Antonio; Pellegrini, Massimo; Pilia, Giuseppe; Forabosco, Antonino

    2002-08-15

    Changes at a single autosomal locus and many X-linked loci have been implicated in women with gonadal dysgenesis [premature ovarian failure (POF) with deficits in ovarian follicles]. For the chromosome 3 locus, a forkhead transcription factor gene (FOXL2) has been identified, in which lesions result in decreased follicles by haploinsufficiency. In contrast, sporadic X; autosomal translocations are distributed at many points on the X, but concentrate in a critical region on Xq. The association of the breakpoints with genes involved in ovarian function is thus far weak (in four analyzed cases) and has not been related to pathology in other POF patients. While many more translocations can be analyzed in detail as the human genome sequence is refined, it remains possible that translocations like X monosomy (Turner syndrome) lead to POF not by interrupting specific genes important in ovarian development, but by causing aberrations in pairing or X-inactivation during folliculogenesis. It is noted that the critical region has unusual features, neighboring the X-inactivation center and including an 18 Mb region of very low recombination. These suggest that chromosome dynamics in the region may be sensitive to structural changes, and when modified by translocations might provoke apoptosis at meiotic checkpoints. Choices among models for the etiology of POF should be feasible based on studies of ovarian follicle development and attrition in mouse models. Studies would prominently include gene expression profiling of developmental-specific pathways in nascent ovaries with controlled levels of Foxl2 and interacting proteins, or with defined changes in the X chromosome.

  6. Hinge and chromoshadow of HP1α participate in recognition of K9 methylated histone H3 in nucleosomes.

    Science.gov (United States)

    Mishima, Yuichi; Watanabe, Makoto; Kawakami, Toru; Jayasinghe, Chanika D; Otani, Junji; Kikugawa, Yusuke; Shirakawa, Masahiro; Kimura, Hiroshi; Nishimura, Osamu; Aimoto, Saburo; Tajima, Shoji; Suetake, Isao

    2013-01-09

    The majority of the genome in eukaryotes is packaged into transcriptionally inactive chromatin. Heterochromatin protein 1 (HP1) is a major player in the establishment and maintenance of heterochromatin. HP1 specifically recognizes a methylated lysine residue at position 9 in histone H3 through its N-terminal chromo domain (CD). To elucidate the binding properties of HP1α to nucleosomes in vitro, we reconstituted nucleosomes containing histone H3 trimethylated at lysine 9. HP1α exhibited high-affinity binding to nucleosomes containing methylated histone H3 in a nucleosome core-number-dependent manner. The hinge region (HR) connecting the CD and C-terminal chromoshadow domain (CSD), and the CSD contributed to the selective binding of HP1α to histone H3 with trimethylated lysine 9 through weak DNA binding and by suppressing the DNA binding, respectively. We propose that not only the specific recognition of lysine 9 methylation of histone H3 by the CD but also the HR and the CSD cooperatively contribute to the selective binding of HP1α to histone H3 lysine 9 methylated nucleosomes.

  7. Cell-free DNA Comprises an In Vivo Nucleosome Footprint that Informs Its Tissues-Of-Origin.

    Science.gov (United States)

    Snyder, Matthew W; Kircher, Martin; Hill, Andrew J; Daza, Riza M; Shendure, Jay

    2016-01-14

    Nucleosome positioning varies between cell types. By deep sequencing cell-free DNA (cfDNA), isolated from circulating blood plasma, we generated maps of genome-wide in vivo nucleosome occupancy and found that short cfDNA fragments harbor footprints of transcription factors. The cfDNA nucleosome occupancies correlate well with the nuclear architecture, gene structure, and expression observed in cells, suggesting that they could inform the cell type of origin. Nucleosome spacing inferred from cfDNA in healthy individuals correlates most strongly with epigenetic features of lymphoid and myeloid cells, consistent with hematopoietic cell death as the normal source of cfDNA. We build on this observation to show how nucleosome footprints can be used to infer cell types contributing to cfDNA in pathological states such as cancer. Since this strategy does not rely on genetic differences to distinguish between contributing tissues, it may enable the noninvasive monitoring of a much broader set of clinical conditions than currently possible.

  8. Topological diversity of chromatin fibers: Interplay between nucleosome repeat length, DNA linking number and the level of transcription

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    Davood Norouzi

    2015-11-01

    Full Text Available The spatial organization of nucleosomes in 30-nm fibers remains unknown in detail. To tackle this problem, we analyzed all stereochemically possible configurations of two-start chromatin fibers with DNA linkers L = 10-70 bp (nucleosome repeat length NRL = 157-217 bp. In our model, the energy of a fiber is a sum of the elastic energy of the linker DNA, steric repulsion, electrostatics, and the H4 tail-acidic patch interaction between two stacked nucleosomes. We found two families of energetically feasible conformations of the fibers—one observed earlier, and the other novel. The fibers from the two families are characterized by different DNA linking numbers—that is, they are topologically different. Remarkably, the optimal geometry of a fiber and its topology depend on the linker length: the fibers with linkers L = 10n and 10n + 5 bp have DNA linking numbers per nucleosome DLk >>-1.5 and -1.0, respectively. In other words, the level of DNA supercoiling is directly related to the length of the inter-nucleosome linker in the chromatin fiber (and therefore, to NRL. We hypothesize that this topological polymorphism of chromatin fibers may play a role in the process of transcription, which is known to generate different levels of DNA supercoiling upstream and downstream from RNA polymerase. A genome-wide analysis of the NRL distribution in active and silent yeast genes yielded results consistent with this assumption.

  9. Relevance of circulating nucleosomes and oncological biomarkers for predicting response to transarterial chemoembolization therapy in liver cancer patients

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    Durner Jürgen

    2011-05-01

    Full Text Available Abstract Background Transarterial chemoembolization (TACE therapy is an effective locoregional treatment in hepatocellular cancer (HCC patients. For early modification of therapy, markers predicting therapy response are urgently required. Methods Here, sera of 50 prospectively and consecutively included HCC patients undergoing 71 TACE therapies were taken before and 3 h, 6 h and 24 h after TACE application to analyze concentrations of circulating nucleosomes, cytokeratin-19 fragments (CYFRA 21-1, alpha fetoprotein (AFP, C-reactive protein (CRP and several liver biomarkers, and to compare these with radiological response to therapy. Results While nucleosomes, CYFRA 21-1, CRP and some liver biomarkers increased already 24 h after TACE, percental changes of nucleosome concentrations before and 24 h after TACE and pre- and posttherapeutic values of AFP, gamma-glutamyl-transferase (GGT and alkaline phosphatase (AP significantly indicated the later therapy response (39 progression versus 32 no progression. In multivariate analysis, nucleosomes (24 h, AP (24 h and TACE number were independent predictive markers. The risk score of this combination model achieved an AUC of 81.8% in receiver operating characteristic (ROC curves and a sensitivity for prediction of non-response to therapy of 41% at 97% specificity, and of 72% at 78% specificity. Conclusion Circulating nucleosomes and liver markers are valuable tools for early estimation of the efficacy of TACE therapy in HCC patients.

  10. Unforced polymer translocation compared to the forced case.

    Science.gov (United States)

    Lehtola, V V; Linna, R P; Kaski, K

    2010-03-01

    We present results for unforced polymer translocation from simulations using Langevin dynamics in two dimensions (2D) to four dimensions and stochastic rotation dynamics supporting hydrodynamic modes in three dimensions (3D). We compare our results to forced translocation and a simplified model where the polymer escapes from an infinite pore. The simple model shows that the scaling behavior of unforced translocation is independent of the dimension of the side to which the polymer is translocating. We find that, unlike its forced counterpart, unforced translocation dynamics is insensitive to pore design. Hydrodynamics is seen to markedly speed up the unforced translocation process but not to affect the scaling relations. Average mean-squared displacement shows scaling with average transition time in unforced but not in forced translocation. The waiting-time distribution in unforced translocation follows closely Poissonian distribution. Our measured transfer probabilities align well with those obtained from an equilibrium theory in 3D, but somewhat worse in 2D, where a polymer's relaxation toward equilibrium with respect to its translocation time is slower. Consequently, in stark contrast to forced translocation, unforced translocation is seen to remain close to equilibrium and shows clear universality.

  11. Financial costs of large carnivore translocations--accounting for conservation.

    Science.gov (United States)

    Weise, Florian J; Stratford, Ken J; van Vuuren, Rudolf J

    2014-01-01

    Human-carnivore conflict continues to present a major conservation challenge around the world. Translocation of large carnivores is widely implemented but remains strongly debated, in part because of a lack of cost transparency. We report detailed translocation costs for three large carnivore species in Namibia and across different translocation scenarios. We consider the effect of various parameters and factors on costs and translocation success. Total translocation cost for 30 individuals in 22 events was $80,681 (US Dollars). Median translocation cost per individual was $2,393, and $2,669 per event. Median cost per cheetah was $2,760 (n = 23), and $2,108 per leopard (n = 6). One hyaena was translocated at a cost of $1,672. Tracking technology was the single biggest cost element (56%), followed by captive holding and feeding. Soft releases, prolonged captivity and orphaned individuals also increased case-specific costs. A substantial proportion (65.4%) of the total translocation cost was successfully recovered from public interest groups. Less than half the translocations were confirmed successes (44.4%, 3 unknown) with a strong species bias. Four leopards (66.7%) were successfully translocated but only eight of the 20 cheetahs (40.0%) with known outcome met these strict criteria. None of the five habituated cheetahs was translocated successfully, nor was the hyaena. We introduce the concept of Individual Conservation Cost (ICC) and define it as the cost of one successfully translocated individual adjusted by costs of unsuccessful events of the same species. The median ICC for cheetah was $6,898 and $3,140 for leopard. Translocations are costly, but we demonstrate that they are not inherently more expensive than other strategies currently employed in non-lethal carnivore conflict management. We conclude that translocation should be one available option for conserving large carnivores, but needs to be critically evaluated on a case-by-case basis.

  12. Financial costs of large carnivore translocations--accounting for conservation.

    Directory of Open Access Journals (Sweden)

    Florian J Weise

    Full Text Available Human-carnivore conflict continues to present a major conservation challenge around the world. Translocation of large carnivores is widely implemented but remains strongly debated, in part because of a lack of cost transparency. We report detailed translocation costs for three large carnivore species in Namibia and across different translocation scenarios. We consider the effect of various parameters and factors on costs and translocation success. Total translocation cost for 30 individuals in 22 events was $80,681 (US Dollars. Median translocation cost per individual was $2,393, and $2,669 per event. Median cost per cheetah was $2,760 (n = 23, and $2,108 per leopard (n = 6. One hyaena was translocated at a cost of $1,672. Tracking technology was the single biggest cost element (56%, followed by captive holding and feeding. Soft releases, prolonged captivity and orphaned individuals also increased case-specific costs. A substantial proportion (65.4% of the total translocation cost was successfully recovered from public interest groups. Less than half the translocations were confirmed successes (44.4%, 3 unknown with a strong species bias. Four leopards (66.7% were successfully translocated but only eight of the 20 cheetahs (40.0% with known outcome met these strict criteria. None of the five habituated cheetahs was translocated successfully, nor was the hyaena. We introduce the concept of Individual Conservation Cost (ICC and define it as the cost of one successfully translocated individual adjusted by costs of unsuccessful events of the same species. The median ICC for cheetah was $6,898 and $3,140 for leopard. Translocations are costly, but we demonstrate that they are not inherently more expensive than other strategies currently employed in non-lethal carnivore conflict management. We conclude that translocation should be one available option for conserving large carnivores, but needs to be critically evaluated on a case-by-case basis.

  13. The disequilibrium of nucleosomes distribution along chromosomes plays a functional and evolutionarily role in regulating gene expression.

    Directory of Open Access Journals (Sweden)

    Peng Cui

    Full Text Available To further understand the relationship between nucleosome-space occupancy (NO and global transcriptional activity in mammals, we acquired a set of genome-wide nucleosome distribution and transcriptome data from the mouse cerebrum and testis based on ChIP (H3-seq and RNA-seq, respectively. We identified a nearly consistent NO patterns among three mouse tissues--cerebrum, testis, and ESCs--and found, through clustering analysis for transcriptional activation, that the NO variations among chromosomes are closely associated with distinct expression levels between house-keeping (HK genes and tissue-specific (TS genes. Both TS and HK genes form clusters albeit the obvious majority. This feature implies that NO patterns, i.e. nucleosome binding and clustering, are coupled with gene clustering that may be functionally and evolutionarily conserved in regulating gene expression among different cell types.

  14. The disequilibrium of nucleosomes distribution along chromosomes plays a functional and evolutionarily role in regulating gene expression

    KAUST Repository

    Cui, Peng

    2011-08-19

    To further understand the relationship between nucleosome-space occupancy (NO) and global transcriptional activity in mammals, we acquired a set of genome-wide nucleosome distribution and transcriptome data from the mouse cerebrum and testis based on ChIP (H3)-seq and RNA-seq, respectively. We identified a nearly consistent NO patterns among three mouse tissues-cerebrum, testis, and ESCs-and found, through clustering analysis for transcriptional activation, that the NO variations among chromosomes are closely associated with distinct expression levels between house-keeping (HK) genes and tissue-specific (TS) genes. Both TS and HK genes form clusters albeit the obvious majority. This feature implies that NO patterns, i.e. nucleosome binding and clustering, are coupled with gene clustering that may be functionally and evolutionarily conserved in regulating gene expression among different cell types. © 2011 Cui et al.

  15. Fibroblast growth factor receptor 2 translocations in intrahepatic cholangiocarcinoma.

    Science.gov (United States)

    Graham, Rondell P; Barr Fritcher, Emily G; Pestova, Ekaterina; Schulz, John; Sitailo, Leonid A; Vasmatzis, George; Murphy, Stephen J; McWilliams, Robert R; Hart, Steven N; Halling, Kevin C; Roberts, Lewis R; Gores, Gregory J; Couch, Fergus J; Zhang, Lizhi; Borad, Mitesh J; Kipp, Benjamin R

    2014-08-01

    Patients with cholangiocarcinoma often present with locally advanced or metastatic disease. There is a need for effective therapeutic strategies for advanced stage cholangiocarcinoma. Recently, FGFR2 translocations have been identified as a potential target for tyrosine kinase inhibitor therapies. This study evaluated 152 cholangiocarcinomas and 4 intraductal papillary biliary neoplasms of the bile duct for presence of FGFR2 translocations by fluorescence in situ hybridization and characterized the clinicopathologic features of cases with FGFR2 translocations. Thirteen (10 women, 3 men; 8%) of 156 biliary tumors harbored FGFR2 translocations, including 12 intrahepatic cholangiocarcinomas (12/96; 13%) and 1 intraductal papillary neoplasm of the bile duct. Histologically, cholangiocarcinomas with FGFR2 translocations displayed prominent intraductal growth (62%) or anastomosing tubular glands with desmoplasia (38%). Immunohistochemically, the tumors with FGFR2 translocations frequently showed weak and patchy expression of CK19 (77%). Markers of the stem cell phenotype in cholangiocarcinoma, HepPar1 and CK20, were negative in all cases. The median cancer-specific survival for patients whose tumors harbored FGFR2 translocations was 123 months compared to 37 months for cases without FGFR2 translocations (P = .039). This study also assessed 100 cholangiocarcinomas for ERBB2 amplification and ROS1 translocations. Of the cases tested, 3% and 1% were positive for ERBB2 amplification and ROS1 translocation, respectively. These results confirm that FGFR2, ERRB2, and ROS1 alterations are potential therapeutic targets for intrahepatic cholangiocarcinoma.

  16. Measurement of background translocation frequencies in individuals with clones

    Energy Technology Data Exchange (ETDEWEB)

    Wade, M.J.

    1996-08-01

    In the leukemia case the unseparated B and T lymphocytes had a high translocation frequency even after 0.0014, respectively. After purging all clones from the data, the translocation frequencies for Bio 8 and Bio 23 were 0.00750.0014 and 0.0073 metaphases were scored for chromosomal aberrations,, specifically reciprocal translocations, using fluorescence in situ hybridization (FISH). Metaphase spreads were used from two healthy, unexposed individuals (not exposed to radiation, chemotherapy or radiotherapy) and one early B- precursor acute lymphocytic leukemia (ALL) patient (metaphase spreads from both separated T lymphocytes and unseparated B and T lymphocytes were scored). All three individuals had an abnormally high translocation frequency. The high translocation frequencies resulted from clonal expansion of specific translocated chromosomes. I show in this thesis that by purging (discounting or removing) clones from the data of unexposed individuals, one can obtain true background translocation frequencies. In two cases, Bio 8 and Bio 23, the measured translocation frequency for chromosomes 1, 2 and 4 was 0.0124 purging all of the clones from the data. This high translocation frequency may be due to a low frequency of some clones and may not be recognized. The separated T lymphocytes had a higher translocation frequency than expected.

  17. Learning a weighted sequence model of the nucleosome core and linker yields more accurate predictions in Saccharomyces cerevisiae and Homo sapiens.

    Science.gov (United States)

    Reynolds, Sheila M; Bilmes, Jeff A; Noble, William Stafford

    2010-07-08

    DNA in eukaryotes is packaged into a chromatin complex, the most basic element of which is the nucleosome. The precise positioning of the nucleosome cores allows for selective access to the DNA, and the mechanisms that control this positioning are important pieces of the gene expression puzzle. We describe a large-scale nucleosome pattern that jointly characterizes the nucleosome core and the adjacent linkers and is predominantly characterized by long-range oscillations in the mono, di- and tri-nucleotide content of the DNA sequence, and we show that this pattern can be used to predict nucleosome positions in both Homo sapiens and Saccharomyces cerevisiae more accurately than previously published methods. Surprisingly, in both H. sapiens and S. cerevisiae, the most informative individual features are the mono-nucleotide patterns, although the inclusion of di- and tri-nucleotide features results in improved performance. Our approach combines a much longer pattern than has been previously used to predict nucleosome positioning from sequence-301 base pairs, centered at the position to be scored-with a novel discriminative classification approach that selectively weights the contributions from each of the input features. The resulting scores are relatively insensitive to local AT-content and can be used to accurately discriminate putative dyad positions from adjacent linker regions without requiring an additional dynamic programming step and without the attendant edge effects and assumptions about linker length modeling and overall nucleosome density. Our approach produces the best dyad-linker classification results published to date in H. sapiens, and outperforms two recently published models on a large set of S. cerevisiae nucleosome positions. Our results suggest that in both genomes, a comparable and relatively small fraction of nucleosomes are well-positioned and that these positions are predictable based on sequence alone. We believe that the bulk of the

  18. Learning a weighted sequence model of the nucleosome core and linker yields more accurate predictions in Saccharomyces cerevisiae and Homo sapiens.

    Directory of Open Access Journals (Sweden)

    Sheila M Reynolds

    Full Text Available DNA in eukaryotes is packaged into a chromatin complex, the most basic element of which is the nucleosome. The precise positioning of the nucleosome cores allows for selective access to the DNA, and the mechanisms that control this positioning are important pieces of the gene expression puzzle. We describe a large-scale nucleosome pattern that jointly characterizes the nucleosome core and the adjacent linkers and is predominantly characterized by long-range oscillations in the mono, di- and tri-nucleotide content of the DNA sequence, and we show that this pattern can be used to predict nucleosome positions in both Homo sapiens and Saccharomyces cerevisiae more accurately than previously published methods. Surprisingly, in both H. sapiens and S. cerevisiae, the most informative individual features are the mono-nucleotide patterns, although the inclusion of di- and tri-nucleotide features results in improved performance. Our approach combines a much longer pattern than has been previously used to predict nucleosome positioning from sequence-301 base pairs, centered at the position to be scored-with a novel discriminative classification approach that selectively weights the contributions from each of the input features. The resulting scores are relatively insensitive to local AT-content and can be used to accurately discriminate putative dyad positions from adjacent linker regions without requiring an additional dynamic programming step and without the attendant edge effects and assumptions about linker length modeling and overall nucleosome density. Our approach produces the best dyad-linker classification results published to date in H. sapiens, and outperforms two recently published models on a large set of S. cerevisiae nucleosome positions. Our results suggest that in both genomes, a comparable and relatively small fraction of nucleosomes are well-positioned and that these positions are predictable based on sequence alone. We believe that the

  19. Formation of complex and unstable chromosomal translocations in yeast.

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    Kristina H Schmidt

    Full Text Available Genome instability, associated with chromosome breakage syndromes and most human cancers, is still poorly understood. In the yeast Saccharomyces cerevisiae, numerous genes with roles in the preservation of genome integrity have been identified. DNA-damage-checkpoint-deficient yeast cells that lack Sgs1, a RecQ-like DNA helicase related to the human Bloom's-syndrome-associated helicase BLM, show an increased rate of genome instability, and we have previously shown that they accumulate recurring chromosomal translocations between three similar genes, CAN1, LYP1 and ALP1. Here, the chromosomal location, copy number and sequence similarity of the translocation targets ALP1 and LYP1 were altered to gain insight into the formation of complex translocations. Among 844 clones with chromosomal rearrangements, 93 with various types of simple and complex translocations involving CAN1, LYP1 and ALP1 were identified. Breakpoint sequencing and mapping showed that the formation of complex translocation types is strictly dependent on the location of the initiating DNA break and revealed that complex translocations arise via a combination of interchromosomal translocation and template-switching, as well as from unstable dicentric intermediates. Template-switching occurred between sequences on the same chromosome, but was inhibited if the genes were transferred to different chromosomes. Unstable dicentric translocations continuously gave rise to clones with multiple translocations in various combinations, reminiscent of intratumor heterogeneity in human cancers. Base substitutions and evidence of DNA slippage near rearrangement breakpoints revealed that translocation formation can be accompanied by point mutations, and their presence in different translocation types within the same clone provides evidence that some of the different translocation types are derived from each other rather than being formed de novo. These findings provide insight into eukaryotic

  20. Fast, Accurate and Automatic Ancient Nucleosome and Methylation Maps with epiPALEOMIX.

    Science.gov (United States)

    Hanghøj, Kristian; Seguin-Orlando, Andaine; Schubert, Mikkel; Madsen, Tobias; Pedersen, Jakob Skou; Willerslev, Eske; Orlando, Ludovic

    2016-12-01

    The first epigenomes from archaic hominins (AH) and ancient anatomically modern humans (AMH) have recently been characterized, based, however, on a limited number of samples. The extent to which ancient genome-wide epigenetic landscapes can be reconstructed thus remains contentious. Here, we present epiPALEOMIX, an open-source and user-friendly pipeline that exploits post-mortem DNA degradation patterns to reconstruct ancient methylomes and nucleosome maps from shotgun and/or capture-enrichment data. Applying epiPALEOMIX to the sequence data underlying 35 ancient genomes including AMH, AH, equids and aurochs, we investigate the temporal, geographical and preservation range of ancient epigenetic signatures. We first assess the quality of inferred ancient epigenetic signatures within well-characterized genomic regions. We find that tissue-specific methylation signatures can be obtained across a wider range of DNA preparation types than previously thought, including when no particular experimental procedures have been used to remove deaminated cytosines prior to sequencing. We identify a large subset of samples for which DNA associated with nucleosomes is protected from post-mortem degradation, and nucleosome positioning patterns can be reconstructed. Finally, we describe parameters and conditions such as DNA damage levels and sequencing depth that limit the preservation of epigenetic signatures in ancient samples. When such conditions are met, we propose that epigenetic profiles of CTCF binding regions can be used to help data authentication. Our work, including epiPALEOMIX, opens for further investigations of ancient epigenomes through time especially aimed at tracking possible epigenetic changes during major evolutionary, environmental, socioeconomic, and cultural shifts.

  1. Roles of histone chaperone CIA/Asf1 in nascent DNA elongation during nucleosome replication.

    Science.gov (United States)

    Ishikawa, Katsuyuki; Ohsumi, Tatsuya; Tada, Shusuke; Natsume, Ryo; Kundu, Lena Rani; Nozaki, Naohito; Senda, Toshiya; Enomoto, Takemi; Horikoshi, Masami; Seki, Masayuki

    2011-10-01

    The nucleosome, which is composed of DNA wrapped around a histone octamer, is a fundamental unit of chromatin and is duplicated during the eukaryotic DNA replication process. The evolutionarily conserved histone chaperone cell cycle gene 1 (CCG1) interacting factor A/anti-silencing function 1 (CIA/Asf1) is involved in histone transfer and nucleosome reassembly during DNA replication. CIA/Asf1 has been reported to split the histone (H3-H4)(2) tetramer into histone H3-H4 dimer(s) in vitro, raising a possibility that, in DNA replication, CIA/Asf1 is involved in nucleosome disassembly and the promotion of semi-conservative histone H3-H4 dimer deposition onto each daughter strand in vivo. Despite numerous studies on the functional roles of CIA/Asf1, its mechanistic role(s) remains elusive because of lack of biochemical analyses. The biochemical studies described here show that a V94R CIA/Asf1 mutant, which lacks histone (H3-H4)(2) tetramer splitting activity, does not form efficiently a quaternary complex with histones H3-H4 and the minichromosome maintenance 2 (Mcm2) subunit of the Mcm2-7 replicative DNA helicase. Interestingly, the mutant enhances nascent DNA strand synthesis in a cell-free chromosomal DNA replication system using Xenopus egg extracts. These results suggest that CIA/Asf1 in the CIA/Asf1-H3-H4-Mcm2 complex, which is considered to be an intermediate in histone transfer during DNA replication, negatively regulates the progression of the replication fork.

  2. A core viral protein binds host nucleosomes to sequester immune danger signals.

    Science.gov (United States)

    Avgousti, Daphne C; Herrmann, Christin; Kulej, Katarzyna; Pancholi, Neha J; Sekulic, Nikolina; Petrescu, Joana; Molden, Rosalynn C; Blumenthal, Daniel; Paris, Andrew J; Reyes, Emigdio D; Ostapchuk, Philomena; Hearing, Patrick; Seeholzer, Steven H; Worthen, G Scott; Black, Ben E; Garcia, Benjamin A; Weitzman, Matthew D

    2016-07-01

    Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

  3. Regulation of Budding Yeast CENP-A levels Prevents Misincorporation at Promoter Nucleosomes and Transcriptional Defects.

    Directory of Open Access Journals (Sweden)

    Erica M Hildebrand

    2016-03-01

    Full Text Available The exclusive localization of the histone H3 variant CENP-A to centromeres is essential for accurate chromosome segregation. Ubiquitin-mediated proteolysis helps to ensure that CENP-A does not mislocalize to euchromatin, which can lead to genomic instability. Consistent with this, overexpression of the budding yeast CENP-A(Cse4 is lethal in cells lacking Psh1, the E3 ubiquitin ligase that targets CENP-A(Cse4 for degradation. To identify additional mechanisms that prevent CENP-A(Cse4 misincorporation and lethality, we analyzed the genome-wide mislocalization pattern of overexpressed CENP-A(Cse4 in the presence and absence of Psh1 by chromatin immunoprecipitation followed by high throughput sequencing. We found that ectopic CENP-A(Cse4 is enriched at promoters that contain histone H2A.Z(Htz1 nucleosomes, but that H2A.Z(Htz1 is not required for CENP-A(Cse4 mislocalization. Instead, the INO80 complex, which removes H2A.Z(Htz1 from nucleosomes, promotes the ectopic deposition of CENP-A(Cse4. Transcriptional profiling revealed gene expression changes in the psh1Δ cells overexpressing CENP-A(Cse4. The down-regulated genes are enriched for CENP-A(Cse4 mislocalization to promoters, while the up-regulated genes correlate with those that are also transcriptionally up-regulated in an htz1Δ strain. Together, these data show that regulating centromeric nucleosome localization is not only critical for maintaining centromere function, but also for ensuring accurate promoter function and transcriptional regulation.

  4. Regulation of Budding Yeast CENP-A levels Prevents Misincorporation at Promoter Nucleosomes and Transcriptional Defects.

    Science.gov (United States)

    Hildebrand, Erica M; Biggins, Sue

    2016-03-01

    The exclusive localization of the histone H3 variant CENP-A to centromeres is essential for accurate chromosome segregation. Ubiquitin-mediated proteolysis helps to ensure that CENP-A does not mislocalize to euchromatin, which can lead to genomic instability. Consistent with this, overexpression of the budding yeast CENP-A(Cse4) is lethal in cells lacking Psh1, the E3 ubiquitin ligase that targets CENP-A(Cse4) for degradation. To identify additional mechanisms that prevent CENP-A(Cse4) misincorporation and lethality, we analyzed the genome-wide mislocalization pattern of overexpressed CENP-A(Cse4) in the presence and absence of Psh1 by chromatin immunoprecipitation followed by high throughput sequencing. We found that ectopic CENP-A(Cse4) is enriched at promoters that contain histone H2A.Z(Htz1) nucleosomes, but that H2A.Z(Htz1) is not required for CENP-A(Cse4) mislocalization. Instead, the INO80 complex, which removes H2A.Z(Htz1) from nucleosomes, promotes the ectopic deposition of CENP-A(Cse4). Transcriptional profiling revealed gene expression changes in the psh1Δ cells overexpressing CENP-A(Cse4). The down-regulated genes are enriched for CENP-A(Cse4) mislocalization to promoters, while the up-regulated genes correlate with those that are also transcriptionally up-regulated in an htz1Δ strain. Together, these data show that regulating centromeric nucleosome localization is not only critical for maintaining centromere function, but also for ensuring accurate promoter function and transcriptional regulation.

  5. The role of histone tails in the nucleosome: a computational study.

    Science.gov (United States)

    Erler, Jochen; Zhang, Ruihan; Petridis, Loukas; Cheng, Xiaolin; Smith, Jeremy C; Langowski, Jörg

    2014-12-16

    Histone tails play an important role in gene transcription and expression. We present here a systematic computational study of the role of histone tails in the nucleosome, using replica exchange molecular dynamics simulations with an implicit solvent model and different well-established force fields. We performed simulations for all four histone tails, H4, H3, H2A, and H2B, isolated and with inclusion of the nucleosome. The results confirm predictions of previous theoretical studies for the secondary structure of the isolated tails but show a strong dependence on the force field used. In the presence of the entire nucleosome for all force fields, the secondary structure of the histone tails is destabilized. Specific contacts are found between charged lysine and arginine residues and DNA phosphate groups and other binding sites in the minor and major DNA grooves. Using cluster analysis, we found a single dominant configuration of binding to DNA for the H4 and H2A histone tails, whereas H3 and H2B show multiple binding configurations with an equal probability. The leading stabilizing contribution for those binding configurations is the attractive interaction between the positively charged lysine and arginine residues and the negatively charged phosphate groups, and thus the resulting charge neutralization. Finally, we present results of molecular dynamics simulations in explicit solvent to confirm our conclusions. Results from both implicit and explicit solvent models show that large portions of the histone tails are not bound to DNA, supporting the complex role of these tails in gene transcription and expression and making them possible candidates for binding sites of transcription factors, enzymes, and other proteins.

  6. Non-sticky translocation of bio-molecules through Tween 20-coated solid-state nanopores in a wide pH range

    Science.gov (United States)

    Li, Xiaoqing; Hu, Rui; Li, Ji; Tong, Xin; Diao, J. J.; Yu, Dapeng; Zhao, Qing

    2016-10-01

    Nanopore-based sensing technology is considered high-throughput and low-cost for single molecule detection, but solid-state nanopores have suffered from pore clogging issues. A simple Tween 20 coating method is applied to ensure long-term (several hours) non-sticky translocation of various types of bio-molecules through SiN nanopores in a wide pH range (4.0-13.0). We also emphasize the importance of choosing appropriate concentration of Tween 20 coating buffer for desired effect. By coating nanopores with a Tween 20 layer, we are able to differentiate between single-stranded DNA and double-stranded DNA, to identify drift-dominated domain for single-stranded DNA, to estimate BSA volume and to observe the shape of individual nucleosome translocation event without non-specific adsorption. The wide pH endurance from 4.0 to 13.0 and the broad types of detection analytes including nucleic acids, proteins, and biological complexes highlight the great application potential of Tween 20-coated solid-state nanopores.

  7. FACT, the Bur kinase pathway, and the histone co-repressor HirC have overlapping nucleosome-related roles in yeast transcription elongation.

    Directory of Open Access Journals (Sweden)

    Jennifer R Stevens

    Full Text Available Gene transcription is constrained by the nucleosomal nature of chromosomal DNA. This nucleosomal barrier is modulated by FACT, a conserved histone-binding heterodimer. FACT mediates transcription-linked nucleosome disassembly and also nucleosome reassembly in the wake of the RNA polymerase II transcription complex, and in this way maintains the repression of 'cryptic' promoters found within some genes. Here we focus on a novel mutant version of the yeast FACT subunit Spt16 that supplies essential Spt16 activities but impairs transcription-linked nucleosome reassembly in dominant fashion. This Spt16 mutant protein also has genetic effects that are recessive, which we used to show that certain Spt16 activities collaborate with histone acetylation and the activities of a Bur-kinase/Spt4-Spt5/Paf1C pathway that facilitate transcription elongation. These collaborating activities were opposed by the actions of Rpd3S, a histone deacetylase that restores a repressive chromatin environment in a transcription-linked manner. Spt16 activity paralleling that of HirC, a co-repressor of histone gene expression, was also found to be opposed by Rpd3S. Our findings suggest that Spt16, the Bur/Spt4-Spt5/Paf1C pathway, and normal histone abundance and/or stoichiometry, in mutually cooperative fashion, facilitate nucleosome disassembly during transcription elongation. The recessive nature of these effects of the mutant Spt16 protein on transcription-linked nucleosome disassembly, contrasted to its dominant negative effect on transcription-linked nucleosome reassembly, indicate that mutant FACT harbouring the mutant Spt16 protein competes poorly with normal FACT at the stage of transcription-linked nucleosome disassembly, but effectively with normal FACT for transcription-linked nucleosome reassembly. This functional difference is consistent with the idea that FACT association with the transcription elongation complex depends on nucleosome disassembly, and that the

  8. Another reptile translocation to a national park

    Directory of Open Access Journals (Sweden)

    W.R. Branch

    1990-10-01

    Full Text Available On 4 May 1988 a sub-adult (50 mm snout-vent length, 42 mm tail Jones' girdled lizard Cordylus tropidosternum jonesi was collected in a pile of wood being off-loaded at the new restcamp in the Karoo National Park, Beaufort West. The wood had been transported by lorry from the Kruger National Park. The specimen is deposited in the herpetological collection of the Port Elizabeth Museum (PEM R 4584. Jones' girdled lizard is a small, arboreal cordylid that shelters under tree bark and in hollow logs. It is common and widely-distributed in the Kruger National Park (Pienaar, Haacke & Jacobsen 1983, The Reptiles of the Kruger National Park, 3rd edition. Pretoria: National Parks Board and adjacent lowveld, being replaced in northern Zimbabwe and East Africa by the nominate race. Hewitt & Power (1913, Transactions of the Royal Society of South Africa 3: 147-176, 1913 reported a similar translocation of the species to Kimberley in association with timber brought to the diamond mining camps. One of us noted recently the ease and danger of the unwitting spread of commensal reptile species into conservation areas (Branch 1978, Koedoe 30: 165, and this is confirmed by this additional example. We recommend that should similar shipments of wood be considered essential, then they be fumigated to prevent the translocation of other alien organisms that may potentially have more dangerous consequences.

  9. Translocation as a species conservation tool: Status and strategy

    Science.gov (United States)

    Griffith, B.; Scott, J.M.; Carpenter, J.W.; Reed, C.

    1989-01-01

    Surveys of recent (1973 to 1986) intentional releases of native birds and mammals to the wild in Australia, Canada, Hawaii, New Zealand, and the United States, were conducted to document current activities, identify factors associated with success, and suggest guidelines for enhancing future work. Nearly 700 translocations were conducted each year. Native game species constituted percent of translocations and were more successful (86 percent) than were translocations of threatened, endangered, or sensitive species (46 percent). Knowledge of habitat quality, location of release area within the species range, number of animals released, program length, and reproductive traits, allowed currect classification of 81 percent of observed translocations as successful or not.

  10. Obstacle Effects on One-Dimensional Translocation of ATPase

    Institute of Scientific and Technical Information of China (English)

    WANG Xian-Ju; AI Bao-Quan; LIU Liang-Gang

    2002-01-01

    We apply a general random walk model to the study of the ATPase's one-dimensional translocation along obstacle biological environment, and show the effects of random obstacles on the ATPase translocation along single stranded DNA. We find that the obstacle environment can reduce the lifetime of ATPase lattice-bound state which results in the inhibition of ATPase activity. We also carry out the ranges of rate constant of ATPase unidirectonal translocation and bidirectional translocation. Our results are consistent with the experiments and relevant theoretical consideration, and can be used to explain some physiological phenomena.

  11. Minichromosome replication in vitro: inhibition of re-replication by replicatively assembled nucleosomes.

    Science.gov (United States)

    Krude, T; Knippers, R

    1994-08-19

    Single-stranded circular DNA, containing the SV40 origin sequence, was used as a template for complementary DNA strand synthesis in cytosolic extracts from HeLa cells. In the presence of the replication-dependent chromatin assembly factor CAF-1, defined numbers of nucleosomes were assembled during complementary DNA strand synthesis. These minichromosomes were then induced to semiconservatively replicate by the addition of the SV40 initiator protein T antigen (re-replication). The results indicate that re-replication of minichromosomes appears to be inhibited by two independent mechanisms. One acts at the initiation of minichromosome re-replication, and the other affects replicative chain elongation. To directly demonstrate the inhibitory effect of replicatively assembled nucleosomes, two types of minichromosomes were prepared: (i) post-replicative minichromosomes were assembled in a reaction coupled to replication as above; (ii) pre-replicative minichromosomes were assembled independently of replication on double-stranded DNA. Both types of minichromosomes were used as templates for DNA replication under identical conditions. Replicative fork movement was found to be impeded only on post-replicative minichromosome templates. In contrast, pre-replicative minichromosomes allowed one unconstrained replication cycle, but re-replication was inhibited due to a block in fork movement. Thus, replicatively assembled chromatin may have a profound influence on the re-replication of DNA.

  12. Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability

    Science.gov (United States)

    Ngo, Thuy T. M.; Yoo, Jejoong; Dai, Qing; Zhang, Qiucen; He, Chuan; Aksimentiev, Aleksei; Ha, Taekjip

    2016-02-01

    Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility.

  13. From chemical embryology to nucleosome patterning - an interview with Roumen G. Tsanev.

    Science.gov (United States)

    Tsanev, Roumen G

    2009-01-01

    Roumen Tsanev was a prominent Bulgarian scientist whose pioneering ideas about the role for chromatin in cell differentiation and development led him to propose the first hypothesis for epigenetic information based on a histone code. To test experimentally his ideas, Dr.Tsanev explored nucleosome structure and heterogeneity and generated seminal data on nucleosome segregation in replicating chromatin. Roumen Tsanev made significant contributions to the understanding of chromatin changes that underlie zygotic gene activation. He identified sperm specific chromatin components tightly bound to DNA and demonstrated that the histone complement of the male pronucleus appears before the onset of DNA synthesis in the mouse zygote. In this interview, Roumen Tsanev talks about his passion for science and literature, reminisces about surmounting the harsh realities in post-war communist Bulgaria through creativity and determination, and explains what led him to propose that histones were carriers of epigenetic information. Dr. Tsanev discusses mathematical models of gene regulation and recalls computer simulations that reveal the non-linearity of genetic networks. He explains how this non-linearity could affect cell proliferation, differentiation, development and evolution.

  14. Arabidopsis FORGETTER1 mediates stress-induced chromatin memory through nucleosome remodeling

    Science.gov (United States)

    Brzezinka, Krzysztof; Altmann, Simone; Czesnick, Hjördis; Nicolas, Philippe; Gorka, Michal; Benke, Eileen; Kabelitz, Tina; Jähne, Felix; Graf, Alexander; Kappel, Christian; Bäurle, Isabel

    2016-01-01

    Plants as sessile organisms can adapt to environmental stress to mitigate its adverse effects. As part of such adaptation they maintain an active memory of heat stress for several days that promotes a more efficient response to recurring stress. We show that this heat stress memory requires the activity of the FORGETTER1 (FGT1) locus, with fgt1 mutants displaying reduced maintenance of heat-induced gene expression. FGT1 encodes the Arabidopsis thaliana orthologue of Strawberry notch (Sno), and the protein globally associates with the promoter regions of actively expressed genes in a heat-dependent fashion. FGT1 interacts with chromatin remodelers of the SWI/SNF and ISWI families, which also display reduced heat stress memory. Genomic targets of the BRM remodeler overlap significantly with FGT1 targets. Accordingly, nucleosome dynamics at loci with altered maintenance of heat-induced expression are affected in fgt1. Together, our results suggest that by modulating nucleosome occupancy, FGT1 mediates stress-induced chromatin memory. DOI: http://dx.doi.org/10.7554/eLife.17061.001 PMID:27680998

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

    Science.gov (United States)

    Ng, Marlee K; Cheung, Peter

    2016-02-01

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

  16. Arginine-phosphate salt bridges between histones and DNA: Intermolecular actuators that control nucleosome architecture

    Science.gov (United States)

    Yusufaly, Tahir I.; Li, Yun; Singh, Gautam; Olson, Wilma K.

    2014-10-01

    Structural bioinformatics and van der Waals density functional theory are combined to investigate the mechanochemical impact of a major class of histone-DNA interactions, namely, the formation of salt bridges between arginine residues in histones and phosphate groups on the DNA backbone. Principal component analysis reveals that the configurational fluctuations of the sugar-phosphate backbone display sequence-specific directionality and variability, and clustering of nucleosome crystal structures identifies two major salt-bridge configurations: a monodentate form in which the arginine end-group guanidinium only forms one hydrogen bond with the phosphate, and a bidentate form in which it forms two. Density functional theory calculations highlight that the combination of sequence, denticity, and salt-bridge positioning enables the histones to apply a tunable mechanochemical stress to the DNA via precise and specific activation of backbone deformations. The results suggest that selection for specific placements of van der Waals contacts, with high-precision control of the spatial distribution of intermolecular forces, may serve as an underlying evolutionary design principle for the structure and function of nucleosomes, a conjecture that is corroborated by previous experimental studies.

  17. Chromatin and Nucleosome Organizations and DNA Replication of Nucleus Reassembled in vitro Using Purified Exogenous DNA and Xenopus Egg Extracts

    Institute of Scientific and Technical Information of China (English)

    张传茂; 张博; 翟中和

    1994-01-01

    It has been demonstrated in the last ten years that the nuclear reassembly may occur in the cell-free systems from frog egg extracts added with exogenous naked DNA. However, there remains an open question : is the cell-free reassembled nucleus structurally similar to the nucleus in the intact cell ? That is, does the cell-free reassembled nucleus contain nucleosomes and chromatin? For this issue, we have designed experiments for identifying the internal structures of the cell-free reassembled nucleus. These experiments show that the nucleus reassembled in vitro also contains chromatin which is composed of typical 10 nm nucleosome fibers of "beads-on-a-string", 30 nm filaments and the next higher-order structures. The digestion experiment with the enzyme micrococcal nuclease has demonstrated that the DNA in the nucleosome of the reconstituted chromatin is about 200 base pairs (bp) in length, of which 165 bp may be in the nucleosome particle, and 35 bp may be in the linker between two particles. Prolongin

  18. Nucleosomes and C1q bound to glomerular endothelial cells serve as targets for autoantibodies and determine complement activation

    NARCIS (Netherlands)

    O'Flynn, J.; Flierman, R.; Pol, P. van der; Meulemans-Rops, L.W.M.; Satchell, S.C.; Mathieson, P.W.; Kooten, C. van; Vlag, J. van der; Berden, J.H.M.; Daha, M.R.

    2011-01-01

    Various studies indicate a role for both anti-nucleosome and anti-C1q autoantibodies in glomerulonephritis in patients with systemic lupus erythematosus. However, a causal relationship between these autoantibodies and the development of lupus nephritis has not been fully established. Since injury of

  19. [THE MODEL OF NUCLEOSOME STRUCTURE BASED ON THE LOCAL ROTATION OF THE NUCLEOHISTONE CHAIN, WHICH INDUCES ITS FOLDING].

    Science.gov (United States)

    Priyatkina, T N

    2015-01-01

    An alternative model to the "double turn of DNA on the histone core" approach is forwarded based on the biochemical, cytological, and crystallographic data on the structural organization of the chromatin units--nucleosomes. The model assumes that the initial structure is a linear nucleohistone cord with a repeating symmetrical histone sequence. The compact (core) particle (a minimal nucleosome) is forming upon a stepwise rotation of DNA (kinks) at the centre and at two symmetrical sites into each repeating fragment stemming from the electrostatic binding of the lysine ε-NH2-groups with the followed one by one phosphates of the sugar-phosphate chain. As a result, we have a rhomboid structure composed of two counter-symmetrical DNA folds stabilized by histone-histone interactions. Based on disposable data, the histone sequence along nucleosome DNA is deduced. The following characteristics of the sequence are considered: continuity, non-overlapping, versatility, and dyadic symmetry in dispose of two every kind histone molecules and the sequence on the whole. The model is in agreement with a topology of nucleosome DNA, as well as the pattern of DNA-histone and histone-histone interactions in chromatin.

  20. KSHV encoded LANA recruits Nucleosome Assembly Protein NAP1L1 for regulating viral DNA replication and transcription

    Science.gov (United States)

    Gupta, Namrata; Thakker, Suhani; Verma, Subhash C.

    2016-09-01

    The establishment of latency is an essential for lifelong persistence and pathogenesis of Kaposi’s sarcoma-associated herpesvirus (KSHV). Latency-associated nuclear antigen (LANA) is the most abundantly expressed protein during latency and is important for viral genome replication and transcription. Replication-coupled nucleosome assembly is a major step in packaging the newly synthesized DNA into chromatin, but the mechanism of KSHV genome chromatinization post-replication is not understood. Here, we show that nucleosome assembly protein 1-like protein 1 (NAP1L1) associates with LANA. Our binding assays revealed an association of LANA with NAP1L1 in KSHV-infected cells, which binds through its amino terminal domain. Association of these proteins confirmed their localization in specific nuclear compartments of the infected cells. Chromatin immunoprecipitation assays from NAP1L1-depleted cells showed LANA-mediated recruitment of NAP1L1 at the terminal repeat (TR) region of the viral genome. Presence of NAP1L1 stimulated LANA-mediated DNA replication and persistence of a TR-containing plasmid. Depletion of NAP1L1 led to a reduced nucleosome positioning on the viral genome. Furthermore, depletion of NAP1L1 increased the transcription of viral lytic genes and overexpression decreased the promoter activities of LANA-regulated genes. These results confirmed that LANA recruitment of NAP1L1 helps in assembling nucleosome for the chromatinization of newly synthesized viral DNA.

  1. Pb distribution and translocation in Jiaozhou Bay

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The trends of distribution, translocation and seasonal change of heavy metal Pb were studied based on the surface and bottom water sampling in Jiaozhou Bay in 1979, and compared with those in 1990's. The results showed that the source of Pb in the bay was from wastewater and sewage in the east of Jiaozhou Bay from ocean vessels. Pb concentration was higher in spring and lower in summer and autumn, and remained stable through sedimentation in the bottom layer. The overall water quality was good in 1970's. Compared with the environmental monitoring data of 1995-1999, Pb pollution had become serious. Therefore, more efforts should be made to protect the bay from Pb pollution.

  2. DNA methylation directly silences genes with non-CpG island promoters and establishes a nucleosome occupied promoter.

    Science.gov (United States)

    Han, Han; Cortez, Connie C; Yang, Xiaojing; Nichols, Peter W; Jones, Peter A; Liang, Gangning

    2011-11-15

    Despite the fact that 45% of all human gene promoters do not contain CpG islands, the role of DNA methylation in control of non-CpG island promoters is controversial and its relevance in normal and pathological processes is poorly understood. Among the few studies which investigate the correlation between DNA methylation and expression of genes with non-CpG island promoters, the majority do not support the view that DNA methylation directly leads to transcription silencing of these genes. Our reporter assays and gene reactivation by 5-aza-2'-deoxycytidine, a DNA demethylating agent, show that DNA methylation occurring at CpG poor LAMB3 promoter and RUNX3 promoter 1(RUNX3 P1) can directly lead to transcriptional silencing in cells competent to express these genes in vitro. Using Nucleosome Occupancy Methylome- Sequencing, NOMe-Seq, a single-molecule, high-resolution nucleosome positioning assay, we demonstrate that active, but not inactive, non-CpG island promoters display a nucleosome-depleted region (NDR) immediately upstream of the transcription start site (TSS). Furthermore, using NOMe-Seq and clonal analysis, we show that in RUNX3 expressing 623 melanoma cells, RUNX3 P1 has two distinct chromatin configurations: one is unmethylated with an NDR upstream of the TSS; another is methylated and nucleosome occupied, indicating that RUNX3 P1 is monoallelically methylated. Together, these results demonstrate that the epigenetic signatures comprising DNA methylation, histone marks and nucleosome occupancy of non-CpG island promoters are almost identical to CpG island promoters, suggesting that aberrant methylation patterns of non-CpG island promoters may also contribute to tumorigenesis and should therefore be included in analyses of cancer epigenetics.

  3. Intrauterine growth restriction perturbs nucleosome depletion at a growth hormone-responsive element in the mouse IGF-1 gene.

    Science.gov (United States)

    McKnight, Robert A; Yost, Christian C; Yu, Xing; Wiedmeier, Julia E; Callaway, Christopher W; Brown, Ashley S; Lane, Robert H; Fung, Camille M

    2015-12-01

    Intrauterine growth restriction (IUGR) is a common human pregnancy complication. IUGR offspring carry significant postnatal risk for early-onset metabolic syndrome, which is associated with persistent reduction in IGF-1 protein expression. We have previously shown that preadolescent IUGR male mice have decreased hepatic IGF-1 mRNA and circulating IGF-1 protein at postnatal day 21, the age when growth hormone (GH) normally upregulates hepatic IGF-1 expression. Here we studied nucleosome occupancy and CpG methylation at a putative growth hormone-responsive element in intron 2 (in2GHRE) of the hepatic IGF-1 gene in normal, sham-operated, and IUGR mice. Nucleosome occupancy and CpG methylation were determined in embryonic stem cells (ESCs) and in liver at postnatal days 14, 21, and 42. For CpG methylation, additional time points out to 2 yr were analyzed. We confirmed the putative mouse in2GHRE was GH-responsive, and in normal mice, a single nucleosome was displaced from the hepatic in2GHRE by postnatal day 21, which exposed two STAT5b DNA binding sites. Nucleosome displacement correlated with developmentally programmed CpG demethylation. Finally, IUGR significantly altered the nucleosome-depleted region (NDR) at the in2GHRE of IGF-1 on postnatal day 21, with either complete absence of the NDR or with a shifted NDR exposing only one of two STAT5b DNA binding sites. An NDR shift was also seen in offspring of sham-operated mothers. We conclude that prenatal insult such as IUGR or anesthesia/surgery could perturb the proper formation of a well-positioned NDR at the mouse hepatic IGF-1 in2GHRE necessary for transitioning to an open chromatin state.

  4. Intestinal translocation of Streptococcus suis type 2 EF+ in pigs

    NARCIS (Netherlands)

    Swildens, B.; Stockhofe-Zurwieden, N.; Meulen, van der J.; Wisselink, H.J.; Nielen, M.; Niewold, T.A.

    2004-01-01

    Sepsis with subsequent multisystem organ failure after translocation of bacteria from the gut is a serious risk associated with stress situations. We showed that intestinal bacterial translocation could be one of the pathways for pathogenic Streptococcus suis infections in the pig. In 24 piglets wei

  5. Label Free Chromosome Translocation Detection with Silicon nanowires

    DEFF Research Database (Denmark)

    Kwasny, Dorota; Andersen, Karsten Brandt; Frøhling, Kasper Bayer;

    is a Fluorescent In Situ Hybridization, which is laborious and involves use of expensive reagents [1]. Here we present a label free technique for detection of chromosome translocations. As a proof of concept detection of chromosome translocation between chromosome 3 (Chr3) and chromosome 9 (Chr9) was chosen....

  6. Inducement of chromosome translocation with small alien segments by irradiating mature female gametes of the whole arm translocation line

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Haynaldia villosa Schur. (syn. Dasypyrum villosum Candargy, 2n=14, VV) has been proved to be an important genetic resource for wheat improvement. The development of translocation with small alien chromosome segments, especially interstitial translocation, will be helpful for better utilization of its useful genes. Up to now, most of the reported Triticum aestivum – H. villosa translocation lines are involved in a whole arm or large alien fragments. In this paper, we report a highly efficient approach for the creation of small chromosome segment translocation lines. Before flowering, the female gametes of wheat-H. villosa 6VS/6AL translocation line were irradiated by 60CO-γ ray at 160 Rad/M dosage rate and three dosages (1600, 1920, 2240 Rad). Anthers were removed from the irradiated florets on the same day and the florets were pollinated with normal fresh pollens of T. aestivum cv. Chinese Spring after 2-3 days. Genomic in situ hybridization (GISH) at mitosis metaphase of root-tip cell of M1 plants was used to detect the chromosome structural changes involving 6VS of H. villosa. Among the 534 M1 plants screened, 97 plants contained small segment chromosome structural changes of 6VS, including 80 interstitial translocation chromosomes, 57 terminal translocation chromosomes and 55 deletion chromosomes. For the 2240 Rad dosage treatment, the inducement frequencies of interstitial translo-cation, terminal translocation and deletion were 21.02%, 14.01%, and 14.65%, respectively, which were much higher than those previously reported. The M2 seeds were obtained by backcrossing of 74 M1 plants involving 146 chromosomes structural changes of 6VS, and it was found that the structural aberrations in the M1 plants could be transmitted to their progenies. Irradiating mature female gametes of whole arm translocation is a new and highly efficient approach for creation of small segment chromosome struc-tural changes, especially for interstitial translocations.

  7. Strandwise translocation of a DNA glycosylase on undamaged DNA

    Energy Technology Data Exchange (ETDEWEB)

    Qi, Yan; Nam, Kwangho; Spong, Marie C.; Banerjee, Anirban; Sung, Rou-Jia; Zhang, Michael; Karplus, Martin; Verdine, Gregory L. (Harvard)

    2012-05-14

    Base excision repair of genotoxic nucleobase lesions in the genome is critically dependent upon the ability of DNA glycosylases to locate rare sites of damage embedded in a vast excess of undamaged DNA, using only thermal energy to fuel the search process. Considerable interest surrounds the question of how DNA glycosylases translocate efficiently along DNA while maintaining their vigilance for target damaged sites. Here, we report the observation of strandwise translocation of 8-oxoguanine DNA glycosylase, MutM, along undamaged DNA. In these complexes, the protein is observed to translocate by one nucleotide on one strand while remaining untranslocated on the complementary strand. We further report that alterations of single base-pairs or a single amino acid substitution (R112A) can induce strandwise translocation. Molecular dynamics simulations confirm that MutM can translocate along DNA in a strandwise fashion. These observations reveal a previously unobserved mode of movement for a DNA-binding protein along the surface of DNA.

  8. Multistep Current Signal in Protein Translocation through Graphene Nanopores

    KAUST Repository

    Bonome, Emma Letizia

    2015-05-07

    © 2015 American Chemical Society. In nanopore sensing experiments, the properties of molecules are probed by the variation of ionic currents flowing through the nanopore. In this context, the electronic properties and the single-layer thickness of graphene constitute a major advantage for molecule characterization. Here we analyze the translocation pathway of the thioredoxin protein across a graphene nanopore, and the related ionic currents, by integrating two nonequilibrium molecular dynamics methods with a bioinformatic structural analysis. To obtain a qualitative picture of the translocation process and to identify salient features we performed unsupervised structural clustering on translocation conformations. This allowed us to identify some specific and robust translocation intermediates, characterized by significantly different ionic current flows. We found that the ion current strictly anticorrelates with the amount of pore occupancy by thioredoxin residues, providing a putative explanation of the multilevel current scenario observed in recently published translocation experiments.

  9. Chaperone-assisted translocation of flexible polymers in three dimensions

    CERN Document Server

    Suhonen, P M

    2016-01-01

    Polymer translocation through a nanometer-scale pore assisted by chaperones binding to the polymer is a process encountered in vivo for proteins. Studying the relevant models by computer simulations is computationally demanding. Accordingly, previous studies are either for stiff polymers in three dimensions or flexible polymers in two dimensions. Here, we study chaperone-assisted translocation of flexible polymers in three dimensions using Langevin dynamics. We show that differences in binding mechanisms, more specifically, whether a chaperone can bind to a single or multiple sites on the polymer, lead to substantial differences in translocation dynamics in three dimensions. We show that the single-binding mode leads to dynamics that is very much like that in the constant-force driven translocation and accordingly mainly determined by tension propagation on the cis side. We obtain $\\beta \\approx 1.26$ for the exponent for the scaling of the translocation time with polymer length. This fairly low value can be ...

  10. Mechanisms underlying stage-1 TRPL channel translocation in Drosophila photoreceptors.

    Directory of Open Access Journals (Sweden)

    Minh-Ha Lieu

    Full Text Available BACKGROUND: TRP channels function as key mediators of sensory transduction and other cellular signaling pathways. In Drosophila, TRP and TRPL are the light-activated channels in photoreceptors. While TRP is statically localized in the signaling compartment of the cell (the rhabdomere, TRPL localization is regulated by light. TRPL channels translocate out of the rhabdomere in two distinct stages, returning to the rhabdomere with dark-incubation. Translocation of TRPL channels regulates their availability, and thereby the gain of the signal. Little, however, is known about the mechanisms underlying this trafficking of TRPL channels. METHODOLOGY/PRINCIPAL FINDINGS: We first examine the involvement of de novo protein synthesis in TRPL translocation. We feed flies cycloheximide, verify inhibition of protein synthesis, and test for TRPL translocation in photoreceptors. We find that protein synthesis is not involved in either stage of TRPL translocation out of the rhabdomere, but that re-localization to the rhabdomere from stage-1, but not stage-2, depends on protein synthesis. We also characterize an ex vivo eye preparation that is amenable to biochemical and genetic manipulation. We use this preparation to examine mechanisms of stage-1 TRPL translocation. We find that stage-1 translocation is: induced with ATP depletion, unaltered with perturbation of the actin cytoskeleton or inhibition of endocytosis, and slowed with increased membrane sterol content. CONCLUSIONS/SIGNIFICANCE: Our results indicate that translocation of TRPL out of the rhabdomere is likely due to protein transport, and not degradation/re-synthesis. Re-localization from each stage to the rhabdomere likely involves different strategies. Since TRPL channels can translocate to stage-1 in the absence of ATP, with no major requirement of the cytoskeleton, we suggest that stage-1 translocation involves simple diffusion through the apical membrane, which may be regulated by release of a

  11. Translocations of amphibians: Proven management method or experimental technique

    Science.gov (United States)

    Seigel, Richard A.; Dodd, C. Kenneth

    2002-01-01

    In an otherwise excellent review of metapopulation dynamics in amphibians, Marsh and Trenham (2001) make the following provocative statements (emphasis added): If isolation effects occur primarily in highly disturbed habitats, species translocations may be necessary to promote local and regional population persistence. Because most amphibians lack parental care, they areprime candidates for egg and larval translocations. Indeed, translocations have already proven successful for several species of amphibians. Where populations are severely isolated, translocations into extinct subpopulations may be the best strategy to promote regional population persistence. We take issue with these statements for a number of reasons. First, the authors fail to cite much of the relevant literature on species translocations in general and for amphibians in particular. Second, to those unfamiliar with current research in amphibian conservation biology, these comments might suggest that translocations are a proven management method. This is not the case, at least in most instances where translocations have been evaluated for an appropriate period of time. Finally, the authors fail to point out some of the negative aspects of species translocation as a management method. We realize that Marsh and Trenham's paper was not concerned primarily with translocations. However, because Marsh and Trenham (2001) made specific recommendations for conservation planners and managers (many of whom are not herpetologists or may not be familiar with the pertinent literature on amphibians), we believe that it is essential to point out that not all amphibian biologists are as comfortable with translocations as these authors appear to be. We especially urge caution about advocating potentially unproven techniques without a thorough review of available options.

  12. 40 CFR 798.5955 - Heritable translocation test in drosophila melanogaster.

    Science.gov (United States)

    2010-07-01

    ... drosophila melanogaster. 798.5955 Section 798.5955 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5955 Heritable translocation test in drosophila melanogaster. (a) Purpose. The heritable translocation test in Drosophila measures the induction of chromosomal translocations in germ cells of...

  13. Bacterial Translocation and Change in Intestinal Permeability in Patients after Abdominal Surgery

    Institute of Scientific and Technical Information of China (English)

    Zhi QIAO; Zhanliang LI; Jiye LI; Lianrong LU; Yi LV; Junyou LI

    2009-01-01

    sely related with bacterial translocation. Intestinal bacterial translocation (most commonly E. coli) might occur at early stage (2 h) after ab-dominal surgery. Postoperative SIRS and infection might bear a close relationship with bacterial translocation.

  14. Poly(dA:dT) tracts: major determinants of nucleosome organization.

    Science.gov (United States)

    Segal, Eran; Widom, Jonathan

    2009-02-01

    Homopolymeric stretches of deoxyadenosine nucleotides (A's) on one strand of double-stranded DNA, referred to as poly(dA:dT) tracts or A-tracts, are overabundant in eukaryotic genomes. They have unusual structural, dynamic, and mechanical properties, and may resist sharp bending. Such unusual material properties, together with their overabundance in eukaryotes, raised the possibility that poly(dA:dT) tracts might function in eukaryotes to influence the organization of nucleosomes at many genomic regions. Recent genome-wide studies strongly confirm these ideas and suggest that these tracts play major roles in chromatin organization and genome function. Here we review what is known about poly(dA:dT) tracts and how they work.

  15. Inducement of chromosome translocation with small alien segments by irradiating mature female gametes of the whole arm translocation line

    Institute of Scientific and Technical Information of China (English)

    CHEN ShengWei; CHEN PeiDu; WANG XiuE

    2008-01-01

    Haynaldia villosa Schur. (syn. Dasypyrum villosum Candargy, 2n=14, VV) has been proved to be an Important genetic resource for wheat improvement. The development of translocation with small alien chromosome segments, especially interstitial translocation, will be helpful for better utilization of its useful genes. Up to now, most of the reported Triticum aestivum - H. villosa translocation lines are involved in a whole arm or large alien fragments. In this paper, we report a highly efficient approach for the creation of small chromosome segment translocation lines. Before flowering, the female gametes of wheat-H, villosa 6VS/6AL trsnslocation line were irradiated by 60Co-γ ray at 160 Rad/M dosage rate and three dosages (1600, 1920, 2240 Rad). Anthers were removed from the irradiated florets on the same day and the florets were pollinated with normal fresh pollens of T. aestivum cv. Chinese Spring after 2-3 days. Genomic in situ hybridization (GISH) at mitosis metaphase of root-tip cell of M1 plants was used to detect the chromosome structural changes involving 6VS of H. villosa. Among the 534 M1 plants screened, 97 plants contained small segment chromosome structural changes of 6VS, including 80 interstitial translocation chromosomes, 57 terminal translocation chromosomes and 55 deletion chromosomes. For the 2240 Rad dosage treatment, the inducement frequencies of interstitial translocation, terminal translocation and deletion were 21.02%, 14.01%, and 14.65%, respectively, which were much higher than those previously reported. The M2 seeds were obtained by bsckcrossing of 74 M1 plants involving 146 chromosomes structural changes of 6VS, and it was found that the structural aberrations in the M1 plants could be transmitted to their progenies. Irradiating mature female gametes of whole arm translocation is a new and highly efficient approach for creation of small segment chromosome structural changes, especially for interstitial translocations.

  16. Histone Methylation Marks on Circulating Nucleosomes as Novel Blood-Based Biomarker in Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Ugur Gezer

    2015-12-01

    Full Text Available Circulating nucleic acids (CNAs are under investigation as a liquid biopsy in cancer as potential non-invasive biomarkers, as stable structure in circulation nucleosomes could be valuable sources for detection of cancer-specific alterations in histone modifications. Our interest is in histone methylation marks with a focus on colorectal cancer, one of the leading cancers respective the incidence and mortality. Our previous work included the analysis of trimethylations of lysine 9 on histone 3 (H3K9me3 and of lysine 20 on histone 4 (H4K20me3 by chromatin immuno- precipitation-related PCR in circulating nucleosomes. Here we asked whether global immunologic measurement of histone marks in circulation could be a suitable approach to show their potential as biomarkers. In addition to H3K9me3 and H4K20me3 we also measured H3K27me3 in plasma samples from CRC patients (n = 63 and cancer free individuals (n = 40 by ELISA-based methylation assays. Our results show that of three marks, the amounts of H3K27me3 (p = 0.04 and H4K20me3 (p < 0.001 were significantly lower in CRC patients than in healthy controls. For H3K9me3 similar amounts were measured in both groups. Areas under the curve (AUC in receiver operating characteristic (ROC curves indicating the power of CRC detection were 0.620 for H3K27me3, 0.715 for H4K20me3 and 0.769 for the combination of both markers. In conclusion, findings of this preliminary study reveal the potential of blood-based detection of CRC by quantification of histone methylation marks and the additive effect of the marker combination.

  17. The changing paradigm: estrogen receptor α recognition on DNA and within the dynamic nature of nucleosomes

    Directory of Open Access Journals (Sweden)

    William M. Scovell

    2015-03-01

    Full Text Available Estrogen receptor alpha (ERα plays a major role in the expression of estrogen-responsive genes. Although its conventional binding characteristics have been considered coincident with & exclusively in the class of steroid hormone receptors, increasing evidence challenges this paradigm. ERα was shown to bind to consensus estrogen response element half-sites (cHERE in DNA in the presence of the ubiquitous, abundant & conserved architectural protein, high mobility group protein 1 (HMGB1. It also binds to direct repeats with various spacers, in addition to everted repeats. These in vitro binding sites have been shown to be active in vivo, with both the binding affinity and transcriptional activity increased in the presence of HMGB1. Surprisingly, ERα does not bind to the optimally oriented cERE at the dyad in rotationally phased and translationally positioned nucleosomes. However, the presence of HMGB1 restructures the nucleosome to facilitate increased ERα accessibility, resulting in sequence-specific estrogen receptor binding. The finding that HMGB1 interacts with unbound ERα provides a unique avenue for enhanced ERα activity and possibly an increase in the extent of targeting at estrogen-responsive genes. The findings are consistent with ERα 1 targeting a much wider selection of genomic response elements (half-sites and inverted, direct and everted repeats and 2 exhibiting characteristics of both steroid and non steroid nuclear receptors. Growing evidence already shows a competition occurs at the DNA level between ERα and the non steroid nuclear hormone receptor, thyroid receptor (TR. Collectively, these reports suggest a less restrictive cataloging for estrogen receptor and a broader paradigm for understanding its role in the regulation of estrogen-responsive genes and influence on non steroid hormone receptor activities.

  18. A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin.

    Science.gov (United States)

    Svensson, J Peter; Shukla, Manu; Menendez-Benito, Victoria; Norman-Axelsson, Ulrika; Audergon, Pauline; Sinha, Indranil; Tanny, Jason C; Allshire, Robin C; Ekwall, Karl

    2015-06-01

    Nucleosome composition actively contributes to chromatin structure and accessibility. Cells have developed mechanisms to remove or recycle histones, generating a landscape of differentially aged nucleosomes. This study aimed to create a high-resolution, genome-wide map of nucleosome turnover in Schizosaccharomyces pombe. The recombination-induced tag exchange (RITE) method was used to study replication-independent nucleosome turnover through the appearance of new histone H3 and the disappearance or preservation of old histone H3. The genome-wide location of histones was determined by chromatin immunoprecipitation-exonuclease methodology (ChIP-exo). The findings were compared with diverse chromatin marks, including histone variant H2A.Z, post-translational histone modifications, and Pol II binding. Finally, genome-wide mapping of the methylation states of H4K20 was performed to determine the relationship between methylation (mono, di, and tri) of this residue and nucleosome turnover. Our analysis showed that histone recycling resulted in low nucleosome turnover in the coding regions of active genes, stably expressed at intermediate levels. High levels of transcription resulted in the incorporation of new histones primarily at the end of transcribed units. H4K20 was methylated in low-turnover nucleosomes in euchromatic regions, notably in the coding regions of long genes that were expressed at low levels. This transcription-dependent accumulation of histone methylation was dependent on the histone chaperone complex FACT. Our data showed that nucleosome turnover is highly dynamic in the genome and that several mechanisms are at play to either maintain or suppress stability. In particular, we found that FACT-associated transcription conserves histones by recycling them and is required for progressive H4K20 methylation.

  19. Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair.

    Science.gov (United States)

    Gursoy-Yuzugullu, Ozge; Ayrapetov, Marina K; Price, Brendan D

    2015-06-16

    The repair of DNA double-strand breaks (DSBs) requires open, flexible chromatin domains. The NuA4-Tip60 complex creates these flexible chromatin structures by exchanging histone H2A.Z onto nucleosomes and promoting acetylation of histone H4. Here, we demonstrate that the accumulation of H2A.Z on nucleosomes at DSBs is transient, and that rapid eviction of H2A.Z is required for DSB repair. Anp32e, an H2A.Z chaperone that interacts with the C-terminal docking domain of H2A.Z, is rapidly recruited to DSBs. Anp32e functions to remove H2A.Z from nucleosomes, so that H2A.Z levels return to basal within 10 min of DNA damage. Further, H2A.Z removal by Anp32e disrupts inhibitory interactions between the histone H4 tail and the nucleosome surface, facilitating increased acetylation of histone H4 following DNA damage. When H2A.Z removal by Anp32e is blocked, nucleosomes at DSBs retain elevated levels of H2A.Z, and assume a more stable, hypoacetylated conformation. Further, loss of Anp32e leads to increased CtIP-dependent end resection, accumulation of single-stranded DNA, and an increase in repair by the alternative nonhomologous end joining pathway. Exchange of H2A.Z onto the chromatin and subsequent rapid removal by Anp32e are therefore critical for creating open, acetylated nucleosome structures and for controlling end resection by CtIP. Dynamic modulation of H2A.Z exchange and removal by Anp32e reveals the importance of the nucleosome surface and nucleosome dynamics in processing the damaged chromatin template during DSB repair.

  20. Translocation of gut flora and its role in sepsis

    Directory of Open Access Journals (Sweden)

    C Vaishnavi

    2013-01-01

    Full Text Available Bacterial translocation is the invasion of indigenous intestinal bacteria through the gut mucosa to normally sterile tissues and the internal organs. Sometimes instead of bacteria, inflammatory compounds are responsible for clinical symptoms as in systemic inflammatory response syndrome (SIRS. The difference between sepsis and SIRS is that pathogenic bacteria are isolated from patients with sepsis but not with those of SIRS. Bacterial translocation occurs more frequently in patients with intestinal obstruction and in immunocompromised patients and is the cause of subsequent sepsis. Factors that can trigger bacterial translocation from the gut are host immune deficiencies and immunosuppression, disturbances in normal ecological balance of gut, mucosal barrier permeability, obstructive jaundice, stress, etc. Bacterial translocation occurs through the transcellular and the paracellular pathways and can be measured both directly by culture of mesenteric lymph nodes and indirectly by using labeled bacteria, peripheral blood culture, detection of microbial DNA or endotoxin and urinary excretion of non-metabolisable sugars. Bacterial translocation may be a normal phenomenon occurring on frequent basis in healthy individuals without any deleterious consequences. But when the immune system is challenged extensively, it breaks down and results in septic complications at different sites away from the main focus. The factors released from the gut and carried in the mesenteric lymphatics but not in the portal blood are enough to cause multi-organ failure. Thus, bacterial translocation may be a promoter of sepsis but not the initiator. This paper reviews literature on the translocation of gut flora and its role in causing sepsis.

  1. Does translocation influence physiological stress in the desert tortoise?

    Science.gov (United States)

    Drake, K.K.; Nussear, K.E.; Esque, T.C.; Barber, A.M.; Vittum, K.M.; Medica, P.A.; Tracy, C.R.; Hunter, K.W.

    2012-01-01

    Wildlife translocation is increasingly used to mitigate disturbances to animals or habitat due to human activities, yet little is known about the extent to which translocating animals causes stress. To understand the relationship between physiological stress and translocation, we conducted a multiyear study (2007–2009) using a population of desert tortoises (Gopherus agassizii) near Fort Irwin, California. Blood samples were collected from adult tortoises in three treatment groups (resident, translocated and control) for 1 year prior to and 2 years after translocation. Samples were analyzed by radioimmunoassay for plasma total corticosterone (CORT), a glucocorticoid hormone commonly associated with stress responses in reptiles. CORT values were analyzed in relation to potential covariates (animal sex, date, behavior, treatment, handling time, air temperature, home-range size, precipitation and annual plant production) among seasons and years. CORT values in males were higher than in females, and values for both varied monthly throughout the activity season and among years. Year and sex were strong predictors of CORT, and translocation explained little in terms of CORT. Based on these results, we conclude that translocation does not elicit a physiological stress response in desert tortoises.

  2. Genetic outcomes from the translocations of the critically endangered woylie

    Institute of Scientific and Technical Information of China (English)

    Carlo PACIONI; Adrian F.WAYNE; Peter B.S.SPENCER

    2013-01-01

    Translocations are an important conservation strategy for many species.However simply observing demographic growth of a translocated population is not sufficient to infer species recovery.Adequate genetic representation of the source population(s) and their long-term viability should also be considered.The woylie Bettongiapenicillata ogilbyi has been subject to more formal translocations for conservation than any other marsupial that,up until recently,has resulted in one of the most successful species recoveries in Australia.We used mitochondrial and nuclear DNA markers to assess the genetic outcomes of translocated woylie populations.These populations have lost genetic variability,differentiated from their source population and the supplementation program on two island populations appears to have failed.We discuss the conservation implications that our results have for managing threatened species,outline some general recommendations for the management of present and future translocations and discuss the appropriate sampling design for the establishment of new populations or captive breeding programs that may mitigate the genetic ‘erosion' seen in our study species.This research provides some practical outcomes and a pmgrnatic understanding of translocation biology.The findings are directly applicable to other translocation programs.

  3. Mode of ATM-dependent suppression of chromosome translocation

    Energy Technology Data Exchange (ETDEWEB)

    Yamauchi, Motohiro, E-mail: motoyama@nagasaki-u.ac.jp [Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Suzuki, Keiji; Oka, Yasuyoshi; Suzuki, Masatoshi; Kondo, Hisayoshi; Yamashita, Shunichi [Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan)

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer We addressed how ATM suppresses frequency of chromosome translocation. Black-Right-Pointing-Pointer We found ATM/p53-dependent G1 checkpoint suppresses translocation frequency. Black-Right-Pointing-Pointer We found ATM and DNA-PKcs function in a common pathway to suppress translocation. -- Abstract: It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs.

  4. Translocation of threatened plants as a conservation measure in China.

    Science.gov (United States)

    Liu, Hong; Ren, Hai; Liu, Qiang; Wen, XiangYing; Maunder, Michael; Gao, JiangYun

    2015-12-01

    We assessed the current status of plant conservation translocation efforts in China, a topic poorly reported in recent scientific literature. We identified 222 conservation translocation cases involving 154 species, of these 87 were Chinese endemic species and 101 (78%) were listed as threatened on the Chinese Species Red List. We categorized the life form of each species and, when possible, determined for each case the translocation type, propagule source, propagule type, and survival and reproductive parameters. A surprisingly large proportion (26%) of the conservation translocations in China were conservation introductions, largely implemented in response to large-scale habitat destruction caused by the Three-Gorge Dam and another hydropower project. Documentation and management of the translocations varied greatly. Less than half the cases had plant survival records. Statistical analyses showed that survival percentages were significantly correlated with plant life form and the type of planting materials. Thirty percent of the cases had records on whether or not individuals flowered or fruited. Results of information theoretic model selection indicated that plant life form, translocation type, propagule type, propagule source, and time since planting significantly influenced the likelihood of flowering and fruiting on the project level. We suggest that the scientific-based application of species conservation translocations should be promoted as part of a commitment to species recovery management. In addition, we recommend that the common practice of within and out of range introductions in nature reserves to be regulated more carefully due to its potential ecological risks. We recommend the establishment of a national office and database to coordinate conservation translocations in China. Our review effort is timely considering the need for a comprehensive national guideline for the newly announced nation-wide conservation program on species with extremely

  5. Effects of MacroH2A and H2A.Z on Nucleosome Dynamics as Elucidated by Molecular Dynamics Simulations.

    Science.gov (United States)

    Bowerman, Samuel; Wereszczynski, Jeff

    2016-01-19

    Eukaryotes tune the transcriptional activity of their genome by altering the nucleosome core particle through multiple chemical processes. In particular, replacement of the canonical H2A histone with the variants macroH2A and H2A.Z has been shown to affect DNA accessibility and nucleosome stability; however, the processes by which this occurs remain poorly understood. In this study, we elucidate the molecular mechanisms of these variants with an extensive molecular dynamics study of the canonical nucleosome along with three variant-containing structures: H2A.Z, macroH2A, and an H2A mutant with macroH2A-like L1 loops. Simulation results show that variant L1 loops play a pivotal role in stabilizing DNA binding to the octamer through direct interactions, core structural rearrangements, and altered allosteric networks in the nucleosome. All variants influence dynamics; however, macroH2A-like systems have the largest effect on energetics. In addition, we provide a comprehensive analysis of allosteric networks in the nucleosome and demonstrate that variants take advantage of stronger interactions between L1 loops to propagate dynamics throughout the complex. Furthermore, we show that posttranslational modifications are enriched at key locations in these networks. Taken together, these results provide, to our knowledge, new insights into the relationship between the structure, dynamics, and function of the nucleosome core particle and chromatin fibers, and how they are influenced by chromatin remodeling factors.

  6. Yeast H2A.Z, FACT complex and RSC regulate transcription of tRNA gene through differential dynamics of flanking nucleosomes.

    Science.gov (United States)

    Mahapatra, Sahasransu; Dewari, Pooran S; Bhardwaj, Anubhav; Bhargava, Purnima

    2011-05-01

    FACT complex is involved in elongation and ensures fidelity in the initiation step of transcription by RNA polymerase (pol) II. Histone variant H2A.Z is found in nucleosomes at the 5'-end of many genes. We report here H2A.Z-chaperone activity of the yeast FACT complex on the short, nucleosome-free, non-coding, pol III-transcribed yeast tRNA genes. On a prototype gene, yeast SUP4, chromatin remodeler RSC and FACT regulate its transcription through novel mechanisms, wherein the two gene-flanking nucleosomes containing H2A.Z, play different roles. Nhp6, which ensures transcription fidelity and helps load yFACT onto the gene flanking nucleosomes, has inhibitory role. RSC maintains a nucleosome abutting the gene terminator downstream, which results in reduced transcription rate in active state while H2A.Z probably helps RSC in keeping the gene nucleosome-free and serves as stress-sensor. All these factors maintain an epigenetic state which allows the gene to return quickly from repressed to active state and tones down the expression from the active SUP4 gene, required probably to maintain the balance in cellular tRNA pool.

  7. Using informative Multinomial-Dirichlet prior in a t-mixture with reversible jump estimation of nucleosome positions for genome-wide profiling.

    Science.gov (United States)

    Samb, Rawane; Khadraoui, Khader; Belleau, Pascal; Deschênes, Astrid; Lakhal-Chaieb, Lajmi; Droit, Arnaud

    2015-12-01

    Genome-wide mapping of nucleosomes has revealed a great deal about the relationships between chromatin structure and control of gene expression. Recent next generation CHIP-chip and CHIP-Seq technologies have accelerated our understanding of basic principles of chromatin organization. These technologies have taught us that nucleosomes play a crucial role in gene regulation by allowing physical access to transcription factors. Recent methods and experimental advancements allow the determination of nucleosome positions for a given genome area. However, most of these methods estimate the number of nucleosomes either by an EM algorithm using a BIC criterion or an effective heuristic strategy. Here, we introduce a Bayesian method for identifying nucleosome positions. The proposed model is based on a Multinomial-Dirichlet classification and a hierarchical mixture distributions. The number and the positions of nucleosomes are estimated using a reversible jump Markov chain Monte Carlo simulation technique. We compare the performance of our method on simulated data and MNase-Seq data from Saccharomyces cerevisiae against PING and NOrMAL methods.

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

    Science.gov (United States)

    Duan, Ming-Rui; Smerdon, Michael J

    2014-03-21

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

  9. [Mechanisms of bacteria translocation in generalized chronic parodontitis].

    Science.gov (United States)

    Bukharin, O V; Usviatsov, B Ia; Doroshina, N B; Kushkinbaeva, D R; Khlopko, Iu A

    2011-01-01

    Peculiarities of behavior reactions of bacteria-symbionts created conditions for the selection of translocators-strains. In microsymbiocenosis of parodontal pockets, from which translocation of bacteria into the blood was observed, the number of signals from intermicrobial communication, inhibiting the expression of the factors of colonization, virulence and persistence, was decreasing. Meanwhile, the number of signals on the increase of the expression of the factors given was increased. In 75% of cases strains-translocators were leaders; they gave more often signals on the inhibition of the growth of other strains-symbionts.

  10. Bacterial translocation - impact on the adipocyte compartment.

    Science.gov (United States)

    Kruis, Tassilo; Batra, Arvind; Siegmund, Britta

    2014-01-01

    Over the last decade it became broadly recognized that adipokines and thus the fat tissue compartment exert a regulatory function on the immune system. Our own group described the pro-inflammatory function of the adipokine leptin within intestinal inflammation in a variety of animal models. Following-up on this initial work, the aim was to reveal stimuli and mechanisms involved in the activation of the fat tissue compartment and the subsequent release of adipokines and other mediators paralleled by the infiltration of immune cells. This review will summarize the current literature on the possible role of the mesenteric fat tissue in intestinal inflammation with a focus on Crohn's disease (CD). CD is of particular interest in this context since the transmural intestinal inflammation has been associated with a characteristic hypertrophy of the mesenteric fat, a phenomenon called "creeping fat." The review will address three consecutive questions: (i) What is inducing adipocyte activation, (ii) which factors are released after activation and what are the consequences for the local fat tissue compartment and infiltrating cells; (iii) do the answers generated before allow for an explanation of the role of the mesenteric fat tissue within intestinal inflammation? With this review we will provide a working model indicating a close interaction in between bacterial translocation, activation of the adipocytes, and subsequent direction of the infiltrating immune cells. In summary, the models system mesenteric fat indicates a unique way how adipocytes can directly interact with the immune system.

  11. Energy-dependent intracellular translocation of proparathormone.

    Science.gov (United States)

    Chu, L L; MacGregor, R R; Cohn, D V

    1977-01-01

    We previously suggested that after synthesis, proparathormone is transferred from rough endoplasmic reticulum to the Golgi region where its conversion to parathormone occurs. We have attempted to define more closely this transfer process. In the first type of study, bovine parathyroid slices were incubated with [3H]leucine for 10 min and then radioisotope labeling was restricted by addition of a large excess of nonradioactive leucine. Under these conditions, more than 90% of the initially labeled proparathormone was converted to parathormone in 40 min. Lowered temperature in the chase period markedly inhibited the conversion. Several chemical agents were employed individually in the chase period to examine their effect on the conversion process. Antimycin A, dinitrophenol, oligomycin, and anaerobiosis (N2) inhibited the conversion, whereas sodium flouride and cycloheximide had no effect. In the second type of study, parathyroid slices were incubated with [3H]leucine for the entire incubation period. Lowered temperature and inhibitors of energy metabolism and microtubular function all lengthened the interval (lag) between the initial synthesis of [3H]parathormone. Cycloheximide, Tris, and chloroquine decreased the rates of protein synthesis and conversion, respectively, but none had any effect on the lag. We interpret the lag to represent the time of transit for proparathormone from rough endoplasmic reticulum to the Golgi region. We conclude that this transfer process is independent of the synthesis of the prohormone and its conversion to the hormone. Moreover, this translocation requires metabolic energy and appears to be mediated by microtubules.

  12. De Novo microdeletion on an inherited Robertsonian translocation chromosome: A cause for dysmorphism in the apparently balanced translocation carrier

    Energy Technology Data Exchange (ETDEWEB)

    Bonthron, D.T.; Smith, S.J.L.; Fantes, J.; Gosden, C.M.

    1993-09-01

    Robertsonian translocations are usually ascertained through abnormal children, making proposed phenotypic effects of apparently balanced translocations difficult to study in an unbiased way. From molecular genetic studies, though, some apparently balanced rearrangments are now known to be associated with phenotypic abnormalities resulting from uniparental disomy. Molecular explanations for other cases in which abnormality is seen in a balanced translocation carrier are being sought. In the present paper, an infant is described who has retarded growth, developmental delay, gross muscular hypotonia, slender habitus, frontal bossing, micrognathia, hooked nose, abundant wispy hair, and blue sclerae. Cytogenetically, she appeared to be a carrier of a balanced, paternally derived 14;21 Robertsonian translocation. Analysis of DNA polymorphisms showed that she had no paternal allele at the D14S13 locus (14q32). Study of additional DNA markers within 14q32 revealed that her previously undescribed phenotype results from an interstitial microdeletion within 14q32. Fluorescent in situ hybridization was used to show that this microdeletion had occurred de novo on the Robertsonian translocation chromosome. These observations may reactivate old suspicions of a causal association between Robertsonian translocations and de novo rearrangements in offspring; a systematic search for similar subcytogentic rearrangements in other families, in which there are phenotypically abnormal children with apparently balanced translocations, may be fruitful. The clinical and molecular genetic data presented also define a new contiguous gene syndrome due to interstitial 14q32 deletion. 42 refs., 4 figs., 1 tab.

  13. DNA-graphene interactions during translocation through nanogaps

    Science.gov (United States)

    Patel, Hiral N.; Carroll, Ian; Lopez, Rodolfo; Sankararaman, Sandeep; Etienne, Charles; Kodigala, Subba Ramaiah; Paul, Mark R.

    2017-01-01

    We study how double-stranded DNA translocates through graphene nanogaps. Nanogaps are fabricated with a novel capillary-force induced graphene nanogap formation technique. DNA translocation signatures for nanogaps are qualitatively different from those obtained with circular nanopores, owing to the distinct shape of the gaps discussed here. Translocation time and conductance values vary by ∼ 100%, which we suggest are caused by local gap width variations. We also observe exponentially relaxing current traces. We suggest that slow relaxation of the graphene membrane following DNA translocation may be responsible. We conclude that DNA-graphene interactions are important, and need to be considered for graphene-nanogap based devices. This work further opens up new avenues for direct read of single molecule activitities, and possibly sequencing. PMID:28158244

  14. Fragility in the 14q21q translocation region

    Directory of Open Access Journals (Sweden)

    Stacy R. Denison

    2002-01-01

    Full Text Available Aphidicolin (APC-induced chromosomal breakage was analyzed for women representing three generations of a single family and carrying a Robertsonian translocation rob(14q21q. Fluorescence in situ hybridization (FISH analysis confirmed the dicentric constitution of the derived chromosome and indicated the absence of beta-satellite signal at the translocation region. Per-individual analysis of metaphases from APC-treated peripheral blood lymphocyte cultures identified significantly nonrandom chromosomal breakage at the translocation region in all three individuals examined. The APC-inducible fragility at the 14q21q translocation region suggests that this rearrangement was the result of chromosomal mutation at fragile site(s in the progenitor chromosomes, or that this fragility was the result of the fusion of nonfragile progenitor chromosomes.

  15. DNA translocations through solid-state plasmonic nanopores.

    Science.gov (United States)

    Nicoli, Francesca; Verschueren, Daniel; Klein, Misha; Dekker, Cees; Jonsson, Magnus P

    2014-12-10

    Nanopores enable label-free detection and analysis of single biomolecules. Here, we investigate DNA translocations through a novel type of plasmonic nanopore based on a gold bowtie nanoantenna with a solid-state nanopore at the plasmonic hot spot. Plasmonic excitation of the nanopore is found to influence both the sensor signal (nanopore ionic conductance blockade during DNA translocation) and the process that captures DNA into the nanopore, without affecting the duration time of the translocations. Most striking is a strong plasmon-induced enhancement of the rate of DNA translocation events in lithium chloride (LiCl, already 10-fold enhancement at a few mW of laser power). This provides a means to utilize the excellent spatiotemporal resolution of DNA interrogations with nanopores in LiCl buffers, which is known to suffer from low event rates. We propose a mechanism based on plasmon-induced local heating and thermophoresis as explanation of our observations.

  16. TRANSLOCATION OF BACTERIA AND ENDOTOXIN IN ORGAN DONORS

    NARCIS (Netherlands)

    van Goor, Harry; Rosman, C; Kooi, K; Wubbels, GH; Bleichrodt, RP

    1994-01-01

    Objective: To determine if bacterial translocation and endotoxin absorption occur in organ donors with an anatomically intact gastrointestinal tract. Design: Case series. Setting: Intensive care units in general and university hospitals. Patients: Twenty-one (multiple) organ donors. Intervention: No

  17. Influence of Methylobacterium on iron translocation in plants.

    Science.gov (United States)

    Bishop, Yvonne M; Barton, Larry L; Johnson, Gordon V

    2011-06-01

    Iron metabolism in plants is essential to maintain optimal growth and iron nutrition is dependent on uptake of iron from the environment and movement of iron in the plant tissues. We have examined the translocation of iron in plant leaves following foliar application of FeEDTA to Vicia faba and Zea mays. Using radiolabeled iron, we observed that iron translocation is stimulated by products of Methylobacterium mesophylicum and by the cytokinin, kinetin. When cytokinins were applied to leaves along with (55)FeEDTA, the rate of iron translocation was greater than in controls without cytokinin addition. Since recent studies indicate that M. mesophylicum is widely distributed in the environment as a pyllospheric bacterium, this organism may have an important role in enhancing translocation of nutrients in plant leaves.

  18. Rapid deamination of cyclobutane pyrimidine dimer photoproducts at TCG sites in a translationally and rotationally positioned nucleosome in vivo.

    Science.gov (United States)

    Cannistraro, Vincent J; Pondugula, Santhi; Song, Qian; Taylor, John-Stephen

    2015-10-30

    Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for T(m)CG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots.

  19. Slowing DNA Translocation in a Nanofluidic Field-Effect Transistor.

    Science.gov (United States)

    Liu, Yifan; Yobas, Levent

    2016-04-26

    Here, we present an experimental demonstration of slowing DNA translocation across a nanochannel by modulating the channel surface charge through an externally applied gate bias. The experiments were performed on a nanofluidic field-effect transistor, which is a monolithic integrated platform featuring a 50 nm-diameter in-plane alumina nanocapillary whose entire length is surrounded by a gate electrode. The field-effect transistor behavior was validated on the gating of ionic conductance and protein transport. The gating of DNA translocation was subsequently studied by measuring discrete current dips associated with single λ-DNA translocation events under a source-to-drain bias of 1 V. The translocation speeds under various gate bias conditions were extracted by fitting event histograms of the measured translocation time to the first passage time distributions obtained from a simple 1D biased diffusion model. A positive gate bias was observed to slow the translocation of single λ-DNA chains markedly; the translocation speed was reduced by an order of magnitude from 18.4 mm/s obtained under a floating gate down to 1.33 mm/s under a positive gate bias of 9 V. Therefore, a dynamic and flexible regulation of the DNA translocation speed, which is vital for single-molecule sequencing, can be achieved on this device by simply tuning the gate bias. The device is realized in a conventional semiconductor microfabrication process without the requirement of advanced lithography, and can be potentially further developed into a compact electronic single-molecule sequencer.

  20. Survival of mountain quail translocated from two distinct source populations

    Science.gov (United States)

    Troy, Ronald J.; Coates, Peter S.; Connelly, John W.; Gillette, Gifford; Delehanty, David J.

    2013-01-01

    Translocation of mountain quail (Oreortyx pictus) to restore viable populations to their former range has become a common practice. Because differences in post-release vital rates between animals from multiple source populations has not been well studied, wildlife and land managers may arbitrarily choose the source population or base the source population on immediate availability when planning translocation projects. Similarly, an understanding of the optimal proportion of individuals from different age and sex classes for translocation would benefit translocation planning. During 2006 and 2007, we captured and translocated 125 mountain quail from 2 ecologically distinct areas: 38 from southern California and 87 from southwestern Oregon. We released mountain quail in the Bennett Hills of south-central Idaho. We radio-marked and monitored a subsample of 58 quail and used them for a 2-part survival analysis. Cumulative survival probability was 0.23 ± 0.05 (SE) at 150 days post-release. We first examined an a priori hypothesis (model) that survival varied between the 2 distinct source populations. We found that source population did not explain variation in survival. This result suggests that wildlife managers have flexibility in selecting source populations for mountain quail translocation efforts. In a post hoc examination, we pooled the quail across source populations and evaluated differences in survival probabilities between sex and age classes. The most parsimonious model indicated that adult male survival was substantially less than survival rates of other mountain quail age and sex classes (i.e., interaction between sex and age). This result suggests that translocation success could benefit by translocating yearling males rather than adult males, perhaps because adult male breeding behavior results in vulnerability to predators

  1. Chromosome-wide nucleosome replacement and H3.3 incorporation during mammalian meiotic sex chromosome inactivation.

    Science.gov (United States)

    van der Heijden, Godfried W; Derijck, Alwin A H A; Pósfai, Eszter; Giele, Maud; Pelczar, Pawel; Ramos, Liliana; Wansink, Derick G; van der Vlag, Johan; Peters, Antoine H F M; de Boer, Peter

    2007-02-01

    In mammalian males, the first meiotic prophase is characterized by formation of a separate chromatin domain called the sex body. In this domain, the X and Y chromosomes are partially synapsed and transcriptionally silenced, a process termed meiotic sex-chromosome inactivation (MSCI). Likewise, unsynapsed autosomal chromatin present during pachytene is also silenced (meiotic silencing of unsynapsed chromatin, MSUC). Although it is known that MSCI and MSUC are both dependent on histone H2A.X phosphorylation mediated by the kinase ATR, and cause repressive H3 Lys9 dimethylation, the mechanisms underlying silencing are largely unidentified. Here, we demonstrate an extensive replacement of nucleosomes within unsynapsed chromatin, depending on and initiated shortly after induction of MSCI and MSUC. Nucleosomal eviction results in the exclusive incorporation of the H3.3 variant, which to date has primarily been associated with transcriptional activity. Nucleosomal exchange causes loss and subsequent selective reacquisition of specific histone modifications. This process therefore provides a means for epigenetic reprogramming of sex chromatin presumably required for gene silencing in the male mammalian germ line.

  2. Atomic structure of anthrax protective antigen pore elucidates toxin translocation.

    Science.gov (United States)

    Jiang, Jiansen; Pentelute, Bradley L; Collier, R John; Zhou, Z Hong

    2015-05-28

    Anthrax toxin, comprising protective antigen, lethal factor, and oedema factor, is the major virulence factor of Bacillus anthracis, an agent that causes high mortality in humans and animals. Protective antigen forms oligomeric prepores that undergo conversion to membrane-spanning pores by endosomal acidification, and these pores translocate the enzymes lethal factor and oedema factor into the cytosol of target cells. Protective antigen is not only a vaccine component and therapeutic target for anthrax infections but also an excellent model system for understanding the mechanism of protein translocation. On the basis of biochemical and electrophysiological results, researchers have proposed that a phi (Φ)-clamp composed of phenylalanine (Phe)427 residues of protective antigen catalyses protein translocation via a charge-state-dependent Brownian ratchet. Although atomic structures of protective antigen prepores are available, how protective antigen senses low pH, converts to active pore, and translocates lethal factor and oedema factor are not well defined without an atomic model of its pore. Here, by cryo-electron microscopy with direct electron counting, we determine the protective antigen pore structure at 2.9-Å resolution. The structure reveals the long-sought-after catalytic Φ-clamp and the membrane-spanning translocation channel, and supports the Brownian ratchet model for protein translocation. Comparisons of four structures reveal conformational changes in prepore to pore conversion that support a multi-step mechanism by which low pH is sensed and the membrane-spanning channel is formed.

  3. Surface modification of graphene nanopores for protein translocation

    Science.gov (United States)

    Shan, Y. P.; Tiwari, P. B.; Krishnakumar, P.; Vlassiouk, I.; Li, W.Z.; Wang, X.W.; Darici, Y.; Lindsay, S.M.; Wang, H. D.; Smirnov, S.; He, J.

    2014-01-01

    Studies of DNA translocation through graphene nanopores have revealed their potential for DNA sequencing. Here we report a study of protein translocation through chemically modified graphene nanopores. A transmission electron microscope (TEM) was used to cut nanopores with diameters between 5-20 nm in multilayer graphene prepared by chemical vapor deposition (CVD). After oxygen plasma treatment, the dependence of the measured ionic current on salt concentration and pH was consistent with a small surface charge induced by the formation of carboxyl groups. While translocation of gold nanoparticles (10 nm) was readily detected through such treated pores of a larger diameter, translocation of protein ferritin was not observed either for oxygen plasma treated pores, or for pores modified with mercaptohexadecanoic acid. Ferritin translocation events were reliably observed after the pores were modified with the phospholipid-PEG (DPPE-PEG750) amphiphile. The ion current signature of translocation events was complex, suggesting that a series of interactions between the protein and pore occur during the process. PMID:24231385

  4. Uptake, translocation, and debromination of polybrominated diphenyl ethers in maize

    Institute of Scientific and Technical Information of China (English)

    Moming Zhao; Shuzhen Zhang; Sen Wang; Honglin Huang

    2012-01-01

    Uptake,translocation and debromination of three polybrominated diphenyl ethers(PBDEs),BDE-28,-47 and-99,in maize were studied in a hydroponic experiment.Roots took up most of the PBDEs in the culture solutions and more highly brominated PBDEs had a stronger uptake capability.PBDEs were detected in the stems and leaves of maize after exposure but rarely detected in the blank control plants.Furthermore,PBDE concentrations decreased from roots to stems and then to leaves,and a very clear decreasing gradient was found in segments upwards along the stem.These altogether provide substantiating evidence for the acropetal translocation of PBDEs in maize.More highly brominated PBDEs were translocated with more difficulty.Radial translocation of PBDEs from nodes to sheath inside maize was also observed.Both acropetal and radial translocations were enhanced at higher transpiration rates,suggesting that PBDE transport was probably driven by the transpiration stream.Debromination of PBDEs occurred in all parts of the maize,and debromination patterns of different parent PBDEs and in different parts of a plant were similar but with some differences.This study for the first time provides direct evidence for the acropetal translocation of PBDEs within plants,elucidates the process of PBDE transport and clarifies the debromination products of PBDEs in maize.

  5. Conflict bear translocation: investigating population genetics and fate of bear translocation in Dachigam National Park, Jammu and Kashmir, India.

    Science.gov (United States)

    Mukesh; Sharma, Lalit Kumar; Charoo, Samina Amin; Sathyakumar, Sambandam

    2015-01-01

    The Asiatic black bear population in Dachigam landscape, Jammu and Kashmir is well recognized as one of the highest density bear populations in India. Increasing incidences of bear-human interactions and the resultant retaliatory killings by locals have become a serious threat to the survivorship of black bears in the Dachigam landscape. The Department of Wildlife Protection in Jammu and Kashmir has been translocating bears involved in conflicts, henceforth 'conflict bears' from different sites in Dachigam landscape to Dachigam National Park as a flagship activity to mitigate conflicts. We undertook this study to investigate the population genetics and the fate of bear translocation in Dachigam National Park. We identified 109 unique genotypes in an area of ca. 650 km2 and observed bear population under panmixia that showed sound genetic variability. Molecular tracking of translocated bears revealed that mostly bears (7 out of 11 bears) returned to their capture sites, possibly due to homing instincts or habituation to the high quality food available in agricultural croplands and orchards, while only four bears remained in Dachigam National Park after translocation. Results indicated that translocation success was most likely to be season dependent as bears translocated during spring and late autumn returned to their capture sites, perhaps due to the scarcity of food inside Dachigam National Park while bears translocated in summer remained in Dachigam National Park due to availability of surplus food resources. Thus, the current management practices of translocating conflict bears, without taking into account spatio-temporal variability of food resources in Dachigam landscape seemed to be ineffective in mitigating conflicts on a long-term basis. However, the study highlighted the importance of molecular tracking of bears to understand their movement patterns and socio-biology in tough terrains like Dachigam landscape.

  6. Apoptosis-related deregulation of proteolytic activities and high serum levels of circulating nucleosomes and DNA in blood correlate with breast cancer progression

    Directory of Open Access Journals (Sweden)

    Kasimir-Bauer Sabine

    2011-01-01

    Full Text Available Abstract Background As cell-free circulating DNA exists predominantly as mono- and oligonucleosomes, the focus of the current study was to examine the interplay of circulating nucleosomes, DNA, proteases and caspases in blood of patients with benign and malignant breast diseases. Methods The concentrations of cell-free DNA and nucleosomes as well as the protease and caspase activities were measured in serum of patients with benign breast disease (n = 20, primary breast cancer (M0, n = 31, metastatic breast cancer (M1, n = 32, and healthy individuals (n = 28 by PicoGreen, Cell Death Detection ELISA, Protease Fluorescent Detection Kit and Caspase-Glo®3/7 Assay, respectively. Results Patients with benign and malignant tumors had significantly higher levels of circulating nucleic acids in their blood than healthy individuals (p = 0.001, p = 0.0001, whereas these levels could not discriminate between benign and malignant lesions. Our analyses of all serum samples revealed significant correlations of circulating nucleosome with DNA concentrations (p = 0.001, nucleosome concentrations with caspase activities (p = 0.008, and caspase with protease activities (p = 0.0001. High serum levels of protease and caspase activities associated with advanced tumor stages (p = 0.009. Patients with lymph node-positive breast cancer had significantly higher nucleosome levels in their blood than node-negative patients (p = 0.004. The presence of distant metastases associated with a significant increase in serum nucleosome (p = 0.01 and DNA levels (p = 0.04, and protease activities (p = 0.008. Conclusion Our findings demonstrate that high circulating nucleic acid concentrations in blood are no indicators of a malignant breast tumor. However, the observed changes in apoptosis-related deregulation of proteolytic activities along with the elevated serum levels of nucleosomes and DNA in blood are linked to breast cancer progression.

  7. The role of anti-nucleosome antibodies in systemic lupus erythematosus. Results of a study of patients with systemic lupus erythematosus and other connective tissue diseases

    Directory of Open Access Journals (Sweden)

    V. Tigano

    2011-09-01

    Full Text Available Objective: The aim of our study was to investigate the prevalence and the disease specificity of anti-nucleosome antibodies in systemic lupus erythematosus and their association with disease activity and renal involvement. Methods: Anti-nucleosome antibodies were measured by ELISA in the sera of patients with systemic lupus erythematosus (SLE (47, rheumatoid arthritis (RA (22, mixed connective tissue disease (MCTD (19, systemic sclerosis (SSc (11 and Siögren’s syndrome (SS (10. Anti-dsDNA antibodies were measured by IIF on Chritidia luciliae. In the patients with SLE serum levels of C3 and C4 complement components were also measured. Sera of 22 healthy individuals were assayed as controls. SLE activity was evaluated by the ECLAM score. Results: Anti-nucleosome antibodies were found in 40 patients with SLE (85.1%, in 10 with RA (45.4%, in 8 with MCTD (42.1%, in 4 with SSc (36.3%, in 1 with SS (10% and in none of the healthy controls. Anti-dsDNA antibodies were found in 23 patients with SLE and were absent in the patients with other CTD and in controls. All the patients with SLE and renal involvement were positive both for anti-dsDNA antibodies and anti-nucleosome antibodies. No significant correlation was observed between anti-nucleosome antibodies and disease activity and renal involvement. Conclusion: Anti-nucleosome antibodies are present in a high percentage of the patients with SLE but they don’t seem to be specific markers of the desease. Our data don’t support a clear correlation between anti-nucleosome antibodies and disease activity and renal involvement.

  8. The effects of translocations on recombination frequency in Caenorhabditis elegans.

    Science.gov (United States)

    McKim, K S; Howell, A M; Rose, A M

    1988-12-01

    In the nematode Caenorhabditis elegans, recombination suppression in translocation heterozygotes is severe and extensive. We have examined the meiotic properties of two translocations involving chromosome I, szT1(I;X) and hT1(I;V). No recombination was observed in either of these translocation heterozygotes along the left (let-362-unc-13) 17 map units of chromosome I. Using half-translocations as free duplications, we mapped the breakpoints of szT1 and hT1. The boundaries of crossover suppression coincided with the physical breakpoints. We propose that DNA sequences at the right end of chromosome I facilitate pairing and recombination. We use the data from translocations of other chromosomes to map the location of pairing sites on four other chromosomes. hT1 and szT1 differed markedly in their effect on recombination adjacent to the crossover suppressed region. hT1 had no effect on recombination in the adjacent interval. In contrast, the 0.8 map unit interval immediately adjacent to the szT1(I;X) breakpoint on chromosome I increased to 2.5 map units in translocation heterozygotes. This increase occurs in a chromosomal interval which can be expanded by treatment with radiation. These results are consistent with the suggestion that the szT1(I) breakpoint is in a region of DNA in which meiotic recombination is suppressed relative to the genomic average. We propose that DNA sequences disrupted by the szT1 translocation are responsible for determining the frequency of meiotic recombination in the vicinity of the breakpoint.

  9. Identification of chromosomal translocation hotspots via scan statistics

    Science.gov (United States)

    Silva, Israel T.; Rosales, Rafael A.; Holanda, Adriano J.; Nussenzweig, Michel C.; Jankovic, Mila

    2014-01-01

    Motivation: The detection of genomic regions unusually rich in a given pattern is an important undertaking in the analysis of next-generation sequencing data. Recent studies of chromosomal translocations in activated B lymphocytes have identified regions that are frequently translocated to c-myc oncogene. A quantitative method for the identification of translocation hotspots was crucial to this study. Here we improve this analysis by using a simple probabilistic model and the framework provided by scan statistics to define the number and location of translocation breakpoint hotspots. A key feature of our method is that it provides a global chromosome-wide nominal control level to clustering, as opposed to previous methods based on local criteria. While being motivated by a specific application, the detection of unusual clusters is a widespread problem in bioinformatics. We expect our method to be useful in the analysis of data from other experimental approaches such as of ChIP-seq and 4C-seq. Results: The analysis of translocations from B lymphocytes with the method described here reveals the presence of longer hotspots when compared with those defined previously. Further, we show that the hotspot size changes substantially in the absence of DNA repair protein 53BP1. When 53BP1 deficiency is combined with overexpression of activation-induced cytidine deaminase, the hotspot length increases even further. These changes are not detected by previous methods that use local significance criteria for clustering. Our method is also able to identify several exclusive translocation hotspots located in genes of known tumor supressors. Availability and implementation: The detection of translocation hotspots is done with hot_scan, a program implemented in R and Perl. Source code and documentation are freely available for download at https://github.com/itojal/hot_scan. Contact: isilva@rockefeller.edu Supplementary information: Supplementary data are available at Bioinformatics

  10. Nondriven Polymer Translocation Through a Nanopore:Scaling for Translocation Time with Chain Length

    Institute of Scientific and Technical Information of China (English)

    LI Hui; ZHANG Jing; LIU Hong; SUN Chia-chung

    2011-01-01

    We investigated the dynamics of the passage for a polymer chain through a nanopore in the absence of any external driving force with Weeks-Chandler-Andersen potential in two-dimensional simulations,in particular,focused our attention on the scaling law of the mean translocation time.We found that the effect of hydrodynamic interactions is the major factor in determining the scaling exponents with increasing pore size.The scaling close to N1+2v was observed when the hydrodynamic interactions were screened in the cases of small pore sizes,while the scaling close to N3v was obtained when the hydrodynamic interactions were present in the cases of large pore sizes.

  11. Nucleosome dynamics and maintenance of epigenetic states of CpG islands

    Science.gov (United States)

    Sneppen, Kim; Dodd, Ian B.

    2016-06-01

    Methylation of mammalian DNA occurs primarily at CG dinucleotides. These CpG sites are located nonrandomly in the genome, tending to occur within high density clusters of CpGs (islands) or within large regions of low CpG density. Cluster methylation tends to be bimodal, being dominantly unmethylated or mostly methylated. For CpG clusters near promoters, low methylation is associated with transcriptional activity, while high methylation is associated with gene silencing. Alternative CpG methylation states are thought to be stable and heritable, conferring localized epigenetic memory that allows transient signals to create long-lived gene expression states. Positive feedback where methylated CpG sites recruit enzymes that methylate nearby CpGs, can produce heritable bistability but does not easily explain that as clusters increase in size or density they change from being primarily methylated to primarily unmethylated. Here, we show that an interaction between the methylation state of a cluster and its occupancy by nucleosomes provides a mechanism to generate these features and explain genome wide systematics of CpG islands.

  12. The yeast histone chaperone hif1p functions with RNA in nucleosome assembly.

    Directory of Open Access Journals (Sweden)

    Amy R Knapp

    Full Text Available Hif1p is an H3/H4-specific histone chaperone that associates with the nuclear form of the Hat1p/Hat2p complex (NuB4 complex in the yeast Saccharomyces cerevisiae. While not capable of depositing histones onto DNA on its own, Hif1p can act in conjunction with a yeast cytosolic extract to assemble nucleosomes onto a relaxed circular plasmid.To identify the factor(s that function with Hif1p to carry out chromatin assembly, multiple steps of column chromatography were carried out to fractionate the yeast cytosolic extract. Analysis of partially purified fractions indicated that Hif1p-dependent chromatin assembly activity resided in RNA rather than protein. Fractionation of isolated RNA indicated that the chromatin assembly activity did not simply purify with bulk RNA. In addition, the RNA-mediated chromatin assembly activity was blocked by mutations in the human homolog of Hif1p, sNASP, that prevent the association of this histone chaperone with histone H3 and H4 without altering its electrostatic properties.These results suggest that specific RNA species may function in concert with histone chaperones to assemble chromatin.

  13. Pluripotency Transcription Factor Oct4 Mediates Stepwise Nucleosome Demethylation and Depletion

    Science.gov (United States)

    Shakya, Arvind; Callister, Catherine; Goren, Alon; Yosef, Nir; Garg, Neha; Khoddami, Vahid; Nix, David; Regev, Aviv

    2015-01-01

    The mechanisms whereby the crucial pluripotency transcription factor Oct4 regulates target gene expression are incompletely understood. Using an assay system based on partially differentiated embryonic stem cells, we show that Oct4 opposes the accumulation of local H3K9me2 and subsequent Dnmt3a-mediated DNA methylation. Upon binding DNA, Oct4 recruits the histone lysine demethylase Jmjd1c. Chromatin immunoprecipitation (ChIP) time course experiments identify a stepwise Oct4 mechanism involving Jmjd1c recruitment and H3K9me2 demethylation, transient FACT (facilitates chromatin transactions) complex recruitment, and nucleosome depletion. Genome-wide and targeted ChIP confirms binding of newly synthesized Oct4, together with Jmjd1c and FACT, to the Pou5f1 enhancer and a small number of other Oct4 targets, including the Nanog promoter. Histone demethylation is required for both FACT recruitment and H3 depletion. Jmjd1c is required to induce endogenous Oct4 expression and fully reprogram fibroblasts to pluripotency, indicating that the assay system identifies functional Oct4 cofactors. These findings indicate that Oct4 sequentially recruits activities that catalyze histone demethylation and depletion. PMID:25582194

  14. MYC translocation partner gene determines survival of patients with large B-cell lymphoma with MYC- or double-hit MYC/BCL2 translocations

    DEFF Research Database (Denmark)

    Pedersen, Mette Ø; Gang, Anne O; Poulsen, Tim S;

    2014-01-01

    In large B-cell lymphoma (LBCL) MYC- and MYC/BCL2 double-hit (DH) translocations have been associated with inferior survival. We hypothesised that the negative prognostic impact of MYC translocation was determined by an immunoglobulin MYC translocation partner gene (IG-MYC), as opposed to a non-i...

  15. Nucleosome acidic patch promotes RNF168- and RING1B/BMI1-dependent H2AX and H2A ubiquitination and DNA damage signaling.

    Directory of Open Access Journals (Sweden)

    Justin W Leung

    2014-03-01

    Full Text Available Histone ubiquitinations are critical for the activation of the DNA damage response (DDR. In particular, RNF168 and RING1B/BMI1 function in the DDR by ubiquitinating H2A/H2AX on Lys-13/15 and Lys-118/119, respectively. However, it remains to be defined how the ubiquitin pathway engages chromatin to provide regulation of ubiquitin targeting of specific histone residues. Here we identify the nucleosome acid patch as a critical chromatin mediator of H2A/H2AX ubiquitination (ub. The acidic patch is required for RNF168- and RING1B/BMI1-dependent H2A/H2AXub in vivo. The acidic patch functions within the nucleosome as nucleosomes containing a mutated acidic patch exhibit defective H2A/H2AXub by RNF168 and RING1B/BMI1 in vitro. Furthermore, direct perturbation of the nucleosome acidic patch in vivo by the expression of an engineered acidic patch interacting viral peptide, LANA, results in defective H2AXub and RNF168-dependent DNA damage responses including 53BP1 and BRCA1 recruitment to DNA damage. The acidic patch therefore is a critical nucleosome feature that may serve as a scaffold to integrate multiple ubiquitin signals on chromatin to compose selective ubiquitinations on histones for DNA damage signaling.

  16. Factors Affecting Polymer Translocation Through a Nanopore in a Membrane

    Institute of Scientific and Technical Information of China (English)

    Tao Chen; Teng Lu; Hao-jun Liang

    2008-01-01

    Monte Carlo simulations were used to study the translocation of a flexible polymer through a pore in a membrane, assuming an attractive interaction between the monomers and the membrane on the trans side of the membrane and no interaction on the cis side. For the case T<Tc (the temperature corresponding to the minimum in the translocation time τ), the value of τ decreases with increasing temperature, whereas for T>Tc, τ increases with increasing temperature. The translocation time depends on the absorbed energy uo in a nontrivial way. The value of τ increases initially upon increasing uo before it begins to decrease. The variation of the translocation time with respect to the solvent quality was also studied. It showed that there is a transition, as the solvent quality improves from "poor" to "good": when εAB<εc (the interaction energy corresponding to the minimum in τ), τ decreases with increasing the value of εAB; when εAB>εc, τ increases with increasing εAB. When the chain length was changed, it was found that when the absorbed energy uo was greater than uc, τ was proportional to N1.602; for uo<uc, τ∝N2.248. As the solvent quality improved from "poor" to "good," the translocation probability increased initially before becoming stable.

  17. Obstructed Bile Duct as a Trigger for Microbe's Translocation?

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To assess the potential mechanisms of bacterial translocation in a murine model of obstructive jaundice. Methods: Adult rats were randomized to be operated on for ligation or sham-ligation of the common bile duct. Bacterial translocation to the mesenteric lymph nodes (MLNs), liver, spleen, portal blood and systemic circulation and bacterial population levels in the ceca were quantitated after 7 and 14 days. The terminal ilea were histologically examined by light and transmission electron microscopy. Results: Bacterial translocation to the MNLs was seen in both 7 (10/17) and 14 (11/18) day ligated animals, but not found in their corresponding controls (both 0/8). No significant difference in the cecal bacterial population levels was found between the ligated groups and their corresponding control groups, also between the two subgroups that were set up within each ligated group according to the presence or absence of bacteria in the MLNs. In the ligated rats, light microscopy demonstrated subepithelial edema in association with infiltration of flammatory cells and, transmission electron microscopy showed that the enterocytes were injured with abnormal microvilli, swollen mitochondria, unclear endoplasmic reticulum and cytoplasm with bubble degeneration. However, the ilea from the controls appeared normal. Conclusions: Obstructive jaundice promotes bacterial translocation in rats. The gut mucosal damage rather than the intestinal bacterial overgrowth may play a crucial role in bacterial translocation.

  18. Nonabsorbable Antibiotics Reduce Bacterial and Endotoxin Translocation in Hepatectomised Rats

    Directory of Open Access Journals (Sweden)

    S. K. Kakkos

    1997-01-01

    Full Text Available There is increasing evidence that septic complications, occurring after major hepatectomies, may be caused by gram negative bacteria, translocating from the gut. We investigated in rats, the effect of extended hepatectomy on the structure and morphology of the intestinal mucosa as well as on the translocation of intestinal bacteria and endotoxins. We also examined the effect of nonabsorbable antibiotics on reducing the intestinal flora and consequently the phenomenon of translocation by administering neomycin sulphate and cefazoline. Hepatectomy was found to increase translocation, while administration of nonabsorbable antibiotics decreased it significantly. In addition, hepatectomy increased the aerobic cecal bacterial population, which normalised in the group receiving antibiotics. Among the histological parameters evaluated, villus height demonstrated a significant reduction after hepatectomy, while the number of villi per cm and the number of mitoses per crypt, remained unchanged. Our results indicate that administration of nonabsorbable antibiotics presents a positive effect on bacterial and endotoxin translocation after extended hepatectomy, and this may be related to reduction of colonic bacterial load as an intraluminal effect of antibiotics.

  19. A somatic origin of homologous Robertsonian translocations and isochromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, W.P.; Bernasconi, F.; Schinzel, A.A. (Univ. of Zurich (Switzerland)); Basaran, S.; Yueksel-Apak, M. (Univ. of Istanbul (Turkey)); Neri, G. (Universita Cattolica, Rome (Italy)); Serville, F. (Hopital d' Enfants Pellegrin, Bordeaux (France)); Balicek, P.; Haluza, R. (Univ. Hospital of Hradeck Kralove, Hradec Kralove (Czech Republic)); Farah, L.M.S. (Escuola Paulista de Medicina, Sao Paulo (Brazil)) (and others)

    1994-02-01

    One t(14q 14q), three t(15q 15q), two t(21q21q), and two t(22q22q) nonmosaic, apparently balanced, de novo Robertsonian translocation cases were investigated with polymorphic markers to establish the origin of the translocated chromosomes. Four cases had results indicative of an isochromosome: one t(14q14q) case with mild mental retardation and maternal uniparental disomy (UPD) for chromosome 14, one t(15q15q) case with the Prader-Willi syndrome and UPD(15), a phenotypically normal carrier of t(22q22q) with maternal UPD(22), and a phenotypically normal t(21q21q) case of paternal UPD(21). All UPD cases showed complete homozygosity throughout the involved chromosome, which is supportive of a postmeiotic origin. In the remaining four cases, maternal and paternal inheritance of the involved chromosome was found, which unambiguously implies a somatic origin. One t(15q15q) female had a child with a ring chromosome 15, which was also of probable postmeiotic origin as recombination between grandparental haplotypes had occurred prior to ring formation. UPD might be expected to result from de novo Robertsonian translocations of meiotic origin; however, all de novo homologous translocation cases, so far reported, with UPD of chromosomes 14, 15, 21, or 22 have been isochromosomes. These data provide the first direct evidence that nonmosaic Robertsonian translocations, as well as isochromosomes, are commonly the result of a mitotic exchange. 75 refs., 1 fig., 4 tabs.

  20. Electrostatics of polymer translocation events in electrolyte solutions.

    Science.gov (United States)

    Buyukdagli, Sahin; Ala-Nissila, T

    2016-07-07

    We develop an analytical theory that accounts for the image and surface charge interactions between a charged dielectric membrane and a DNA molecule translocating through the membrane. Translocation events through neutral carbon-based membranes are driven by a competition between the repulsive DNA-image-charge interactions and the attractive coupling between the DNA segments on the trans and the cis sides of the membrane. The latter effect is induced by the reduction of the coupling by the dielectric membrane. In strong salt solutions where the repulsive image-charge effects dominate the attractive trans-cis coupling, the DNA molecule encounters a translocation barrier of ≈10 kBT. In dilute electrolytes, the trans-cis coupling takes over image-charge forces and the membrane becomes a metastable attraction point that can trap translocating polymers over long time intervals. This mechanism can be used in translocation experiments in order to control DNA motion by tuning the salt concentration of the solution.

  1. Meiotic behaviour of evolutionary sex-autosome translocations in Bovidae.

    Science.gov (United States)

    Vozdova, Miluse; Ruiz-Herrera, Aurora; Fernandez, Jonathan; Cernohorska, Halina; Frohlich, Jan; Sebestova, Hana; Kubickova, Svatava; Rubes, Jiri

    2016-09-01

    The recurrent occurrence of sex-autosome translocations during mammalian evolution suggests common mechanisms enabling a precise control of meiotic synapsis, recombination and inactivation of sex chromosomes. We used immunofluorescence and FISH to study the meiotic behaviour of sex chromosomes in six species of Bovidae with evolutionary sex-autosome translocations (Tragelaphus strepsiceros, Taurotragus oryx, Tragelaphus imberbis, Tragelaphus spekii, Gazella leptoceros and Nanger dama ruficollis). The autosomal regions of fused sex chromosomes showed normal synapsis with their homologous counterparts. Synapsis in the pseudoautosomal region (PAR) leads to the formation of characteristic bivalent (in T. imberbis and T. spekii with X;BTA13/Y;BTA13), trivalent (in T. strepsiceros and T. oryx with X/Y;BTA13 and G. leptoceros with X;BTA5/Y) and quadrivalent (in N. dama ruficollis with X;BTA5/Y;BTA16) structures at pachynema. However, when compared with other mammals, the number of pachynema lacking MLH1 foci in the PAR was relatively high, especially in T. imberbis and T. spekii, species with both sex chromosomes involved in sex autosome translocations. Meiotic transcriptional inactivation of the sex-autosome translocations assessed by γH2AX staining was restricted to their gonosomal regions. Despite intraspecies differences, the evolutionary fixation of sex-autosome translocations among bovids appears to involve general mechanisms ensuring sex chromosome pairing, synapsis, recombination and inactivation.

  2. Multiscale modeling of biopolymer translocation through a nanopore

    CERN Document Server

    Fyta, M G; Kaxiras, E; Succi, S; Fyta, Maria; Melchionna, Simone; Kaxiras, Efthimios; Succi, Sauro

    2007-01-01

    We employ a multiscale approach to model the translocation of biopolymers through nanometer size pores. Our computational scheme combines microscopic Langevin molecular dynamics (MD) with a mesoscopic lattice Boltzmann (LB) method for the solvent dynamics, explicitly taking into account the interactions of the molecule with the surrounding fluid. Both dynamical and statistical aspects of the translocation process were investigated, by simulating polymers of various initial configurations and lengths. For a representative molecule size, we explore the effects of important parameters that enter in the simulation, paying particular attention to the strength of the molecule-solvent coupling and of the external electric field which drives the translocation process. Finally, we explore the connection between the generic polymers modeled in the simulation and DNA, for which interesting recent experimental results are available.

  3. Prenatal diagnosis of an autosomal translocation with regular trisomy 21.

    Science.gov (United States)

    Tunca, Yusuf; Deveci, M Salih; Koc, Altug; Kaya, Halide; Alanbay, Ibrahim; Coksuer, Hakan; Dede, Murat

    2013-06-01

    The coincidence of trisomy 21 and a structural rearrangement is very rare, and even it has not been reported as a prenatal diagnosis yet. In this article, we present an autosomal translocation carrier fetus with trisomy 21: 47,XX,+21, t(3;8)(p21;q24). Although the coincidence of reciprocal translocation and trisomy may be seen in reciprocal translocation carrier families, de novo cases are extremely rare. The presented case is diagnosed by amniocentesis, which was performed because of abnormal fetal ultrasonographic findings and increased trisomy 21 risk at maternal serum screening test. The postmortem pathologic examination of the fetus revealed that the findings of hypertelorism and right lung with two lobes are interesting novel findings of our cases associated with the breakpoints 3p21 and 8q24.

  4. Enhancing nuclear translocation: perspectives in inhaled corticosteroid therapy.

    Science.gov (United States)

    Hakim, Amir; Usmani, Omar S

    2015-01-01

    Corticosteroids are widely used in the treatment of asthma and chronic obstructive pulmonary disease (COPD). In contrast to their use in mild-to-moderate asthma, they are less efficacious in improving lung function and controlling the underlying inflammation in COPD. In most clinical trials, corticosteroids have shown little benefit in COPD, but have shown a greater clinical effect in combination with long-acting bronchodilators. Impaired corticosteroid activation of the glucocorticoid receptor (GR) has been reported in corticosteroid-insensitive individuals. Reversal of corticosteroid-insensitivity by enhancing GR nuclear translocation is a potential therapeutic target. Preclinical studies suggest members of the nuclear receptor superfamily may facilitate glucocorticoid receptor nuclear translocation. Unravelling the mechanisms that govern GR nuclear translocation may identify novel therapeutic targets for reversing corticosteroid-insensitivity.

  5. High-speed detection of DNA translocation in nanopipettes.

    Science.gov (United States)

    Fraccari, Raquel L; Ciccarella, Pietro; Bahrami, Azadeh; Carminati, Marco; Ferrari, Giorgio; Albrecht, Tim

    2016-04-14

    We present a high-speed electrical detection scheme based on a custom-designed CMOS amplifier which allows the analysis of DNA translocation in glass nanopipettes on a microsecond timescale. Translocation of different DNA lengths in KCl electrolyte provides a scaling factor of the DNA translocation time equal to p = 1.22, which is different from values observed previously with nanopipettes in LiCl electrolyte or with nanopores. Based on a theoretical model involving electrophoresis, hydrodynamics and surface friction, we show that the experimentally observed range of p-values may be the result of, or at least be affected by DNA adsorption and friction between the DNA and the substrate surface.

  6. Mechanism for translocation of fluoroquinolones across lipid membranes

    DEFF Research Database (Denmark)

    Cramariuc, O.; Rog, T.; Javanainen, M.

    2012-01-01

    Classical atom-scale molecular dynamics simulations, constrained free energy calculations, and quantum mechanical (QM) calculations are employed to study the diffusive translocation of ciprofloxacin (CPFX) across lipid membranes. CPFX is considered here as a representative of the fluoroquinolone...... antibiotics class. Neutral and zwitterionic CPFX coexist at physiological pH, with the latter being predominant. Simulations reveal that only the neutral form permeates the bilayer, and it does so through a novel mechanism that involves dissolution of concerted stacks of zwitterionic ciprofloxacins....... Subsequent QM analysis of the observed molecular stacking shows the important role of partial charge neutralization in the stacks, highlighting how the zwitterionic form of the drug is neutralized for translocation. The findings propose a translocation mechanism in which zwitterionic CPFX molecules approach...

  7. Scintigraphic visualization of bacterial translocation in experimental strangulated intestinal obstruction

    Energy Technology Data Exchange (ETDEWEB)

    Galeev, Yu.M.; Popov, M.V.; Salato, O.V. [Siberian Branch of Russian Academy of Medical Science, Research Centre of Reparative and Restorative Surgery, East Siberian Research Centre, 100 Yubileyniy, P.O. Box 23, Irkutsk (Russian Federation); Lishmanov, Yu.B. [Siberian Branch of Russian Academy of Medical Science, Research and Development Institute of Cardiology, Tomsk Research Centre, Tomsk (Russian Federation); Grigorev, E.G.; Aparcin, K.A. [Siberian Branch of Russian Academy of Medical Science, Research Centre of Reparative and Restorative Surgery, East Siberian Research Centre, 100 Yubileyniy, P.O. Box 23, Irkutsk (Russian Federation); Irkutsk State Medical University, Department of Hospital Surgery, Irkutsk (Russian Federation)

    2009-11-15

    The purpose of this study was to obtain scintigraphic images depicting translocation of {sup 99m}Tc-labelled Escherichia coli bacteria through the intestinal barrier and to quantify this process using methods of nuclear medicine. Thirty male Wistar rats (including 20 rats with modelled strangulated intestinal obstruction and 10 healthy rats) were used for bacterial scintigraphy. {sup 99m}Tc-labelled E. coli bacteria ({sup 99m}Ts-E. coli) with an activity of 7.4-11.1 MBq were administered into a section of the small intestine. Scintigraphic visualization of bacterial translocation into organs and tissues of laboratory animals was recorded in dynamic (240 min) and static (15 min) modes. The number of labelled bacteria, which migrated through the intestinal barrier, was quantified by calculating the translocation index (TI). Control indicated no translocation of {sup 99m}Ts-E. coli administered into the intestine through the parietes of the small intestine's distal part in healthy animals. Animals with strangulated obstruction demonstrated different migration strength and routes of labelled bacteria from strangulated and superior to strangulation sections of the small intestine. {sup 99m}Ts-E. coli migrated from the strangulated loop into the peritoneal cavity later causing systemic bacteraemia through peritoneal resorption. The section of the small intestine, which was superior to the strangulation, demonstrated migration of labelled bacteria first into the portal and then into the systemic circulation. The strangulated section of the small intestine was the main source of bacteria dissemination since the number of labelled bacteria, which migrated from this section significantly, exceeded that of the area superior to the strangulation section of the small intestine (p = 0.0003). Bacterial scintigraphy demonstrated the possibility of visualizing migration routes of labelled bacteria and quantifying their translocation through the intestinal barrier. This

  8. Spontaneous modification of the oxoglutarate translocator in vivo.

    Science.gov (United States)

    Duyckaerts, C; Sluse-Goffart, C M; Sluse, F E; Gosselin-Rey, C; Liébecq, C

    1984-07-16

    In studying the oxoglutarate translocator of rat-heart mitochondria over many years, we have observed an unexpected decrease in its efficiency. It has been divided by 2.48 +/- 0.07, (S.E.M.) for the exchange of external oxoglutarate for internal malate at 2 degrees C when the internal-malate concentration is 4 mM and is accompanied by an increase in its concentration (multiplied by 1.61 +/- 0.02, S.E.M.). The affinity of the external sites of the translocator for the external oxoglutarate is unchanged as well as the binding and kinetic cooperativities of the external oxoglutarate. This shows that the external side of the translocator has not been modified and suggests that its central part has not been modified either. The apparent Michaelis constant of the internal malate is increased (multiplied by 1.74 +/- 0.23, S.E.M.) suggesting that the translocator has been modified on its matricial side. Some control experiments show that a change in the diet of the rats, despite its effect on the fatty-acid content of the mitoplasts, is probably not responsible for the observed modification. As it is nevertheless very likely that changes of the oxoglutarate translocator have occurred in vivo, it is proposed that the observed modification has a genetic origin. The existence of two antagonist changes which are not directly related suggests that one of them is a response of the organism against the other; thus the oxoglutarate translocator may play a regulatory rôle in certain physiological conditions.

  9. Mode of ATM-dependent suppression of chromosome translocation.

    Science.gov (United States)

    Yamauchi, Motohiro; Suzuki, Keiji; Oka, Yasuyoshi; Suzuki, Masatoshi; Kondo, Hisayoshi; Yamashita, Shunichi

    2011-12-09

    It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs.

  10. Hard Sphere Diffusion Behaviour of Polymer Translocating through Interacting Pores

    Institute of Scientific and Technical Information of China (English)

    SUN Li-Zhen; LUO Meng-Bo

    2008-01-01

    The translocation of polymer chain through a small pore from a high concentration side (cis side) to a low concentration side (trans side) is simulated by using Monte Carlo technique. The effect of the polymer-pore interaction on the translocation is studied. We find a special interaction at which the decay of the number of polymer chain, N, at the cis side obeys Fick's law, i.e. N decreases exponentially with time. The behaviour is analogous to the diffusion of hard sphere.

  11. Patterns of nucleosomal organization in the alc regulon of Aspergillus nidulans: roles of the AlcR transcriptional activator and the CreA global repressor.

    Science.gov (United States)

    Mathieu, Martine; Nikolaev, Igor; Scazzocchio, Claudio; Felenbok, Béatrice

    2005-04-01

    We have studied the chromatin organization of three promoters of the alc regulon of Aspergillus nidulans. No positioned nucleosomes are seen in the aldA (aldehyde dehydrogenase) promoter under any physiological condition tested by us. In the alcA (alcohol dehydrogenase I) and alcR (coding for the pathway-specific transcription factor) promoters, a pattern of positioned nucleosomes is seen under non-induced and non-induced repressed conditions. While each of these promoters shows a specific pattern of chromatin restructuring, in both cases induction results in loss of nucleosome positioning. Glucose repression in the presence of inducer results in a specific pattern of partial positioning in the alcA and alcR promoters. Loss of nucleosome positioning depends absolutely on the AlcR protein and it is very unlikely to be a passive result of the induction of transcription. In an alcR loss-of-function background and in strains carrying mutations of the respective AlcR binding sites of the alcA and alcR promoters, nucleosomes are fully positioned under all growth conditions. Analysis of mutant AlcR proteins establishes that all domains needed for transcriptional activation and chromatin restructuring are included within the first 241 residues. The results suggest a two-step process, one step resulting in chromatin restructuring, a second one in transcriptional activation. Partial positioning upon glucose repression shows a specific pattern that depends on the CreA global repressor. An alcR loss-of-function mutation is epistatic to a creA loss-of-function mutation, showing that AlcR does not act by negating a nucleosome positioning activity of CreA.

  12. 对于核小体定位检测方法的比较研究%A Comparative Study on Different Methods of Nucleosome Positioning

    Institute of Scientific and Technical Information of China (English)

    陈文辉

    2012-01-01

    In eukaryotes,Nucleosomes are the basic unit of DNA packaging,consisting of a segment of DNA wound around a histone protein core.Although the nucleosome is a very stable protein-DNA complex,it is not static and those DNA which are nucleosome free seems more easily to binding proteins which involved in transcriptional regulation.And this would suggest that there may have some interactions between nucleosome positioning and transcriptional regulation.In this article we will discuss the advantages and disadvantages between different methos of nucleosome positioning.And Studying the different methods of nucleosome positioning may contributes to further understanding the range of its application.%在真核细胞中,核小体是组成染色质的基本结构单位,是由DNA紧密缠绕在组蛋白八聚体上所形成的一个复合体结构。而DNA与组蛋白的结合并不是固定不变的,没有核小体结合的DNA区域易于各种调节蛋白的接近与结合。因此人们怀疑核小体的定位与基因的转录调节之间存在某种内在联系。对现行的核小体定位的检测方法进行了归类,并对其优缺点进行了分析整理。对更深入的探索核小体定位检测方法的应用有一定意义。

  13. Reduced Nuclease Activity of Apurinic/Apyrimidinic Endonuclease (APE1) Variants on Nucleosomes: IDENTIFICATION OF ACCESS RESIDUES.

    Science.gov (United States)

    Hinz, John M; Mao, Peng; McNeill, Daniel R; Wilson, David M

    2015-08-21

    Non-coding apurinic/apyrimidinic (AP) sites are generated at high frequency in genomic DNA via spontaneous hydrolytic, damage-induced or enzyme-mediated base release. AP endonuclease 1 (APE1) is the predominant mammalian enzyme responsible for initiating removal of mutagenic and cytotoxic abasic lesions as part of the base excision repair (BER) pathway. We have examined here the ability of wild-type (WT) and a collection of variant/mutant APE1 proteins to cleave at an AP site within a nucleosome core particle. Our studies indicate that, in comparison to the WT protein and other variant/mutant enzymes, the incision activity of the tumor-associated variant R237C and the rare population variant G241R are uniquely hypersensitive to nucleosome complexes in the vicinity of the AP site. This defect appears to stem from an abnormal interaction of R237C and G241R with abasic DNA substrates, but is not simply due to a DNA binding defect, as the site-specific APE1 mutant Y128A, which displays markedly reduced AP-DNA complex stability, did not exhibit a similar hypersensitivity to nucleosome structures. Notably, this incision defect of R237C and G241R was observed on a pre-assembled DNA glycosylase·AP-DNA complex as well. Our results suggest that the BER enzyme, APE1, has acquired distinct surface residues that permit efficient processing of AP sites within the context of protein-DNA complexes independent of classic chromatin remodeling mechanisms.

  14. Drosophila Yemanuclein and HIRA cooperate for de novo assembly of H3.3-containing nucleosomes in the male pronucleus.

    Directory of Open Access Journals (Sweden)

    Guillermo A Orsi

    Full Text Available The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM, the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male pronucleus. yem loss of function alleles affect male pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation.

  15. The structure of the nucleosome core particle of chromatin in chicken erythrocytes visualized by using atomic force microscopy

    Institute of Scientific and Technical Information of China (English)

    ZHAOHUI; YIZHANG; 等

    1999-01-01

    The structure of the nuclosome core particle of chromatin in chicken erythrocytes has been examined by using AFM.The 146 bp of DNA wrapped twice around the core histone octamer are clearly visualized.Both the ends of entry/exit of linker DNA are also demonstrated.The dimension of the nucleosome core particles is - 1-4 nm in height and - 13-22 nm in width.In addition,superbeads (width of - 48-57 nm,height of - 2-3 nm )are occasionally revealed,two turns of DNA around the core particles are also detected.

  16. Ubiquitous human ‘master’ origins of replication are encoded in the DNA sequence via a local enrichment in nucleosome excluding energy barriers

    Science.gov (United States)

    Drillon, Guénola; Audit, Benjamin; Argoul, Françoise; Arneodo, Alain

    2015-02-01

    As the elementary building block of eukaryotic chromatin, the nucleosome is at the heart of the compromise between the necessity of compacting DNA in the cell nucleus and the required accessibility to regulatory proteins. The recent availability of genome-wide experimental maps of nucleosome positions for many different organisms and cell types has provided an unprecedented opportunity to elucidate to what extent the DNA sequence conditions the primary structure of chromatin and in turn participates in the chromatin-mediated regulation of nuclear functions, such as gene expression and DNA replication. In this study, we use in vivo and in vitro genome-wide nucleosome occupancy data together with the set of nucleosome-free regions (NFRs) predicted by a physical model of nucleosome formation based on sequence-dependent bending properties of the DNA double-helix, to investigate the role of intrinsic nucleosome occupancy in the regulation of the replication spatio-temporal programme in human. We focus our analysis on the so-called replication U/N-domains that were shown to cover about half of the human genome in the germline (skew-N domains) as well as in embryonic stem cells, somatic and HeLa cells (mean replication timing U-domains). The ‘master’ origins of replication (MaOris) that border these megabase-sized U/N-domains were found to be specified by a few hundred kb wide regions that are hyper-sensitive to DNase I cleavage, hypomethylated, and enriched in epigenetic marks involved in transcription regulation, the hallmarks of localized open chromatin structures. Here we show that replication U/N-domain borders that are conserved in all considered cell lines have an environment highly enriched in nucleosome-excluding-energy barriers, suggesting that these ubiquitous MaOris have been selected during evolution. In contrast, MaOris that are cell-type-specific are mainly regulated epigenetically and are no longer favoured by a local abundance of intrinsic NFRs

  17. Ubiquitous human 'master' origins of replication are encoded in the DNA sequence via a local enrichment in nucleosome excluding energy barriers.

    Science.gov (United States)

    Drillon, Guénola; Audit, Benjamin; Argoul, Françoise; Arneodo, Alain

    2015-02-18

    As the elementary building block of eukaryotic chromatin, the nucleosome is at the heart of the compromise between the necessity of compacting DNA in the cell nucleus and the required accessibility to regulatory proteins. The recent availability of genome-wide experimental maps of nucleosome positions for many different organisms and cell types has provided an unprecedented opportunity to elucidate to what extent the DNA sequence conditions the primary structure of chromatin and in turn participates in the chromatin-mediated regulation of nuclear functions, such as gene expression and DNA replication. In this study, we use in vivo and in vitro genome-wide nucleosome occupancy data together with the set of nucleosome-free regions (NFRs) predicted by a physical model of nucleosome formation based on sequence-dependent bending properties of the DNA double-helix, to investigate the role of intrinsic nucleosome occupancy in the regulation of the replication spatio-temporal programme in human. We focus our analysis on the so-called replication U/N-domains that were shown to cover about half of the human genome in the germline (skew-N domains) as well as in embryonic stem cells, somatic and HeLa cells (mean replication timing U-domains). The 'master' origins of replication (MaOris) that border these megabase-sized U/N-domains were found to be specified by a few hundred kb wide regions that are hyper-sensitive to DNase I cleavage, hypomethylated, and enriched in epigenetic marks involved in transcription regulation, the hallmarks of localized open chromatin structures. Here we show that replication U/N-domain borders that are conserved in all considered cell lines have an environment highly enriched in nucleosome-excluding-energy barriers, suggesting that these ubiquitous MaOris have been selected during evolution. In contrast, MaOris that are cell-type-specific are mainly regulated epigenetically and are no longer favoured by a local abundance of intrinsic NFRs encoded in

  18. Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins

    DEFF Research Database (Denmark)

    Foulk, M. S.; Urban, J. M.; Casella, Cinzia;

    2015-01-01

    Nascent strand sequencing (NS-seq) is used to discover DNA replication origins genome-wide, allowing identification of features for their specification. NS-seq depends on the ability of lambda exonuclease (lambda-exo) to efficiently digest parental DNA while leaving RNA-primer protected nascent...... are not general determinants for origin specification but may play a role for a subset. Interestingly, we observed a periodic spacing of G4 motifs and nucleosomes around the peak summits, suggesting that G4s may position nucleosomes at this subset of origins. Finally, we demonstrate that use of Na+ instead of K...

  19. Unassisted translocation of large polypeptide domains across phospholipid bilayers.

    Science.gov (United States)

    Brambillasca, Silvia; Yabal, Monica; Makarow, Marja; Borgese, Nica

    2006-12-01

    Although transmembrane proteins generally require membrane-embedded machinery for integration, a few can insert spontaneously into liposomes. Previously, we established that the tail-anchored (TA) protein cytochrome b(5) (b5) can posttranslationally translocate 28 residues downstream to its transmembrane domain (TMD) across protein-free bilayers (Brambillasca, S., M. Yabal, P. Soffientini, S. Stefanovic, M. Makarow, R.S. Hegde, and N. Borgese. 2005. EMBO J. 24:2533-2542). In the present study, we investigated the limits of this unassisted translocation and report that surprisingly long (85 residues) domains of different sequence and charge placed downstream of b5's TMD can posttranslationally translocate into mammalian microsomes and liposomes at nanomolar nucleotide concentrations. Furthermore, integration of these constructs occurred in vivo in translocon-defective yeast strains. Unassisted translocation was not unique to b5 but was also observed for another TA protein (protein tyrosine phosphatase 1B) whose TMD, like the one of b5, is only moderately hydrophobic. In contrast, more hydrophobic TMDs, like synaptobrevin's, were incapable of supporting unassisted integration, possibly because of their tendency to aggregate in aqueous solution. Our data resolve long-standing discrepancies on TA protein insertion and are relevant to membrane evolution, biogenesis, and physiology.

  20. Concentration Polarization in Translocation of DNA through Nanopores and Nanochannels

    NARCIS (Netherlands)

    Das, Siddhartha; Dubsky, Pavel; Berg, van den Albert; Eijkel, J.C.T.

    2012-01-01

    In this Letter we provide a theory to show that high-field electrokinetic translocation of DNA through nanopores or nanochannels causes large transient variations of the ionic concentrations in front and at the back of the DNA due to concentration polarization (CP). The CP causes strong local conduc

  1. Three cases of mosaicism for balanced reciprocal translocations

    NARCIS (Netherlands)

    Leegte, B; Sikkema-Raddatz, B; Hordijk, R; Bouman, K; van Essen, T; Castedo, S; de Jong, B

    1998-01-01

    Mosaicism for a balanced reciprocal translocation (BRTM) is rare. As far as we know only 26 cases of BRTM, demonstrated in lymphocyte cultures, have been described, five of which had an abnormal phenotype. Prenatally three confirmed cases with a normal phenotypic outcome have been described. Here we

  2. Bioenergetic aspects of the translocation of macromolecules across bacterial membranes

    NARCIS (Netherlands)

    Palmen, Ronald; Driessen, Arnold J.M.; Hellingwerf, K

    1994-01-01

    Bacteria are extremely versatile in the sense that they have gained the ability to transport all three major classes of biopolymers through their cell envelope: proteins, nucleic acids, and polysaccharides. These macromolecules are translocated across membranes in a large number of cellular processe

  3. Driven translocation of a polymer: Fluctuations at work

    NARCIS (Netherlands)

    Dubbeldam, J.L.A.; Rostiashvii, V.G.; Milchev, A.; Vilgis, T.A.

    2013-01-01

    The impact of thermal fluctuations on the translocation dynamics of a polymer chain driven through a narrow pore has been investigated theoretically and by means of extensive molecular dynamics (MD) simulation. The theoretical consideration is based on the so-called velocity Langevin (V-Langevin) eq

  4. Macular pigment and fixation after macular translocation surgery

    NARCIS (Netherlands)

    Reinhard, Jens; Kanis, Martijn J.; Berendschot, Tos T. J. M.; Schoen, Christiane; Gelisken, Faik; Trauzettel-Klosinski, Susanne; Bartz-Schmidt, Karl U.; Zrenner, Eberhart

    2010-01-01

    Background After full macular translocation (MT) surgery with 3608 retinotomy, the fovea is rarely identifiable. Our aim was to verify the position of the fovea, to determine how patients fixate after MT and to examine distribution and optical density of macular pigment ( MP). Methods 9 patients aft

  5. Chromosome 14 translocations in non-Burkitt lymphomas

    Energy Technology Data Exchange (ETDEWEB)

    Fukuhara, S.; Rowley, J.D.

    1978-01-01

    Chromosome studies were performed on malignant cells obtained from 27 patients with non-Burkitt lymphomas. A marker chromosome affecting the long arm of No. 14 (14q+) was the single most frequent abnormality and was noted in 17 of these patients. The frequency of the 14q+ marker varied with the type of lymphoma. For patients with malignant lymphoma, histiocytic, the frequency was 5 or 8; for mixed-cell type, 1 of 3; for poorly differentiated lymphocytic, 8 of 8; for well-differentiated lymphocytic, 0.3; for lymphoblastic, 0 of 1; for Hodgkin's disease, 2 of 3; and for mycosis fungoides, 1 of 1. The donor chromosome involved in the 14q translocation was identified in 12 cases; certain chromosomes appeared to be affected more frequently than others. Although the break point was band 14q32 in most cases, the exact location of the receptor site on 14q was not always consistent. The distal part of 14q24 was also involved as a receptor site in at least one translocation. These findings suggest that, in some types of lymphoid malignancy, cells with a 14q translocation have a proliferative advantage over cells with other chromosome rearrangements. The presence of the 14q translocation may be important in the future for the distinction among morphologically different, but functionally comparable, subgroups of lymphoid malignancies.

  6. Translocation of Polymer Chains Through a Channel with Complex Geometries

    Institute of Scientific and Technical Information of China (English)

    Zhi-yong Yang; Lin-xi Zhang; Jun Cheng

    2008-01-01

    The elastic behavior of a single chain transporting through complex channel which can be seen as the combination of three different channels (left channel, middle channel, and right channel, respectively) is investigated using the new pruned-enriched Rosenbluth method with importance sampling. The elastic force during the translocation process is calculated. At the entrance into the middle channel, there is the first plateau in the curve of the elastic force f (f0) versus x, here x represents the position of the first monomer along the x-axis direction. When the first monomer moves to a certain position, a second plateau is observed with the elastic force f<0, which represents spontaneous translocation. The free energy difference between the subchain in the right channel and the subchain in the left channel may drive the trauslocation. The influence of chain length and width of the left and right channels on the translocation process are also investigated. From the simulation results, more detailed explanations for the reason why the component translocation time is not the same for different channels can be presented.

  7. Genetic counseling in carriers of reciprocal translocations involving two autosomes

    Directory of Open Access Journals (Sweden)

    Bahareh Pourjafari

    2012-01-01

    Couples in which one partner is the carrier of such balanced translocation have increased risks of infertility, recurrent abortion, and delivery of chromosomally abnormal offspring. Genetic counseling of such couples, therefore, presents a unique challenge and should be considered in dealing with such families.

  8. Markers of immunity and bacterial translocation in cirrhosis

    DEFF Research Database (Denmark)

    Mortensen, Christian

    2015-01-01

    , in 38 patients with ascites, we found no association between bDNA and immunity, in contrast to some previous findings. In the final paper, exploring one possible translocation route, we hypothesized a difference in bDNA levels between the blood from the veins draining the gut on one hand and the liver...

  9. Centrifugally driven microfluidic disc for detection of chromosomal translocations

    DEFF Research Database (Denmark)

    Brøgger, Anna Line; Kwasny, Dorota; Bosco, Filippo G.

    2012-01-01

    and prognosis of patients. In this work we demonstrate a novel, centrifugally-driven microfluidic system for controlled manipulation of oligonucleotides and subsequent detection of chromosomal translocations. The device is fabricated in the form of a disc with capillary burst microvalves employed to control...

  10. 40 CFR 798.5460 - Rodent heritable translocation assays.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 31 2010-07-01 2010-07-01 true Rodent heritable translocation assays. 798.5460 Section 798.5460 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC...) Species. The mouse is the species generally used, and is recommended. (ii) Age. Healthy sexually...

  11. Dietary nitrite improves insulin signaling through GLUT4 translocation.

    Science.gov (United States)

    Jiang, Hong; Torregrossa, Ashley C; Potts, Amy; Pierini, Dan; Aranke, Mayank; Garg, Harsha K; Bryan, Nathan S

    2014-02-01

    Diabetes mellitus type 2 is a syndrome of disordered metabolism with inappropriate hyperglycemia owing to a reduction in the biological effectiveness of insulin. Type 2 diabetes is associated with an impaired nitric oxide (NO) pathway that probably serves as the key link between metabolic disorders and cardiovascular disease. Insulin-mediated translocation of GLUT4 involves the PI3K/Akt kinase signal cascade that results in activation of endothelial NO synthase (eNOS). eNOS is dysfunctional during diabetes. We hypothesize that loss of eNOS-derived NO terminates the signaling cascade and therefore cannot activate GLUT4 translocation and that dietary nitrite may repair this pathway. In this study, we administered 50mg/L sodium nitrite to db/db diabetic mice for 4 weeks. After 4 weeks treatment, the db/db mice experienced less weight gain, improved fasting glucose levels, and reduced insulin levels. Cell culture experiments using CHO-HIRc-myc-GLUT4eGFP cell lines stably expressing insulin receptor and myc-GLUT4eGFP protein, as well as L6 skeletal muscle cells stably expressing rat GLUT4 with a Myc epitope (L6-GLUT4myc), showed that NO, nitrite, and GSNO stimulate GLUT4 translocation independent of insulin, which is inhibited by NEM. Collectively our data suggest that nitrite improves insulin signaling through restoration of NO-dependent nitrosation of GLUT4 signaling translocation. These data suggest that NO-mediated nitrosation of GLUT4 by nitrite or other nitrosating agents is necessary and sufficient for GLUT4 translocation in target tissue. Description of this pathway may justify a high-nitrate/nitrite diet along with the glycemic index to provide a safe and nutritional regimen for the management and treatment of diabetes.

  12. Biosecurity for Translocations: Cirl Bunting (Emberiza cirlus), Fisher's Estuarine Moth (Gortyna borelii lunata), Short-Haired Bumblebee (Bombus subterraneus) and Pool Frog (Pelophylax lessonae) Translocations as Case Studies.

    Science.gov (United States)

    Vaughan-Higgins, R J; Masters, N; Sainsbury, A W

    2016-08-04

    Exposure to parasites in conservation translocations increases the risks to recipient and translocated populations from disease, and therefore there has been interest in implementing biosecurity methods. Using four case examples we described how biosecurity was applied in practical translocation scenarios prior to and during a translocation and also post-release. We implemented biosecurity, including quarantine barriers, at specific points in the translocation pathway where hazards, identified by the disease risk analysis, had the potential to induce disease. Evidence that biosecurity protected translocated and recipient populations, included an absence of mortality associated with high-risk non-native parasites, a reduction in mortality associated with endemic parasites, the absence of high-risk pathogenic parasites, or associated diseases, at the destination; and the apparent absence of diseases in closely related species at the destination site. The biosecurity protocols did not alter the level or duration of translocated species confinement and therefore probably did not act as a stressor. There is a monetary cost involved in biosecurity but the epidemiological evidence suggests that conservation translocation managers should carefully consider its use. Breakdowns in quarantine have occurred in human hospitals despite considerable investment and training for health professionals, and we therefore judge that there is a need for training in the objectives and maintenance of quarantine barriers in conservation translocations. Biosecurity protocols for conservation translocations should be continually updated in response to findings from disease risk analysis and post-release disease surveillance and we recommend further studies to evaluate their effectiveness.

  13. Microbial Translocation in HIV Infection is Associated with Dyslipidemia, Insulin Resistance, and Risk of Myocardial Infarction

    DEFF Research Database (Denmark)

    Pedersen, Karin Kaereby; Pedersen, Maria; Trøseid, Marius;

    2013-01-01

    Microbial translocation has been suggested to be a driver of immune activation and inflammation. We hypothesized that microbial translocation may be related to dyslipidemia, insulin resistance, and the risk of coronary heart disease in HIV-infected individuals....

  14. FACT Disrupts Nucleosome Structure by Binding H2A-H2B with Conserved Peptide Motifs.

    Science.gov (United States)

    Kemble, David J; McCullough, Laura L; Whitby, Frank G; Formosa, Tim; Hill, Christopher P

    2015-10-15

    FACT, a heterodimer of Spt16 and Pob3, is an essential histone chaperone. We show that the H2A-H2B binding activity that is central to FACT function resides in short acidic regions near the C termini of each subunit. Mutations throughout these regions affect binding and cause correlated phenotypes that range from mild to lethal, with the largest individual contributions unexpectedly coming from an aromatic residue and a nearby carboxylate residue within each domain. Spt16 and Pob3 bind overlapping sites on H2A-H2B, and Spt16-Pob3 heterodimers simultaneously bind two H2A-H2B dimers, the same stoichiometry as the components of a nucleosome. An Spt16:H2A-H2B crystal structure explains the biochemical and genetic data, provides a model for Pob3 binding, and implies a mechanism for FACT reorganization that we confirm biochemically. Moreover, unexpected similarity to binding of ANP32E and Swr1 with H2A.Z-H2B reveals that diverse H2A-H2B chaperones use common mechanisms of histone binding and regulating nucleosome functions.

  15. lncRNA-Induced Nucleosome Repositioning Reinforces Transcriptional Repression of rRNA Genes upon Hypotonic Stress

    Directory of Open Access Journals (Sweden)

    Zhongliang Zhao

    2016-03-01

    Full Text Available The activity of rRNA genes (rDNA is regulated by pathways that target the transcription machinery or alter the epigenetic state of rDNA. Previous work has established that downregulation of rRNA synthesis in quiescent cells is accompanied by upregulation of PAPAS, a long noncoding RNA (lncRNA that recruits the histone methyltransferase Suv4-20h2 to rDNA, thus triggering trimethylation of H4K20 (H4K20me3 and chromatin compaction. Here, we show that upregulation of PAPAS in response to hypoosmotic stress does not increase H4K20me3 because of Nedd4-dependent ubiquitinylation and proteasomal degradation of Suv4-20h2. Loss of Suv4-20h2 enables PAPAS to interact with CHD4, a subunit of the chromatin remodeling complex NuRD, which shifts the promoter-bound nucleosome into the transcriptional “off” position. Thus, PAPAS exerts a “stress-tailored” dual function in rDNA silencing, facilitating either Suv4-20h2-dependent chromatin compaction or NuRD-dependent changes in nucleosome positioning.

  16. DCCD inhibits protein translocation into plasma membrane vesicles from Escherichia coli at two different steps.

    OpenAIRE

    1987-01-01

    In vitro translocation of periplasmic and outer membrane proteins into inverted plasma membrane vesicles from Escherichia coli was completely prevented by the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD). DCCD was inhibitory to both co- and post-translational translocations, suggesting an involvement of the H+-translocating F1F0-ATPase in either mode of transport. This was verified by (i) the dependence of efficient co-translational translocation upon a low salt, i.e. F1-containin...

  17. Meiotic behaviour and spermatogenesis in male mice heterozygous for translocation types also occurring in man

    NARCIS (Netherlands)

    Nijhoff, J.H.

    1981-01-01

    In this thesis a start was made with meiotic observations of mouse translocation types - a Robertsonian translocation and a translocation between a metacentric and an acrocentric chromosome - which also occur in man. It is generally accepted that, when no chromosomal rearrangements are involved, man

  18. The in vitro reconstitution of nucleosome and its binding patterns with HMG1/2 and HMG14/17 proteins

    Institute of Scientific and Technical Information of China (English)

    JIA HUA HU; JIE JIANG; YING HUA MA; NA YANG; MAO HU ZHANG; MIN WU; JIAN FEI; LI HE GUO

    2003-01-01

    Using atomic force microscopy (AFM), the dynamic process of the in vitro nucleosome reconstitution followed by slow dilution from high salt to low salt was visualized. Data showed that the histone octamers were dissociated from DNA at 1M NaCl. When the salt concentration was slowly reduced to 650 mMand 300 mM, the core histones bound to the naked DNA gradually. Once the salt concentration was reduced to 50 mM the classic "beads-on-a-string" structure was clearly visualized. Furthermore, using the technique of the in vitro reconstitution ofnucleosome,the mono- and di- nucleosomes were assembled in vitro with both HS2core (-10681 to -10970 bp) and NCR2 (-372to -194 bp) DNA sequences in the 5'flanking sequence of human b-globin gene. Data revealed that HMG 1/2 and HMG 14/17 proteins binding to both DNA sequences are changeable following the assembly and disassembly of nucleosomes. We suggest that the changeable binding patterns of HMG 14/17 and HMG1/2 proteins with these regulatory elements may be critical in the process of nucleosome assembly, recruitment of chromatin-modifying activities, and the regulation of human b-globin gene expression.

  19. Mimotopes for lupus-derived anti-DNA and nucleosome-specific autoantibodies selected from random peptide phage display libraries: facts and follies.

    NARCIS (Netherlands)

    Dieker, J.W.C.; Sun, Y.J.; Jacobs, C.W.M.; Putterman, C.; Monestier, M.; Muller, S.; Vlag, J. van der; Berden, J.H.M.

    2005-01-01

    Autoantibodies against chromatin are the most characteristic serological feature in SLE patients. Anti-dsDNA and nucleosome-specific antibodies are associated with glomerulonephritis, the most serious manifestation of SLE. Identification of peptides mimicking conformational epitopes (so-called mimot

  20. Effects of macroH2A and H2A.Z on nucleosome structure and dynamics as elucidated by molecular dynamics simulations

    CERN Document Server

    Bowerman, Samuel

    2015-01-01

    Eukaryotes tune the transcriptional activity of their genome by altering the nucleosome core particle through multiple chemical processes. In particular, replacement of the canonical H2A histone with the variants macroH2A and H2A.Z has been shown to affect DNA accessibility and nucleosome stability; however, the processes by which this occurs remain poorly understood. Here, we elucidate the molecular mechanisms of these variants with an extensive molecular dynamics study of the canonical nucleosome along with three variant-containing structures: H2A.Z, macroH2A, and an H2A mutant with macroH2A-like L1 loops. Simulation results show that variant L1 loops play a pivotal role in stabilizing DNA binding to the octamer through direct interactions, core structural rearrangements, and altered allosteric networks in the nucleosome. All variants influence dynamics; however, macroH2A-like systems have the largest effect on energetics. In addition, we provide a comprehensive analysis of allosteric networks in the nucleo...

  1. Development of NuQ nucleosome blood tests for the detection of colon cancer.

    Science.gov (United States)

    Micallef, Jake; Wilson, Hannah L

    2014-01-01

    Dr Jake Micallef speaks to Hannah L Wilson, Commissioning Editor: Dr Micallef has 20 years of experience in research and development and in the management of early-stage biotechnical companies, including the manufacture of biotechnology products and the establishment of manufacturing operations. Dr Micallef gained this experience while working for WHO over a 10-year period from 1985. While working for WHO, Dr Micallef developed new diagnostic products in the areas of reproductive health and cancer. In 1990 he commenced development of a new diagnostic technology platform for WHO that was launched in 1992 and supported 13 tests. Dr Micallef also initiated and implemented in-house manufacture (previously outsourced to Abbott Diagnostics Inc., Dartford, UK) and worldwide distribution of these products for WHO. In 1990, he started a 'not-for-profit' WHO company, Immunometrics Ltd (London, UK), which marketed and distributed those diagnostic products worldwide. In 1999 Dr Micallef studied for an MBA and went on to co-found Gene Expression Technologies Ltd (London, UK) in 2001 where he successfully lead the development of the chemistry of the GeneICE technology and implemented the manufacture of GeneICE molecules. He also played a major role in business development and procured a GeneICE contract with Bayer Pharmaceuticals (Leverkusen, Germany). From 2004 to 2007, he taught 'science and enterprise' to science research workers from four universities at CASS Business School (London, UK) before joining Cronos Therapeutics (London, UK) in 2004. In 2006 Cronos was listed in the UK on AIM, becoming ValiRx. Dr Micallef continued to work as Technical Officer for ValiRx, where he in-licensed the Hypergenomics and Nucleosomics technologies and co-founded ValiBio SA (Namur, Belgium), which is now Belgian Volition SA, a subsidiary of Singapore Volition. Dr Micallef was educated at King's College London (UK; BSc, Biology and Chemistry, 1977; PhD Physical Chemistry, 1981), St Thomas

  2. 核小体定位的转录调控功能研究进展%Transcriptional Regulation Functions of Nucleosome Positioning: a Survey

    Institute of Scientific and Technical Information of China (English)

    刘辉; 壮子恒; 关佶红; 周水庚

    2012-01-01

    核小体是真核生物染色质的基本组成单位,组蛋白八聚体在DNA双螺旋上精确位置称为核小体定位.核小体定位已被证实在基因转录调控、DNA复制与修复、调控进化等过程中扮演着重要的角色.随着染色质免疫共沉淀-芯片(ChIP-chip)与染色质免疫共沉淀-测序(ChIP-seq)等高通量技术的出现,已测定了多种模式生物全基因组核小体定位图谱,掀起了一股核小体定位及其功能的研究热潮,并取得了一定的成果.本文介绍了核小体定位的概念,总结了核小体在启动了与编码区域内定位的基本模式.在此基础上,综述了核小体定位在转录起始、转录延伸、基因表达模式多样化以及可变剪接等方面的功能研究进展.%Nucleosome is the building unit of eukaryotic chromatin, the location of histone octamer on the DNA sequence is called nucleosome positioning. With the advent of high throughout technologies such as ChIP-chip and ChIP-seq. large-scale nucleosome positioning atlas of multiple model organisms have been measured, which attract many researchers to investigate nucleosome positioning and its functions on transcriptional regulation. In this paper, we first introduce the concept of nucleosome positioning and summarize the regular patterns of nucleosome positioning on genetic region, then review the major advances of functions of nucleosome positioning on transcriptional initiation, elongation, divergence of expression patterns and alternative splicing.

  3. Predictive and prognostic value of circulating nucleosomes and serum biomarkers in patients with metastasized colorectal cancer undergoing Selective Internal Radiation Therapy

    Directory of Open Access Journals (Sweden)

    Fahmueller Yvonne

    2012-01-01

    Full Text Available Abstract Background Selective Internal Radiation Therapy (SIRT is a new and effective locoregional anticancer therapy for colorectal cancer patients with liver metastases. Markers for prediction of therapy response and prognosis are needed for the individual management of those patients undergoing SIRT. Methods Blood samples were prospectively and consecutively taken from 49 colorectal cancer patients with extensive hepatic metastases before, three, six, 24 and 48 h after SIRT to analyze the concentrations of nucleosomes and further laboratory parameters, and to compare them with the response to therapy regularly determined 3 months after therapy and with overall survival. Results Circulating nucleosomes, cytokeratin-19 fragments (CYFRA 21-1, carcinoembryonic antigen (CEA, C-reactive protein (CRP and various liver markers increased already 24 h after SIRT. Pretherapeutical levels of CYFRA 21-1, CEA, cancer antigen 19-9 (CA 19-9, asparate-aminotransferase (AST and lactate dehydrogenase (LDH as well as 24 h values of nucleosomes were significantly higher in patients suffering from disease progression (N = 35 than in non-progressive patients (N = 14. Concerning overall survival, CEA, CA 19-9, CYFRA 21-1, CRP, LDH, AST, choline esterase (CHE, gamma-glutamyl-transferase, alkaline phosphatase, and amylase (all 0 h, 24 h and nucleosomes (24 h were found to be prognostic relevant markers in univariate analyses. In multivariate Cox-Regression analysis, the best prognostic model was obtained for the combination of CRP and AST. When 24 h values were additionally included, nucleosomes (24 h further improved the existing model. Conclusion Panels of biochemical markers are helpful to stratify pretherapeutically colorectal cancer patients for SIR-therapy and to early estimate the response to SIR-therapy.

  4. 转录起始位点核小体定位的研究进展%Advances on Nucleosome Mapping Around TSS

    Institute of Scientific and Technical Information of China (English)

    王成爱

    2014-01-01

    核小体定位是参与真核生物基因表达调控的一种重要的表观遗传因素,深刻影响基因转录、DNA复制与修复等生物学过程。对于在许多基因位点,比如转录起始位点(TSS)、转录因子结合位点(TFBS)等处的核小体定位已有不少报道。主要介绍了核小体的定位特性,综述了转录起始位点处核小体的定位特征,分别从序列依赖性因素和DNA甲基化、组蛋白变体及修饰、染色质重塑、可变剪接等表观遗传因素较为详细地概括了转录起始位点核小体定位的研究进展。%Nucleosome mapping,an important epigenetic factor participating in regulating eukaryotic gene expression, deeply affects lots of biological processes,including gene transcription,DNA replication,DNA repair and so forth. The nucleosome mapping around TSS and TFBS has been reported properties of nucleosome mapping were introduced in this paper. Features in nucleosome mapping around TSS were summarized. Advances on nucleosome mapping around TSS from both the DNA sequence-dependent factor and some epigenetic factors including DNA methylation, variants and modification of histone, chromation remodeling,alternative splicing were reviewed.

  5. Gut flora and bacterial translocation in chronic liver disease

    Institute of Scientific and Technical Information of China (English)

    John Almeida; Sumedha Galhenage; Jennifer Yu; Jelica Kurtovic; Stephen M Riordan

    2006-01-01

    Increasing evidence suggests that derangement of gut flora is of substantial clinical relevance to patients with cirrhosis. Intestinal bacterial overgrowth and increased bacterial translocation of gut flora from the intestinal lumen, in particular, predispose to an increased potential for bacterial infection in this group. Recent studies suggest that, in addition to their role in the pathogenesis of overt infective episodes and the clinical consequences of sepsis, gut flora contributes to the pro-inflammatory state of cirrhosis even in the absence of overt infection.Furthermore, manipulation of gut flora to augment the intestinal content of lactic acid-type bacteria at the expense of other gut flora species with more pathogenic potential may favourably influence liver function in cirrhotic patients. Here we review current concepts of the various inter-relationships between gut flora, bacterial translocation, bacterial infection, pro-inflammatory cytokine production and liver function in this group.

  6. Studying DNA translocation in nanocapillaries using single molecule fluorescence

    CERN Document Server

    Thacker, Vivek V; Hernández-Ainsa, Silvia; Bell, Nicholas A W; Keyser, Ulrich F; 10.1063/1.4768929

    2013-01-01

    We demonstrate simultaneous measurements of DNA translocation into glass nanopores using ionic current detection and fluorescent imaging. We verify the correspondence between the passage of a single DNA molecule through the nanopore and the accompanying characteristic ionic current blockage. By tracking the motion of individual DNA molecules in the nanocapillary perpendicular to the optical axis and using a model, we can extract an effective mobility constant for DNA in our geometry under high electric fields.

  7. The pathological effect of bacterial translocation to the Henssge Nomogram

    OpenAIRE

    Ivanka, Ján

    2012-01-01

    This article presents the results of measurements of the influence of pathological bacterial translocation on the intestinal wall of the area, measured per recta, and its influence on the course of a Henssge Nomogram. The gram-positive /negative bacteria which influence temperature measurements and the subsequent regressive non-stationary temperature data of biological objects when establishing the moment of death are described in a lucid, synoptic form. Based upon forensic praxis, profession...

  8. Particles translocate from the vagina to the oviducts and beyond

    Energy Technology Data Exchange (ETDEWEB)

    Wehner, A.P.; Hall, A.S.; Weller, R.E.; Lepel, E.A.; Schirmer, R.E.

    1985-03-01

    To investigate whether particles deposited in the vagina translocate to the oviducts, 0.3 ml of a 4% bone black suspension was deposited in the posterior vaginal fornix of each of five cynomolgus monkeys (Macaca fascicularis) during their mid-menstrual cycle. Simultaneously, each animal received 10 units of oxytocin by intramuscular injection. The oviducts of three animals were removed 1 hr after administration of the bone black, while those of the remaining two animals were removed 72 hr after dosing. The removed oviducts were flushed with Hank's solution and then with collagenase solution. The solutions were collected in clean vials and filtered. The filters were examined for bone black particles by light microscopy, as were filters through which solution blanks (negative controls) had been passed. Particles resembling bone black were found on all filters. There were no appreciable differences in the number or shape of these particles between the solution-blank filters and the oviduct-flush filters. The particles on both the solution-blank filters and on the oviduct-flush filters probably originated from environmental contamination by ubiquitous carbon particles. While these results suggested that no translocation took place, translocation could not be ruled out with certainty in the absence of quantitative analyses. A more definitive pilot study was then conducted with two dosed monkeys and one control, using talc labelled by neutron activation to circumvent the problem of environmental contamination. Gamma-Ray analysis of tissue and peritoneal lavage samples for the radionuclides /sup 46/Sc, /sup 59/Fe and /sup 60/Co indicated that no measurable quantities (i.e. greater than 0.5 micrograms) of talc translocated from the deposition site in the vagina to the uterine cavity and beyond.

  9. Subcellular distribution and translocation of radionuclides in plants

    Energy Technology Data Exchange (ETDEWEB)

    Gouthu, S.; Weginwar, R.; Arie, Tsutomu; Ambe, Shizuko; Ozaki, Takuo; Enomoto, Shuichi; Ambe, Fumitoshi; Yamaguchi, Isamu

    1999-09-01

    The subcellular distribution of radionuclides in Glycine max Merr. (soybean) and Cucumis sativus L. (cucumber) and translocation of plant absorbed radionuclides with growth in soybean were studied. More than 60% of cellular incorporated Rb{sup {minus}83}, Sr{sup {minus}85}, Mn{sup {minus}54}, Nb{sup {minus}95}, and Se{sup {minus}75} remained in the supernatant fraction; 55% and 20% of Cr{sup {minus}51} was bound to soybean and cucumber cell wall fractions, respectively; 70% or more of Be{sup {minus}7}, Y{sup {minus}88}, and Fe{sup {minus}59} was fixed in the chloroplast fraction; and approx. 10% of Sc{sup {minus}46}, Fe{sup {minus}59}, V{sup {minus}48}, and As were fixed in the mitochondrial fraction. Translocation of nuclides within the soybean plant at different stages of growth has been determined. Vanadium, Y{sup {minus}88}, Be{sup {minus}7}, Se{sup {minus}75}, Nb{sup {minus}95}, Sc{sup {minus}46}, Cr{sup {minus}51}, and Zr{sup {minus}88} were predominantly accumulated in the root. Although the total percentage of plant uptake of Sc{sup {minus}46}, Zr{sup {minus}88}, Nb{sup {minus}95}, Sc{sup {minus}46}, and Cr{sup {minus}51} was high, because of low mobility and translocation to shoot, their accumulation in the fruit fraction was negligible. The translocation of mobile nuclides in plants was demonstrated clearly by Rb{sup {minus}83}, Zn{sup {minus}65}, and Fe{sup {minus}59}. Data on the nuclide fraction mobilized from vegetative parts into edible parts was used to assess the percentage of accumulated radionuclides in plants that may reach humans through beans.

  10. "Translocal Express" juba täna! / Rael Artel

    Index Scriptorium Estoniae

    Artel, Rael, 1980-

    2009-01-01

    27. märtsil algab Kumu Kunstimuuseumis "Public Preparation'i" ("Avalik ettevalmistus") sarja rahvusvaheline seminar "Translocal Express. Golden Age" ("Translokaalne ekspress. Kuldaeg"), kus on kõne all ajalookirjutamise ja kollektiivse mälu roll praegu domineerivas natsionalistlikus diskursuses ja selle käsitlemine kaasaegses kunstis. Seminaril on lähtutud eelkõige kunstnike Martin Krenni (Viin) ja Kristina Normani teoste tutvustamisest

  11. Black bears in Arkansas: Characteristics of a successful translocation

    Science.gov (United States)

    Smith, Kimberly G.; Clark, Joseph D.

    1994-01-01

    In 1958, the Arkansas Game and Fish Commission began translocating black bears (Ursus americanus) from Minnesota to the Interior Highlands (Ozark and Ouachita mountains) of Arkansas where bears had been extirpated early in this century. This project continued for 11 years with little public imput, during which time an estimated 254 bears were released. We estimate there are now >2,500 bears in the Interior Highlands of Arkansas, Missouri, and Oklahoma, making it one of the most successful translocations of a Carnivora. Factors that contributed to the success include use of wild-captured animals, elimination of major factors associated with extirpation, release into prime habitats within the former range, multiple release sites, release of 20–40 animals/year for eight years, and release of mostly males prior to release of mostly females. Studies on two allopatric populations demonstrate that they are now diverging in some demographic characteristics, including litter size, cub survivorship, and adult sex-ratio. Translocation of black bears to the Interior Highlands is successful in terms of numbers of animals, but it will not be truly successful until people accept black bears as part of the regional fauna. To that end, those associated with management and research of bears in Arkansas are now focussing on public education and control of nuisance bears.

  12. Dominant-lethal mutations and heritable translocations in mice

    Energy Technology Data Exchange (ETDEWEB)

    Generoso, W.M.

    1983-01-01

    Chromosome aberrations are a major component of radiation or chemically induced genetic damage in mammalian germ cells. The types of aberration produced are dependent upon the mutagen used and the germ-cell stage treated. For example, in male meiotic and postmeiotic germ cells certain alkylating chemicals induce both dominant-lethal mutations and heritable translocations while others induce primarily dominant-lethal mutations. Production of these two endpoints appears to be determined by the stability of alkylation products with the chromosomes. If the reaction products are intact in the male chromosomes at the time of sperm entry, they may be repaired in fertilized eggs. If repair is not effected and the alkylation products persist to the time of pronuclear chromosome replication, they lead to chromatid-type aberrations and eventually to dominant-lethality. The production of heritable translocations, on the other hand, requires a transformation of unstable alkylation products into suitable intermediate lesions. The process by which these lesions are converted into chromosome exchange within the male genome takes place after sperm enters the egg but prior to the time of pronuclear chromosome replication (i.e., chromosome-type). Thus, dominant-lethal mutations result from both chromatid- and chromosome-type aberrations while heritable translocations result primarily from the latter type. DNA target sites associated with the production of these two endpoints are discussed.

  13. Detection of Kinase Translocation Using Microfluidic Electroporative Flow Cytometry

    Science.gov (United States)

    Lu, Chang; Wang, Jun; Bao, Ning; Paris, Leela; Wang, Hsiang-Yu; Geahlen, Robert

    2008-03-01

    Translocation of a protein between different subcellular compartments is a common event during signal transduction in living cells. Detection of these events has been largely carried out based on imaging of a low number of cells and subcellular fractionation/Western blotting. These conventional techniques either lack the high throughput desired for probing an entire cell population or provide only the average behaviors of cell populations without information from single cells. Here we demonstrate a new tool, referred to as microfluidic electroporative flow cytometry, to detect the translocation of an EGFP-tagged tyrosine kinase, Syk, to the plasma membrane in B cells at the level of the cell population. We combine electroporation with flow cytometry and observe the release of intracellular kinase out of the cells during electroporation. We found that the release of the kinase was strongly influenced by its subcellular localization. Cells stimulated through the antigen receptor have a fraction of the kinase at the plasma membrane and retain more kinase after electroporation than do cells without stimulation and translocation. This tool will have utility for kinase-related drug discovery and tumor diagnosis and staging.

  14. DegP Chaperone Suppresses Toxic Inner Membrane Translocation Intermediates

    Science.gov (United States)

    Braselmann, Esther; Chaney, Julie L.; Champion, Matthew M.

    2016-01-01

    The periplasm of Gram-negative bacteria includes a variety of molecular chaperones that shepherd the folding and targeting of secreted proteins. A central player of this quality control network is DegP, a protease also suggested to have a chaperone function. We serendipitously discovered that production of the Bordetella pertussis autotransporter virulence protein pertactin is lethal in Escherichia coli ΔdegP strains. We investigated specific contributions of DegP to secretion of pertactin as a model system to test the functions of DegP in vivo. The DegP chaperone activity was sufficient to restore growth during pertactin production. This chaperone dependency could be relieved by changing the pertactin signal sequence: an E. coli signal sequence leading to co-translational inner membrane (IM) translocation was sufficient to suppress lethality in the absence of DegP, whereas an E. coli post-translational signal sequence was sufficient to recapitulate the lethal phenotype. These results identify a novel connection between the DegP chaperone and the mechanism used to translocate a protein across the IM. Lethality coincided with loss of periplasmic proteins, soluble σE, and proteins regulated by this essential stress response. These results suggest post-translational IM translocation can lead to the formation of toxic periplasmic folding intermediates, which DegP can suppress. PMID:27626276

  15. DegP Chaperone Suppresses Toxic Inner Membrane Translocation Intermediates.

    Science.gov (United States)

    Braselmann, Esther; Chaney, Julie L; Champion, Matthew M; Clark, Patricia L

    2016-01-01

    The periplasm of Gram-negative bacteria includes a variety of molecular chaperones that shepherd the folding and targeting of secreted proteins. A central player of this quality control network is DegP, a protease also suggested to have a chaperone function. We serendipitously discovered that production of the Bordetella pertussis autotransporter virulence protein pertactin is lethal in Escherichia coli ΔdegP strains. We investigated specific contributions of DegP to secretion of pertactin as a model system to test the functions of DegP in vivo. The DegP chaperone activity was sufficient to restore growth during pertactin production. This chaperone dependency could be relieved by changing the pertactin signal sequence: an E. coli signal sequence leading to co-translational inner membrane (IM) translocation was sufficient to suppress lethality in the absence of DegP, whereas an E. coli post-translational signal sequence was sufficient to recapitulate the lethal phenotype. These results identify a novel connection between the DegP chaperone and the mechanism used to translocate a protein across the IM. Lethality coincided with loss of periplasmic proteins, soluble σE, and proteins regulated by this essential stress response. These results suggest post-translational IM translocation can lead to the formation of toxic periplasmic folding intermediates, which DegP can suppress.

  16. Patch-clamp detection of macromolecular translocation along nuclear pores

    Directory of Open Access Journals (Sweden)

    Bustamante J.O.

    1998-01-01

    Full Text Available The present paper reviews the application of patch-clamp principles to the detection and measurement of macromolecular translocation along the nuclear pores. We demonstrate that the tight-seal 'gigaseal' between the pipette tip and the nuclear membrane is possible in the presence of fully operational nuclear pores. We show that the ability to form a gigaseal in nucleus-attached configurations does not mean that only the activity of channels from the outer membrane of the nuclear envelope can be detected. Instead, we show that, in the presence of fully operational nuclear pores, it is likely that the large-conductance ion channel activity recorded derives from the nuclear pores. We conclude the technical section with the suggestion that the best way to demonstrate that the nuclear pores are responsible for ion channel activity is by showing with fluorescence microscopy the nuclear translocation of ions and small molecules and the exclusion of the same from the cisterna enclosed by the two membranes of the envelope. Since transcription factors and mRNAs, two major groups of nuclear macromolecules, use nuclear pores to enter and exit the nucleus and play essential roles in the control of gene activity and expression, this review should be useful to cell and molecular biologists interested in understanding how patch-clamp can be used to quantitate the translocation of such macromolecules into and out of the nucleus

  17. Growth factor deprivation induces cytosolic translocation of SIRT1

    Science.gov (United States)

    Meng, Chengbo; Xing, Da; Wu, Shengnan; Huang, Lei

    2010-02-01

    Sirtuin type 1 (SIRT1), a NAD+-dependent histone deacetylases, plays a critical role in cellular senescence, aging and longevity. In general, SIRT1 is localized in nucleus and is believed as a nuclear protein. Though overexpression of SIRT1 delays senescence, SIRT1-protein levels decline naturally in thymus and heart during aging. In the present studies, we investigated the subcellular localization of SIRT1 in response to growth factor deprivation in African green monkey SV40-transformed kidney fibroblast cells (COS-7). Using SIRT1-EGFP fluorescence reporter, we found that SIRT1 localized to nucleus in physiological conditions. We devised a model enabling cell senescence via growth factor deprivation, and we found that SIRT1 partially translocated to cytosol under the treatment, suggesting a reduced level of SIRT1's activity. We found PI3K/Akt pathway was involved in the inhibition of SIRT1's cytosolic translocation, because inhibition of these kinases significantly decreased the amount of SIRT1 maintained in nucleus. Taken together, we demonstrated that growth factor deprivation induces cytosolic translocation of SIRT1, which suggesting a possible connection between cytoplasm-localized SIRT1 and the aging process.

  18. Single Nanoparticle Translocation Through Chemically Modified Solid Nanopore

    Science.gov (United States)

    Tan, Shengwei; Wang, Lei; Liu, Hang; Wu, Hongwen; Liu, Quanjun

    2016-02-01

    The nanopore sensor as a high-throughput and low-cost technology can detect single nanoparticle in solution. In the present study, the silicon nitride nanopores were fabricated by focused Ga ion beam (FIB), and the surface was functionalized with 3-aminopropyltriethoxysilane to change its surface charge density. The positively charged nanopore surface attracted negatively charged nanoparticles when they were in the vicinity of the nanopore. And, nanoparticle translocation speed was slowed down to obtain a clear and deterministic signal. Compared with previous studied small nanoparticles, the electrophoretic translocation of negatively charged polystyrene (PS) nanoparticles (diameter ~100 nm) was investigated in solution using the Coulter counter principle in which the time-dependent nanopore current was recorded as the nanoparticles were driven across the nanopore. A linear dependence was found between current drop and biased voltage. An exponentially decaying function ( t d ~ e -v/v0 ) was found between the duration time and biased voltage. The interaction between the amine-functionalized nanopore wall and PS microspheres was discussed while translating PS microspheres. We explored also translocations of PS microspheres through amine-functionalized solid-state nanopores by varying the solution pH (5.4, 7.0, and 10.0) with 0.02 M potassium chloride (KCl). Surface functionalization showed to provide a useful step to fine-tune the surface property, which can selectively transport molecules or particles. This approach is likely to be applied to gene sequencing.

  19. Spatial behaviour and survival of translocated wild brown hares

    Directory of Open Access Journals (Sweden)

    Fischer, C.

    2012-01-01

    Full Text Available The fragility of many populations of brown hares in Western Europe is a concern for managers, hunters and naturalists. We took advantage of a locally high density population to use wild individuals to restock areas where the species had disappeared or was close to disappearing. The aim of the project was to assess the evolution of the spatial behaviour after release using radio–tracking. Over 150 wild brown hares were translocated, one third of which were fitted with radio collars. In addition, fifteen individuals were radio–tagged and released back into the source population as a control. Most individuals settled in less than two months and their seasonal home range, once settled, was similar to that observed in the source population. Mean duration of tracking was not significantly different between the two groups. Moreover, two years after the last translocation, tagged individuals can still be observed, but most hares present are not tagged, which indicates natural reproduction of the released individuals. The translocation of wild individuals thus appears to give encouraging results.

  20. Growth hormone reduces mortality and bacterial translocation in irradiated rats

    Energy Technology Data Exchange (ETDEWEB)

    Gomez-de-Segura, I.A.; Miguel, E. de [`La Paz` Hospital, Madrid (Spain). Dept. of Experimental Surgery; Prieto, I. [`La Paz` Hospital, Madrid (Spain). Dept. of General and Digestive Surgery; Grande, A.G. [`La Paz` Hospital, Madrid (Spain). Dept. of Oncology Radiotherapy; Garcia, P.; Mendez, J. [`La Paz` Hospital, Madrid (Spain). Dept. of Clinical Biochemistry; Guerra, A. [`La Paz` Hospital, Madrid (Spain). Dept. of Microbiology

    1998-09-01

    Growth hormone stimulates the growth of intestinal mucosa and may reduce the severity of injury caused by radiation. Male Wistar rats underwent abdominal irradiation (12 Gy) and were treated with either human growth hormone (hGH) or saline, and sacrificed at day 4 or 7 post-irradiation. Bacterial translocation, and the ileal mucosal thickness, proliferation, and disaccharidase activity were assessed. Mortality was 65% in irradiated animals, whereas hGH caused a decrement (29%, p<0.05). Bacterial translocation was also reduced by hGH (p<0.05). Treating irradiated rats with hGH prevented body weight loss (p<0.05). Mucosal thickness increased faster in irradiated hGH-treated animals. The proliferative index showed an increment in hGH-treated animals (p<0.05). Giving hGH to irradiated rats prevented decrease in sucrose activity, and increment in lactase activity. In conclusion, giving hGH to irradiated rats promotes the adaptative process of the intestine and acute radiation-related negative effects, including mortality, bacterial translocation, and weight loss. (orig.)

  1. Dieldrin uptake and translocation in plants growing in hydroponic medium.

    Science.gov (United States)

    Murano, Hirotatsu; Otani, Takashi; Seike, Nobuyasu; Sakai, Mizuki

    2010-01-01

    It has been known that the Cucurbitaceae family takes up a large amount of persistent organic pollutants from soils and that the translocation of those compounds in cucurbits is higher than those in non-cucurbits. To understand the persistent organic pollutant uptake mechanisms of plant species, we compared the dieldrin absorption and transportation potentials of several plants in hydroponic medium. Sorghum (Sorghum vulgare Moench), sunflower (Helianthus annuus L.), soybean (Glycine max), komatsuna (Brassica rapa var. peruviridis), white-flowered gourd (Lagenaria siceraria var. hispida), cucumber (Cucumis sativus L.), and zucchini (Cucurbita pepo L.) were grown in a dieldrin-added hydroponic medium for 10 d, and then the amount of dieldrin in their shoots and roots was measured. All of the roots contained dieldrin, whereas only the cucurbits (white-flowered gourd, cucumber, and zucchini) contained considerable amounts of dieldrin in their shoots. The dieldrin uptake to the roots depended on the concentration of the n-hexane soluble components in the roots, regardless of whether the dieldrin in the roots was translocated to shoots or not. The dieldrin uptake from the solution to the roots was thought to be due to a passive response, such as adsorption on the roots. The translocation of dieldrin from the roots to the shoots was probably through the xylems. The amounts of dieldrin in the shoots per transpiration rates were higher for cucurbits than for non-cucurbits. It seems likely that cucurbits have uptake mechanisms for hydrophobic organic chemicals.

  2. Gq-mediated Akt translocation to the membrane: a novel PIP3-independent mechanism in platelets.

    Science.gov (United States)

    Badolia, Rachit; Manne, Bhanu Kanth; Dangelmaier, Carol; Chernoff, Jonathan; Kunapuli, Satya P

    2015-01-01

    Akt is an important signaling molecule regulating platelet aggregation. Akt is phosphorylated after translocation to the membrane through Gi signaling pathways by a phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent mechanism. However, Akt is more robustly phosphorylated by thrombin compared with adenosine 5'-diphosphate in platelets. This study investigated the mechanisms of Akt translocation as a possible explanation for this difference. Stimulation of washed human platelets with protease-activated receptor agonists caused translocation of Akt to the membrane rapidly, whereas phosphorylation occurred later. The translocation of Akt was abolished in the presence of a Gq-selective inhibitor or in Gq-deficient murine platelets, indicating that Akt translocation is regulated downstream of Gq pathways. Interestingly, phosphatidylinositol 3-kinase (PI3K) inhibitors or P2Y12 antagonist abolished Akt phosphorylation without affecting Akt translocation to the membrane, suggesting that Akt translocation occurs through a PI3K/PIP3/Gi-independent mechanism. An Akt scaffolding protein, p21-activated kinase (PAK), translocates to the membrane after stimulation with protease-activated receptor agonists in a Gq-dependent manner, with the kinetics of translocation similar to that of Akt. Coimmunoprecipitation studies showed constitutive association of PAK and Akt, suggesting a possible role of PAK in Akt translocation. These results show, for the first time, an important role of the Gq pathway in mediating Akt translocation to the membrane in a novel Gi/PI3K/PIP3-independent mechanism.

  3. Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining.

    Science.gov (United States)

    Ghezraoui, Hind; Piganeau, Marion; Renouf, Benjamin; Renaud, Jean-Baptiste; Sallmyr, Annahita; Ruis, Brian; Oh, Sehyun; Tomkinson, Alan E; Hendrickson, Eric A; Giovannangeli, Carine; Jasin, Maria; Brunet, Erika

    2014-09-18

    Breakpoint junctions of the chromosomal translocations that occur in human cancers display hallmarks of nonhomologous end-joining (NHEJ). In mouse cells, translocations are suppressed by canonical NHEJ (c-NHEJ) components, which include DNA ligase IV (LIG4), and instead arise from alternative NHEJ (alt-NHEJ). Here we used designer nucleases (ZFNs, TALENs, and CRISPR/Cas9) to introduce DSBs on two chromosomes to study translocation joining mechanisms in human cells. Remarkably, translocations were altered in cells deficient for LIG4 or its interacting protein XRCC4. Translocation junctions had significantly longer deletions and more microhomology, indicative of alt-NHEJ. Thus, unlike mouse cells, translocations in human cells are generated by c-NHEJ. Human cancer translocations induced by paired Cas9 nicks also showed a dependence on c-NHEJ, despite having distinct joining characteristics. These results demonstrate an unexpected and striking species-specific difference for common genomic rearrangements associated with tumorigenesis.

  4. The nucleosome (histone-DNA complex is the TLR9-specific immunostimulatory component of Plasmodium falciparum that activates DCs.

    Directory of Open Access Journals (Sweden)

    Nagaraj M Gowda

    Full Text Available The systemic clinical symptoms of Plasmodium falciparum infection such as fever and chills correspond to the proinflammatory cytokines produced in response to the parasite components released during the synchronized rupture of schizonts. We recently demonstrated that, among the schizont-released products, merozoites are the predominant components that activate dendritic cells (DCs by TLR9-specific recognition to induce the maturation of cells and to produce proinflammatory cytokines. We also demonstrated that DNA is the active constituent and that formation of a DNA-protein complex is essential for the entry of parasite DNA into cells for recognition by TLR9. However, the nature of endogenous protein-DNA complex in the parasite is not known. In this study, we show that parasite nucleosome constitute the major protein-DNA complex involved in the activation of DCs by parasite nuclear material. The parasite components were fractionated into the nuclear and non-nuclear materials. The nuclear material was further fractionated into chromatin and the proteins loosely bound to chromatin. Polynucleosomes and oligonucleosomes were prepared from the chromatin. These were tested for their ability to activate DCs obtained by the FLT3 ligand differentiation of bone marrow cells from the wild type, and TLR2(-/-, TLR9(-/- and MyD88(-/- mice. DCs stimulated with the nuclear material and polynucleosomes as well as mono- and oligonucleosomes efficiently induced the production of proinflammatory cytokines in a TLR9-dependent manner, demonstrating that nucleosomes (histone-DNA complex represent the major TLR9-specific DC-immunostimulatory component of the malaria parasite nuclear material. Thus, our data provide a significant insight into the activation of DCs by malaria parasites and have important implications for malaria vaccine development.

  5. 两种模式生物核小体定位比较研究%A comparative study of nucleosome positioning pattern in two model organisms

    Institute of Scientific and Technical Information of China (English)

    卢英; 丰继华; 单秋甫; 陈攀峰; 单增辉

    2014-01-01

    在对两种模式生物酵母与果蝇胚胎期核小体定位进行研究时,发现不同物种间以及同一物种中不同表达模式基因上的核小体分布呈现出差显著异性。在总体上,转录起始位点附近的酵母核小体NFR区域比果蝇的NFR短。经基因中心对齐后,酵母与果蝇胚胎期沉默型基因的核小体缺失区域的两个边界中间处共同呈现了一个明确有着均匀间隔的核小体数n,且随着基因长度L的变长其周期性特性逐渐变模糊,但果蝇的图谱表现的更为复杂。结果表明,从单细胞酵母生物到多细胞果蝇生物间基因组的进化过程中,核小体组织的演化既有变异性,也具有保守性。%By studying nucleosome positioning during embryonic period of two model organisms, Yeast and Drosophila, we found that nucleosome are differently distributed along genome-wide genes which either belong to different species or the same species in different expression patterns. In general, nucleosome free regions ( NFR) regions around the transcription start sites in yeast are narrower than that in Drosophila. If aligned by the gene center, there always are a well-defined number n of uniformly spaced nucleosomes in the middle of NFRs for embryo silence genes in both species, and the characteristics of these uniformly spaced nucleosomes gradually go fuzzy with the gene length L being longer , while drosophila presents more complexity and ambiguity than yeast relatively. The results show that the evolution of genome nucleosomes organization holds both variability and conservatism from unicellular organism to the multicellular organism.

  6. Nucleosomes correlate with in vivo progression pattern of de novo methylation of p16 CpG islands in human gastric carcinogenesis.

    Directory of Open Access Journals (Sweden)

    Zhe-Ming Lu

    Full Text Available BACKGROUND: The exact relationship between nucleosome positioning and methylation of CpG islands in human pathogenesis is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we characterized the nucleosome position within the p16 CpG island and established a seeding methylation-specific PCR (sMSP assay based on bisulfite modification to enrich the p16 alleles containing methylated-CpG at the methylation "seeding" sites within its intron-1 in gastric carcinogenesis. The sMSP-positive rate in primary gastric carcinoma (GC samples (36/40 was significantly higher than that observed in gastritis (19/45 or normal samples (7/13 (P<0.01. Extensive clone sequencing of these sMSP products showed that the density of methylated-CpGs in p16 CpG islands increased gradually along with the severity of pathological changes in gastric tissues. In gastritis lesions the methylation was frequently observed in the region corresponding to the exon-1 coding-nucleosome and the 5'UTR-nucleosome; the methylation was further extended to the region corresponding to the promoter-nucleosome in GC samples. Only few methylated-CpG sites were randomly detected within p16 CpG islands in normal tissues. The significantly inversed relationship between the p16 exon-1 methylation and its transcription was observed in GC samples. An exact p16 promoter-specific 83 bp-MSP assay confirms the result of sMSP (33/55 vs. 1/6, P<0.01. In addition, p16 methylation in chronic gastritis lesions significantly correlated with H. pylori infection; however, such correlation was not observed in GC specimens. CONCLUSIONS/SIGNIFICANCE: It was determined that de novo methylation was initiated in the coding region of p16 exon-1 in gastritis, then progressed to its 5'UTR, and ultimately to the proximal promoter in GCs. Nucleosomes may function as the basic extension/progression unit of de novo methylation of p16 CpG islands in vivo.

  7. Translocation of positively and negatively charged polystyrene nanoparticles in an in vitro placental model.

    Science.gov (United States)

    Kloet, Samantha K; Walczak, Agata P; Louisse, Jochem; van den Berg, Hans H J; Bouwmeester, Hans; Tromp, Peter; Fokkink, Remco G; Rietjens, Ivonne M C M

    2015-10-01

    To obtain insight in translocation of nanoparticles across the placental barrier, translocation was studied for one positively and two negatively charged polystyrene nanoparticles (PS-NPs) of similar size in an in vitro model. The model consisted of BeWo b30 cells, derived from a human choriocarcinoma grown on a transwell insert forming a cell layer that separates an apical from a basolateral compartment. PS-NPs were characterized with respect to size, surface charge, morphology and protein corona. Translocation of PS-NPs was not related to PS-NP charge. Two PS-NPs were translocated across the BeWo transwell model to a lower extent than amoxicillin, a model compound known to be translocated over the placental barrier to only a limited extent, whereas one PS-NP showed a slightly higher translocation. Studies on the effect of transporter inhibitors on the translocation of the PS-NPs indicated that their translocation was not mediated by known transporters and mainly dependent on passive diffusion. It is concluded that the BeWo b30 model can be used as an efficient method to get an initial qualitative impression about the capacity of NPs to translocate across the placental barrier and set priorities in further in vivo studies on translocation of NPs to the fetus.

  8. Efficient induction of Wheat-agropyron cristatum 6P translocation lines and GISH detection.

    Directory of Open Access Journals (Sweden)

    Liqiang Song

    Full Text Available The narrow genetic background restricts wheat yield and quality improvement. The wild relatives of wheat are the huge gene pools for wheat improvement and can broaden its genetic basis. Production of wheat-alien translocation lines can transfer alien genes to wheat. So it is important to develop an efficient method to induce wheat-alien chromosome translocation. Agropyroncristatum (P genome carries many potential genes beneficial to disease resistance, stress tolerance and high yield. Chromosome 6P possesses the desirable genes exhibiting good agronomic traits, such as high grain number per spike, powdery mildew resistance and stress tolerance. In this study, the wheat-A. cristatum disomic addition was used as bridge material to produce wheat-A. cristatum translocation lines induced by (60Co-γirradiation. The results of genomic in situ hybridization showed that 216 plants contained alien chromosome translocation among 571 self-pollinated progenies. The frequency of translocation was 37.83%, much higher than previous reports. Moreover, various alien translocation types were identified. The analysis of M2 showed that 62.5% of intergeneric translocation lines grew normally without losing the translocated chromosomes. The paper reported a high efficient technical method for inducing alien translocation between wheat and Agropyroncristatum. Additionally, these translocation lines will be valuable for not only basic research on genetic balance, interaction and expression of different chromosome segments of wheat and alien species, but also wheat breeding programs to utilize superior agronomic traits and good compensation effect from alien chromosomes.

  9. Hyaluronan synthase mediates dye translocation across liposomal membranes

    Directory of Open Access Journals (Sweden)

    Medina Andria P

    2012-01-01

    Full Text Available Abstract Background Hyaluronan (HA is made at the plasma membrane and secreted into the extracellular medium or matrix by phospolipid-dependent hyaluronan synthase (HAS, which is active as a monomer. Since the mechanism by which HA is translocated across membranes is still unresolved, we assessed the presence of an intraprotein pore within HAS by adding purified Streptococcus equisimilis HAS (SeHAS to liposomes preloaded with the fluorophore Cascade Blue (CB. Results CB translocation (efflux was not observed with mock-purified material from empty vector control E. coli membranes, but was induced by SeHAS, purified from membranes, in a time- and dose-dependent manner. CB efflux was eliminated or greatly reduced when purified SeHAS was first treated under conditions that inhibit enzyme activity: heating, oxidization or cysteine modification with N-ethylmaleimide. Reduced CB efflux also occurred with SeHAS K48E or K48F mutants, in which alteration of K48 within membrane domain 2 causes decreased activity and HA product size. The above results used liposomes containing bovine cardiolipin (BCL. An earlier study testing many synthetic lipids found that the best activating lipid for SeHAS is tetraoleoyl cardiolipin (TO-CL and that, in contrast, tetramyristoyl cardiolipin (TM-CL is an inactivating lipid (Weigel et al, J. Biol. Chem. 281, 36542, 2006. Consistent with the effects of these CL species on SeHAS activity, CB efflux was more than 2-fold greater in liposomes made with TO-CL compared to TM-CL. Conclusions The results indicate the presence of an intraprotein pore in HAS and support a model in which HA is translocated to the exterior by HAS itself.

  10. Mitochondrial function in Antarctic nototheniids with ND6 translocation.

    Directory of Open Access Journals (Sweden)

    Felix C Mark

    Full Text Available Fish of the suborder Notothenioidei have successfully radiated into the Southern Ocean and today comprise the dominant fish sub-order in Antarctic waters in terms of biomass and species abundance. During evolution in the cold and stable Antarctic climate, the Antarctic lineage of notothenioids developed several unique physiological adaptations, which make them extremely vulnerable to the rapid warming of Antarctic waters currently observed. Only recently, a further phenomenon exclusive to notothenioid fish was reported: the translocation of the mitochondrial gene encoding the NADH Dehydrogenase subunit 6 (ND6, an indispensable part of complex I in the mitochondrial electron transport system.This study investigated the potential physiological consequences of ND6 translocation for the function and thermal sensitivity of the electron transport system in isolated liver mitochondria of the two nototheniid species Notothenia coriiceps and Notothenia rossii, with special attention to the contributions of complex I (NADH DH and complex II (Succinate DH to oxidative phosphorylation. Furthermore, enzymatic activities of NADH:Cytochrome c Oxidoreductase and Cytochrome C Oxidase were measured in membrane-enriched tissue extracts.During acute thermal challenge (0-15°C, capacities of mitochondrial respiration and enzymatic function in the liver could only be increased until 9°C. Mitochondrial complex I (NADH Dehydrogenase was fully functional but displayed a higher thermal sensitivity than the other complexes of the electron transport system, which may specifically result from its unique amino acid composition, revealing a lower degree of stability in notothenioids in general. We interpret the translocation of ND6 as functionally neutral but the change in amino acid sequence as adaptive and supportive of cold stenothermy in Antarctic nototheniids. From these findings, an enhanced sensitivity to ocean warming can be deduced for Antarctic notothenioid fish.

  11. Mitochondrial function in Antarctic nototheniids with ND6 translocation.

    Science.gov (United States)

    Mark, Felix C; Lucassen, Magnus; Strobel, Anneli; Barrera-Oro, Esteban; Koschnick, Nils; Zane, Lorenzo; Patarnello, Tomaso; Pörtner, Hans O; Papetti, Chiara

    2012-01-01

    Fish of the suborder Notothenioidei have successfully radiated into the Southern Ocean and today comprise the dominant fish sub-order in Antarctic waters in terms of biomass and species abundance. During evolution in the cold and stable Antarctic climate, the Antarctic lineage of notothenioids developed several unique physiological adaptations, which make them extremely vulnerable to the rapid warming of Antarctic waters currently observed. Only recently, a further phenomenon exclusive to notothenioid fish was reported: the translocation of the mitochondrial gene encoding the NADH Dehydrogenase subunit 6 (ND6), an indispensable part of complex I in the mitochondrial electron transport system.This study investigated the potential physiological consequences of ND6 translocation for the function and thermal sensitivity of the electron transport system in isolated liver mitochondria of the two nototheniid species Notothenia coriiceps and Notothenia rossii, with special attention to the contributions of complex I (NADH DH) and complex II (Succinate DH) to oxidative phosphorylation. Furthermore, enzymatic activities of NADH:Cytochrome c Oxidoreductase and Cytochrome C Oxidase were measured in membrane-enriched tissue extracts.During acute thermal challenge (0-15°C), capacities of mitochondrial respiration and enzymatic function in the liver could only be increased until 9°C. Mitochondrial complex I (NADH Dehydrogenase) was fully functional but displayed a higher thermal sensitivity than the other complexes of the electron transport system, which may specifically result from its unique amino acid composition, revealing a lower degree of stability in notothenioids in general. We interpret the translocation of ND6 as functionally neutral but the change in amino acid sequence as adaptive and supportive of cold stenothermy in Antarctic nototheniids. From these findings, an enhanced sensitivity to ocean warming can be deduced for Antarctic notothenioid fish.

  12. Effect of probiotics on enterocyte bacterial translocation in vitro.

    Science.gov (United States)

    Mattar, A F; Drongowski, R A; Coran, A G; Harmon, C M

    2001-05-01

    Enteral probiotics such as Lactobacillus casei GG (LGG) have been used in the treatment of a variety of intestinal disorders in infants and children, including diarrhea, malabsorption, and Clostridium difficile colitis. We have previously demonstrated that the probiotic bacterium LGG has an inhibitory effect on bacterial translocation (BT) in a neonatal rabbit model. However, this in-vivo model is limited for investigating the cellular and molecular mechanisms responsible for probiotic inhibition of BT. The purpose of this study was to determine the efficacy of LGG in reducing the rate of Escherichia coli C25 (E. coli C25) translocation using an in-vitro enterocyte cell-culture model. Human colonic carcinoma (Caco-2) enterocytes were seeded in porous filters in the apical chamber of a two-chamber cell-culture system and grown for 14 days to confluence. The monolayers were incubated at 37 degrees C with LGG for 180 min. Non-adherent LGG was washed away prior to a 120-min incubation period with 10(5) CFU E. coli C25. E. coli that had translocated across the enterocyte monolayer were quantified by growing basal-chamber media samples on gram-negative bacteria-specific MacConkey's agar. In order to determine monolayer integrity, transepithelial electrical resistance (TEER) was measured across Caco-2 cells treated with LGG and E. coli. Statistical analysis was by ANOVA with P probiotic bacterium LGG inhibits BT of E. coli C25 in a dose-dependent manner in an in-vitro cell-culture model. This model should be valuable in investigating the cellular and molecular mechanisms involved in the inhibition of pathological enteral bacteria by probiotic agents.

  13. Driven polymer translocation in good and bad solvent: effects of hydrodynamics and tension propagation

    CERN Document Server

    Moisio, Jaakko E; Linna, Riku P

    2016-01-01

    We investigate the driven polymer translocation through a nanometer-scale pore in the presence and absence of hydrodynamics both in good and bad solvent. We measure tension of the polymer segment on the {\\it cis} side of the pore in t