The Experts below are selected from a list of 303 Experts worldwide ranked by ideXlab platform
Jeffrey J. Hayes - One of the best experts on this subject based on the ideXlab platform.
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A nucleosome-free region locally abrogates histone H1–dependent restriction of Linker DNA accessibility in chromatin
The Journal of biological chemistry, 2018Co-Authors: Laxmi N. Mishra, Jeffrey J. HayesAbstract:Eukaryotic genomes are packaged into Linker-oligonucleosome assemblies, providing compaction of genomic DNA and contributing to gene regulation and genome integrity. To define minimal requirements for initial steps in the transition of compact, closed chromatin to a transcriptionally active, open state, we developed a model in vitro system containing a single, unique, "target" nucleosome in the center of a 25-nucleosome array and evaluated the accessibility of the Linker DNA adjacent to this target nucleosome. We found that condensation of H1-lacking chromatin results in ∼60-fold reduction in Linker DNA accessibility and that mimics of acetylation within all four core histone tail domains of the target nucleosome synergize to increase accessibility ∼3-fold. Notably, stoichiometric binding of histone H1 caused >2 orders of magnitude reduction in accessibility that was marginally diminished by histone acetylation mimics. Remarkably, a nucleosome-free region (NFR) in place of the target nucleosome completely abrogated H1-dependent restriction of Linker accessibility in the immediate vicinity of the NFR. Our results suggest that Linker DNA is as inaccessible as DNA within the nucleosome core in fully condensed, H1-containing chromatin. They further imply that an unrecognized function of NFRs in gene promoter regions is to locally abrogate the severe restriction of Linker DNA accessibility imposed by H1s.
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Nucleosome Linker DNA contacts and induces specific folding of the intrinsically disordered H1 carboxyl-terminal domain.
Molecular and cellular biology, 2011Co-Authors: Tamara L. Caterino, He Fang, Jeffrey J. HayesAbstract:Linker histones play essential roles in the chromatin structure of higher eukaryotes. While binding to the surface of nucleosomes is directed by an ∼ 80-amino-acid-residue globular domain, the structure and interactions of the lysine-rich ∼ 100-residue C-terminal domain (CTD), primarily responsible for the chromatin-condensing functions of Linker histones, are poorly understood. By quantitatively analyzing binding of a set of H1 CTD deletion mutants to nucleosomes containing various lengths of Linker DNA, we have identified interactions between distinct regions of the CTD and nucleosome Linker DNA at least 21 bp from the edge of the nucleosome core. Importantly, partial CTD truncations caused increases in H1 binding affinity, suggesting that significant entropic costs are incurred upon binding due to CTD folding. van't Hoff entropy/enthalpy analysis and intramolecular fluorescent resonance energy transfer (FRET) studies indicate that the CTD undergoes substantial nucleosome-directed folding, in a manner that is distinct from that which occurs upon H1 binding to naked DNA. In addition to defining critical interactions between the H1 CTD and Linker DNA, our data indicate that the H1 CTD is an intrinsically disordered domain and provide important insights into the biological function of this protein.
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Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Sajad Hussain Syed, Jeffrey J. Hayes, Jan Bednar, Dimitar Angelov, Manu Shubhdarshan Shukla, Damien Goutte-gattat, Nils Becker, Sam Meyer, Ralf Everaers, Stefan DimitrovAbstract:Despite the key role of the Linker histone H1 in chromatin structure and dynamics, its location and interactions with nucleosomal DNA have not been elucidated. In this work we have used a combination of electron cryomicroscopy, hydroxyl radical footprinting, and nanoscale modeling to analyze the structure of precisely positioned mono-, di-, and trinucleosomes containing physiologically assembled full-length histone H1 or truncated mutants of this protein. Single-base resolution *OH footprinting shows that the globular domain of histone H1 (GH1) interacts with the DNA minor groove located at the center of the nucleosome and contacts a 10-bp region of DNA localized symmetrically with respect to the nucleosomal dyad. In addition, GH1 interacts with and organizes about one helical turn of DNA in each Linker region of the nucleosome. We also find that a seven amino acid residue region (121-127) in the COOH terminus of histone H1 was required for the formation of the stem structure of the Linker DNA. A molecular model on the basis of these data and coarse-grain DNA mechanics provides novel insights on how the different domains of H1 interact with the nucleosome and predicts a specific H1-mediated stem structure within Linker DNA.
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preferential interaction of the core histone tail domains with Linker DNA
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Dimitar Angelov, Joseph M Vitolo, Vesco Mutskov, Stefan Dimitrov, Jeffrey J. HayesAbstract:Within chromatin, the core histone tail domains play critical roles in regulating the structure and accessibility of nucleosomal DNA within the chromatin fiber. Thus, many nuclear processes are facilitated by concomitant posttranslational modification of these domains. However, elucidation of the mechanisms by which the tails mediate such processes awaits definition of tail interactions within chromatin. In this study we have investigated the primary DNA target of the majority of the tails in mononucleosomes. The results clearly show that the tails bind preferentially to “Linker” DNA, outside of the DNA encompassed by the nucleosome core. These results have important implications for models of tail function within the chromatin fiber and for in vitro structural and functional studies using nucleosome core particles.
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Linker DNA and H1-dependent reorganization of histone-DNA interactions within the nucleosome.
Biochemistry, 1998Co-Authors: Kyu-min Lee, Jeffrey J. HayesAbstract:We have employed a site-directed photochemical cross-linking procedure to precisely map interactions between nucleosomal DNA and the C-terminal tail of core histone H2A. We find that this tail has the potential to contact multiple sites within the nucleosome and that these contacts are dependent upon the configuration of the complex. This tail contacts DNA near the dyad axis within nucleosome core particles but rearranges to a site near the edge of the nucleosomal DNA when Linker DNA is present. Moreover, in the presence of Linker histone H1 the contacts near the edge of the nucleosome but not at the dyad are further rearranged. In addition, we present further evidence for the suggestion that the binding of Linker histone causes a subtle but global change in core histone-DNA interactions within the nucleosome [Usachenko, S. I., Gavin, I. M., and Bavykin, S. G. (1996) J. Biol. Chem. 271, 3831-3836].
Donald J. Hnatowich - One of the best experts on this subject based on the ideXlab platform.
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in vitro investigations of tumor targeting with 99m tc labeled antisense DNA
The Journal of Nuclear Medicine, 2001Co-Authors: Yu-min Zhang, Yi Wang, Ning Liu, Zhihong Zhu, Mary Rusckowski, Donald J. HnatowichAbstract:One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with γ- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an antisense mechanism. Methods: An 18mer uniform phosphorothioate DNA antisense to the messenger RNA (mRNA) of the type I regulatory subunit α of cyclic adenosine monophosphate-dependent protein kinase A (RIα) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/Linker and investigated in vitro in cell culture. Results: By surface plasmon resonance, the association kinetics between native (i.e., without amine/Linker) DNA and MAG3-amide/Linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/Linker-DNA, and MAG3-amide/Linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of 99mTc-MAG3-DNA was lower than that of 35S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an antisense effect was suggested by 3 findings: an increased accumulation of 35S- or 99mTc-labeled antisense versus sense DNA, an increased accumulation of 99mTc-antisense DNA in another RIα-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of 99mTc-antisense DNA with increasing dosage of antisense DNA. Higher than expected cellular accumulations of about 105 antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5′-triphosphate into RNA in cells exposed to the antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the antisense DNA but not to the control DNA. Conclusion: This evidence suggests tumor cell accumulation by an antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates.
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In vitro investigations of tumor targeting with (99m)Tc-labeled antisense DNA.
Journal of nuclear medicine : official publication Society of Nuclear Medicine, 2001Co-Authors: Yu-min Zhang, Yi Wang, Ning Liu, Zhihong Zhu, Mary Rusckowski, Donald J. HnatowichAbstract:UNLABELLED One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with gamma- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an antisense mechanism. METHODS An 18mer uniform phosphorothioate DNA antisense to the messenger RNA (mRNA) of the type I regulatory subunit alpha of cyclic adenosine monophosphate-dependent protein kinase A (RI alpha) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/Linker and investigated in vitro in cell culture. RESULTS By surface plasmon resonance, the association kinetics between native (i.e., without amine/Linker) DNA and MAG3-amide/Linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/Linker-DNA, and MAG3-amide/Linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of (99m)Tc-MAG3-DNA was lower than that of (35)S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an antisense effect was suggested by 3 findings: an increased accumulation of (35)S- or (99m)Tc-labeled antisense versus sense DNA, an increased accumulation of (99m)Tc-antisense DNA in another RI alpha-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of (99m)Tc-antisense DNA with increasing dosage of antisense DNA. Higher than expected cellular accumulations of about 10(5) antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5'-triphosphate into RNA in cells exposed to the antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the antisense DNA but not to the control DNA. CONCLUSION This evidence suggests tumor cell accumulation by an antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates.
Naoto Nemoto - One of the best experts on this subject based on the ideXlab platform.
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One-Pot Preparation of mRNA/cDNA Display by a Novel and Versatile Puromycin-Linker DNA
ACS Combinatorial Science, 2011Co-Authors: Yuki Mochizuki, Manish Biyani, Sachika Tsuji-ueno, Koichi Nishigaki, Yuzuru Husimi, Miho Suzuki, Naoto NemotoAbstract:A rapid, easy, and robust preparation method for mRNA/cDNA display using a newly designed puromycin-Linker DNA is presented. The new Linker is structurally simple, easy to synthesize, and cost-effective for use in “in vitro peptide and protein selection”. An introduction of RNase T1 nuclease site to the new Linker facilitates the easy recovery of mRNA/cDNA displayed protein by an improvement of the efficiency of ligating the Linker to mRNAs and efficient release of mRNA/cDNA displayed protein from the solid-phase (magnetic bead). For application demonstration, affinity selections were successfully performed. Furthermore, we introduced a “one-pot” preparation protocol to perform mRNA display easy. Unlike conventional approaches that require tedious and downstream multistep process including purification, this protocol will make the mRNA/cDNA display methods more practical and convenient and also facilitate the development of next-generation, high-throughput mRNA/cDNA display systems amenable to automation.
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one pot preparation of mrna cDNA display by a novel and versatile puromycin Linker DNA
ACS Combinatorial Science, 2011Co-Authors: Yuki Mochizuki, Manish Biyani, Koichi Nishigaki, Yuzuru Husimi, Miho Suzuki, Sachika Tsujiueno, Naoto NemotoAbstract:A rapid, easy, and robust preparation method for mRNA/cDNA display using a newly designed puromycin-Linker DNA is presented. The new Linker is structurally simple, easy to synthesize, and cost-effective for use in “in vitro peptide and protein selection”. An introduction of RNase T1 nuclease site to the new Linker facilitates the easy recovery of mRNA/cDNA displayed protein by an improvement of the efficiency of ligating the Linker to mRNAs and efficient release of mRNA/cDNA displayed protein from the solid-phase (magnetic bead). For application demonstration, affinity selections were successfully performed. Furthermore, we introduced a “one-pot” preparation protocol to perform mRNA display easy. Unlike conventional approaches that require tedious and downstream multistep process including purification, this protocol will make the mRNA/cDNA display methods more practical and convenient and also facilitate the development of next-generation, high-throughput mRNA/cDNA display systems amenable to automation.
Jörg Langowski - One of the best experts on this subject based on the ideXlab platform.
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Rigid assembly and Monte Carlo models of stable and unstable chromatin structures: the effect of nucleosomal spacing
Theoretical Chemistry Accounts, 2010Co-Authors: Frank Aumann, Jörg Langowski, Jürgen Sühnel, Stephan DiekmannAbstract:Coarse-grained models are used to assess the packing of the 30-nm chromatin fiber. First, rigid assembly models for nucleosomal repeats from 155 to 211 bp are built using the crystal structure of the mononucleosome and attached straight stretches of B-DNA. The resulting fiber conformations are analyzed for static clashes and classified into stable and unstable structures. The effect of flexibility and thermal fluctuations is then taken into account by conducting Monte Carlo simulations of chromatin fiber models. Here the DNA is approximated by a flexible polymer chain with Debye–Hückel electrostatics, the geometry of the Linker DNA connecting the nucleosomes is based on a two-angle zigzag model, and nucleosomes are represented by flat ellipsoids interacting via an attractive Gay–Berne potential. Unstable fibers occur at a particular repeat length period of 10 bp. Also, the regions of densely compacted fibers repeat at intervals of 10 bp. Besides one- and two-start helical zigzag structures, we show evidence for possible three-start structures, which have not been reported in experiments yet. Finally, we show that a local opening of the Linker DNA at the nucleosome core—as probably occurs upon histone acetylation—leads to more open and flexible structures.
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Trajectory of nucleosomal Linker DNA studied by fluorescence resonance energy transfer.
Biochemistry, 2001Co-Authors: Katalin Tóth, Nathalie Brun, Jörg LangowskiAbstract:While the structure of the nucleosome core is known in atomic detail, the precise geometry of the DNA beyond the core particle is still unknown. We have used fluorescence resonance energy transfer (FRET) for determining the end-to-end distance of DNA fragments assembled with histones into nucleosomes. The DNA of a length of 150-220 bp was labeled with rhodamine-X on one end and fluorescein or Alexa 488 on the other. Assembling nucleosomes on these DNA fragments leads to a measurable energy transfer. The end-to-end distance computed from the FRET increases from 60 ( 5A at 150 bp to 75 ( 5 A at 170 bp without measurable change above it. These distances are compatible with different geometries of the Linker DNA, all having in common that no crossing can be observed up to 220 bp. Addition of H1 histone leads to an increase in energy transfer, indicating a compaction of the Linker DNA toward the nucleosome. The packing of the DNA into chromosomes in the eukaryotic cell has been an unresolved puzzle for many years. At the lowest level of compaction, DNA is bound to histones into nucleosomes (1, 2). This structure is quite well understood: about two turns of DNA are wrapped around the histone octamer core, one histone octamer and 146 bp of DNA form a biochemically stable unit called the core particle, and the spacing between successive nucleosomes is 160-220 bp depending on the cell type and species. The crystal structures of the histone octamer ( 3) and of the full nucleosome core particle (4) have been solved at atomic resolution.
Yu-min Zhang - One of the best experts on this subject based on the ideXlab platform.
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in vitro investigations of tumor targeting with 99m tc labeled antisense DNA
The Journal of Nuclear Medicine, 2001Co-Authors: Yu-min Zhang, Yi Wang, Ning Liu, Zhihong Zhu, Mary Rusckowski, Donald J. HnatowichAbstract:One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with γ- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an antisense mechanism. Methods: An 18mer uniform phosphorothioate DNA antisense to the messenger RNA (mRNA) of the type I regulatory subunit α of cyclic adenosine monophosphate-dependent protein kinase A (RIα) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/Linker and investigated in vitro in cell culture. Results: By surface plasmon resonance, the association kinetics between native (i.e., without amine/Linker) DNA and MAG3-amide/Linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/Linker-DNA, and MAG3-amide/Linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of 99mTc-MAG3-DNA was lower than that of 35S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an antisense effect was suggested by 3 findings: an increased accumulation of 35S- or 99mTc-labeled antisense versus sense DNA, an increased accumulation of 99mTc-antisense DNA in another RIα-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of 99mTc-antisense DNA with increasing dosage of antisense DNA. Higher than expected cellular accumulations of about 105 antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5′-triphosphate into RNA in cells exposed to the antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the antisense DNA but not to the control DNA. Conclusion: This evidence suggests tumor cell accumulation by an antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates.
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In vitro investigations of tumor targeting with (99m)Tc-labeled antisense DNA.
Journal of nuclear medicine : official publication Society of Nuclear Medicine, 2001Co-Authors: Yu-min Zhang, Yi Wang, Ning Liu, Zhihong Zhu, Mary Rusckowski, Donald J. HnatowichAbstract:UNLABELLED One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with gamma- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an antisense mechanism. METHODS An 18mer uniform phosphorothioate DNA antisense to the messenger RNA (mRNA) of the type I regulatory subunit alpha of cyclic adenosine monophosphate-dependent protein kinase A (RI alpha) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/Linker and investigated in vitro in cell culture. RESULTS By surface plasmon resonance, the association kinetics between native (i.e., without amine/Linker) DNA and MAG3-amide/Linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/Linker-DNA, and MAG3-amide/Linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of (99m)Tc-MAG3-DNA was lower than that of (35)S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an antisense effect was suggested by 3 findings: an increased accumulation of (35)S- or (99m)Tc-labeled antisense versus sense DNA, an increased accumulation of (99m)Tc-antisense DNA in another RI alpha-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of (99m)Tc-antisense DNA with increasing dosage of antisense DNA. Higher than expected cellular accumulations of about 10(5) antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5'-triphosphate into RNA in cells exposed to the antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the antisense DNA but not to the control DNA. CONCLUSION This evidence suggests tumor cell accumulation by an antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates.