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Journal ArticleDOI

Distinctive patterns of histone H4 acetylation are associated with defined sequence elements within both heterochromatic and euchromatic regions of the human genome

01 Feb 1998-Nucleic Acids Research (Oxford University Press)-Vol. 26, Iss: 4, pp 994-1001
TL;DR: All acetylated histone H4 isoforms were depleted in non-coding, simple repeat DNA in heterochromatin, though the extent of depletion varied with the type of heterochromaatin and with the isoform.
Abstract: The pattern of histone H4 acetylation in different genomic regions has been investigated by immunoprecipitating oligonucleosomes from a human lymphoblastoid cell line with antibodies to H4 acetylated at lysines 5, 8, 12 or 16. DNA from antibody-bound or unbound chromatin was assayed by slot blotting. Pol I and pol II transcribed genes located in euchromatin were shown to have levels of H4 acetylation at lysines 5, 8 and 12 equivalent to those in input chromatin, but to be slightly enriched in H4 acetylated at lysine 16. In no case did the acetylation level correlate with actual or potential transcriptional activity. All acetylated histone H4 isoforms were depleted in non-coding, simple repeat DNA in heterochromatin, though the extent of depletion varied with the type of heterochromatin and with the isoform. Two single copy genes that map within or adjacent to blocks of paracentric heterochromatin are depleted in H4 acetylated at lysines 5, 8 and 12, but not 16. Consensus sequences of repetitive elements of the Alu family (SINES, enriched in R bands) were associated with H4 that was more highly acetylated at all four lysines than input chromatin, while H4 associated with Kpn I elements (LINES, enriched in G bands) was significantly underacetylated.

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Citations
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Journal ArticleDOI
TL;DR: Recent progress in understanding how histone variants lead to changes in chromatin structure and dynamics to carry out specific functions is discussed.
Abstract: The modification of histone plays a crucial role in regulating chromatin states that conserve transcription programs and provide a mechanism for chromatin states to be maintained as cells proliferate. A large number of factors and protein complexes are now known to be involved in regulating the dynamic states of the modification and variants of histone. A fraction of histones are nonallelic variants that have specific expression localization, and species-distribution patterns. Here we discuss recent progress in understanding how histone variants lead to changes in chromatin structure and dynamics to carry out specific functions.

4 citations

Dissertation
12 Jan 2009
TL;DR: Le laboratoire a identifie 6 familles independantes avec un diabete insipide nephrogenique (DIN) lie a l'X portant de grandes deletions en amont du gene de l'AVPR2, d'etudier l'expression « tissu specifique » de ce gene.
Abstract: Le controle de la transcription constitue le principal niveau de la regulation de l'expression des genes dans les cellules eucaryotes. Le laboratoire a identifie 6 familles independantes avec un diabete insipide nephrogenique (DIN) lie a l'X portant de grandes deletions en amont du gene de l'AVPR2. Dans chacune de ces familles, les genes AVPR2 et AQP2 sont intacts et les hommes sont atteints de DIN lie a l'X dans sa forme renale « classique ». Le sequencage et l'analyse de 61 kilobases en amont et en aval de l'AVPR2 ont permis l'identification de 6 zones deletees chez 6 familles independantes, dont 5 zones de taille superieure a 7 kilo bases, et une zone, de 102 paires de bases, commune a l'ensemble des deletions. Chez le patient porteur de cette deletion, les recepteurs V2 ne sont pas exprimes dans le tubule collecteur mais le sont au niveau des cellules endotheliales. Notre travail est de tenter de comprendre les mecanismes regulateurs du locus de l'AVPR2, et d'etudier l'expression « tissu specifique » de ce gene. Les etudes realisees dans le systeme Hprt, confirment le role activateur de la sequence de 102 pb. Les experiences in vitro indiquent que cet effet depende du contexte extracellulaire, de la nature des cellules, ainsi que du promoteur de l'AVPR2. Les proteines liant potentiellement l'une des extremites de la deletion a revele la presence, soit de proteines regulatrices, soit de sequences inconnues, toutes exprimees dans le rein. A terme, ces etudes, ainsi que celles en decoulant, permettront de positionner l'AVPR2 comme une cible de choix dans le traitement des diabetes insipides, centraux et nephrogeniques, par therapie genique.

3 citations

Dissertation
01 Jan 2008
TL;DR: All human and rodent SINEs studied here were found to be bound by transcription factors TFIIIB and TFIIIC at comparable levels with actively transcribed genes, suggesting that the occupancy is not affected by methylated DNA or DNA methylation-dependent components of chromatin.
Abstract: Despite the abundance of the templates, both human and rodent SINEs are normally expressed at a very low level. DNA methylation-mediated silencing has been proposed as a possible cause of their transcriptional repression. The effect of DNA methylation and the effect of DNA methylation-dependent methyl-CpG-binding domain proteins (MBD proteins) on SINE transcription were studied here. It was shown that both human and rodent SINEs are bound by MeCP2, MBD1 and MBD2. Both human and rodent SINEs were also shown to be occupied by HDAC1, HDAC2 and a component of SWI/SNF complex, Brahma. Human Alus were also found to be occupied by components of two corepressor complexes, SIN3 and NuRD. Whether MBD proteins repress SINE transcription in a DNA methylation-dependent manner was further investigated using systems with low or near absent DNA methylation and, in the case of MeCP2 protein, by its direct removal. MeCP2 was found to have no repressive effect on B1 and B2 expression. RT-PCR analysis showed no increase in B1 and B2 RNA levels in MeCP2 null mice kidneys. ChIP analysis of Dnmt1n/n p53-/- embryonic fibroblasts, which have less than 5% of the normal DNA methylation level, showed significant reduction in MeCP2 and MBD2 binding, confirming that their presence is DNA methylation-dependant. RT-PCR comparison of Dnmt1+/+ p53-/- and Dnmt1n/n p53-/- cells, however, detected no increase in B1 or B2 RNA levels. This was consistent with results obtained from MeCP2 null mice, where lack of MeCP2 did not result in increased B1 and B2 expression and with a previous study involving human Alus (Yu et al., 2001). MBD2 also does not seem to repress SINE activity, as its release following loss of DNA methylation did not result in increased SINE RNA levels. Strikingly, all human and rodent SINEs studied here were found to be bound by transcription factors TFIIIB and TFIIIC at comparable levels with actively transcribed genes. Some RNA polymerase III was also detected, but at levels significantly lower than on active genes, suggesting a defect in RNA polymerase III loading onto SINEs. This occupancy of the transcriptional complex was comparable in cells with normal levels of DNA methylation and in cells with significantly reduced levels of DNA methylation, suggesting that the occupancy is not affected by methylated DNA or DNA methylation-dependent components of chromatin. Indeed, removal of 50% of histone H1 did not result in increased B1 or B2 expression in this study. The fact that all tested SINEs are occupied by TFIIIB and TFIIIC also brings an unprecedented insight into the number of these transcription factors present in the cell.

3 citations


Cites background from "Distinctive patterns of histone H4 ..."

  • ...Acetylated histones H3 and H4 were observed on Alus in other studies (Hakimi et al., 2002; Johnson et al., 1998), but the presence of HDACs have never been described before....

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Dissertation
01 Jan 2006
TL;DR: It is shown that increased histone H4 acetylation at replication origin loci occurs after treatment with the histone deacetylase inhibitor TSA and coincides with a loss of specific initiation site selection both withinorigin loci and throughout the genome, suggesting a physiological role for histone acetylated in controlling the initiation of DNA synthesis at specific genomic sites.
Abstract: Kemp, Michael G. Ph.D., Biomedical Sciences Program, Wright State University, 2006. Regulation of DNA Replication Initiation by Histone Acetylation and the DNA Unwinding Element Binding Protein DUE-B. Duplication of the genome during S phase of the mitotic cell cycle begins at thousands of sites along chromosomes termed origins of replication. Although many of the essential protein components catalyzing events at these sites are known and are conserved throughout eukaryotes, the likelihood or efficiency of initiation of DNA synthesis at any given genomic site is expected to be influenced by other novel factors, including aspects of chromatin and DNA structure. Here I show that increased histone H4 acetylation at replication origin loci occurs after treatment with the histone deacetylase inhibitor TSA and coincides with a loss of specific initiation site selection both within origin loci and throughout the genome. Furthermore, new replication initiation sites become activated or used with greater frequency after treatment with TSA, and TSA promotes the activation of replication origins earlier during the S phase of the cell cycle. These data suggest a physiological role for histone acetylation in controlling the initiation of DNA synthesis at specific

2 citations

Book ChapterDOI
01 Jan 2006
TL;DR: This review focuses on the diverse sub-cellular distribution, substrate specificity, and cellular functions of sirtUins with particular emphasis on the biology of mammalian sirtuins.
Abstract: Sirtuins are NAD-dependent protein deacetylases found in organisms ranging from bacteria to humans that share sequence homology with the yeast transcriptional regulator Sir2. In eukaryotes, sirtuins regulate The first two authors contributed equally to this review. transcriptional repression, recombination, cell cycle division, microtubule organization, and cellular responses to DNA-damaging agents. Sir2 proteins have also been implicated in regulating the molecular mechanisms of aging. Eukaryotic sirtuins contain a core catalytic domain and variable amino- and carboxyl-terminal extensions that regulate their subcellular localizations and catalytic activity. This review focuses on the diverse sub-cellular distribution, substrate specificity, and cellular functions of sirtuins with particular emphasis on the biology of mammalian sirtuins.

2 citations

References
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Book
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol

215,169 citations

BookDOI
01 Jan 1979
TL;DR: The Chromatin Pattern in Situ: Dependence upon Cell Cycle, Preimplantation and Development, and Cellular Aging in Vitro, and Generalized Biological Effects.
Abstract: of Part A.- Section I: What is the Chromatin?.- Properties and Composition of Isolated Chromatin.- Expressed and Nonexpressed Portions of the Genome: Their Separation and Their Characterization.- Discussion.- Section II: Physical, Chemical and Biological Techniques for Studying Nucleosome, Chromatin, Chromosome and Nuclei.- Electron Microscopy: A Tool for Visualizing Chromatin.- Transcriptional Control of Native Chromatin.- Circular Dichroism of DNA, Protein and Chromatin.- Important Hydrodynamic and Spectroscopic Techniques in the Field of Chromatin Structure.- Preparation and Analysis of Core Particles and Nucleosomes: A Conveinient Method For Studying the Protein Composition of Nucleosomes Using Protamine-Release into Triton-Acid-Urea Gels.- The Interaction of Histones with DNA: Equilibrium Binding Studies.- Nucleosome Shape and Structure in Solution from Flow Birefringence.- Scattering and Diffraction by Neutrons and X-rays in the Study of Chromatin.- Nuclear Magnetic Resonance Studies of Nucleic Acids and Proteins.- Techniques for Cytochemical Studies of the Nucleus and its Substructures.- Chromatin Study in Situ: I. Image Analysis.- Chromatin Study in Situ: II. Static and Flow Microfluorimetry.- Chromatin Study in Situ: III. Differential Effects of Feulgen Hydrolysis.- Scanning and Flow Photometry of Chromosomes.- Discussion.- Section III: Various Levels of Chromatin Organization and Mechanisms for Transcriptional Control.- Histones Assembly and Their Structural Role for Nucleosome Core.- Nuclease Digestion and the Structure of Chromatin.- Reconstitution of Nucleosomes.- Conformation of Polynucleosomes in Low Ionic Strength Solution.- Chromatin Structure: Relation of Nucleosomes of DNA Sequences.- Histone Complexes, Nucleosomes, Chromatin and Cell-Cycle Dependent Modification of Histones.- Evidence for Superstructures of Wet Chromatin.- Chromatin Fractionation and the Properties of Transcriptionally Active Regions of Chromatin.- Chromatin Reconstitution and Non-Histone Proteins.- Discussion.- Section IV: Structure-Function of the Genetic Apparatus and Cell Cycle, Aging, Neoplastic Transformation, Differentiation, Chemical Carcinogenesis.- The Structure and Function of Chromatin in Lower Eukaryotes.- Chromatin Structure from Angstrom to Micorn Levels, and Its Relationship to Mammalian Cell Proliferation.- Chromatin Pattern in Situ: Dependence upon Cell Cycle, Preimplantation and Development, and Cellular Aging in Vitro.- Neoplastic Transformation: The Relevance of in Vitro Studies for the Understanding of Tumor Pathenogenesis and Neoplastic Growth.- Cell Differentiation and Malignancy in Leukemia.- Cellular Morphometry in Transformation, Differentiation and Aging.- Basic Mechanisms in Chemical Carcinogenesis.- Carcinogen Induced Alteration in Gene Packing and Its Possible Significance in Carcinogenesis.- Covalent Binding of a Carcinogen to DNA as a Probe of Chromatin Structure.- Carcinogenesis, DNA Repair and Chromatin.- Electromagnetic Induction of Electrochemical Information at Cell Surfaces: Application to Chromatin Structure Modification.- Discussion.- Section V: Review and Summary of the Genetic Apparatus.- Session I: Basic Components of the Genetic Apparatus.- Session II: The Second Level of Organization - Chromatin.- Session III: The Third Level of Organization.- Session IV: Generalized Biological Effects.

1,058 citations

Book
01 Jan 1987
TL;DR: The induction and enumeration of antibody-forming cells in vitro and the development of human B lymphoblastoid cell lines using epstein are studied.
Abstract: Preparation of lymphocytes and accessory cells Preparation of lymphocyte subpopulations Fractionation of lymphocytes by immunomagnetic beads Immunofluorescence and immunohisto-chemistry The induction and enumeration of antibody-forming cells in vitro In vitro culture of T cell lines and clones Generation of human B lymphoblastoid cell lines using epstein Limiting dilution analysis Lymphocyte proliferation assays Assays for interleukins and other related factors Biochemical characterization of lymphocyte surface antigens

185 citations

Journal ArticleDOI
TL;DR: Clear localization with oligonucleotides from alphoid (centromeric sequences), simple sequence (satellite) DNAs, a variety of Alu-dispersed repeated sequences, and oligon nucleotides derived from the Tetrahymena and Trypanosoma telomere-specific sequences is obtained.
Abstract: Oligonucleotides were annealed to complementary sequences in fixed human metaphase chromosomes and extended with DNA polymerase. The newly synthesized fragments were labeled by incorporating bio-11-dU

100 citations

Book
01 Jan 2000
TL;DR: The objective is to establish a protocol for quantification of antigen-specific T-cells HLA -peptide tetrameric complexes and investigate the role of T-cell reprograming in the selection of lymphocytes for HLA typing.
Abstract: Preface Preparation of lymphocytes and idenfication of lymphocyte subpopulations Immunohistochemistry of lymphoid organs T and B-cell hybridomas Murine T-cell culture Human CD4+ T-cell culture Human Cytotoxic T-cell culture Limiting dilution analysis for quantification of antigen-specific T-cells HLA -peptide tetrameric complexes Expansion of human T-cells for immunotheraphy HLA typing Characterisation of lymphocyte surface markers Apoptosis assays for lymphocytes Thymic organ culture Index

29 citations