Distinctive patterns of histone H4 acetylation are associated with defined sequence elements within both heterochromatic and euchromatic regions of the human genome
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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TL;DR: It is shown that the association of Gcn5 with other proteins in two native yeast complexes, Ada and SAGA (Spt-Ada-Gcn5-acetyltransferase), directly confers upon Gcn 5 the ability to acetylate an expanded set of lysines on H3.
371 citations
TL;DR: It is found that in vivo, GCN5 is required either directly or indirectly for the acetylation of several sites in H3 and H4 in addition to those recognized by the recombinant enzyme in vitro.
Abstract: The Gcn5p histone acetyltransferase exhibits a limited substrate specificity in vitro. However, neither the specificity of this enzyme in vivo nor the importance of particular acetylated residues to transcription or cell growth are well defined. To probe these questions, we mutated specific lysines in the N-termini of histones H3 and H4 and examined the effects of these mutations in yeast strains with and without functional GCN5. We found that in vivo, GCN5 is required either directly or indirectly for the acetylation of several sites in H3 and H4 in addition to those recognized by the recombinant enzyme in vitro. Moreover, in the absence of GCN5, cells accumulate in G2/M indicating that Gcn5p functions are important for normal cell-cycle progression. Mutation of K14 in H3, which serves as the major target of recombinant Gcn5p acetylation in vitro, confers a strong, synthetic growth defect in gcn5 cells. Synergistic growth defects were also observed in gcn5 cells carrying mutations in lysine pairs (K8/K16 or K5/K12) in histone H4. Strikingly, simultaneous mutation of K14 in H3 and K8 and K16 in H4 to arginine, or deletion of either the H3 or the H4 N-terminal tail, results in the death of gcn5 cells. Mutation of these same three sites to glutamine is not lethal. Indeed, this combination of mutations largely bypasses the need for GCN5 for transcriptional activation by Gal4-VP16, supporting an important role for histone acetylation in Gcn5p-mediated regulation of transcription. Our data indicate that acetylation of particular lysines in histones H3 and H4 serves both unique and overlapping functions important for normal cell growth, and that a critical overall level of histone acetylation is essential for cell viability.
328 citations
Cites background from "Distinctive patterns of histone H4 ..."
...…of K5 and K12 is linked to histone deposition into newly synthesized chromatin during S phase in several species (Sobel et al., 1995), and acetylation of K16 is commonly enriched in transcriptionally active and potentially active chromatin (Turner, 1993; Roth and Allis, 1996; Johnson et al., 1998)....
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..., 1995), and acetylation of K16 is commonly enriched in transcriptionally active and potentially active chromatin (Turner, 1993; Roth and Allis, 1996; Johnson et al., 1998)....
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TL;DR: A multistep model for gene activation is suggested; localization away from centromeric heterochromatin is required to achieve general hyperacetylation and an open chromatin structure of the locus, whereas a mechanism involving LCR/promoter histone H3 hyperacetolation is required for high-level transcription of the beta-globin genes.
Abstract: We have investigated the mechanism, structural correlates, and cis-acting elements involved in chromatin opening and gene activation, using the human beta-globin locus as a model. Full transcriptional activity of the human beta-globin locus requires the locus control region (LCR), composed of a series of nuclease hypersensitive sites located upstream of this globin gene cluster. Our previous analysis of naturally occurring and targeted LCR deletions revealed that chromatin opening and transcriptional activity in the endogenous beta-globin locus are dissociable and dependent on distinct cis-acting elements. We now report that general histone H3/H4 acetylation and relocation of the locus away from centromeric heterochromatin in the interphase nucleus are correlated and do not require the LCR. In contrast, LCR-dependent promoter activation is associated with localized histone H3 hyperacetylation at the LCR and the transcribed beta-globin-promoter and gene. On the basis of these results, we suggest a multistep model for gene activation; localization away from centromeric heterochromatin is required to achieve general hyperacetylation and an open chromatin structure of the locus, whereas a mechanism involving LCR/promoter histone H3 hyperacetylation is required for high-level transcription of the beta-globin genes.
321 citations
TL;DR: The treatment of cancer through the development of new therapies is one of the most important challenges of the authors' time, and the modulation of epigenetic mechanisms enables the alteration of cellular phenotype without altering the genotype.
Abstract: The treatment of cancer through the development of new therapies is one of the most important challenges of our time. The decoding of the human genome has yielded important insights into the molecular basis of physical disorders, and in most cases a connection between failures in specific genes and the resulting clinical symptoms can be made. The modulation of epigenetic mechanisms enables, by definition, the alteration of cellular phenotype without altering the genotype. The information content of a single gene can be crucial or harmful, but the prerequisite for a cellular effect is active gene transcription. To this end, epigenetic mechanisms play a very important role, and the transcription of a given gene is directly influenced by the modification pattern of the surrounding histone proteins as well as the methylation pattern of the DNA. These processes are effected by different enzymes which can be directly influenced through the development of specific modulators. Of course, all genetic information is written as a four-character code in DNA. However, epigenetics describes the art of reading between the lines.
292 citations
TL;DR: This result identifies histone acetylation as a central factor in the dynamic regulation of chromatin accessibility during interphase and a reversible change of the chromatin conformation to a uniform 60-100 nm pore size distribution was observed upon increased histoneacetylation.
Abstract: In eukaryotes, the interaction of DNA with proteins and supramolecular complexes involved in gene expression is controlled by the dynamic organization of chromatin inasmuch as it defines the DNA accessibility. Here, the nuclear distribution of microinjected fluorescein-labeled dextrans of 42 kDa to 2.5 MDa molecular mass was used to characterize the chromatin accessibility in dependence on histone acetylation. Measurements of the fluorescein-dextran sizes were combined with an image correlation spectroscopy analysis, and three different interphase chromatin condensation states with apparent pore sizes of 16-20 nm, 36-56 nm and 60-100 nm were identified. A reversible change of the chromatin conformation to a uniform 60-100 nm pore size distribution was observed upon increased histone acetylation. This result identifies histone acetylation as a central factor in the dynamic regulation of chromatin accessibility during interphase. In mitotic chromosomes, the chromatin exclusion limit was 10-20 nm and independent of the histone acetylation state.
280 citations
Cites background from "Distinctive patterns of histone H4 ..."
..., 2001); and (4) a decreased level of histone H3 and H4 acetylation (Jeppesen et al., 1992; Jiang et al., 2004; Johnson et al., 1998)....
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...…al., 2003; Peters and Schubeler, 2005; Zinner et al., 2005); (3) a preferential binding of HP1 and HP1 (Maison and Almouzni, 2004; Minc et al., 1999; Nielsen et al., 2001); and (4) a decreased level of histone H3 and H4 acetylation (Jeppesen et al., 1992; Jiang et al., 2004; Johnson et al., 1998)....
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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
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
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