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: The methods currently in use in the laboratory to isolate and immunoprecipitate native chromatin from cultured cells, and to isolation and analyze immunop Recipitated protein and DNA are outlined.
244 citations
TL;DR: The three‐dimensional (3D) structure of the Escherichia coli multidrug transporter EmrE is determined by electron cryomicroscopy of 2D crystals, including data to 7.0 Å resolution.
Abstract: The small multidrug resistance family of transporters is widespread in bacteria and is responsible for bacterial resistance to toxic aromatic cations by proton-linked efflux. We have determined the three-dimensional (3D) structure of the Escherichia coli multidrug transporter EmrE by electron cryomicroscopy of 2D crystals, including data to 7.0 A resolution. The structure of EmrE consists of a bundle of eight transmembrane α-helices with one substrate molecule bound near the centre. The substrate binding chamber is formed from six helices and is accessible both from the aqueous phase and laterally from the lipid bilayer. The most remarkable feature of the structure of EmrE is that it is an asymmetric homodimer. The possible arrangement of the two polypeptides in the EmrE dimer is discussed based on the 3D density map.
217 citations
TL;DR: In this article, trichostatin A (TSA) treatment was shown to increase the acetylation level of histone H4 globally and at these initiation sites, leading to a more dispersive pattern of DNA replication initiation.
Abstract: Eukaryotic chromatin structure limits the initiation of DNA replication spatially to chromosomal origin zones and temporally to the ordered firing of origins during S phase. Here, we show that the level of histone H4 acetylation correlates with the frequency of replication initiation as measured by the abundance of short nascent DNA strands within the human c-myc and lamin B2 origins, but less well with the frequency of initiation across the b-globin locus. Treatment of HeLa cells with trichostatin A (TSA) reversibly increased the acetylation level of histone H4 globally and at these initiation sites. At all three origins, TSA treatment transiently promoted a more dispersive pattern of initiations, decreasing the abundance of nascent DNA at previously preferred initiation sites while increasing the nascent strand abundance at lower frequency genomic initiation sites. When cells arrested in late G1 were released into TSA, they completed S phase more rapidly than untreated cells, possibly due to the earlier initiation from late-firing origins, as exemplified by the b-globin origin. Thus, TSA may modulate replication origin activity through its effects on chromatin structure, by changing the selection of initiation sites, and by advancing the time at which DNA synthesis can begin at some initiation sites.
217 citations
Cites background from "Distinctive patterns of histone H4 ..."
...The extent of the increase in AcH4 level varied among the sites investigated, suggesting that there may be differences in HDAC or HAT localization or activity among the sites, as has been proposed previously (66,77)....
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TL;DR: A marked synergistic effect of histone hyperacetylation and DNA demethylation in the reactivation of FMR1 full mutations is shown, showing a 2- to 5-fold increase in FMR 1 mRNA levels obtained with 5-azadC alone.
Abstract: Most fragile X syndrome patients have expansion of a (CGG) n sequence with >200 repeats (full mutation) in the FMR1 gene responsible for this condition. Hypermethylation of the expanded repeat and of the FMR1 promoter is almost always present and apparently suppresses transcription, resulting in absence of the FMR1 protein. We recently showed that transcriptional reactivation of FMR1 full mutations can be achieved by inducing DNA demethylation with 5-azadeoxycytidine (5-azadC). The level of histone acetylation is another important factor in regulating gene expression; therefore, we treated lymphoblastoid cell lines of non-mosaic full mutation patients with three drugs capable of Inducing histone hyperacetylation. We observed a consistent, although modest, reactivation of the FMR1 gene with 4-phenylbutyrate, sodium butyrate and trichostatin A, as shown by RT-PCR. However, we report that combining these drugs with 5-azadC results in a 2- to 5-fold increase in FMR1 mRNA levels obtained with 5-azadC alone, thus showing a marked synergistic effect of histone hyperacetylation and DNA demethylation in the reactivation of FMR1 full mutations.
216 citations
TL;DR: In this paper, real-time PCR analysis of breast and prostate cancer cell lines demonstrated that PPARdelta expression was increased 1.5 to 3.2-fold after three hours stimulation with the natural vitamin D receptor (VDR) agonist, 1α,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3).
196 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
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