Showing papers on "Nucleosome published in 1988"

Specific glucocorticoid receptor binding to DNA reconstituted in a nucleosome.

Abstract: We have reconstituted a nucleosome with core histones from rat liver using a restriction fragment containing a sequence from the mouse mammary tumour virus (MTV) long terminal repeat (LTR). This sequence harbours glucocorticoid responsive elements (GREs) which mediate glucocorticoid hormone induction of transcription from the MTV promoter via glucocorticoid receptor (GR) binding. Exonuclease III and DNase I footprinting demonstrated that the reconstituted nucleosome was specifically located between positions -219 and -76. A nucleosome was previously shown to be located at a similar or identical position in the MTV promoter in situ and to be structurally altered upon glucocorticoid hormone induction. We demonstrated, by DNase I footprinting, that GR is able to bind sequence specifically to the DNA in the in vitro assembled nucleosome. No evidence for unfolding of the nucleosome was obtained, but the DNase I footprinting pattern demonstrated GR induced local alterations in the DNA.

Depletion of histone H4 and nucleosomes activates the PHO5 gene in Saccharomyces cerevisiae.

Abstract: We have previously constructed a yeast strain (UKY403) whose sole histone H4 gene is under control of the GAL1 promoter. This yeast arrests in G2 upon glucose treatment as a result of histone H4 depletion. The yeast PHO5 gene contains phase nucleosomes covering promoter (UAS) sequences in the PHO5 repressed state and it has been suggested that nucleosomes prevent the binding of positively acting factors to these UAS sequences. Using UKY403 we examined the length of polynucleosomes and nucleosome phasing in the PHO5 upstream region by the use of micrococcal nuclease and indirect end-labeling. It was found that glucose arrest led to a severe disruption in PHO5 chromatin structure and that most nucleosomes had their position altered or were lost from the PHO5 promoter region. Cell undergoing nucleosome depletion synthesized large quantities of accurate PHO5 transcripts even under repressive, high inorganic phosphate conditions. Histone H4 depletion did not appear to affect the repression or activation of another inducible yeast gene, CUP1. Arrest with landmarks in early G1 (in the cell division cycle mutant cdc28) or in various stages of G2 (in cdc15, cdc17 and cdc20) does not activate PHO5; nor does arrest due to chromosome topology changes (in top2 or the top1top2 topoisomerase mutants). cdc14, which has its arrest landmark at a similar point in the cell cycle as cdc15, does derepress PHO5. However, since it also leads to derepression of CUP1 it is probably functioning through an independent mechanism. Therefore, our data suggest that nucleosomes regulate PHO5 transcription.

Protein/DNA architecture of the DNase I hypersensitive region of the Drosophila hsp26 promoter

Abstract: Genomic footprinting on the Drosophila hsp26 promoter in isolated nuclei has shown that a TATA box binding factor is present before and after induction by heat shock, while three of the seven heat shock consensus sequences 5' of the gene are occupied (presumably by heat shock factor, HSF) specifically on heat shock. The sites of HSF interaction are separated by greater than 200 bp of which approximately 150 bp are bound to the surface of a nucleosome. The juxtaposition of these various macromolecules on the DNA suggests a basis for the major DNase I hypersensitive site 5' of hsp26 and a novel tertiary structure for the promoter complex.

A highly basic histone H4 domain bound to the sharply bent region of nucleosomal DNA

Abstract: A nucleosomal core particle is composed of two each of histones H2A, H2B, H3 and H4 located inside the particle with ∼147 base pairs (bp) of DNA wrapped around the octamer in about 1.8 turns of a left-handed superhelix1,2. The path of the superhelix is not smooth; the DNA is sharply bent, or kinked, at positions symmetrically disposed at a distance of about one and four double-helical turns in both directions from the nucleosomal dyad axis (designated as sites ±1 and ±4 respectively3). This non-uniform bending is considered archetypal to other DNA-protein complexes, but its mechanism is not clear (reviewed in ref. 4). DNA-histone chemical cross-linking within the core particle has revealed strong binding of each of the two histone H4 molecules to DNA at a distance of 1.5 helical turns either side of the nucleosomal dyad axis (sites ±1.5)5,6. In each of these sites, a single flexible domain of H4 was previously shown to contact three points7, at about nucleotides 55 and 65 on one strand and nucleotide 88 on the complementary strand, numbering from the 5′ terminus of each 147-base strand; these three locations are closely juxtaposed across the highly compressed1 minor and major grooves (Fig. 1). Here we report that the amino-acid residue of histone H4 cross-linked at the 1.5 site is histidine-18, embedded in a highly basic cluster Lys-Arg-His-Arg-Lys-Val-Leu-Arg which is probably involved in the sharp bending of the DNA double helix at the ±1 sites.

Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity.

Abstract: Undiluted extracts from eggs or oocytes of Xenopus laevis support the assembly of chromatin with physiologically spaced nucleosomes Micrococcal nuclease and DNase I digestion experiments show that nucleosome formation as well as supercoiling of circular DNA concomitant to assembly do not require ATP or Mg2+ However these factors are essential for the stability and the physiological spacing of the assembled chromatin gamma-S-ATP can substitute for ATP in this process With topoisomers of defined linking number topological interconversions proceed by steps of unity, both in vitro as well as in vivo, indicating that topoisomerase I is dominantly acting in this process Novobiocin sensitivity occurred only with diluted extracts and was unrelated to an inhibition of topoisomerase II Finally, nucleosome assembly occurs efficiently on linear DNA although the assembled DNA is less stable than with circular DNA From these results we propose that mature chromatin is formed in a two-step reaction In the first step, nucleosome deposition occurs independently of ATP and Mg2+ Thus, nucleosome formation can be uncoupled from their spacing In this step, topoisomerase activity is involved in the relaxation of the topological constraints generated by chromatin assembly rather than in the process of assembly itself The second step, requiring ATP and Mg2+, generates properly spaced chromatin

Characterization of the repressed 5S DNA minichromosomes assembled in vitro with a high-speed supernatant of Xenopus laevis oocytes.

Abstract: We describe an in vitro system, based on the Xenopus laevis oocyte supernatant of Glikin et al. (G. Glikin, I. Ruberti, and A. Worcel, Cell 37:33-41, 1984), that packages DNA into minichromosomes with regularly spaced nucleosomes containing histones H3, H4, H2A, and H2B but no histone H1. The same supernatant also assembles the 5S RNA transcription complex; however, under the conditions that favor chromatin assembly, transcription is inhibited and a phased nucleosome forms over the 5S RNA gene. The minichromosomes that are fully loaded with nucleosomes remain refractory to transcriptional activation by 5S RNA transcription factors. Our data suggest that this repression is caused by a nucleosome covering the 5S RNA gene and that histone H1 is not required for regular nucleosome spacing or for gene repression in this system.

Footprinting of linker histones H5 and H1 on the nucleosome.

Abstract: DNase I has been used to footprint the linker histones H5 and H1 on the nucleosome of chicken erythrocyte chromatin. Rate constants have been derived for digestion at the principal sites of attack on chromatosome length DNA (168 bp), located about 10 bp apart, and compared with those observed for linker histone-depleted chromatosomes. Complete protection was found for site S7 on the dyad axis and decreasing partial protection seen at symmetrically positioned sites on each side of S7. Strong, but not complete protection was noted at S14, the site corresponding to the end of the core particle, situated less than 1/4 of a turn away from the dyad. Uniform partial protection was observed for sites S2, S3, S4 and S10, S12 on the far side of the chromatosome. The simplest interpretation of these results is that the globular domain of H5/H1 is responsible for the protection at S7, whilst extended N- and C-domains give rise to the partial protection at sites away from the dyad axis.

An erythrocyte-specific protein that binds to the poly(dG) region of the chicken beta-globin gene promoter.

Abstract: The promoter region of the chicken adult beta-globin gene contains a sequence of 16 deoxyguanosine residues located at a nucleosome boundary in tissues where the gene is inactive. In definitive erythrocytes that express the beta-globin gene, the nucleosome is displaced, the G-string and adjacent sequences are occupied by sequence-specific DNA-binding proteins, and a nuclease hypersensitive domain is generated in this region. To gain insight into the role of the G-string in this series of events, we have examined the proteins that bind to it. Using the gel mobility shift assay and a monoclonal antibody that blocks specific binding to the G-string, we have identified a specific protein, BGP1, that is found only in chicken erythroid cells and appears at the same time, or shortly before, the changes in chromatin structure. The antibody interacts strongly with BGP1 and cross-reacts weakly with Sp1. Although both BGP1 and Sp1 require Zn2+ for their DNA-binding activity, these proteins differ in their binding-site specificities, chromatographic properties, and molecular weights. In contrast to Sp1, which is found in a wide variety of cell types, BGP1 is restricted to erythrocytes and is most abundant in definitive erythrocytes. Thus, its presence corresponds to the tissue- and stage-specific occupancy of the G-string in vivo.

At least 60 ADP-ribosylated variant histones are present in nuclei from dimethylsulfate-treated and untreated cells.

Abstract: The level of histone adenosine diphospho (ADP) ribosylation was studied in isolated nuclei from mouse myeloma cells in culture. The cells were treated with dimethylsulfate (DMS), a DNA-methylating agent, and histones were analyzed using two-dimensional gel electrophoresis. Seventeen or more bands probably representing mono-to heptadeca (ADP-ribosylated) histones could be visualized for each major variant histone. DMS treatment, by increasing the number of chromatin sites undergoing repair, greatly enhanced histone ADP-ribosylation. When histones were labeled in a cell lysate rather than in isolated nuclei, mono- and oligo(ADP-ribosylated) histone forms prevailed. The presence of approximately 87 ADP-ribosylated variant histone forms in cell lysates and of approximately 170 in isolated nuclei is shown for the first time in this work. Previous studies show multiple ADP-ribosylated forms for only histone H1. The theoretical number of variegated nucleosomes is thus much higher than previously thought, provided that histone-histone contacts are not disrupted at up to a certain level of histone ADP-ribosylation.

Architecture of Eukaryotic Genes

Abstract: I. DNA Structure and Gene Structure Structural and Topological Polymorphism of DNA The Molecular Architecture of Plant Genes: A Phylogenetic Perspective Transfer, Structure and Expression of Foreign Genes in Plants Enhancers and Transacting Factors II. DNA-Affine Proteins and Their Genes Histones, Histone Variants and Postsynthetic Histone Modifications Histones and HMG Proteins of Higher Plants The Role of Histone H1 in Nucleosomes and Chromatin Fibers The HMG Proteins and Their Genes Recent Progress in the Study of Nonhistone Chromosomal Proteins Histone Genes in Higher Plants III. DNA-Protein Interactions Nucleosomes and Transcription Nucleosome Positioning 'in vivo' and 'in vitro' Mechanisms of Nucleosome Positioning Tested in Minichromosomes of Yeast 'Saccaromyces cerevisiae' IV. Chromatin Chromatin of Active and Inactive Genes Chromatin Structure of Plant Genes Chromatin Domains and Gene Expression_Different Chromatin Conformations Characterize the Various Functional States of the Chicken Lysozyme Gene Regulatory Function and Molecular Structure of DNase I_Hypersensitive Elements in the Chromatin Domain of a Gene Organization and Length Heterogeneity of Plant Ribosomal RNA Genes Chromatin Structure and Expression of Plant Ribosomal RNA Genes Architecture of the 5' Flanking Regions of the Maize Adh Genes V. DNA Methylation and Transcription Eukaryotic Gene Inactivation by Sequence-Specific Promoter Methylation and the Release of the Transcription Block DNA Methylation and its Effect on Transformed Gene Expression Enzymology of DNA Methylation in Mammalian Cells VI. Nuclear Matrix Nuclear Matrix Nuclear Scaffold and the Higher-order Folding of Eukaryotic DNA VII. Chromosomes Three-Dimensional Organization of Interphase Chromosomes in Polytene Nuclei of 'Drosophila melanogaster' Phased Approaches to Mapping the Physical Structure of Entire Chromosomes VIII. Perspectives of Chromatin Research Perspectives of Chromatin Research A Special Case for Plant Molecular Biology.

Structure of the chromatin axis during transcription

Abstract: A new isolation procedure for polytene chromosomes has been developed which permits visualization of the native chromatin template of transcriptionally active genes. The Balbiani ring genes in the salivary glands of Chironomus tentans have been analyzed specifically: these genes are exceptionally long (37 kb) and very active in transcription. The most abundant configuration of the template is an extended fiber, approximately 5 nm in diameter. When the distance between adjacent RNA polymerases is unusually long, the template is packed into a 10 nm fiber. Occasionally, the fiber can further fold into a loose coil forming a more or less distinct 30 nm fiber. It is concluded that a large part of the chromatin axis is in a fully extended form during transcription of the Balbiani ring genes. However, if a given segment of the template is not continuously occupied by RNA polymerases it can be packed into a single nucleosome, into a string of nucleosomes (the thin fiber) or even into a supercoiled string of nucleosomes (the thick fiber). A comprehensive model based on the opposing topological effects of nucleosome disassembly and DNA melting caused by the RNA polymerase, is presented to account for the observed dynamic behavior of the chromatin template.

The effect of histone gene deletions on chromatin structure in Saccharomyces cerevisiae.

Abstract: As a way of studying nucleosome assembly and maintenance in Saccharomyces cerevisiae, mutants bearing deletions or duplications of the genes encoding histones H2A and H2B were analyzed. Previous genetic analysis had shown that only one of these mutants exhibited dramatic and pleiotropic phenotypes. This mutant was also the only one that contained disrupted chromatin, suggesting that the original phenotypes were attributable to alterations in chromosome structure. The chromatin disruption in the mutant, however, did not extend over the entire genome, but rather was localized to specific regions. Thus, while the arrangement of nucleosomes over the HIS4 and GAL1 genes, the telomeres, and the long terminal repeats (delta sequences) of Ty retrotransposons appeared essentially normal, nucleosomes over the CYH2 and UBI4 genes and the centromere of chromosome III were dramatically disrupted. The observation that the mutant exhibited localized chromatin disruptions implies that the assembly or maintenance of nucleosomes differs over different parts of the yeast genome.

Chromatin assembly in vitro and in vivo.

Abstract: The assembly of nucleosomes and higher-order chromatin structures has been extensively studied in vitro. Provided that non-specific charge interactions are controlled, all the information for correct assembly is found to be inherent in the macromolecular components. Cellular extracts which can assemble chromatin in vitro with nucleosomes correctly spaced on the DNA have been studied in detail and also used to investigate the role of chromatin structure in transcription. However, the mechanisms of chromatin assembly in vivo are still controversial.

The chromatin structure at the promoter of a glyceraldehyde phosphate dehydrogenase gene from Saccharomyces cerevisiae reflects its functional state.

Abstract: The chromatin structure of TDH3, one of three genes encoding glyceraldehyde phosphate dehydrogenases in Saccharomyces cerevisiae, was analyzed by nuclease digestion. A large hypersensitive region was found at the TDH3 promoter extending from the RNA initiation site at position -40 to position -560. This hypersensitive domain is nucleosome free and includes all putative cis-acting regulatory DNA elements. It is equally present in cells grown on fermentable as well as nonfermentable carbon sources. In a mutant which lacks the trans-activating protein GCR1 and which as a consequence expresses TDH3 at less than 5% of the wild-type level, the chromatin structure is different. Hypersensitivity between -40 and -370 is lost, due to the deposition of nucleosomes on a stretch that is nucleosome free in wild-type cells. Hypersensitivity is retained, however, further upstream (from -370 to -560). A similarly altered chromatin structure, as in a ger1 mutant, is found in wild-type cells when they approach stationary phase. This is the first evidence for a growth-dependent regulation of the TDH3 promoter.

Selective production of autoantibodies in graft-vs-host-induced and spontaneous murine lupus. Predominant reactivity with histone regions accessible in chromatin.

Abstract: The injection of (C57BL/6 X DBA/2)F1 mice with parental DBA/2 lymphoid cells leads to a lupus-like disease in which IgG autoantibodies are targeted to certain nuclear and cell surface antigens. To investigate further the extent of antibody diversity in this graft-vs-host (GVH) model, we studied the specificity of antihistone antibodies induced by the GVH reaction. High levels of IgG antibodies to histones H1 and H2B were detected whereas responses to H2A, H3, and H4 were only marginally elevated above pre-GVH levels. Immunoblotting analysis further revealed that the response to H2B was focused on epitopes that most likely reside in the N-terminal region. In contrast, F1 mice immunized with H2B/RNA complexes in adjuvant produced antibodies to the N terminus as well as to other regions of the H2B molecule. Thus, the antihistone response stimulated by the GVH reaction is only a fraction of the potentially activatable B cell repertoire. We also determined whether antibodies that arise spontaneously in genetically predisposed lupus strains were restricted in their histone reactivity. The response to core histones was highly variable among individual animals of the NZB/NZW and MRL-lpr/lpr strains despite their inbred nature. However, nearly all mice exhibited a preferential reactivity for epitopes in histone regions that are lost after partial trypsin digestion of chromatin. These data demonstrating autoantibody responses that are limited to particular histone regions support a mechanism by which B cells are selectively activated in murine lupus. The predominant production of antibodies to histone regions that are exposed in nucleosomes raises the possibility that chromatin is an antigenic stimulus for histone-specific B cells in this disease.

The effect of poly(ADP-ribose) on interactions of DNA with histones H1, H3 and H4

Abstract: The competition between poly(ADP-ribose) and DNA for binding of the histones H1, H3 and H4 was studied, using a membrane filter-binding test. Poly(ADP-ribose) differently affected the interaction between DNA and the individual histones. While poly(ADP-ribose) effectively competed with DNA for binding of histone H4, it equally competed with DNA for binding of histone H3 and only inefficiently competed with DNA for binding of histone H1. Moreover, preformed complexes were correspondingly affected by the addition of competing polynucleotides, thereby also indicating the reversibility of complex formation. The competition capacity of DNA for histone H4 binding did not depend on DNA size. Competition experiments with poly(A) also indicated that poly(ADP-ribose) preferentially affected DNA-histone H4 interaction. The significance of the differing binding properties is discussed with regard to the possible molecular function of poly(ADP-ribose), especially with regard to its potential effect on nucleosome structure.