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PubMed1997

Nucleosome packaging and nucleosome positioning of genomic DNA.

The goals of this study were to assess the extent to which bulk genomic DNA sequences contribute to their own packaging in nucleosomes and to reveal the relationship between nucleosome packaging and positioning. Using a competitive nucleosome reconstitution assay, we found that at least 95% of bulk DNA sequences have an affinity for histone octamer in nucleosomes that is similar to that of randomly synthesized DNA; they contribute little to their own packaging at the level of individual nucleosomes. An equation was developed that relates the measured free energy to the fractional occupancy of specific nucleosome positions. Evidently, the bulk of eukaryotic genomic DNA is also not evolved or constrained for significant sequence-directed nucleosome positioning at the level of individual nucleosomes. Implications for gene regulation in vivo are discussed.

Animals↗
PubMed1996

Formation of triple-stranded DNA at d(GA.TC)n sequences prevents nucleosome assembly and is hindered by nucleosomes.

Simple repeating d(GA.TC)n DNA sequences are frequently found at eukaryotic promoters, and in some cases they have been shown to be nucleosome free in vivo. These sequences show a high degree of structural polymorphism and are capable of adopting several types of non-B-DNA conformations. Here we show that the structural versatility of these sequences affects their ability to be packed into nucleosomes. Nucleosome assembly onto short double-stranded DNA fragments carrying d(GA.TC)n sequences of different length (n = 10 and n = 22) is very efficient. However, when the simple repeating sequence is forming a [CT(GA.TC)] triplex, nucleosome assembly is either prevented, as in the case of the d(GA.TC)22 sequence, or results in the destabilization of the triple-stranded conformation, as in the case of the d(GA.TC)10 sequence. Similarly, formation of triple-stranded DNA is hindered when the sequence is organized as nucleosomes. Efficient formation of triplex DNA occurs only at relatively high ionic strength (0.6 M NaCl), when the nucleosome is partially destabilized, and results in the disruption of the nucleosomal particle. These results indicate that nucleosome assembly and triplex formation are competing processes.

Base Sequence↗
PubMed1995

Modified curved DNA that could allow local DNA underwinding at the nucleosomal pseudodyad fails to position a nucleosome in vivo.

In competitive in vitro reconstitution experiments synthetic DNA composed of tandem repeats of the repetitive sequence (A/T)3NN(G/C)3NN, specifically the 20 bp 'TG sequence' (5'-TCGGTGTTAGAGCCTGTAAC-3'), was reported to associate with the histone octamer with an affinity higher than that of nucleosomally derived DNA. However, at least two groups have independently shown that tandem repeats of the TG sequence do not accommodate a stably positioned nucleosome in vivo. It was suggested that the anisotropic flexibility of the TG sequence, governed by a 10 bp sequence periodicity, is incompatible with the required underwinding of the DNA helix at the nucleosome pseudodyad while maintaining a bending preference that can be accommodated in the remainder of the nucleosome. Here we test this hypothesis directly by studying the in vivo nucleosomal structure of modified TG sequences designed to accommodate underwinding at the pseudodyad. We show that these modifications are not sufficient to allow stable incorporation of the TG sequence repeat into a nucleosome in vivo, but do note invasion from one end of the TG heptamer of a translationally random but rotationally constrained nucleosome. We discuss possible reasons for the absence of nucleosomes from the TG sequence in vivo.

Base Sequence↗
PubMed1994

A histone-binding protein, nucleoplasmin, stimulates transcription factor binding to nucleosomes and factor-induced nucleosome disassembly.

The binding of a GAL4-AH, USF or Sp1 to nucleosome cores was stimulated by the presence of the histone-binding protein, nucleoplasmin. Stimulation of factor binding by nucleoplasmin was specific for nucleosome reconstituted DNA and was not mimicked by non-specific histone sinks (i.e. polyglutamate or RNA). Upon GAL4-AH binding, nucleoplasmin specifically removed histones H2A and H2B from the nucleosome which enhanced the subsequent loss of the H3/H4 tetramers onto competing DNA. Thus, nucleoplasmin participated in the complete conversion of nucleosome cores to transcription factor-DNA complexes. These data indicate that proteins which bind histones can increase transcription factor binding to nucleosomal DNA and that transcription factor binding can initiate nucleosome disassembly. Similar activities of histone-binding proteins may participate in the displacement of nucleosomes at enhancers and promoters in vivo.

Base Sequence↗
PubMed1985

Primary organization of nucleosomes. Interaction of non-histone high mobility group proteins 14 and 17 with nucleosomes, as revealed by DNA-protein crosslinking and immunoaffinity isolation.

The binding sites for histones and high mobility group proteins (HMG) 14 and 17 have been located on DNA in the nucleosomal cores and H1/H5-containing nucleosomes. The nucleosomes were specifically associated with two molecules of the non-histone proteins HMG 14 and/or HMG 17 when followed by DNA-protein crosslinking and immunoaffinity isolation of the crosslinked HMG-DNA complexes. HMGs 14 and 17 were shown to be crosslinked in a similar manner to each core DNA strand at four sites: to both 3' and 5' DNA ends and also at distances of about 25 and 125 nucleotides from the 5' termini of the DNA. These sites are designated as HMG(143), (0), (25) and (125). The site HMG(125) is located at the place where no significant histone-DNA crosslinking was observed. The HMG(125) and HMG(25) sites lie opposite one another on the complementary DNA strands across the minor DNA groove and are placed, similarly to histones, on the inner side of the DNA superhelix in the nucleosome. The crosslinking of HMG 17 to the 3' ends of the DNA is much weaker than that of HMG 14. These data indicate that each of two molecules of HMG 14 and/or HMG 17 is bound to the double-stranded core DNA at two discrete sites: to the 3' and 5' ends of the DNA and at a distance of 20 to 25 base-pairs from each DNA terminus inside the nucleosome on a histone-free DNA region. Binding of HMG 14 or 17 does not induce any detectable rearrangement of histones on DNA and both HMGs seem to choose the same sites for attachment in nucleosomal cores and H1/H5-containing nucleosomes.

Animals↗
PubMed1991

The curvature vector in nucleosomal DNAs and theoretical prediction of nucleosome positioning.

Using our model for predicting DNA superstructures from the sequence, the average distribution of the phases of curvature along the sequences of the set of the 177 nucleosomal DNAs investigated by Satchwell et al. (J. Mol. Biol. 191 (1986) 659) was calculated. The diagram obtained shows very significant features which allow the visualization of the intrinsic nucleosomal superstructure characterized by two quasi-parallel tracts of a flat left-handed superhelical turn connected by a left-handed inflection in a perpendicular direction; such a superstructure appears to be closely related to the nucleosome model of Travers and Klug (Phil. Trans. R. Soc. Lond. 317 (1987) 537). The nucleosomal curvature phase diagram was then adopted as a sensitive determinant for the nucleosome virtual positioning in DNAs via correlation function, obtaining a good agreement with the experimental mapping of SV40 regulatory region as recently investigated by Ambrose et al. (J. Mol. Biol. 209 (1989) 255). This analysis shows also the presence of a constant phase relation between the virtual nucleosome positions which suggests its possible implication in the nucleosome condensation in chromatin.

Base Sequence↗
PubMed2000

Upstream nucleosomes and Rgr1p are required for nucleosomal repression of transcription.

The mechanisms of transcription repression and derepression in vivo are not fully understood. We have obtained evidence that begins to clarify the minimum requirements for counteracting nucleosomal repression in vivo. Location of the TATA element near the nucleosome dyad does not block RNA polymerase II transcription in vivo if there is a nucleosome-free region located immediately upstream. However, location of the TATA element similarly within the nucleosome does block transcription if the region upstream of it is nucleosome bound. Histone H4 depletion derepresses transcription in the latter case, supporting the idea that the nucleosomes are responsible for the repression. These results raise the intriguing possibility that the minimum requirement for derepression of transcription in vivo is a nucleosome-free region upstream of the core promoter. Importantly, we find that a C-terminal deletion in RGR1, a component of the mediator/holoenzyme complex and a global repressor, can also derepress transcription.

Chromosomal Proteins, Non-Histone↗
PubMed1998

Nucleosome structural transition during chromatin unfolding is caused by conformational changes in nucleosomal DNA.

We have recently reported that certain core histone-DNA contacts are altered in nucleosomes during chromatin unfolding (Usachenko, S. I., Gavin I. M., and Bavykin, S. G. (1996) J. Biol. Chem. 271, 3831-3836). In this work, we demonstrate that these alterations are caused by a conformational change in the nucleosomal DNA. Using zero-length protein-DNA cross-linking, we have mapped histone-DNA contacts in isolated core particles at ionic conditions affecting DNA stiffness, which may change the nucleosomal DNA conformation. We found that the alterations in histone-DNA contacts induced by an increase in DNA stiffness in isolated core particles are identical to those observed in nucleosomes during chromatin unfolding. The change in the pattern of micrococcal nuclease digestion of linker histone-depleted chromatin at ionic conditions affecting chromatin compaction also suggests that the stretching of the linker DNA may alter the nucleosomal DNA conformation, resulting in a structural transition in the nucleosome which may play a role in rendering the nucleosome competent for transcription and/or replication.

Animals↗
PubMed1994

The cooperative binding of chromosomal protein HMG-14 to nucleosome cores is reduced by single point mutations in the nucleosomal binding domain.

Mutants of human chromosomal protein HMG-14 were generated by site directed mutagenesis and used to study functional domains in this protein. A replacement of serine by cysteine at position 7 did not affect the binding of the protein to nucleosome cores. The sulfhydryl group in the nucleosome-bound protein is accessible to modifying agents suggesting that position 7 in the protein is not in close contact with either the DNA or the histones in the core particles. Under cooperative binding conditions, replacements of alanine by proline at position 21, or of lysine by cysteine at position 26, decreased the affinity of the protein for nucleosome cores 6.7- and 3-fold respectively. In contrast, the non-cooperative mode of binding was only minimally affected. A replacement of glutamic acid by glutamine at position 76 caused only minor changes in the binding of the protein to the cores. The results indicate that single point mutations, which change either the conformation or change in the nucleosomal binding domain of the protein, significantly reduce the ability of the HMG-14 protein to bind to nucleosome cores. We suggest that in chromatin the protein binds to nucleosomes in a cooperative manner and that upon binding to nucleosomes the protein acquires a distinct conformation.

Alanine↗
PubMed1995

Triple helix DNA alters nucleosomal histone-DNA interactions and acts as a nucleosome barrier.

Oligonucleotides which form triple helical complexes on double-stranded DNA have been previously reported to selectively inhibit transcription both in vitro and in vivo by physically blocking RNA polymerase or transcription factor access to the DNA template. Here we show that a 16mer oligonucleotide, which forms triple helix DNA by binding to a 16 bp homopurine segment, alters the formation of histone-DNA contacts during in vitro nucleosome reconstitution. This effect was DNA sequence-specific and required the oligonucleotide to be present during in vitro nucleosome reconstitution. Binding of the triple helix oligonucleotide on a 199 bp mouse mammary tumour virus promoter DNA fragment with a centrally located triplex DNA resulted in interruption of histone-DNA contacts flanking the triplex DNA segment. When nucleosome reconstitution is carried out on a longer, 279 bp DNA fragment with an asymmetrically located triplex site, nucleosome formation occurred at the border of the triple helical DNA. In this case the triplex DNA functioned as a nucleosome barrier. We conclude that triplex DNA cannot be accommodated within a nucleosome context and thus may be used to site-specifically manipulate nucleosome organization.

Base Sequence↗
PubMed1996

Binding of the wheat basic leucine zipper protein EmBP-1 to nucleosomal binding sites is modulated by nucleosome positioning.

To investigate interactions of the basic leucine zipper transcription factor EmBP-1 with its recognition sites in nucleosomal DNA, we reconstituted an abscisic acid response element and a high-affinity binding site for EmBP-1 into human and wheat nucleosome cores in vitro. DNA binding studies demonstrated that nucleosomal elements can be bound by EmBP-1 at reduced affinities relative to naked DNA. EmBP-1 affinity was lowest when the recognition sites were positioned near the center of the nucleosome. Binding was achieved with a truncated DNA binding domain; however, binding of full-length EmBP-1 caused additional strong DNase I hypersensitivity flanking the binding sites. Similar results were observed with nucleosomes reconstituted with either human or wheat histones, demonstrating a conserved mechanism of transcription factor-nucleosome interactions. We conclude that positioning of recognition sequences on a nucleosome may play an important role in regulating interactions of EmBP-1 with its target sites in plant cells.

Abscisic Acid↗
PubMed1985

Fractionation of newly replicated nucleosomes by density labeling and rate zonal centrifugation for the analysis of the deposition sites of newly synthesized nucleosomal core histones.

We have found that partial resolution of newly replicated nucleosomes can be achieved by rate zonal centrifugation through sucrose density gradients preformed in heavy water. Nucleosome samples were obtained from MH-134SC cells density labeled with 5-iododeoxyuridine in the presence of suitable isotopic precursors. The method is simple and can be performed under conditions that do not destabilize the nucleosome structure. This gave us an exciting opportunity to study the deposition sites of newly synthesized histones. Nucleosomes were obtained from cells pulse-labeled simultaneously with 5-iododeoxyuridine and [3H]lysine for the rate zonal analysis. Proteins in the resulting fractions were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and visualized by silver staining and fluorography. The distribution of newly synthesized H2A and H2B coincided closely with that of bulk nucleosomes. The distribution of newly synthesized H3 and H4 was shifted to the bottom sides of the bulk nucleosome peaks, but not so far as to the putative peaks of newly replicated (dense) nucleosomes. This means that newly synthesized histones are deposited on DNA in disproportionate amounts and that their sites of deposition are not restricted to newly replicated DNA.

Binding Sites↗
PubMed2006

Diagnostic value of anti-nucleosome antibodies in the assessment of disease activity of systemic lupus erythematosus: a prospective study comparing anti-nucleosome with anti-dsDNA antibodies.

OBJECTIVE: To determine the diagnostic value of anti-nucleosome antibodies in the assessment of clinically active systemic lupus erythematosus (SLE) and active nephritis. METHODS: A 12 month prospective study of 87 patients diagnosed with SLE. At each evaluation, disease activity was scored by SLE Disease Activity Index and Lupus Activity Criteria Count, and blood samples were collected for laboratory tests. Autoantibodies were detected by ELISA. RESULTS: Nearly all patients were female (96.6%). The mean age was 33 years and the mean disease duration was 60.7 months. About half the patients presented with nephritis (49.4%) and active SLE (50.6%) at the first clinical examination. During the study period, the prevalence of active SLE decreased from 50.6% to 29.1%. The prevalence of anti-nucleosome and anti-dsDNA antibodies was 40.0%-58.6% and 10.9%-21.8%, respectively, throughout the study period. The sensitivity of anti-nucleosome and anti-dsDNA antibodies for active SLE was 72.7%-100% and 31.3%-54.8%, respectively. The specificity of anti-nucleosome and anti-dsDNA antibodies for active SLE was 66.7%-83.7% and 88.7%-100%, respectively. The sensitivity and specificity of anti-nucleosome antibodies for active nephritis were 32.0%-67.5% and 46.2%-67.3%, respectively. The sensitivity and specificity for anti-dsDNA antibodies for active nephritis were 16.0%-35.4% and 85.1-97.5%, respectively. CONCLUSION: Anti-nucleosome antibodies are more sensitive than anti-dsDNA antibodies to active SLE and active nephritis. Thus, anti-nucleosome antibody reactivity may be a useful marker in the diagnosis and assessment of active SLE.

Adult↗
PubMed1997

Nucleosome-dependent escape of tumor cells from natural-killer-mediated lysis: nucleosomes are taken up by target cells and act at a postconjugation level.

Our previous data suggested that chromatin fragments released from dead cells into the extracellular medium could be involved in the impairment of natural-killer (NK)-mediated cytotoxicity reported in cancer patients. In the present study, an inhibition of the NK-mediated lysis was obtained in vitro by nucleosome addition to different tumor target cells, independently of their sensitivity to NK-mediated lysis. We observed a rapid endocytosis and degradation of nucleosomes by K562 tumor target cells and (although to a much lesser extent) a binding to a subpopulation of lymphocytes. Nucleosomes impaired neither the conjugation step nor the expression of adhesion molecules at the effector (CD11a, CD18, CD2) or target (CD54, CD58) cell surface. On the contrary, flow-cytometry analysis of the conjugation suggested that nucleosomes might stabilize the conjugates. Investigations of the killing process showed that nucleosomes decreased the NK cytotoxic potential without modifying Ca2+-dependent lethal-hit-delivery kinetics. The cytotoxic potential was not restored by increasing the available magnesium and calcium concentrations in the extracellular medium. Taken together, the results suggest that the inhibition of NK-mediated lysis by nucleosomes may result from alterations of the NK mechanism at the postconjugation level and after lethal-hit delivery. Hence, the inhibition could involve a delay in the recycling of effector cells, or a resistance of tumor target cells to NK cells.

Biological Transport↗
PubMed1989

Formation, stability and core histone positioning of nucleosomes reassembled on bent and other nucleosome-derived DNA.

DNA originating from chicken erythrocyte mononucleosomes was cloned and sequenced. The properties of nucleosome reconstruction were compared for two cloned inserts, selected on account of their interesting sequence organization, length and difference in DNA bending. Cloned fragment 223 (182 base-pairs) carries alternatively (A)3-4 and (T)4-5 runs approximately every ten base-pairs and is bent; cloned fragment 213 (182 base-pairs) contains a repeated C4-5ATAAGG consensus sequence and is apparently not bent. Our experiments indicate the preference of the bent DNA fragment 223 over fragment 213 to associate in vitro with an octamer of histones under stringent conditions. We provide evidence that the in vitro nucleosome formation is hampered in the case of fragment 213, whereas the reconstituted nucleosomes were equally stable once formed. For the correct determination of the positioning of the histone octamer with regard to the two nucleosome-derived cloned DNA sequences, the complementary use of micrococcal nuclease, exonuclease III and DNase I is a prerequisite. No unique, but rotationally related, positions of the histone octamer were found on these nucleosome-derived DNA fragments. The sequence-dependent anisotropic flexibility, as well as intrinsic bending of the DNA, resulting in a rotational setting of the DNA fragments on the histone core, seems to be a strong determinant for the allowed octamer positions, Exonuclease III digestion indicates a different histone-DNA association when oligo(d(C.G)n) stretches are involved. The apparent stagger near oligo(d(A.T)n) stretches generated by DNase I digestion on reconstituted nucleosome 223 was found to be inverted from the normal two-base 3' overhang to a two-base 5' overhang. Two possibilities of the oligo(d(A.T)n) minor groove location relative to the histone core are envisaged to explain this anomaly in stagger.

Animals↗
PubMed1980

Reversible phosphorylation of a nucleosome binding protein that stimulates transcription of nucleosome deoxyribonucleic acid.

A nonhistone chromosomal protein (D-55) of Mr 55000 has been isolated in homogeneous form from calf thymus by using the standard salt-extraction procedures for the isolation of nonhistone chromosomal proteins followed by hydroxyapatite chromatography. D-55 is further characterized as coming from the group of nonhistone chromosomal proteins easily phosphorylated by an endogenous nuclear protein kinase. The kinase incorporates 1 mol of phosphate per mol of protein from [gamma-32P]ATP. The unphosphorylated form of D-55 binds to DNA, histones, and nucleosomes. Phosphorylation of D-55 does not significantly alter the binding of D-55 to DNA but greatly enhances its binding to histones and nucleosomes. Binding of D-55 to reconstituted nucleosomes enhances transcription of the nucleosome DNA by E. coli RNA polymerase by approximately 100-fold, to a level approximately 4 times that observed with naked calf thymus DNA as template. Phosphorylation of D-55 abolishes this enhancement. Binding of D-55 produces no apparent alteration in nucleosome structure as assayed by nuclease digestion patterns. In contrast, phospho-D55 alters nucleosome structure significantly.

Amino Acids↗
PubMed2004

Positioning of a nucleosome on mouse satellite DNA inserted into a yeast plasmid is determined by its DNA sequence and an adjacent nucleosome.

It has earlier been shown that multiple positioning of nucleosomes on mouse satellite DNA is determined by its nucleotide sequence. To clarify whether other factors, such as boundary ones, can affect the positionings, we modified the environment of satellite DNA monomer by inserting it into a yeast plasmid between inducible GalCyc promoter and a structural region of the yeast FLP gene. We have revealed that the positions of nucleosomes on satellite DNA are identical to those detected upon reconstruction in vitro. The positioning signal (GAAAAA sequence) of satellite DNA governs nucleosome location at the adjacent nucleotide sequence as well. Upon promoter induction the nucleosome, translationally positioned on the GalCyc promoter, transfers to the satellite DNA and its location follows the positioning signal of the latter. Thus, the alternatives of positioning of a nucleosome on satellite DNA are controlled by its nucleotide sequence, though the choice of one of them is determined by the adjacent nucleosome.

Animals↗
PubMed2003

DNA-PK is activated by nucleosomes and phosphorylates H2AX within the nucleosomes in an acetylation-dependent manner.

Eukaryotic DNA is organized into nucleosomes and higher order chromatin structure, which plays an important role in the regulation of many nuclear processes including DNA repair. Non-homologous end-joining, the major pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells, is mediated by a set of proteins including DNA-dependent protein kinase (DNA-PK). DNA-PK is comprised of a large catalytic subunit, DNA-PKcs, and its regulatory subunit, Ku. Current models predict that Ku binds to the ends of broken DNA and DNA-PKcs is recruited to form the active kinase complex. Here we show that DNA-PK can be activated by nucleosomes through the ability of Ku to bind to the ends of nucleosomal DNA, and that the activated DNA-PK is capable of phosphorylating H2AX within the nucleosomes. Histone acetylation has little effect on the steps of Ku binding to nucleosomes and subsequent activation of DNA-PKcs. However, acetylation largely enhances the phosphorylation of H2AX by DNA-PK, and this acetylation effect is observed when H2AX exists in the context of nucleosomes but not in a free form. These results suggest that the phosphorylation of H2AX, known to be important for DSB repair, can be regulated by acetylation and may provide a mechanistic basis on which to understand the recent observations that histone acetylation critically functions in repairing DNA DSBs.

Acetylation↗