Arrangement Of Nucleosomes Research Articles

In vitro assembly of the CENP-B/alpha-satellite DNA/core histone complex: CENP-B causes nucleosome positioning.

We have studied the nucleosome structure formed from alpha-satellite DNA bound with CENP-B and core histones, in order to develop a previous proposal that the CENP-B dimer may play a critical role in the assembly of higher order structures of the human centromere by juxtaposing CENP-B boxes in long alpha-satellite arrays. The dimeric structure of CENP-B was sufficiently stable to bundle together two 3.5 kbp DNA fragments when each DNA contained a CENP-B box. When the same length of DNA included two CENP-B boxes, the intra-molecular interaction with the CENP-B dimer predominated, resulting in the formation of loop structures. The in vitro assembly of CENP-B/alpha-satellite DNA/core histone complexes with the aid of nucleosome assembly protein-1 (NAP-1) permitted an investigation into the nucleosome arrangement in alpha-satellite DNA with CENP-B bound to CENP-B boxes. Footprint analyses with micrococcal nuclease (MNase) revealed that CENP-B causes nucleosome positioning between pairs of CENP-B boxes with unique hypersensitive sites created on both sides. We propose that CENP-B functions as a structural factor in the centromere region in order to establish a unique, centromere specific pattern of nucleosome positioning.

Monte Carlo Simulation of the Production of Short DNA Fragments by Low-Linear Energy Transfer Radiation Using Higher-Order DNA Models

A realistic DNA target model has been developed and implemented in the biophysical simulation code PARTRAC. It describes five levels of the B-DNA structure (nucleotides, DNA helices, nucleosomes, chromatin fiber structure and chromatin fiber loops) on an atomic level for the whole genome inside a mammalian cell nucleus. The model is capable of describing regular solenoidal, crossed-linker or zigzag structures as well as repeating stochastic arrangements of nucleosomes in the chromatin fiber. Electron tracks resulting from monoenergetic electrons with energies up to 100 keV and from 220 kVp X rays, starting at random positions in the cell, were superimposed on four DNA target models with different chromatin fiber structures. The yields of SSBs, DSBs and short single- and double-stranded DNA fragments were determined from spatial coincidences with strand atoms. Two parameters of the model-the energy necessary to create an SSB and the distance between two breaks that would be scored as a DSB-were adapted to equate simulated and measured strand break yields after X irradiation of human fibroblast cells. The integral fractions of short single- and double-stranded fragments were rather similar for all condensed chromatin fiber structures; they agreed with experimental data for DNA fragments below 2 kbp. The simulated fragment size distributions in the range from 0.1 to 1.5 kbp reflected the fiber structure irrespective of strandedness or electron energy. The distributions using a stochastic arrangement of nucleosomes in the chromatin fiber were found to be in better accordance with experimental data than those obtained with regular fiber structures.

Human Protein MCM6 on HeLa Cell Chromatin

Minichromosome maintenance (Mcm) proteins perform essential functions regulating the replication of chromatin. Human cells, like other eukaryotic cells, express at least six Mcm proteins conserved in the central region. We have earlier described the primary structures of five human Mcm proteins, but the primary structure of the sixth human Mcm protein, MCM6, was identified only recently. We now use antibodies, specific for the MCM6 protein, to assess its intranuclear distribution. We find that a fraction of MCM6 protein occurs in the nucleosol, forming multiprotein complexes with other Mcm proteins. More importantly, we use for the first time micrococcal nuclease as a tool to investigate the association of MCM6 protein with chromatin. After short digestion times, a considerable fraction of the MCM6 protein is released from chromatin as a multiprotein complex that includes other Mcm proteins as well. In addition, fractions of MCM3 and MCM6 proteins are released by nuclease digestion as monomeric proteins indicating that at least these two Mcm proteins may also occur as single molecules on chromatin. The data also suggest that the chromatin regions with bound Mcm proteins are more vulnerable to nuclease attack than bulk chromatin and may therefore differ in the arrangement of nucleosomes.

Uncurved-DNA Signals are Important for Translational Positioning of Nucleosomes

We describe here a possible mechanism for non-random arrangement of nucleosomes along genomic DNA by short stretches of uncurved DNA. Available biochemical data on nucleosome positioning on different DNA sequences were analyzed. A strong correlation between location of nucleosome borders and location of uncurved sequences along DNA was observed. Tested sequences include satellites, 5S RNA genes and 5′ regions of different genes.

DNA sequence encodes information for nucleosome array formation

We have examined the effects of base sequence on nucleosome array formation using randomly selected chicken genomic DNA sequences. DNA clones were assembled into chromatin under identical conditions using a defined in vitro system capable of generating physiologically spaced nucleosomes on some sequences. The nucleosome arrangements in native chromatin on the selected sequences were also examined in liver nuclei. Variations in nucleosome ladders were found among the different sequences that were similar in vitro and in nuclei. Differences in both the degree of regularity of nucleosome arrays and in the value of the nucleosome repeat length were observed. Analysis of an approximately 100 kilobase-pair contiguous region using cosmid clones suggested that well-ordered regions of chromatin, generally less than two kilobase-pairs in extent, alternate with less-ordered regions. This mosaic arrangement for chromatin organization appears to be largely a consequence of information encoded in the DNA base sequence. Nucleosome ordering with a 210 base-pair periodicity in a highly ordered ten-nucleosome array appeared to result from linker histone-dependent alignment with respect to each of two positioned nucleosomes, approximately 1000 base-pairs apart.

Visualization and Analysis of Chromatin by Scanning Force Microscopy

The use of the scanning force microscope (SFM) to visualize and analyze chromatin fiber structures is presented. Protocols to prepare chromatin fibers for SFM imaging of fibers in air and in buffer are first discussed. Next, the conditions for acquiring high-quality SFM images such as optimal instrumental parameters, appropriate deposition substrates, and adequate procedures of sample deposition are described. It is shown that analysis and quantitation of the SFM images support an irregular, three-dimensional arrangement of nucleosomes in the native chromatin fiber. This structure is lost in linker histone-depleted fibers, which show, instead, a beads-on-a-string structure. Molecular modeling of the chromatin fiber structures and computer simulation of the SFM imaging process indicate that the natural variability of the linker length may be the major determinant of the structural irregularity of the native chromatin fiber. Removal of linker histones (H1/H5) may change the amount of DNA wrapped around the histone octamer, which in turn may induce the transition from a three-dimensional irregular helix to an extended beads-on-a-string structure. Studies of trinucleosomes indicate that both the average successive nucleosome center-to-center distance and the average angle between two successive linkers increase upon the removal of linker histone.

DNA sequence and nucleosome placement on the murine fibroblast growth factor-4 gene.

A large portion of the 5' flanking region of the fibroblast growth factor-4 (FGF-4) gene has been sequenced, but only 38 bp of sequence information past the end of the last exon of this gene has been described. In this study, a total of 2769 bp of nucleotide sequence down-stream of the last exon of FGF-4 (GenBank #U43515) was determined by a combination of deletional subcloning and primer walking. In addition, we have used the theoretical algorithm of Calladine and Drew to predict the rotational positioning of nucleosomes throughout the entire FGF-4 gene.

Nucleosomal Arrangement of HIV-1 DNA: Maps Generated from an Integrated Genome and an EBV-based Episomal Model

ACH-2 cells, an immortalized human T-cell line, contain a single integrated copy of the HIV-1 provirus. Here, the structure of HIV-1 chromatin was probed using a DNA cleavage reagent. Nuclei were isolated from ACH-2 cells and treated with methidiumpropyl-EDTA (MPE)- iron(II) to produce limited DNA cleavage. Primers were selected at ∼300 bp intervals along the HIV-1 DNA, and sites of preferential cleavage were mapped by carrying out 50 cycles of primer extension using a thermo-stable DNA polymerase in the presence of [ 32P]dATP. By comparing the resulting cleavage pattern with patterns derived from human cell lines not containing HIV-1 sequences, it was possible to map the arrangement of nucleosomes across the integrated HIV-1 genome. Particularly regular spacing was seen in the 3′ end of the pol and env coding regions, and several extended blocks spared of nucleosomes were found in gag and pol, the largest being a |similar|450 bp region in gag. For comparison, and to examine nucleosome placement on HIV-1 DNA when it is not integrated, overlapping segments of HIV-1 DNA were cloned into an EBV-oriP plasmid, grown as stable episomes in a human B lymphoblastoid cell line, and the same analysis using MPE-iron(II) cleavage and primer extension carried out. The major features of nucleosome placement on these EBV/HIV minichromosomes was very similar to that observed in the integrated HIV-1 genome arguing for a strong sequence dependence for nucleosome placement along HIV-1 DNA.

Clusters of DNA Damage Induced by Ionizing Radiation: Formation of Short DNA Fragments. II. Experimental Detection

The basic 30-nm chromatin fiber in the mammalian cell consists of an unknown (possibly helical) arrangement of nucleosomes, with about 1.2 kb of DNA per 10-nm length of fiber. Track-structure considerations suggest that interactions of single delta rays or high-LET particles with the chromatin fiber might result in the formation of multiple lesions spread over a few kilobases of DNA (see the accompanying paper: W.R. Holley and A. Chatterjee, Radiat. Res. 145, 188-199, 1996). In particular, multiple DNA double-strand breaks and single-strand breaks may form. To test this experimentally, primary human fibroblasts were labeled with [3H]thymidine and exposed at 0 degrees C to X rays or accelerated nitrogen or iron ions in the LET range of 97-440 keV/microns. DNA was isolated inside agarose plugs and subjected to agarose gel electrophoresis under conditions that allowed good separation of 0.1-2 kb size DNA. The bulk of DNA remained in the well or migrated only a small distance into the gel. It was found that DNA fragments in the expected size range were formed linearly with dose with an efficiency that increased with LET. A comparison of the yield of such fragments with the yield of total DNA double-strand breaks suggests that for the high-LET ions a substantial proportion (20-90%) of DNA double-strand breaks are accompanied within 0.1-2 kb by at least one additional DNA double-strand break. It is shown that these results are in good agreement with theoretical calculations based on treating the 30-nm chromatin fiber as the target for ionizing particles. Theoretical considerations also predict that the clusters will contain numerous single-strand breaks and base damages. It is proposed that such clusters be designated "regionally multiply damaged sites." Postirradiation incubation at 37 degrees C resulted in a decline in the number of short DNA fragments, suggesting a repair activity. The biological significance of regionally multiply damaged sites is presently unknown.

NF-kappa B-mediated chromatin reconfiguration and transcriptional activation of the HIV-1 enhancer in vitro.

NF-kappa B is a potent inducible transcription factor that regulates many genes in activated T cells. In this report we examined the ability of different subunits of NF-kappa B to enhance HIV-1 transcription in vitro with chromatin templates. We find that the p65 subunit of NF-kappa B is a strong transcriptional activator of nucleosome-assembled HIV-1 DNA, whereas p50 does not activate transcription, and that p65 activates transcription synergistically with Sp1 and distal HIV-1 enhancer-binding factors (LEF-1, Ets-1, and TFE-3). These effects were observed with chromatin, but not with nonchromatin templates. Furthermore, binding of either p50 or p65 with Sp1 induces rearrangement of the chromatin to a structure that resembles the one reported previously for integrated HIV-1 proviral DNA in vivo. These results suggest that p50 and Sp1 contribute to the establishment of the nucleosomal arrangement of the uninduced provirus in resting T cells, and that p65 activates transcription by recruitment of the RNA polymerase II transcriptional machinery to the chromatin-repressed basal promoter.

The Arabidopsis Adh gene exhibits diverse nucleosome arrangements within a small DNase I-sensitive domain.

The alcohol dehydrogenase (Adh) gene from Arabidopsis shows enhanced sensitivity to DNase I in cells that express the gene. This generalized sensitivity to DNase I is demarcated by position -500 on the 5' side and the end of the mRNA on the 3' side. Thus, the gene defined as the promoter and mRNA coding region corresponds very closely in size with the gene defined as a nuclease-sensitive domain. This is a remarkably close correspondence between a sensitive domain and a eukaryotic transcriptional unit, because previously reported DNase I-sensitive domains include large regions of DNA that are not transcribed. Nucleosomes are present in the coding region of the Adh gene when it is expressed, indicating that the transcriptional elongation process causes nucleosome disruption rather than release of nucleosomes from the coding region. In addition, the regulatory region contains a loosely positioned nucleosome that is separated from adjacent nucleosomes by internucleosomic DNA segments longer than the average linker DNA in bulk chromatin. This specific array of nucleosomes coexists with bound transcription factors that could contribute to the organization of the nucleosome arrangement. These results enhance our understanding of the complex interactions among DNA, nucleosomes, and transcription factors during gene expression in plants.

The role of a positioned nucleosome at the Drosophila melanogaster hsp26 promoter.

The regulatory region of Drosophila melanogaster hsp26 includes a positioned nucleosome located between the two DNase I hypersensitive (DH) sites that encompass the critical heat shock elements (HSEs). To test the role of this nucleosome in regulated expression, transgenic flies containing hsp26-lacZ fusion genes with alterations in the nucleosome-associated region have been generated. The positioned nucleosome is associated with a DNA sequence that does not itself contain any critical regulatory elements for heat shock-inducible expression. The nucleosome-associated sequence can be deleted, reversed, duplicated or replaced by a random sequence with no significant effect on DH site formation and gene expression. Analyses of hsp26 and hsp70 transgenes with spacing changes within the promoter region indicate that the location of the (CT)n.(GA)n elements dictates the location of DH site formation. Wrapping the DNA between the regulatory elements around a nucleosome is as effective for gene expression as placing the regulatory elements close to each other. A loss of inducible gene expression was observed when the nucleosome-associated DNA was replaced with sequences which appear to misdirect nucleosome placement. The results indicate considerable flexibility in the spacing between DH regulatory sites.

Quiescent viral genomes in human fibroblasts after infection with herpes simplex virus type 1 Vmw65 mutants

The development and utilization of a tissue culture system for the analysis of quiescent, nonreplicating herpes simplex virus type 1 (HSV-1) genomes is described. It was demonstrated previously that the HSV-1 Vmw65 mutant in1814, which is impaired for immediate early (IE) transcription, was retained for many days in human fetal lung (HFL) fibroblasts in a quiescent 'latent' state. Molecular analysis of the viral genome was not possible, however, due to residual expression of IE proteins and consequent cytotoxicity at high m.o.i. In the study reported here, IE transcription was reduced further by pretreatment of cells with interferon-alpha (IFN-alpha) and by the use of mutant in1820, a derivative of in1814 in which the Vmw110 promoter was replaced by the Moloney murine leukaemia virus (Momulv) enhancer. The Momulv enhancer was not expressed under IE conditions; thus in1820 was more impaired for replication than in1814 and behaved as if deficient for both Vmw65 and Vmw110. In cells pretreated with IFN-alpha and subsequently infected with in1820 cytotoxicity was overcome, enabling a tissue culture system to be developed in which all cells stably retained at least one quiescent viral genome. To assist the analysis of gene expression, in1820 was further modified by insertion of the Escherichia coli lacZ gene controlled by the human cytomegalovirus enhancer (mutant in1883) or the HSV-1 immediate early Vmw110 promoter (in1884). Expression of beta-galactosidase was not detected after infection of IFN-alpha-pretreated cells with in1883 or in1884 but could be induced in almost all cells containing a viral genome, by superinfection of cultures. In1820-derived viruses were retained for at least 9 days and were not reactivated by subculture of cells. A regular arrangement of nucleosomes, as found in cellular chromatin, was not detected on the viral genome at the thymidine kinase locus. The non-linear genome was a template for reactivation with no requirement for prior conversion to a linear form. A small number of remaining linear genomes resulted from incomplete uncoating of input virus.

DNA sequence affects nucleosome ordering on replicating plasmids in transfected COS-1 cells and in vitro.

Nucleosome ordering on a variety of replicating plasmids, assembled into chromatin in transfected COS-1 cells, was studied by micrococcal nuclease digestion of isolated nuclei. Generally, no more than three well defined multiples of a unit nucleosome repeat, which resembled the first three bands of the (187 +/- 5 base pair (bp)) cellular chromatin ladder, could be detected in constructs that contained a near-minimal SV40 replication origin. In contrast, constructs that additionally contained the SV40 early region exhibited significantly more regular nucleosome arrangements. In some cases, eight to nine multiples of a 203 +/- 5-bp repeat could be resolved. The presence of the SV40 early region was necessary for physiological nucleosome alignment over the SV40 late and ori regions, or onto adjacent pBR327 DNA. In an in vitro chromatin assembly system, using purified chicken erythrocyte histones plus polyglutamic acid, a portion of SV40 DNA became packaged into a highly ordered 200 +/- 5-bp nucleosome array, which encompassed the early region and extended for about 2800 bp. The data suggest that in the cell nucleus, nucleosome ordering on SV40 DNA might spread from sequences in the early region, close to where transcription terminates, probably as a consequence of histone H1-nucleosome interactions.

Transitions between in situ and isolated chromatin.

We show that the mechanism by which chromatin displaying higher-order structure is usually isolated from nuclei involves a transition to an extended nucleosomal arrangement. After being released from nuclei, chromatin must refold in order to produce the typical chromatin fibers observed in solution. For starfish sperm chromatin with a long nucleosome repeat (222 bp), isolated fibers are significantly wider than those in the nucleus, indicating that the refolding process does not regenerate the native higher-order structure. We also propose that for typical eukaryotic nuclei, the concept that the native state of the (inactive) bulk of the genome is a chromatin fiber with defined architecture be reconsidered.

Fine analysis of the chromatin structure of yeast RNA polymerase II transcription terminators

In order to study the functional structure of the transcription terminators and the mechanism of termination, a survey of the chromatin structure, including the location of DNase I hypersensitive sites and the nucleosome arrangement, of yeast ADHI and FLP terminators was made. The results show that there is no relationship between the function of the terminators and the existence of DNase I hypersensitive sites. However, it is found that there is always a nucleosome at the immediate upstream of the transcriptional termination sites. As a control, the chromatin structures of the pBR322 DNA fragments on the yeast shutter vectors are also investigated at the same time. The random nucleosome arrangement on lhe bacterial DNA in yeast agrees with the published reports. A new hypothesis, about the mechanism of transcriptional termination is put forward and the reason of different nucleosome arrangement on the DNAs which are originally from different species in yeast is discussed.

Nucleosomal organization of chromatin in sperm nuclei of the bivalve mollusc Aulacomya ater

The sperm nuclei of Aulacomya ater, family Mitylidae, contain three proteins (X, Aa5 and Aa6) which are specific to this cell type coexisting with a set of five somatic-type histones. Information about the chromatin structure resulting from this kind of association is scarce. Therefore, we have probed the structure of this sperm chromatin through digestion with micrococcal nuclease in combination with salt fractionation. The data obtained have allowed us to propose a nucleosomal arrangement for this chromatin. However, two types of nucleosomes would be present in agreement with their protein components.

Effects of DNA sequence and histone-histone interactions on nucleosome placement

Using competitive reconstitution, we have refined the parameters for the binding of histone octamers to artificial nucleosome-positioning sequences of the form: (A/T3nn(G/C)3nn. We find that the optimal period between flexible segments is approximately 10.1 base-pairs, supporting the view that the DNA on the nucleosome surface is overwound. The strongest requirement for flexible DNA is near the protein dyad. However, we see no indication of changes in DNA helical repeat in this region. Using a series of repetitive sequences, we confirm that neither all A/T-rich nor all G/C-rich regions are identical in promoting nucleosome formation. Surprisingly, A/T-rich segments containing the TpA step, subject to purine-purine clash in the minor groove, favor nucleosome formation over sequences lacking this step. Short tracts of adenine residues are found to position on the histone surface like other A/T-rich regions, in the manner predicted by the direction of their sequence-directed bends as determined by electrophoretic methods. Tracts containing five adenine residues are extremely aniostropic in their flexibility and are strongly detrimental to nucleosome formation when positioned for major groove compression. Longer adenine tracts are found to position near the ends of the nucleosomal DNA. However, other positions may be occupied by an A12 tract, with only a minor penalty in the free energy of nucleosome formation. Overall, reconstituted nucleosome positions are translationally degenerate, suggesting a weak dependence on DNA flexibility for nucleosome positioning. Dinucleosomal reconstitutions on tandem dimers of the 5 S RNA gene of Lytechinus variegatus demonstrate a weak phasing dependence for the interaction between nucleosomes. This interaction is maximal for the 202 base-pair repeat and suggests a co-operative mechanism for the formation of ordered nucleosomal arrays based on a combination of DNA flexibility and nucleosome-nucleosome interactions.

Correlation between dinucleotide periodicities and nucleosome positioning on mouse satellite DNA

Previous studies demonstrated 16 well-defined nucleosome locations (A-P) on a tandemly repeated prototype 234 base pair (bp) mouse satellite repeat unit. We have aligned the A-P fragments to search for DNA sequence elements that might contribute to nucleosome placement at these positions. Our results demonstrate a strikingly regular, uninterrupted, periodic pattern for the AA dinucleotide occurrences along the entire length of the aligned fragments. The periodicity of the AA occurrences is about 9.7 bp. The pattern exhibits a local minimum at position 74, near the nucleosome dyad axis of symmetry. Other dinucleotides--including AC: GT, CA: TG, and CC: GG--are also placed periodically, but their patterns of occurrence are less regular and less frequent than AA. The calculated spacings between consecutive preferred nucleosome locations on mouse satellite DNA are nearly identical, corresponding to multiples of 9.7 bp. The correlation between the periodicity of dinucleotide occurrences and the average spacing of nucleosome positions suggests that the preferred nucleosome locations recur at intervals that may correspond to the DNA helical repeat in the mouse satellite nucleosomes, and that the histone octamers sample (or slip along) the duplex in steps of 9.7 bp during nucleosome formation on mouse satellite DNA.

The multiple codes of nucleotide sequences.

Nucleotide sequences carry genetic information of many different kinds, not just instructions for protein synthesis (triplet code). Several codes of nucleotide sequences are discussed including: (1) the translation framing code, responsible for correct triplet counting by the ribosome during protein synthesis; (2) the chromatin code, which provides instructions on appropriate placement of nucleosomes along the DNA molecules and their spatial arrangement; (3) a putative loop code for single-stranded RNA-protein interactions. The codes are degenerate and corresponding messages are not only interspersed but actually overlap, so that some nucleotides belong to several messages simultaneously. Tandemly repeated sequences frequently considered as functionless "junk" are found to be grouped into certain classes of repeat unit lengths. This indicates some functional involvement of these sequences. A hypothesis is formulated according to which the tandem repeats are given the role of weak enhancer-silencers that modulate, in a copy number-dependent way, the expression of proximal genes. Fast amplification and elimination of the repeats provides an attractive mechanism of species adaptation to a rapidly changing environment.