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PubMed1986

Reversible changes in the nucleosomal organization of a human H4 histone gene during the cell cycle.

The organization of nucleosomes associated with a cell cycle regulated human H4 histone gene was examined in synchronized HeLa S3 cells. At various times during the cell cycle, nuclei were digested with micrococcal nuclease, and the nucleosomal pattern of the gene was obtained by Southern blot analysis using radiolabeled human histone H4 gene probes. We have detected reversible changes during the cell cycle in the chromatin structure of this gene, as reflected by the shortening of the nucleosomal spacing after replication and the peak of transcription. This variation is also observed when DNA and protein syntheses are inhibited. By using a probe that comprises 250 base pairs (bp) of the coding region and 240 bp of the 5' end of the gene, containing the promoter and DNase I sensitive sequences, we also have observed a general disruption of the nucleosomal organization, which is reflected by a degeneration of the characteristic nucleosomal ladder produced by micrococcal nuclease digestion. This modification coincides with the replication and active transcription of the gene (early S phase), which recovers its regular nucleosomal appearance when both processes have been completed, although the nucleosome linker length is shortened. When the probe utilized comprises the distal 3' end of the gene, there is no disruption of the nucleosomal pattern, but the linker region also exhibits a shortened length. A non-cell cycle regulated gene (beta-globin) does not exhibit such modifications in any of the situations analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Cycle↗

PubMedDate not supplied

[Structural-functional organization of the SV40 virus chromosome. III. Nucleosome organization of free mini-chromosomes].

Micrococcal nuclease digestion and hybridization end labeling procedure have been used for analysis of nucleosomal organization of SV40 minichromosomes. Usual oligonucleosomal pattern containing long oligonucleosomes has been observed after digestion and DNA electrophoresis. All the regions of the minichromosome, including the "regulatory" one, are involved in nucleosome structure, as judged by hybridization analysis. This is valid at least for the major minichromosome fraction. On the other hand, the nucleosome ordering turned out to be higher in certain genome regions, for example around the site where the replication terminates. Data implying the existence of several discrete nucleosome "frames" in this region have been obtained. Possible artefacts due to micrococcal nuclease sequence-specificity are discussed.

Autoradiography↗

PubMed1990

c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts display more decondensed nucleosomal organization than normal fibroblasts.

We have compared the nucleosomal organization of c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts with that of normal fibroblasts by using micrococcal nuclease (MNase) as a probe for the chromatin structure. The bulk chromatin from asynchronously and exponentially growing ras-transformed cells was much more sensitive to MNase digestion than chromatin from the normal cells. Southern hybridization analyses of the MNase digests with probes specific for the ornithine decarboxylase (odc) and c-myc genes showed that the coding and/or 3' end regions of these growth-inducible genes carry a nucleosomal organization both in ras-transformed and normal cells. Studies with cells synchronized by serum starvation showed that in both cell lines the nucleosomal organization of chromatin is relatively condensed at the quiescent state, becomes highly decondensed during the late G1 phase of the cell cycle, and starts again to condense during the S phase. However, in ras-transformed cells the decondensation state stayed much longer than in normal cells. Moreover, irrespective of the phase of the cell cycle the bulk chromatin as well as that of the odc and c-myc genes was more sensitive to MNase digestion in the ras-transformed cell than in the normal fibroblast. Decondensation of the chromatin was also observed in the normal c-Ha-ras protooncogene-transfected cells, but to a lesser extent than in the mutant ras-transformed cells. Whether the increased degree of chromatin decondensation plays a regulatory role in the increased expression of many growth-related genes in the ras-transformed cells remains an interesting object of further study.

Animals↗

PubMed1991

The effect of the simple repeating d(CG.GC)n, d(CA.GT)n, and d(A.T)n DNA sequences on the nucleosomal organization of SV40 minichromosomes.

The effect of several simple repeating DNA sequences--d(CG.GC)5, d(CA.GT)30, and d(A.T)60--on the nucleosomal organization of the SV40 minichromosome is analyzed. These three different sequences were cloned at the Hpa II site of SV40 (position 346) which occurs at the 3' border of the nucleosome-free SV40 control region. Our results show that neither the d(A.T)60 sequence nor the d(CG.GC)5 sequence appear to have any relevant effect on the nucleosomal organization of the region of the minichromosome surrounding the inserted repeated sequence. Both sequences are hypersensitive to micrococcal nuclease cleavage in the minichromosome, indicating that they are not organized into nucleosomes. On the other hand, the d(CA.GT)30 sequence is found organized as nucleosomes and causes the delocation of nucleosomes in the minichromosomal region close to the inserted repeated sequence.

Chromosomes↗

PubMed1996

Histones associated with non-nucleosomal rat ribosomal genes are acetylated while those bound to nucleosome-organized gene copies are not.

Acetylation of histones bound to rat rRNA genes has been studied relative to their organization in chromatin, either as canonical nucleosomes, containing the inactive copies, or as anucleosomal nonrepeating structures, corresponding to the transcribed genes (Conconi, A., Widmer, R. M., Koller, T., and Sogo, J. M. (1989) Cell 57, 753-761). Nuclei from butyrate-treated rat tumor cells were irradiated with a UV laser to cross-link proteins to DNA, and the purified covalent complexes were immunofractionated by an antibody that specifically recognized the acetylated histones. Upon probing with sequences coding for mature rat 28 S RNA, DNA of the antibody-bound complexes was 5-20-fold enriched relative to the total rat DNA. Since the laser cross-links histones to DNA in both active and inactive genes, one cannot distinguish which one of them, or both, are bound to acetylated histones. Alternatively, purified mononucleosomes were immunofractionated, but DNA from the antibody-bound monosomes was not enriched in coding rDNA. Taken together, these results suggest that nucleosome-organized rRNA genes are bound to nonmodified histones and that the acetylated histones are associated with the active, anucleosomal gene copies.

Acetylation↗

PubMed1997

Nucleosomal organization of the rat liver satellite DNA-containing chromatin during aging.

Nucleosomal organization of the satellite DNA-containing chromatin of the liver of young (18 +/- 2 weeks) and old (100 +/- 5 weeks) rats was examined by nucleases and satellite I DNA probe. The satellite DNA-containing chromatin exhibits lower accessibility to endogenous endonucleases in old rats. The nucleosomal repeat length of this chromatin as investigated from digestion with endogenous endonucleases and MNase differs remarkably from bulk chromatin, though it does not alter with age. However, age-dependent loss of satellite DNA is apparent from hybridization results. Furthermore, DNase I analysis of the satellite DNA-containing chromatin at nucleosomal level reveals a relatively loose organization in young rats than old ones. It also shows an altered 10 bp periodicity as compared to bulk chromatin in both ages. These findings establish organizational differences between rat liver bulk and satellite DNA-containing chromatin. They further show that repeat length and altered 10 bp periodicity are similar in young and old, but accessibility to nucleases declines with age.

Aging↗

PubMed1998

DNA superstructural features and nucleosomal organization of the two centromeres of Kluyveromyces lactis chromosome 1 and Saccharomyces cerevisiae chromosome 6.

Superstructural features of the Kluyveromyces lactis chromosome 1 (KlCEN1) and of the Saccharomyces cerevisiae chromosome 6 (SCEN6) centromeric DNAs were evaluated using a theoretical method, developed by our group, and experimentally measured by gel electrophoretic retardation. Both methods show that, in spite of the remarkable AT richness of the two centromeric sequences, their curvature is not very high. However the peculiar sequence features of the two centromeres allow to organize highly stable nucleosomes, with a free energy about that of the nucleosome formed on the 5S RNA gene. The good agreement between experimental and theoretical evaluation of nucleosome free energies as well as of their multiple positioning shows that in centromeres both DNA curvature and flexibility are relevant in determining nucleosomal features.

Centromere↗

PubMed1987

Nucleosomal organization of a BPV minichromosome containing a human H4 histone gene.

To address the relationship between chromatin structure and histone gene expression, the nucleosomal organization of a cell cycle-dependent human H4 histone gene in a bovine papilloma virus (BPV) minichromosome was examined. The nucleosome repeat length of the human H4 histone gene, maintained as a stable episome in a C127 mouse cell line designated I-8, was compared with that of the chromosomal copy of the H4 gene in human (HeLa) cells. In both cell lines, the H4 histone gene is predominantly expressed during the S phase of the cell cycle. The nucleosome repeat length of total HeLa cell and C127 mouse cell chromatin was similarly examined. Nuclei were digested with micrococcal nuclease and the DNA was fractionated electrophoretically, transferred to nitrocellulose filters and hybridized with radiolabelled (32P) cloned DNA probes. The nucleosome repeat length of the H4 gene, as an episome in the C127 mouse cell (153 +/- 8) and as an integrated copy in a HeLa cell (163 +/- 10) was considerably shorter than total genomic host cell (C127) (190 +/- 5) or HeLa cell chromatin (183 +/- 7). Our results indicate that the episomal H4 histone gene is packaged as chromatin. Moreover, the shortened nucleosome repeat length of the H4 gene, both as an episome or integrated chromosome sequence, suggests that the repeat length is characteristic of the gene and may be functionally related to its cell cycle regulated expression.

Animals↗

PubMed2026

Maintenance of nucleosome organization through replication and transcription counteracts aberrant coalescence of active chromatin.

Nucleosomes with their associated modifications organize and regulate the genome. It is unclear how this is integrated with the requirement of replication and transcription to access the DNA template without jeopardizing chromatin function. Here, we reveal a unified requirement for the histone chaperone FACT in mediating nucleosome disruption and reassembly during mammalian replication and transcription. Upon acute FACT depletion, replisome and RNA polymerase progression is halted genome wide, and chromatin structure in their wake collapses, with reduced nucleosome occupancy, irregular spacing, and intermediate assemblies. Chromatin states deteriorate as modified histones are lost due to a lack of histone recycling. Chromatin fiber disorder further manifests in the 3D genome, triggering active genes to coalesce in aberrant microcompartments. Similarly, aberrant compartments form in cells failing to maintain chromatin fiber structure through replication. Nucleosome organization therefore dynamically regulates genome architecture, guarding against spurious chromatin aggregation.

Nucleosomes↗

PubMedDate not supplied

[Nucleosomal organization of chromatin from sperm of the bivalve mollusk Swiftopecten swifti].

Composition of basic chromosomal proteins from sperm of the bivalve mollusc Swiftopecten swifti is specific: (1) somatic histone H1 is replaced for a sperm-specific one containing three subfractions, enriched in Arg; (2) low molecular weight sperm-specific basic protein (S protein) is present in the chromatin additionally to the histones. It is shown that chromatin from the mollusc sperm has typical nucleosomal organization, however the DNA repeat length is markedly increased (225 bp) on account of the linker region of the nucleosome. It is found using two-dimensional electrophoresis that fraction of the mononucleosomes is heterogeneous and represented by four electrophoretic subfractions which differ in protein composition and length of DNA they contain. Subfraction which differ in protein composition and length of DNA they contain. Subfractions of mononucleosomes in the order of increase of electrophoretic mobility contain along with core histones: (1)--all H1 subfractions, protein X, protein S; (2)--subfraction H1"', protein X, protein S; (3)--protein S. The particles containing only core histones correspond to the most rapidly migrating band. The data show that protein S is, like histone H1, possibly bound to the linker DNA. Interdependence between the presence of sperm-specific non-core (-linker?) proteins, the increase in size of linker DNA and compaction of sperm chromatin is suggested.

Amino Acids↗

PubMed1981

Detailed analysis of the nucleosomal organization of transcribed DNA in yeast chromatin.

The precise chromatin structure of actively transcribed DNA in yeast has been analyzed by electrophoretic transfer of high-resolution staphylococcal nuclease and DNase I chromatin digest DNA patterns to DBM paper and hybridization with active sequence probes. The DNA patterns of the transcribed DNA sequences resemble the DNA patterns produced by digestion of bulk yeast nucleosomes. Hence, these active sequences must be arranged in "typical" nucleosome structures. Furthermore, in details of the structure, the active sequence nucleosomes look almost exactly like the average yeast nucleosome in repeat length, in the length of DNA associated with the core particle, in the amount and type of heterogeneity found within and between the oligomeric and monomeric repeat lengths of DNA, in the occurrence of discrete spacer lengths including the characteristic five nucleotide increments (i.e., 5, 15, 25, ... base pairs), and in the length of DNA between yeast nucleosomes. Early in digestion, there are some differences: increases in peak breadths (i.e., in the distribution of spacer lengths) and some preferential release of monomer DNA. These results suggest that transcribed DNA can exist in the typical (yeast) type of nucleosome organization and thus that active chromatin regions do not necessarily require profound structural rearrangements. The slight differences noted are consistent with some slight, mainly spacer, modification in the vicinity of the transcription event itself.

Alkaline Phosphatase↗

PubMed2005

Correlation between alterations in nucleosomal organization of LINEs in the promoter of cytochrome P450 2B1/2 gene and induction of CYP 2B1/2B2 mRNA expression by phenobarbitone in rat liver.

Mammalian genome contains a high proportion of repetitive DNA with a sizeable fraction of Long Interspersed Nuclear Elements (LINEs). LINEs have been functionally implicated in the organization and evolution of mammalian genome. However, functions of LINEs in the promoter region and gene expression are poorly understood. Here, we report two small, conserved LINE sequences (3P and 5P) that occur as multiple copies of inverted repeats in the rat Cytochrome P450 2B1/2B2 (CYP 2B1/2) gene promoter. Using 3P or 5P as a single primer, the CYP 2B1/2 promoter DNA was amplified by PCR from the rat genome. Phenobarbitone (PB), a prototype xenobiotic drug, strongly induced CYP 2B1/2 mRNA expression in the rat liver. 3P and 5P LINE sequences showed an alteration in the nucleosomal organization in the CYP 2B1/2 promoter after 2, 4, and 6 h of PB induction, and the promoter was mostly devoid of nucleosomes during the induction. Reorganization of nucleosomes associated with these LINE sequences were strongly correlated with induction of CYP 2B1/2 mRNA expression by PB in vivo. Our results strongly suggest that these LINE sequences in CYP 2B1/2 gene promoter(s) may facilitate transcriptional activation of the gene(s) by PB through retention and reorganization of nucleosomes. This might be a novel function of LINEs in the mammalian genome that correlates the chromatin structure with gene expression during drug metabolism.

Animals↗

PubMed2002

Nucleosome organization on Kluyveromyces lactis centromeric DNAs.

The preferential assembly of specialized nucleosomes on budding yeast centromeres can be due either to the higher stability of specialized centromeric nucleosomes and/or to the lower stability of canonical centromeric nucleosomes with respect to bulk nucleosomes. We have evaluated the thermodynamic stability of canonical nucleosomes, assembled on Kluyveromyces lactis centromeric DNAs, with a competitive reconstitution assay and a theoretical method recently developed by us. The results, obtained by both methods, show that all five known centromeric DNAs from K. lactis are able to organize canonical nucleosomes, characterized by higher stability with respect those of bulk DNA. With 'footprinting' and theoretical prediction, based on sequence-dependent DNA elasticity, we have found that centromeric canonical nucleosomes are characterized by nucleosome dyad axis multiple positioning, rotationally phased. The isoenergetic nucleosome multiple positions are relevant in understanding the transition from canonical to specialized nucleosomes in interacting with centromere protein complexes. The satisfactory agreement between the results obtained from theoretical and experimental methods shows that sequence-dependent centromeric DNA elasticity has a main role in nucleosome thermodynamic stability and positioning.

Centromere↗

PubMed1991

Influence of DNA topology and histone tails in nucleosome organization on pBR322 DNA.

Recently, we have found that the assembly of nucleosomes reconstituted on negatively supercoiled DNA is cooperative. In the present paper the role of DNA topology and of histone tails in nucleosome assembly was explored. Reconstituted minichromosomes on relaxed DNA at different histone/DNA ratios (R) were assayed by topological analysis and electron microscopy visualization. Both methods show a linear relationship between average nucleosome number (N) and R. This suggests that in the case of relaxed DNA, cooperative internucleosomal interactions are small or absent. The influence of histone tails in nucleosome assembly was studied on minichromosomes reconstituted with trypsinized histone octamer on negatively supercoiled DNA by topological analysis. The topoisomers distribution, after trypsinization, dramatically changes, indicating that nucleosome-nucleosome interactions are remarkably decreased. These results show that, in chromatin folding, in addition to the well known role of histone H1, the interactions between histone octamer tails and DNA are also of importance.

Chromatin↗

PubMed1997

DNA recognition and nucleosome organization.

The affinity of a DNA sequence for the histone octamer in a core nucleosome depends on the intrinsic flexibility of the DNA. This parameter can be affected both by the sequence-dependent conformational preferences of individual base steps and by the nature and location of the exocyclic groups of the DNA bases. By adopting highly preferred conformations particular types of base step can influence the rotational positioning of the DNA on the surface of the histone octamer. The asymmetry of the next higher order of chromatin structure is determined in part by the asymmetric binding of the globular domain of histone H5 to the core nucleosome.

Binding Sites↗

PubMed1984

Nucleosomal organization of a part of chromatin in mollusc sperm nuclei with a mixed basic protein composition.

The structural organization of mature sperm chromatin from three representatives of the Mytilidae family has been studied. The acid-soluble proteins in these species nuclei are primarily sperm-specific (approximately 80%) with the remainder being core histones. Previously, we have shown that the mature sperm nuclei of these molluscs are compact, dense structures formed by interaction of the spermspecific proteins with DNA (1). Here we show that: a) although the histones are minor chromatin protein fraction, they still organize a part (20-25%) of the total DNA into nucleosomes; b) one of the sperm-specific proteins, different from somatic H1 or H5 histones participates in the formation of the beaded structures.

Animals↗

PubMed2005

Patterns of nucleosomal organization in the alc regulon of Aspergillus nidulans: roles of the AlcR transcriptional activator and the CreA global repressor.

We have studied the chromatin organization of three promoters of the alc regulon of Aspergillus nidulans. No positioned nucleosomes are seen in the aldA (aldehyde dehydrogenase) promoter under any physiological condition tested by us. In the alcA (alcohol dehydrogenase I) and alcR (coding for the pathway-specific transcription factor) promoters, a pattern of positioned nucleosomes is seen under non-induced and non-induced repressed conditions. While each of these promoters shows a specific pattern of chromatin restructuring, in both cases induction results in loss of nucleosome positioning. Glucose repression in the presence of inducer results in a specific pattern of partial positioning in the alcA and alcR promoters. Loss of nucleosome positioning depends absolutely on the AlcR protein and it is very unlikely to be a passive result of the induction of transcription. In an alcR loss-of-function background and in strains carrying mutations of the respective AlcR binding sites of the alcA and alcR promoters, nucleosomes are fully positioned under all growth conditions. Analysis of mutant AlcR proteins establishes that all domains needed for transcriptional activation and chromatin restructuring are included within the first 241 residues. The results suggest a two-step process, one step resulting in chromatin restructuring, a second one in transcriptional activation. Partial positioning upon glucose repression shows a specific pattern that depends on the CreA global repressor. An alcR loss-of-function mutation is epistatic to a creA loss-of-function mutation, showing that AlcR does not act by negating a nucleosome positioning activity of CreA.

Aspergillus nidulans↗

PubMed1996

The affinity of nuclear factor 1 for its DNA site is drastically reduced by nucleosome organization irrespective of its rotational or translational position.

A DNA-bending sequence has been used for in vitro reconstitution of nucleosomes in order to direct a nuclear factor 1 (NF-1) binding site into different nucleosome positions. By this strategy nucleosomes were obtained that had one of two rotational positions of the NF-1 binding site, one oriented toward the periphery and the other toward the histone octamer, translationally positioned 50 and 45 base pairs, respectively, from the nucleosome dyad. The affinity of partially purified NF-1 for these nucleosomal targets was compared with its affinity for free DNA by dimethylsulfate methylation protection and DNase I footprinting assays. The binding affinity of NF-1 to all nucleosomal targets was reduced 100-300-fold compared with its affinity for free DNA. The two rotational settings of the NF-1 site showed the same binding affinity for NF-1 as did other nucleosome constructs in which the NF-1 binding site was translationally positioned from 10 to 40 base pairs from the nucleosome dyad. We conclude that the nucleosomal inhibition of NF-1 binding is an inherent characteristic of NF-1 since another transcription factor, the glucocorticoid receptor, is able to bind to its DNA site in a nucleosome.

Base Sequence↗