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Results for “Chromatin loop”

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PubMed1999

Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances.

The probability that two sites on a linear DNA molecule will contact each other by looping depends on DNA flexibility. Although the flexibility of naked DNA in vitro is well characterized, looping in chromatin is poorly understood. By extending existing theory, we present a single equation that describes DNA looping over all distances. We also show that DNA looping in vitro can be measured accurately by FLP recombination between sites from 74 bp to 15 kb apart. In agreement with previous work, a persistence length of 50 nm was determined. FLP recombination of the same substrates in mammalian cells showed that chromatin increases the flexibility of DNA at short distances, giving an apparent persistence length of 27 nm.

Base Sequence↗
PubMed2004

Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription.

The colinearity of genes in Hox clusters suggests a role for chromosome structure in gene regulation. We reveal programmed changes in chromatin structure and nuclear organization upon induction of Hoxb expression by retinoic acid. There is an early increase in the histone modifications that are marks of active chromatin at both the early expressed gene Hoxb1, and also at Hoxb9 that is not expressed until much later. There is also a visible decondensation of the chromatin between Hoxb1 and Hoxb9 at this early stage. However, a further change in higher-order chromatin structure, looping out of genes from the chromosome territory, occurs in synchrony with the execution of the gene expression program. We suggest that higher-order chromatin structure regulates the expression of the HoxB cluster at several levels. Locus-wide changes in chromatin structure (histone modification and chromatin decondensation) may establish a transcriptionally poised state but are not sufficient for the temporal program of gene expression. The choreographed looping out of decondensed chromatin from chromosome territories may then allow for activation of high levels of transcription from the sequence of genes along the cluster.

Animals↗
PubMed2001

Chromatin remodelling and DNA replication: from nucleosomes to loop domains.

Organization of DNA into chromatin is likely to participate in the control of the timing and selection of DNA replication origins. Reorganization of the chromatin is carried out by chromatin remodelling machines, which may affect the choice of replication origins and efficiency of replication. Replication itself causes a profound rearrangement in the chromatin structure, from nucleosomes to DNA loop domains, allowing to retain or switch an epigenetic state. The present review considers the effects of chromatin remodelling on replication and vice versa.

Animals↗
PubMed1995

The nuclear matrix and the regulation of chromatin organization and function.

Nuclear DNA is organized into loop domains, with the base of the loop being bound to the nuclear matrix. Loops with transcriptionally active and/or potentially active genes have a DNase I-sensitive chromatin structure, while repressed chromatin loops have a condensed configuration that is essentially invisible to the transcription machinery. Core histone acetylation and torsional stress appear to be responsible for the generation and/or maintenance of the open potentially active chromatin loops. The transcriptionally active region of the loop makes several dynamic attachments with the nuclear matrix and is associated with core histones that are dynamically acetylated. Histone acetyltransferase and deacetylase, which catalyze this rapid acetylation and deacetylation, are bound to the nuclear matrix. Several transcription factors are components of the nuclear matrix. Histone acetyltransferase, deacetylase, and transcription factors may contribute to the dynamic attachment of the active chromatin domains with the nuclear matrix at sites of ongoing transcription.

Amino Acid Sequence↗
PubMed2004

[Dynamics of structural changes of Paramecium caudatum and Bursaria truncatella macronuclear chromatin and nucleoli under hypotonic conditions].

Dynamics of structural changes of nucleoli, complex nucleolar aggregates and chromatin bodies in macronuclei (Ma) of ciliates Paramecium candatum and Bursaria truncatella under hypotonic conditions was studied. It was shown that after a 3 min hypotonic treatment nuclei swelled and became highly vacuolated. 3D-reconstruction showed that such nucleoli were formed by nucleolonema-like threads about 100-200 nm in thickness. Intranucleolar chromatin bodies decompacted, but remained bound with fibrillar component of the nucleolus by thin fibres about 10 nm thick. After 6 min hypotonic treatment the nucleolar material loosened and had a "gauze", or network-like appearence. After 10 min hypotonic treatment nucleoli dissociated completely. It was shown that a transition of chromatin bodies from completely compact to partially and fully decompacted state occurred cooperatively in different regions of Ma. In particular, chromatin bodies in the central part of complex nucleolar aggregates decompacted much faster than those in the Ma karyoplasm. It evidences for a specific, well-ordered chromatin organization in Ma. Prolonged hypotonic treatment led to a complete dissociation of Ma components; fibres 6-10 nm thick were solely observed in such preparations. Such fibres may represent remnant structures of the nuclear matrix. Dynamics of Ma chromatin bodies decompaction correlates well with that of chromomeres in the nuclei of higher eukaryotes. Our data confirm that chromatin 100-200 nm bodies in the ciliate Ma are analogues of chromomeres--looped discrete chromatin domains, observed in the nuclei of higher eukaryotes.

Animals↗
PubMed1998

Disassembly of genome of higher eukaryotes: pulsed-field gel electrophoretic study of initial stages of chromatin and DNA degradation in rat liver and thymus nuclei by VM-26 and selected proteases.

Nuclei were treated with VM-26, topoisomerase II inhibitor, or exogenous proteases. Initial stages of multi-step chromatin degradation were analyzed by pulsed-field gel (PFG) electrophoresis. VM-26 induced the total genome breakdown into discrete chromatin fragments containing 0.3 Mbp DNA. Treatment of nuclei with recombinant cathepsin B and chymotrypsin also revealed 0.3-Mbp DNA fragments which were mediated by endonucleolysis. Longer incubation of nuclei with chymotrypsin exhibited the appearance of 0.05-Mbp DNA fragments and their oligomers. Proteases might trigger the detachment of chromatin from nuclear matrix and contribute to the release or activation of the endonucleolytic activity leading to the initial degradation of the DNA into high-molecular weight fragments. The fragments observed correspond in size to the suggested chromatin higher-order structures. The presented data imply that the initial stages of chromatin degradation represents in reality the genome disassembly into two basic units of genome topology--0.3-Mbp DNA chromatin loop-domains which are presumably the bits of biological information, and fundamental 0.05-Mbp DNA loopsize units which might generate the functional loops of different sizes.

Animals↗
PubMed1993

Interchange and intra-nuclear architecture.

For chromatin lesions to interact to form exchanges of any sort, it is obvious that contact between them must be made. However, the probability of such interaction is conditioned by other factors like time, initial separation, metabolic activity, and, in the case of chemically induced lesions, scheduled DNA synthesis. The irradiated nucleus was, for a long time, regarded as a "bag of broken chromosomes" with the severed ends free to move around and find partners with which to form illegitimate reunions. Many of these would be seen at following metaphase as intra- and interchanges. Evidence is rapidly accumulating which indicates that this picture of the nucleus is false. We know now that chromosomes occupy highly localised domains with limited movement, and that there is no massive intermingling; that much of the chromatin is compacted and splinted with proteins and so precluded from exchange-type contact; that most of the chromatin is looped and "fixed" into an intra-nuclear protein scaffold or skeleton; that some chromatin is spun-out and associated with the nuclear envelope in the vicinity of the pore-complexes. Thus it would appear that movement, in the sense envisaged by early workers, is curtailed, and that only a proportion (probably a small proportion) of the chromatin is actually "at-risk" with respect to interchange formation. Where then does interchange take place? Are the "sites" pre-existent, or can proximity requirements be realised after radiation exposure? In what ways will the intra-nuclear architecture influence exchange? These are some of the questions which are considered in this paper.

Animals↗
PubMed1985

Nuclear volume and chromatin organization in some radiosensitive and radioresistant mammalian cells.

Nuclear dimensions in mammalian cells appear as a determining factor of chromatin organization and cellular radiosensitivity. Most radioresistant interphase cells have a nuclear volume (Vn) of 75 to 2700 micron 3, that could allow both the topological organization of chromatin as loops attached to the inner surface of the nuclear envelope and the unfolding of condensed chromatin within the topological constraints existing along the DNA molecule. In contrast, the radiosensitive small lymphocytes, with Vn values of 20 to 65 micron 3, seem to comprise significant amounts of highly condensed chromatin and dispose of an uncompleted topological organization of DNA, which may cause their incapacity to perform replication and transcription of DNA as well as the repair of radiation damage at a cell level. The indications are that radiosensitivity (1/D37) of animal cells, containing a similar quantity of DNA, should be directly proportional to 1/nuclear volume (1/Vn). However, DNA is unevenly distributed within the nuclear space, according to a partial ordering of interphase chromosomes; and it appears that radiosensitivity increases in zones of high DNA or chromatin density.

Animals↗
PubMed1980

Folding up genes and chromosomes.

The basic chromatin fiber is composed of a tandem array of nucleosomes, adjacent nucleosomes being separated by a variable amount of DNA. In vivo, this fiber is folded to form a higher-order fiber with a diameter of 200 to 300 A. The distribution of nucleosomes within the higher-order fiber has been analyzed in negatively stained preparations of chromatin fibers released from metaphase cells or interphase nuclei by mechanical disruption. The fiber appears to be composed to discrete packing domains in each of which there is a high density of closely apposed nucleosomes; within these regions several discrete packing patterns are observed. Adjacent domains are connected by regions of the fiber which are thinner and contain a lower density of nucleosomes. From these observations we suggest that the 200 to 300 A fiber is mosaic and that domains differ from each other in composition so as to confer sequence specificity upon the structure. In addition, compositional differences may have relevance to the functional state of the underlying genes; thus, higher order structure of a region is related to its functional state. Above this level of organization, studies on the early stages of chromatin folding in meiotic prophase suggest that 200 to 300 A fibers are organized as loops which emanate from the longitudinal axis of the chromosome, in a manner consistent with a model for mitotic chromosome folding by means of radial loop formation. Active ribosomal RNA genes are identifiable in these preparations and appear to be organized in a similar fashion; each active gene appears to comprise a loop of chromatin.

Animals↗
PubMed1990

Tightly-bound form of poly(ADP-ribose)polymerase in the higher order of chromatin organization.

A tightly-bound form of poly(ADP-ribose)polymerase is present, within the third level of rat testis chromatin structure, both in the loops and in chromatin matrix. When chromatin matrix was extensively digested with DNAaseI, only little residual enzymatic activity remained in the insoluble fraction, the extent of DNA hydrolysis being well correlated to the progressive loss of the poly(ADP-ribose)polymerase activity. These findings suggest that the tightly-bound form of the enzyme is not an intrinsic protein component of chromatin matrix but is only indirectly located in this structure, being rather associated to the attachment points of loop DNA on the matrix.

Animals↗
PubMed1993

Scanning electron microscopy of the centromeric region of L-cell chromosomes after treatment with Hoechst 33258 combined with 5-bromodeoxyuridine.

When mouse L-cells were treated with a combination of 5-bromodeoxyuridine (BrdUrd) and Hoechst 33258, the metaphase chromosomes revealed under-condensation of the chromatin fibers in the sister centromeres. The application of the osmium-thiocarbohydrazide technique to the air-dried chromosome preparations made it possible to elucidate the ultrastructure of the under-condensed centromeric region at the level of the 30 nm chromatin fiber. Scanning electron microscopy revealed that the under-condensed region consisted of a coiled fiber with a diameter of about 400 nm, and a gyre diameter of approximately 600 nm. The coiled fiber was composed of the 30 nm chromatin fiber loops. These findings indicate that a continuous coiled structure, which is the final higher order structure of the condensed chromatin fiber, exists throughout the entire length of the mouse L-cell metaphase chromosome.

Animals↗
PubMed1981

Structure of the mammalian kinetochore.

The structure of the mammalian trilaminar kinetochore was investigated using stereo electron microscopy of chromosomes in hypotonic solutions which unraveled the chromosome but maintained microtubules. Mouse and Chinese hamster ovary cells were arrested in Colcemid and allowed to reform microtubules after Colcemid was removed. Recovered cells were then swelled, lysed or spread in hypotonic solutions which contained D2O to preserve microtubules. The chromosomes were observed in thin and thick sections and as whole mounts using high voltage electron microscopy. Bundles of microtubules were seen directly attached to chromatin, indicating that the kinetochore outer layer represents a differential arrangement of chromatin, continuous with the body of the chromosome. In cells fixed wihout pretreatment, the outer layer could be seen to be composed of hairpin loops of chromatin stacked together to form a solid layer. The hypotonically-induced unraveling of the outer layer was found to be reversible, and the typical 300 nm thick disk reformed when cells were returned to isotonic solutions. Short microtubules, newly nucleated after Colcemid removal, were found not to be attached to the kinetochore out layer, but were situated in the fibrous corona on the external surface of the outer layer. This was verified by observation of thick sections in stereo which made it possible to identify microtubules ends within the section. Thus, kinetochore microtubules are nucleated within the fibrous corona, and subsequently become attached to the outer layer.

Animals↗
PubMed2005

Whole-genome views of chromatin structure.

DNA in eukaryotes is packed into chromatin. The basic component of chromatin is the nucleosome consisting of DNA wrapped around a histone octamer. Inside the cell nucleus, chromatin is folded into higher-order structures through various mechanisms, including repositioning of nucleosomes along the DNA, packing of nucleosomes into more condensed 3-dimensional configurations, looping of chromatin fibres, and tethering of chromosomal regions to nuclear structures. Over the past few years, new microarray-based methods have been developed for the genome-wide mapping of various aspects of chromatin structure. These methods are beginning to provide insights into the different types of chromatin and the architectural principles that govern the 3-dimensional organisation of the genome inside the nucleus.

Cell Nucleus↗
PubMed1998

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.

DNA↗
PubMed1993

Metaphase chromosome and nucleoid differences between CHO-K1 and its radiosensitive derivative xrs-5.

The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive mutant isolated from CHO-K1 cells. The radiation sensitivity is associated with a defect in DNA double-strand break rejoining. Chromatin structure also appears altered in xrs-5 cells compared with the parental CHO-K1 cells. Metaphase chromosomes from xrs-5 are more condensed in appearance than CHO-K1 chromosomes. The overcondensed look is not the result of colcemid sensitivity. Electron microscopy studies suggest that xrs-5 metaphase chromosomes have larger loops of chromatin extending out from the chromosome core. There are also differences between CHO-K1 and xrs-5 cells in the size and fluorescence pattern of ethidium bromide-stained nucleoid preparations. These results suggest that there is a fundamental difference between CHO-K1 and xrs-5 in either the organization of the supercoiled loops of DNA attached to the nuclear matrix or in the nature of the proteins that attach the DNA to the matrix. These alterations in chromosome structure may underlie, in part, the radiation sensitivity of xrs-5 cells.

Animals↗
PubMed1985

[Effect of the supranucleosomal chromatin organization on histone-DNA interrelations].

It has been demonstrated by the method of competitive displacement of own chromatin histone by excess total histone that chromatin dispersity influence the strength of histone-DNA interactions in a medium of physiological ionic strength. Histone NI was removed from chromatin after the quantity of total histone added to chromatin was equivalent to that existing in chromatin. The proportion of histones H2A and H2B removed from chromatin was increased after mechanical of ultrasonic degradation of chromatin at 5-20-fold excess of total extra-histone. In some histone preparations, the removal of histones H2A and H2B was not detectable at even 200-fold excess of total histone. This may be explained by strengthening histone-DNA interactions in superhelical loops of chromatin.

Animals↗
PubMed2001

DNA looping in the RNA polymerase I enhancesome is the result of non-cooperative in-phase bending by two UBF molecules.

The so-called upstream binding factor (UBF) is required for the initial step in formation of an RNA polymerase I initiation complex. This function of UBF correlates with its ability to induce the ribosomal enhancesome, a structure which resembles in its mass and DNA content the nucleosome of chromatin. DNA looping in the enhancesome is probably the result of six in-phase bends induced by the HMG boxes of a UBF dimer. Here we show that insertion/deletion mutations in the basic peptide linker lying between the N-terminal dimerisation domain and the first HMG box of Xenopus UBF prevent the DNA looping characteristic of the enhancesome. Using these mutants we demonstrate that (i) the enhancesome structure does not depend on tethering of the entering and exiting DNA duplexes, (ii) UBF monomers induce hemi-enhancesomes, bending the DNA by 175 +/- 24 degrees and (iii) two hemi-enhancesomes are precisely phased by UBF dimerisation. We use this and previous data to refine the existing enhancesome model and show that HMG boxes 1 and 2 of UBF lie head-to-head along the DNA.

Amino Acid Motifs↗
PubMed2002

SATB1 targets chromatin remodelling to regulate genes over long distances.

Eukaryotic chromosomes are organized inside the nucleus in such a way that only a subset of the genome is expressed in any given cell type, but the details of this organization are largely unknown. SATB1 ('special AT-rich sequence binding 1'), a protein found predominantly in thymocytes, regulates genes by folding chromatin into loop domains, tethering specialized DNA elements to an SATB1 network structure. Ablation of SATB1 by gene targeting results in temporal and spatial mis-expression of numerous genes and arrested T-cell development, suggesting that SATB1 is a cell-type specific global gene regulator. Here we show that SATB1 targets chromatin remodelling to the IL-2Ralpha ('interleukin-2 receptor alpha') gene, which is ectopically transcribed in SATB1 null thymocytes. SATB1 recruits the histone deacetylase contained in the NURD chromatin remodelling complex to a SATB1-bound site in the IL-2Ralpha locus, and mediates the specific deacetylation of histones in a large domain within the locus. SATB1 also targets ACF1 and ISWI, subunits of CHRAC and ACF nucleosome mobilizing complexes, to this specific site and regulates nucleosome positioning over seven kilobases. SATB1 defines a class of transcriptional regulators that function as a 'landing platform' for several chromatin remodelling enzymes and hence regulate large chromatin domains.

Binding Sites↗