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At least 253 records · Page 14Linked to original sources
PubMed1994

Radiation sensitivity correlates with changes in DNA supercoiling and nucleoid protein content in cells of three Chinese hamster cell lines.

We have investigated the composition of nuclear matrix proteins and DNA supercoiling characteristics of cell lines expressing altered radiation sensitivity. Chinese hamster ovary cell lines 4364 (wild-type), XR-1 (DSB repair-deficient, radiosensitive) and XR-122 (a radioresistant variant of XR-1 bearing human chromosome 5) were used as a model to study the relationship between intrinsic radiation sensitivity and the level of DNA supercoiling ability within chromatin loops and the composition of nuclear matrix proteins. Analysis of the ability of DNA loop domains to undergo changes in DNA supercoiling in the presence of DNA damage revealed that the degree of inhibition of loop rewinding was greater in the radiation-sensitive cells (XR-1) compared to the radiation-resistant cells (4364 and XR-122). Furthermore, the loop-rewinding characteristics correlated inversely with the clonogenic survival of these cells after exposure to ionizing radiation. Since DNA loops are anchored to the nuclear matrix by protein-DNA anchor points, a study of the nuclear matrix proteins by high-resolution 2D-PAGE was conducted for these cells to determine whether differential inhibition of loop rewinding could be due to differences in the DNA loop-protein anchor points in these cells. The XR-1 cells showed an overall absence of 13 proteins compared to the 4364 cells. Of these 13, 5 were restored in XR-122 cells. These results are consistent with the hypothesis that stability of the DNA loop domains in the presence of DNA damage contributes to the expression of potentially lethal damage by ionizing radiation.

Animals↗
PubMedDate not supplied

[Structure of the interphase chromatin in the ciliata Bursaria truncatella macronucleus. II. Loop organization of inactive chromatin clumps].

Electron microscopic study of chromatin organization in isolated macronuclei of a ciliate Bursaria truncatella showed macronuclear chromatin to be organized in compact clumps 120--180 nm in diameter linked with each other by one or several chromatin fibres. Macronucleus being dispersed in a solution of low ionic strength, radial loops basically of nucleosomal structure start appearing around chromatin clumps. Long-time dispersing of macronuclear chromatin brings complete decompactization of chromatin clumps into a set of nucleosome fibres. The way the fibres of interphase chromatin are packed in a chromatin clump is discussed.

Cell Nucleus↗
PubMed1983

The structure of inactive interphase macronuclear chromatin of the ciliate Bursaria truncatella. Radial loops in the structure of chromatin clumps.

The organization of chromatin in macronuclei of Bursaria truncatella cells that completed their growth and differentiation was electron microscopically studied. The data obtained showed that (1) inactive macronuclear chromatin was organized in compact chromatin clumps 120 to 180 nm in diameter linked by one or several chromatin fibres, and (2) in low salt buffer the chromatin clumps gradually unraveled, radial loops of supranucleosomal or, more often, nucleosomal structure appearing around chromatin clumps. Upon prolonged incubation in low salt buffer chromatin clumps were completely transformed into nucleosomal fibres. The data obtained evidenced in favour of a loop-packed structure of chromatin clumps.

Animals↗
PubMed1984

Structural organization of macronuclear chromatin of the ciliate Bursaria truncatella in resting cysts and at excysting.

The structural studies of macronuclear chromatin of the ciliate Bursaria truncatella showed that in resting cysts practically all macronuclear chromatin was arranged in compact clumps 100 to 300 nm in size. By means of negative staining the internal structure of chromatin clumps was revealed; the chromatin clumps were shown to consist of subunits corresponding in size to nucleosomes. Upon incubation in low salt buffer the chromatin clumps decondensed forming loop-shaped chromatin fibres suggesting a similar structural organization of both the chromatin clumps of cysts and the chromatin clumps of grown and differentiated Bursaria truncatella cells [8, 9, 16]. We established that at excysting there occurred natural decondensation of the chromatin clumps resulting in loop-shaped chromatin fibres around the clumps. This in all probability was due to activation of macronuclear chromatin. Sometimes short transcription units of nonribosomal genes (0,2-1,3 micron in contour length) with closely located RNP-fibrils (more than 20 fibrils per 1 micron) were observed on the loops. We suggest that such genes respond to the synthesis of proteins involved in regulation of excysting and/ or subsequent cell growth and differentiation.

Animals↗
PubMed1999

Single-strand breaks in agarose-embedded chromatin of nonapoptotic cells.

Loop-size chromatin fragmentation frequently observed upon apoptotic cell death is thought to be initiated by ss nicks. Here we show that the agarose-embedded, deproteinized chromatin of normal, non-apoptotic murine and human cells, as well as yeast protoplasts, falls apart to 50-300 kb ss fragments upon heat denaturation, as revealed by urea-TAE field-inversion agarose gel electrophoresis resolving ss and ds fragments alike. These data were in line with S1digestion experiments. The nicks (gaps) observed are best explained either by enzymatic cleavages occurring upon cell lysis instantaneously or by preexisting discontinuities becoming manifest upon heat denaturation. These discontinuities go unnoticed in the usual nondenaturaing circumstances but seem to be inevitably present in any DNA preparation. The loop-size ds DNA fragmentation in apoptosis may be based on these pre-existing or "ready-to-go" (upon cell lysis) ss discontinuities of the normal cellular chromatin.

Animals↗
PubMed2006

X-ray crystal structure of MENT: evidence for functional loop-sheet polymers in chromatin condensation.

Most serpins are associated with protease inhibition, and their ability to form loop-sheet polymers is linked to conformational disease and the human serpinopathies. Here we describe the structural and functional dissection of how a unique serpin, the non-histone architectural protein, MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein), participates in DNA and chromatin condensation. Our data suggest that MENT contains at least two distinct DNA-binding sites, consistent with its simultaneous binding to the two closely juxtaposed linker DNA segments on a nucleosome. Remarkably, our studies suggest that the reactive centre loop, a region of the MENT molecule essential for chromatin bridging in vivo and in vitro, is able to mediate formation of a loop-sheet oligomer. These data provide mechanistic insight into chromatin compaction by a non-histone architectural protein and suggest how the structural plasticity of serpins has adapted to mediate physiological, rather than pathogenic, loop-sheet linkages.

Animals↗
PubMed2005

Gene activation and deactivation related changes in the three-dimensional structure of chromatin.

Chromatin in the interphase nucleus is dynamic, decondensing where genes are activated and condensing where they are silenced. Local chromatin remodelling to a more open structure during gene activation is followed by changes in nucleosome distribution through the action of the transcriptional machinery. This leads to chromatin expansion and looping out of whole genomic regions. Such chromatin loops can extend beyond the chromosome territory. As several studies point to the location of transcription sites inside chromosome territories as well as at their periphery, extraterritorial loops cannot simply be a mechanism for making transcribed genes accessible to the transcriptional machinery and must occur for other reasons. The level of decondensation within an activated region varies greatly and probably depends on the density of activated genes and the number of engaged RNA polymerases. Genes that are silenced during development form a more closed chromatin structure. Specific histone modifications are correlated with gene activation and silencing, and silenced genes may become associated with heterochromatin protein 1 homologues or with polycomb group complexes. Several levels of chromatin packaging are found in the nucleus relating to the different functions of and performed by active genes; euchromatic and heterochromatic regions and the models explaining higher-order chromatin structure are still disputed.

Animals↗
PubMed2005

Nick-forming sequences may be involved in the organization of eukaryotic chromatin into approximately 50 kbp loops.

Phenomena involving the disassembly of chromosomes to approximately 50 kbp double-stranded fragments upon protein denaturing treatments of normal and apoptotic mammalian nuclei as well as yeast protoplasts may be an indication of special, hypersensitive regions positioned regularly at loop-size intervals in the eukaryotic chromatin. Here we show evidence in yeast cell systems that loop-size fragmentation can occur in any phase of the cell cycle and that the plating efficiency of these cells is approximately 100%. The possibility of sequence specificity was investigated within the breakpoint cluster region (bcr) of the human MLL gene, frequently rearranged in certain leukemias. Our data suggest that DNA isolated from yeast cultures or mammalian cell lines carry nicks or secondary structures predisposing DNA for a specific nicking activity, at non-random positions. Furthermore, exposure of MLL bcr-carrying plasmid DNA to S1 nuclease or nuclear extracts or purified topoisomerase II elicited cleavages at the nucleotide positions of nick formation on human genomic DNA. These data support the possibility that certain sequence elements are preferentially involved in the cleavage processes responsible for the en masse disassembly of chromatin to loop-size fragments upon isolation of DNA from live eukaryotic cells.

Apoptosis↗
PubMed1995

Evidence for the organization of chromatin in megabase pair-sized loops arranged along a random walk path in the human G0/G1 interphase nucleus.

We determined the folding of chromosomes in interphase nuclei by measuring the distance between points on the same chromosome. Over 25,000 measurements were made in G0/G1 nuclei between DNA sequences separated by 0.15-190 megabase pairs (Mbp) on three human chromosomes. The DNA sequences were specifically labeled by fluorescence in situ hybridization. The relationship between mean-square interphase distance and genomic separation has two linear phases, with a transition at approximately 2 Mbp. This biphasic relationship indicates the existence of two organizational levels at scales > 100 kbp. On one level, chromatin appears to be arranged in large loops several Mbp in size. Within each loop, chromatin is randomly folded. On the second level, specific loop-attachment sites are arranged to form a supple, backbonelike structure, which also shows characteristic random walk behavior. This random walk/giant loop model is the simplest model that fully describes the observed large-scale spatial relationships. Additional evidence for large loops comes from measurements among probes in Xq28, where interphase distance increases and then locally decreases with increasing genomic separation.

Cell Cycle↗
PubMedDate not supplied

[Structure of the macronucleus chromatin of the ciliate Bursaria truncatella. III. Chromatin organization in resting cysts and during excysting].

Structural organization of macronuclear chromatin of a ciliate Bursaria truncatella was studied electronmicroscopically by means of Miller's technique and negative staining of resting cysts and at excysting. In resting cysts practically all the macronuclear chromatin was shown to be organized into compact chromatin clumps 100-300 nm in size. At excysting a natural decompactization of the chromatin clumps occurred and radial loop-shaped chromatin fibres appeared around the clumps. Sometimes transcription units with a relatively small contour length of transcribed region and with high RNA-polymerase density were observed on the loops. The responsibility of such genes for the synthesis of proteins taking part in regulation of excystment and/or subsequent cell growth and differentiation is discussed.

Animals↗
PubMed1997

Radioprotection against the formation of DNA double-strand breaks in cellular DNA but not native cellular chromatin by the polyamine spermine.

The complexing of histones with DNA and the resulting condensation of chromatin protect mammalian cell DNA from radiation-induced strand breakage. In recent studies of SV40 DNA and minichromosomes, marked radioprotection was afforded by spermine through polyamine-induced compaction and aggregation (Newton et al., Radiat. Res. 145, 776-780, 1996). To evaluate the contribution of polyamines to the radioprotection of cellular chromatin, intact V79 cells, nuclei (native chromatin) and chromatin that was partially or completely stripped of histones were treated with spermine or putrescine and gamma-irradiated while embedded in agarose plugs, and induction of double-strand breaks was determined by pulsed-field gel electrophoresis. In the absence of added spermine, the order of radiosensitivity was: dehistonized chromatin (DNA loops anchored to the nuclear matrix) > chromatin depleted of histone H1 > chromatin partially depleted of histone H1 > native chromatin > intact cells. Spermine at concentrations below 1 mM was without effect on strand breakage in any of the preparations, except for limited radioprotection of H1-depleted chromatin. Increasing radioprotection with increasing concentration (1-10 mM) was provided to dehistonized chromatin by spermine but not by putrescine, a polyamine that does not compact DNA or chromatin. Significant radioprotection by spermine was also found for H1-depleted relaxed chromatin at concentrations > or = 1 mM. In contrast, no radioprotection by spermine (up to 10 mM) was observed for any of the chromatin preparations containing all histones. These observations support the hypothesis proposed by Newton et al. that spermine protects DNA against radiation damage via polyamine-induced compaction and aggregation. With removal of histone H1, the exposed chromatin develops the ability to be protected by spermine. However, the absence of radioprotection of native chromatin by spermine is consistent with a role for histones as the major radioprotectors of cellular DNA and the differential radiosensitivity of decondensed compared to condensed cellular chromatin resulting from the effects of factors other than polyamines.

Animals↗
PubMed2025

Barrier effects on the kinetics of cohesin-mediated loop extrusion.

Chromosome organization mediated by structural maintenance of chromosome complexes is crucial in many organisms. Cohesin extrudes chromatin into loops that are thought to lengthen until it is obstructed by CTCF proteins. In complex cellular environments, the loop extrusion machinery may encounter other chromatin-binding proteins. How these proteins interfere with the cohesin-meditated extrusion process is largely unexplored, but recent experiments have shown that some proteins serve as physical barriers that block cohesin translocation. Other proteins containing a cohesin-interaction motif serve as chemical barriers to induce cohesin pausing through interactions with it. Here, we develop an analytically solvable approach for the loop extrusion model incorporating barriers to investigate the effect of the barrier on the passive extrusion process. To further quantify the impact of barriers, we calculate the mean looping time it takes for cohesin to translocate to form a stable loop before dissociation. Our finding reveals that the physical barrier can accelerate the loop formation, and the degree of acceleration is closely related to the impedance strength of the physical barrier. In particular, the synergy of the cohesin loading site and the physical barrier site accelerates loop formation more significantly. The proximity of the cohesin loading site to the barrier site facilitates the rapid formation of stable loops in long genomes, which implies loop extrusion and chromatin-binding proteins might shape functional genomic organization. Conversely, chemical barriers consistently impede loop formation, with increasing impedance strength of the chemical barrier leading to longer loop formation time. Our study contributes to a more comprehensive understanding of the complexity of the loop extrusion process, providing a new perspective on the potential mechanisms of gene regulation.

Cohesins↗
PubMedDate not supplied

[The effect of benz(a)pyrene on the thermal characteristics of DNA in vivo and in vitro].

Thermal properties of DNA-benz(a)pyrene complex and chromatin within liver cells in BALB/c mice and Macaca fascicularis monkeys after benz(a)pyrene administration were studied using a highly sensitive differential scanning microcalorimeter designed for investigations of dilute biopolymer solutions and complex biological systems. It was shown that benz(a)pyrene (BP) had different efforts on DNA in vivo and in vitro. It was established that at a molar ratio r < 0.03 BP/DNA bp, benz(a)pyrene served as a stabilizing but at higher concentrations as a destabilizing factor of DNA. It was found that BP damaged liver DNA stronger than bone marrow and spleen DNA of a given animal in vivo. Based on analysis of heat redistribution at the heat absorption stages corresponding to denaturation of inactive and active chromatin, we concluded that BP is capable of causing specific breaks in the DNA chain of inactive chromatin and unfolding the whole domain-loop of chromatin, which should lead to uncontrolled genome activation and, therefore, to carcinogenesis.

Animals↗
PubMed2006

DNA translocation and loop formation mechanism of chromatin remodeling by SWI/SNF and RSC.

ATP-dependent chromatin-remodeling complexes (remodelers) modulate gene transcription by regulating the accessibility of highly packaged genomic DNA. However, the molecular mechanisms involved at the nucleosomal level in this process remain controversial. Here, we monitor the real-time activity of single ySWI/SNF or RSC complexes on single, stretched nucleosomal templates under tensions above 1 pN forces. We find that these remodelers can translocate along DNA at rates of approximately 13 bp/s and generate forces up to approximately 12 pN, producing DNA loops of a broad range of sizes (20-1200 bp, average approximately 100 bp) in a nucleosome-dependent manner. This nucleosome-specific activity differs significantly from that on bare DNA observed under low tensions and suggests a nucleosome-remodeling mechanism through intranucleosomal DNA loop formation. Such loop formation may provide a molecular basis for the biological functions of remodelers.

Adenosine Triphosphate↗
PubMed1991

A matrix/scaffold attachment region binding protein: identification, purification, and mode of binding.

Matrix/scaffold attachment regions (MARs/SARs) partition chromatin into functional loop domains. Here we have identified a chicken protein that selectively binds to MARs from the chicken lysozyme locus and to MARs from Drosophila, mouse, and human genes. This protein, named ARBP (for attachment region binding protein), was purified to homogeneity and shown to bind to MARs in a cooperative fashion. ARBP is an abundant nuclear protein and a component of the internal nuclear network. Deletion mutants indicate that multiple AT-rich sequences, if contained in a minimal approximately 350 bp MAR fragment, can lead to efficient binding of ARBP. Furthermore, dimerization mutants show that, to bind ARBP efficiently, MAR sequences can act synergistically over large distances, apparently with the intervening DNA looping out. The binding characteristics of ARBP to MARs reproduce those of unfractionated matrix preparations, suggesting that ARBP is an important nuclear element for the generation of functional chromatin loops.

Animals↗
PubMed2006

Linear connection of condensing chromosomes in nuclei of synchronized CHO cells.

Reversibly permeabilized cells allowed the analysis of intermediates of large-scale chomatin condensation in a cell cycle-dependent manner. This paper summarizes major intermediates of chromatin condensation and visualizes connectivity between different forms of chromosomes. At the early S phase the veil-like fibrillary chromatin is supercoiled to form chromatin bodies representing the earliest visible chromosomes. Supercoiling results in a chromatin fiber, which turns to rope (thick fiber) forming loops and chromatin ribbon. The elongated shape of prechromosomes indicates that they are arranged head to tail. Bent forms (loop, c-, and v-shaped structures) of interphase chromosomes open at the end of S phase. Linear arrangement of chromosomes was observed to the end of the condensation process, suggesting that connectivity of chromosomes is maintained throughout the cell cycle.

Animals↗