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Results for “chromatin compaction”

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At least 19 recordsLinked to original sources

PubMed1989

Two mechanisms of chromatin compaction.

2 types of chromatin compaction were studied by cytochemical and ultrastructural approach. The 1st type was induced by cultivating transformed cells with dimethylsulfoxide. It caused cell transition into a more differentiated state coupled with the appearance of multiple small chromatin condensates. The 2nd type was induced by nonionic detergent Triton X-100 and/or saline penetration inside the nuclei. It was found to be connected with hypercompaction of chromocenters with their integral areas left unincreased. Both types of chromatin compaction differ in their capacity to induce toluidine blue polymerization on DNA substrate and in stability of DNA towards an early step of acid hydrolysis. The relation of the 2 mechanisms of chromatin compaction to different ways of DNA folding is discussed.

Animals↗

PubMed1996

Three-dimensional analysis of the arrangement of compact chromatin in the nucleus of G0 rat lymphocytes.

The arrangement of compact chromatin of G0 lymphocytes was studied in three-dimensional reconstructions of the ensemble of the chromatin and of individual compact chromatin bodies. Rat spleen was serially cut and sections were contrasted with procedures preferential for DNA. Electron microscopy images were digitized, processed, and displayed using a commercial software package, complemented by a system for three-dimensional reconstruction and analysis developed by us on an IBM-compatible microcomputer provided with an image acquisition board. The reconstructions showed a continuous layer of compact chromatin in contact with the nuclear envelope that prevents the automatic recognition of individual chromatin clumps. The ensemble of the arrangement of compact chromatin was found to be very similar in different lymphocytes. After morphological filtering procedures, the initial mass was divided into individual bodies of compact chromatin, which were tagged. Most of these bodies contact the nuclear envelope. The number of bodies as well as the number of contacts with the envelope are similar and correspond to a haploid number of chromosomes. The largest body is always the one containing nucleolus-associated chromatin. When the cell has two nucleoli, the nucleolus-associated chromatin bodies contact the envelope in diametrically opposed areas. This feature was also described in rat liver cells. It is concluded that: (a) the individualized compact chromatin bodies do not correspond to an entire chromosome or to a pair of chromosomes; (b) the arrangement of compact chromatin is not identical in each G0 lymphocyte, but there are patterns that are repeated with limited changes; and (c) there are common features that appear in different cell types of individuals of the same species.

Animals↗

PubMed2001

Relationship between chromatin compactness and dye uptake for in situ chromatin stained with DAPI.

BACKGROUND: This study investigated the relationship between chromatin compactness, which is directly related to chromatin condensation, and DAPI uptake. Materials and Methods For the structural characterization of in situ chromatin, we used fluorescence microscopy and differential scanning calorimetry on calf thymocytes. The compactness of nuclear chromatin was altered by permeabilizing native cells with NP40 detergent. A time-dependent analysis of detergent effects was performed by acquiring nuclear images at different time intervals after permeabilization. In order to compare nuclei of different sizes, we implemented a geometrical correction in the calculation of the integrated fluorescence intensity. For a quantitative evaluation of chromatin condensation we introduced two new parameters, "average chromatin packing ratio" and "average dye spatial density." RESULTS: This approach allowed us to estimate the effects of NP40 detergent at the level of in situ chromatin. Detergent effects could be modulated by changing the ionic composition of buffer. Moreover, changes of chromatin condensation induced by detergent were inversely related to modifications of nuclear volume. CONCLUSIONS: The combination of complementary information obtained by fluorescence microscopy, supported by a proper geometrical correction, and differential calorimetry allowed us to interpret the patterns of fluorescence intensities inside the nucleus in terms of chromatin structure.

Animals↗

PubMed2003

Chromatin compaction by human MeCP2. Assembly of novel secondary chromatin structures in the absence of DNA methylation.

MeCP2 is a transcriptional repressor that contains an N-terminal methylated DNA-binding domain, a central transcription regulation domain, and a C-terminal domain of unknown function. Whereas current models of MeCP2 function evoke localized recruitment of histone deacetylases to specific methylated regions of the genome, it is unclear whether MeCP2 requires DNA methylation to bind to chromatin or whether MeCP2 binding influences chromatin structure in the absence of other proteins. To address these issues, we have characterized the complexes formed between MeCP2 and biochemically defined nucleosomal arrays. At molar ratios near 1 MeCP2/nucleosome, unmethylated nucleosomal arrays formed both extensively condensed ellipsoidal particles and oligomeric suprastructures. Furthermore, MeCP2-mediated chromatin compaction occurred in the absence of monovalent or divalent cations, in distinct contrast to all other known chromatin-condensing proteins. Analysis of specific missense and nonsense MeCP2 mutants indicated that the ability to condense chromatin resides in region(s) of the protein other than the methylated DNA-binding domain. These data demonstrate that MeCP2 assembles novel secondary chromatin structures independent of DNA modification and suggest that the ability of MeCP2 to silence chromatin may be related in part to its effects on large-scale chromatin organization.

Chromatin↗

PubMed2025

Free energy spectroscopy reveals the mechanistic landscape of chromatin compaction.

Eukaryotic genomic DNA is repeatedly wrapped into nucleosome spools: the basic building block of chromatin. This organization regulates the physical accessibility of the genome to gene transcription, replication, and repair regulatory factors. Chromatin compaction is controlled by multivalent weak interactions, resulting in a complicated conformational landscape that remains challenging to characterize. This work reports a method for characterizing chromatin compaction, Free Energy Spectroscopy (FES), which is based on DNA nanotechnology and transmission electron microscopy. This method experimentally determines the chromatin compaction free energy landscape in terms of end-to-end distance and nucleosome stacking interactions. By deconvolving the free energy landscapes of partially and fully compact tetranucleosomes, FES revealed three separate mechanisms by which linker histones reshape the compaction energetics to condense chromatin. This study establishes FES as a method with the potential to help answer a broad range of mechanistic questions about genome and epigenome function.

DNA nanotechnology↗

PubMed1994

[A position effect variegation model of chromatin compaction].

A position effect variegation (PEV) model of chromatin compaction is proposed, based on the concept of a statistical distribution of compaction protein (CP) molecules around compaction initiation centers (CICs). The principles of the model are as follows: (1) CICs are present in both hetero- and euchromatin, and (2) different CP molecules interact not only with DNA but also with one another, forming a multimeric complex. When a certain level of DNA-protein binding is exceeded, heterochromatic domains are formed. The model suggests that continuous and discontinuous chromatin compaction resulting from PEV is due to an irregular CIC distribution along the chromosome.

Animals↗

PubMed2002

A complex rearrangement involving simultaneous translocation and inversion is associated with a change in chromatin compaction.

Detailed fluorescence in situ hybridisation analysis of a previously described translocation revealed it to be a more complex rearrangement consisting of both a translocation and a paracentric inversion with an apparent coincident breakpoint at 16p13.3, t(14;16)(p32;p13.3) inv16(p13.3p12.1). This unusual three-breakpoint rearrangement was not obvious from examination of G-banding. Such rearrangements may be undiagnosed in cytogenetic studies. The presence of an interstitial deletion of 16p was unlikely as the rearranged chromosome contained probes distributed along the short arm of chromosome 16. Fluorescence in situ hybridisation studies suggested that the inverted segment was smaller in size than that on the normal chromosome. Measurements of distances between probes on metaphase chromosomes confirmed that there was differential compaction of the inverted portion on 16p. The inverted region was significantly reduced in size by 21% compared with the same region on the normal chromosome 16. The size reduction across the region was non-uniform, with one region showing a 55% increase in compaction. The change in compaction was also associated with a change in the lateral position of a probe on the chromatids. The finding that a single chromosome breakpoint can change the compaction of chromatin over an extensive region has implications for models of the structure of metaphase chromosomes. Possible explanations are either a localized severe disruption of DNA packaging over relatively short distances (hundreds of kilobases) or a more generalized change that extends over many megabases. These results raise the important possibility that chromosome breaks may result in a more global change in DNA compaction across large segments of a chromosome.

Chromatin↗

PubMed2001

Chemical agents that promote chromatin compaction radiosensitize tumour cells.

PURPOSE: Previous studies indicated that cells whose chromatin is naturally compacted at the time of radiation are hypersensitive to radiation-induced killing, primarily by single-hit inactivation. Some chemicals that are known to promote chromatin compaction in interphase cells are here investigated for their radiosensitizing potential. MATERIALS AND METHODS: Okadaic acid (OA), a protein phosphatase inhibitor, fostriecin (FC), a topoisomerase II inhibitor and trichostatin A (TSA), a histone deacetylase inhibitor, were reported to promote chromatin compaction in mammalian cells. Asynchronous populations of HT-29 (human colon carcinoma) cells were exposed to various concentrations of OA, FC and TSA for various times before irradiation with various doses of Cs-137 gamma-rays and toxicity and radiosensitization were measured. Induced chromatin compaction was visualized by electron microscopy (EM). Histone 1 (H1) and histone 3 (H3) phosphorylation was measured by Western blotting, whole-cell fluorescence microscopy and confocal microscopy. RESULTS: OA and FC produced significant radiosensitization at 2 Gy after short (2 h) exposures. These chemical treatments also produced increased phosphorylation of H3 and increased chromatin compaction as measured by EM. A 2-h exposure of cells to TSA had no effect on cell radiosensitivity, histone phosphorylation or chromatin condensation. However, a 16-h exposure to TSA produced significant radiosensitization, histone phosphorylation and chromatin condensation, presumably by secondary mechanisms. CONCLUSIONS: These data are consistent with the hypothesis that compacted chromatin is a hypersensitive target for radiation killing. Furthermore, the modulation of chromatin conformation by drugs selectively in tumour cells might radiosensitize tumours whose cells are intrinsically radioresistant.

Alkenes↗

PubMed2001

Chromatin compaction and tumor cell radiosensitivity at 2 gray.

Mammalian cells at mitosis, differentiated lymphocytes, and some radiation-hypersensitive mutants in interphase contain all or a measurable portion of their chromatin in condensed/compacted form and are hypersensitive to ionizing radiation by the mechanism described by single-hit inactivation kinetics (alpha). These observations led to the investigation as to whether compacted chromatin in interphase is the target that determines the widely variable alpha-parameters and surviving fractions of 2 Gy (SF2Gy) measured for human tumor cell lines. Six cell lines whose SF2Gy ranged from 0.29 to 0.73 were used for this study. Their different radiosensitivities were associated mainly with differences in their single-hit inactivation parameters (alpha). Electron microscope images of interphase nuclei were optically scanned, and the pixel densities were digitized for quantitative analyses. A significant correlation between the percentage of nuclear pixels with densities similar to those found in mitotic chromosomes (percent compacted chromatin) and the alpha-inactivation parameters was observed. Digital analyses of electron and/or confocal microscope images of chromatin in interphase tumor cells in biopsy specimens could become a rapid assay for predicting the intrinsic radiosensitivity of tumor clonogens. This research has also identified some inhibitors of protein (histone) phosphatases that promote chromatin compaction and radiosensitize cells to 2-Gy dose fractions.

Chromatin↗

PubMed1975

The influence of chromatin compactness on the stoichiometry of the Feulgen-Schiff procedure studied in model films. II. Investigations on films containing condensed or swollen chicken erythrocyte nuclei.

As models for different states of chromatin compactness, nuclei from chicken erythrocytes were isolated and either osmotically swollen or kept as condensed as possible. Both types of nuclei were then fixed and incorporated into polyacrylamide films. Hydrolysis with 5 N HCl and staining with Schiff's reagent of these model films were studied using several parameters. The phosphate content of the films was analyzed as a parameter for the depolymerization losses and the staining with Schiff's reagent as a parameter for the apurinic acid (APA) content. The loss of ultraviolet absorbance from the films and the accumulation of ultraviolet absorbing substances in the hydrolyzing acid were monitored as parameters for the progress of hydrolysis. Conversion of the generated aldehyde groups to APA-Schiff chromophore is shown to take place with the same stoichiometry for both types of nuclei as well as for DNA in model films. It is further shown that the nuclei- and DNA-films are suitable models for investigating the influence of chromatin compactness on the course of the Feulgen-Schiff reaction. For the most compact form of chromatin studied, a very high reduction in staining intensity of up to 40% could be demonstrated after certain normally applied hydrolysis times. This is due primarily to a decrease with a factor of 2.3 of the depurination rate constants of these models (from 0.030/min to 0.013/min). Therefore prolonged hydrolysis periods are required to obtain the same APA concentrations, but then depolymerization processes cause losses of nuclear material. The differences in depurination rates could be explained by a decrease in [H3O]+ in the neighborhood of the purine-sugar linkages, caused by the presence of fixed positive charges form the protein components of the chromatin. These findings may explain the cytophotometrically determined differences in chromophore yield of 10-20% found in the nuclei of cells with different states of compactness of their chromatin. The descending part of the Feulgen hydrolysis curve represents the depolymerization of APA and loss by diffusion of the reaction products. In the Appendix, cytophotometric data of cells have been analyzed to show that this part of the hydrolysis curve may be used to estimate the acid stability of chromatin complexes. The depurination and depolymerization rates found closely correspond with the data obtained from the model films.

Animals↗

PubMed2003

High levels of CUG-initiated FGF-2 expression cause chromatin compaction, decreased cardiomyocyte mitosis, and cell death.

Fibroblast growth factor 2 (FGF-2) is a multifunctional mitogen present in CUG-and AUG-initiated forms, referred to as 'hi' and 'lo' FGF-2, respectively. We have used an adenoviral vector to express the predominantly nuclear human 'hi' FGF-2 and examined the relationship between expression levels, mitotic entry, cell number and chromatin compaction of cardiac myocytes, over 1-3 days in culture. At a multiplicity of infection (m.o.i.) of 50, levels of 'hi' FGF-2 (assessed by Western blotting) and mitotic index (fraction of myocyte nuclei staining positive for phosphorylated histone H3) paralleled each other, becoming maximal at 2 days. At 200 m.o.i., maximal expression of 'hi' FGF-2 (approximately double that at 50 m.o.i) was achieved at 2 days and coincided with decreased mitotic index and increased chromatin compaction. At 3 days compaction was maximal, mitotic index was minimal, and cell numbers decreased, accompanied by the appearance of DNA laddering, an indicator of apoptosis. Overall, the lower dose of 'hi' FGF-2 and early time points favored a proliferative phenotype while the higher dose, and later time points, promoted chromatin compaction, inhibition of proliferation and cell death.

Adenoviridae↗

PubMed2001

Dicentric chromosome stretching during anaphase reveals roles of Sir2/Ku in chromatin compaction in budding yeast.

We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction. Escherichia coli lac operator DNA was placed between two centromeres on a conditional dicentric chromosome in budding yeast cells and made visible by expression of a lac repressor-green fluorescent fusion protein. Centromeres on the same chromatid of a dicentric chromosome attach to opposite poles approximately 50% of the time, resulting in chromosome bridges during anaphase. In cells deleted for yKU70, yKU80, or SIR2, a 10-kb region of the dicentric chromosome stretched along the spindle axis to a length of 6 microm during anaphase. On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation. Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the dicentric chromosome. These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a chromosome experiencing tension or DNA damage. The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA.

Anaphase↗

PubMed2003

Repair of UV lesions in silenced chromatin provides in vivo evidence for a compact chromatin structure.

Genes positioned close to telomeres in yeast are silenced by a heterochromatin-like structure containing Sir proteins. To investigate whether silencing also affects DNA repair, we studied removal of UV lesions by photolyase and nucleotide excision repair (NER) in strains containing the URA3 gene inserted 2 kilobases from a telomere. URA3 was transcriptionally active in sir3delta mutants, partially silenced in SIR3 cells, or completely silenced by overexpression of SIR3 or deletion of RPD3. The active URA3 showed efficient repair by both pathways. Fast repair of the promoter and 3' end by photolyase reflected a non-nucleosomal structure. Partial silencing had no remarkable effect on photolyase but reduced repair by NER, indicating differential accessibility for the two repair reactions. Complete silencing inhibits NER and photolyase in the coding region as well as in the promoter and the 3'-end. Conventional nuclease footprinting analyses revealed subtle changes in the promoter proximal nucleosome under partially silenced conditions but a pronounced reorganization of chromatin extending over the whole gene in silenced chromatin. Thus, both repair systems are sensitive to chromatin changes associated with silencing and provide direct evidence for a compact structure of heterochromatin.

DNA Repair↗

PubMed2002

Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4.

The transcription factors HNF3 (FoxA) and GATA-4 are the earliest known to bind the albumin gene enhancer in liver precursor cells in embryos. To understand how they access sites in silent chromatin, we assembled nucleosome arrays containing albumin enhancer sequences and compacted them with linker histone. HNF3 and GATA-4, but not NF-1, C/EBP, and GAL4-AH, bound their sites in compacted chromatin and opened the local nucleosomal domain in the absence of ATP-dependent enzymes. The ability of HNF3 to open chromatin is mediated by a high affinity DNA binding site and by the C-terminal domain of the protein, which binds histones H3 and H4. Thus, factors that potentiate transcription in development are inherently capable of initiating chromatin opening events.

Amino Acid Motifs↗

PubMed1987

Correlation between molecular clock ticking, codon usage fidelity of DNA repair, chromosome banding and chromatin compactness in germline cells.

The vertebrate genome is built of long DNA regions, relatively homogeneous in GC content, which likely correspond to bands on stained chromosomes. Large differences in composition have been found among DNA regions belonging to the same genome. They are paralleled by differences in codon usage in genes differently localized. The hypothesis presented here asserts that these differences in composition are caused by different mutational bias of alpha and beta DNA polymerases, these polymerases being involved to different extents in the repair of DNA lesions in compact and relaxed chromatin, respectively, in germline cells.

Animals↗

PubMed2026

Enhanced chromatin compaction is associated with de novo expression of a nuclear microprotein, global loss of H3 acetylation and local transcriptional changes in retinal rod photoreceptors.

We have limited understanding of how aging alters gene expression and remodels cellular architecture in post-mitotic neurons. The inverted nuclear organization of mouse rod photoreceptors provides a unique model to gain mechanistic insights into age-associated decline in neuronal function. We have generated and integrated multi-omic datasets including 3D-genome topology, histone modifications, chromatin accessibility, DNA methylation and transcriptome of rod photoreceptors from young- and aged-mice. We show that aging drives global chromatin compaction, with regional alterations enriched at active chromatin. Epigenomic and transcriptional changes broadly correlate with chromatin dynamics as validated by high resolution microscopy. We uncover a megabase-sized genomic region with multi-level alterations, including de novo transcription of Gm7239, which encodes a functional microprotein carrying histone acetyltransferase-inhibitor domain. Overexpression of Gm7239 is associated with global loss of histone H3 acetylation, highlighting a potential new axis of genomic regulation in aging. Finally, we identify multiple significant local transcriptional alterations in non-annotated regions and genes associated with age-related macular degeneration. Our studies link age-related chromatin landscape changes with gene expression that may influence rod function and vulnerability to diseases.

Journal Article↗

PubMed2003

[Inactivation of reporter genes by cloned heterochromatic repeats of Drosophila melanogaster is accompanied by chromatin compaction].

Cloned Stellate heterochromatic repeats caused unstable mosaic inactivation (position effect variegation; PEV) of the reporter gene mini-white. A number of known protein modifiers of the classical position effect induced by large heterochromatin blocks do not affect the expression of mini-white. This raises the question as to the specificity of chromatin compaction around the reporter gene. The inactivation of the mini-white gene has been found to be accompanied by a decrease in its methylation catalyzed by Escherichia coli dam-methyltransferase expressed in the genome of Drosophila. However, no changes in the nucleosome organization of mini-white have been found.

Animals↗

PubMed1997

Alleviation of histone H1-mediated transcriptional repression and chromatin compaction by the acidic activation region in chromosomal protein HMG-14.

Histone H1 promotes the generation of a condensed, transcriptionally inactive, higher-order chromatin structure. Consequently, histone H1 activity must be antagonized in order to convert chromatin to a transcriptionally competent, more extended structure. Using simian virus 40 minichromosomes as a model system, we now demonstrate that the nonhistone chromosomal protein HMG-14, which is known to preferentially associate with active chromatin, completely alleviates histone H1-mediated inhibition of transcription by RNA polymerase II. HMG-14 also partially disrupts histone H1-dependent compaction of chromatin. Both the transcriptional enhancement and chromatin-unfolding activities of HMG-14 are mediated through its acidic, C-terminal region. Strikingly, transcriptional and structural activities of HMG-14 are maintained upon replacement of the C-terminal fragment by acidic regions from either GAL4 or HMG-2. These data support the model that the acidic C terminus of HMG-14 is involved in unfolding higher-order chromatin structure to facilitate transcriptional activation of mammalian genes.

Amino Acid Sequence↗