FINE STRUCTURAL ALTERATIONS OF INTERPHASE NUCLEI OF LYMPHOCYTES STIMULATED TO GROWTH ACTIVITY IN VITRO

This report describes fine structural changes of interphase nuclei of human peripheral blood lymphocytes stimulated to growth by short-term culture with phytohemagglutinin. Chromatin is found highly labile, its changes accompanying the sequential increases of RNA and DNA synthesis which are known to occur in lymphocyte cultures. In "resting" lymphocytes, abundant condensed chromatin appears as a network of large and small aggregates. Early in the response to phytohemagglutinin, small aggregates disappear during increase of diffuse chromatin regions. Small aggregates soon reappear, probably resulting from disaggregation of large masses of condensed chromatin. Loosened and highly dispersed forms then appear prior to the formation of prophase chromosomes. The loosened state is found by radioautography to be most active in DNA synthesis. Small nucleoli of resting lymphocytes have concentric agranular, fibrillar, and granular zones with small amounts of intranucleolar chromatin. Enlarging interphase nucleoli change chiefly (1) by increase in amount of intranucleolar chromatin and alteration of its state of aggregation and (2) by increase in granular components in close association with fibrillar components.     

Formation of strand breaks in the DNA ofγ-irradiated chromatin

Summary Strand breaks have been determined by sedimentation on sucrose gradients in the DNA of chromatin irradiated after isolation from Chinese hamster lung fibroblasts. The yields of double-strand and single-strand breaks are similar to those found in the DNA of irradiated mammalian cells. Irradiation of isolated chromatin in the presence of the radical scavenger tertiary butanol indicates that at least 65% of single-strand breaks and 56% of double-strand breaks can be attributed to the action of hydroxyl radicals. The results indicate the influence of chromosomal proteins in modifying radiation damage to DNA and suggest that the mechanisms for the induction of strand breaks in the DNA of isolated chromatin may be comparable to those operating in the intact cell.     

Photobleaching of GFP-labeled H2AX in chromatin: H2AX has low diffusional mobility in the nucleus

The Ser-139 phosphorylated form of replacement histone H2AX (gamma-H2AX) is induced within large chromatin domains by double-strand DNA breaks (DSBs) in mammalian chromosomes. This modification is known to be important for the maintenance of chromosome stability. However, the mechanism of gamma-H2AX formation at DSBs and its subsequent elimination during DSB repair remains unknown. gamma-H2AX formation and elimination could occur by direct phosphorylation and dephosphorylation of H2AX in situ in the chromatin. Alternatively, H2AX molecules could be phosphorylated freely in the nucleus, diffuse into chromatin regions containing DSBs and then diffuse out after DNA repair. In this study we show that free histone H2AX can be efficiently phosphorylated in vitro by nuclear extracts and that free gamma-H2AX can be dephosphorylated in vitro by the mammalian protein phosphatase 1-alpha. We made N-terminal fusion constructs of H2AX with green fluorescent protein (GFP) and studied their diffusional mobility in transient and stable cell transfections. In the absence or presence of DSBs, only a small fraction of GFP-H2AX is redistributed after photobleaching, indicating that in vivo this histone is essentially immobile in chromatin. This suggests that gamma-H2AX formation in chromatin is unlikely to occur by diffusion of free histone and gamma-H2AX dephosphorylation may involve the mammalian protein phosphatase 1alpha.     

Characteristics of the biochemical manifestation of the process of thymic lymphocyte death following UV irradiation

Thymus lymphocyte death caused by UV-radiation is a function of radiation dose. Thymocyte death induced by UV-light, in contrast to gamma-radiation, is not accompanied by chromatin degradation. With UV-irradiation, irreparable DNA breaks are not responsible for thymocyte death. Cycloheximide, but not cysteamine, prevents the UV-induced cell death.     

Effect of chromium(III) on nucleolar RNA synthesis

The enhancement of hepatic nucleolar RNA synthesis induced by Cr(III) in partially hepatectomized rats and its mechanisms are described. Cr(III)-administered (0.5 mg Cr/kg, ip) and then partially hepatectomized rats were significantly enhanced in the hepatic nucleolar RNA synthesis at the very early stage of liver regeneration. This enhancement was caused both by the induction of newly found nucleolar Cr-bound protein of 70 kD (Cr-p70) and by the activation of nucleolar chromatin, both of which arose from nuclear accumulation of Cr together with partial hepatectomy. Studies on the mechanism of this enhancement indicated that the Cr-p70 bound to the activated nucleolar chromatin and loosened its higher-order structure, resulting in an increase of the B-form fraction of chromatin DNA. The degree of this loosening well correlated with the amount of Cr-p70 bound to chromatin and also with the extent of elevation of RNA synthesis. Some molecular species of nonhistone proteins in chromatin were found to play an important role in the interaction to Cr-p70. These results suggest a possibility that the action of Cr is involved in cell proliferation process.     

Induction of CAF-1 expression in response to DNA strand breaks in quiescent human cells

Genome stability in eukaryotic cells is maintained through efficient DNA damage repair pathways, which have to access and utilize chromatin as their natural template. Here we investigate the role of chromatin assembly factor 1 (CAF-1) and its interacting protein, PCNA, in the response of quiescent human cells to DNA double-strand breaks (DSBs). The expression of CAF-1 and PCNA is dramatically induced in quiescent cells upon the generation of DSBs by the radiomimetic drug bleocin (a bleomycin compound) or by ionizing radiation. This induction depends on DNA-PK. CAF-1 and PCNA are recruited to damaged chromatin undergoing DNA repair of single- and double-strand DNA breaks by the base excision repair and nonhomologous end-joining pathways, respectively, in the absence of extensive DNA synthesis. CAF-1 prepared from repair-proficient quiescent cells after induction by bleocin mediates nucleosome assembly in vitro. Depletion of CAF-1 by RNA interference in bleocin-treated quiescent cells in vivo results in a significant loss of cell viability and an accumulation of DSBs. These results support a novel and essential role for CAF-1 in the response of quiescent human cells to DSBs, possibly by reassembling chromatin following repair of DNA strand breaks.     

A role for the Saccharomyces cerevisiae Rtt109 histone acetyltransferase in R-loop homeostasis and associated genome instability

The stability of the genome is occasionally challenged by the formation of DNA–RNA hybrids and R-loops, which can be influenced by the chromatin context. This is mainly due to the fact that DNA–RNA hybrids hamper the progression of replication forks, leading to fork stalling and, ultimately, DNA breaks. Through a specific screening of chromatin modifiers performed in the yeast Saccharomyces cerevisiae, we have found that the Rtt109 histone acetyltransferase is involved in several steps of R-loop-metabolism and their associated genetic instability. On the one hand, Rtt109 prevents DNA–RNA hybridization by the acetylation of histone H3 lysines 14 and 23 and, on the other hand, it is involved in the repair of replication-born DNA breaks, such as those that can be caused by R-loops, by acetylating lysines 14 and 56. In addition, Rtt109 loss renders cells highly sensitive to replication stress in combination with R-loop-accumulating THO-complex mutants. Our data evidence that the chromatin context simultaneously influences the occurrence of DNA–RNA hybrid-associated DNA damage and its repair, adding complexity to the source of R-loop-associated genetic instability.     

The influence of chromatin structure on the frequency of radiation-induced DNA strand breaks: a study using nuclear and nucleoid monolayers

To assess the influence of chromatin structure on the frequency of radiation-induced DNA strand breaks, the alkaline unwinding technique was applied to nuclear and nucleoid monolayers. These chromatin substrates were prepared by treating human fibroblasts grown as monolayers with the nonionic detergent Triton X-100 and varying concentrations of cations. The chromatin structure was modified either by a stepwise removal of DNA-bound proteins by extraction in increasing concentrations of monovalent salt, or by the addition or deletion of mono- and divalent cations to condense or decondense the chromatin, respectively. It was found that the stepwise removal of DNA-bound proteins from the chromatin dramatically increased the frequency of radiation-induced DNA strand breaks. The DNA-bound proteins showed a qualitative difference in their ability to protect the DNA where proteins removed by salt concentrations above 1.0 M exerted the greatest protection. Furthermore, the frequency of radiation-induced DNA strand breaks was found to be 6 times lower in condensed chromatin than in decondensed chromatin and about 80 times lower than in protein-depleted chromatin. It is concluded that the presence of DNA-bound proteins and the folding of the chromatin into higher-order structures protect the DNA against radiation-induced strand breaks.     

THE FINE STRUCTURE AND CHEMICAL COMPOSITION OF NUCLEI DURING SPERMIOGENESIS IN THE HOUSE CRICKET : I. Initial Stages of Differentiation and the Loss of Nonhistone Protein

The early stages of nuclear differentiation in spermatids of the house cricket are described with regard to the fine structural elements and chemical components which occur. Particular attention is given to the loss of nonhistone protein from the nucleus and its relation to chromatin structure. Granular elements about 25 to 80 mµ in diameter, and fibers about 8 mµ in diameter occur in the earliest spermatid nucleus. The fibers are found in diffuse and condensed chromatin while granules are found only in diffuse material. DNA and histone parallel the chromatin fibers in distribution, while nonhistone protein and RNA parallel the granules in distribution. The granules and most of the nonhistone protein are lost, simultaneously, after the early spermatid stage. The protein loss occurs without detectable change in the structure of chromatin fibers. Chromatin fibers first show a structural change in mid spermiogenesis, when they become thicker and very contorted. Unusually thin fibers (about 5 mµ) also appear in mid spermatid nuclei; they are apparently composed of nonhistone protein and free of DNA and histone.     

Radiolysis of chromatin extracted from cultured mammalian cells: production of alkali-labile strand damage in DNA.

Chromatin has been isolated from cultured Chinese-hamster lung fibroblasts as an expanded aqueous gel. The DNA in isolated chromatin has been examined by sedimentation on alkaline sucrose gradients. The average molecular weight of the DNA has been determined to be 50 million. gamma-irradiation of isolated chromatin degrades the DNA to lower molecular weight. The yield of single-strand breaks in the DNA is 0.02 single-strand breaks per krad-10(6) dalton, calculated from a dose-range of &--400 krad and covering a DNA molecular weight range of 2 X 10(7)-1.4 X 10(5). There is a considerable difference in the efficiency of the formation of single-strand breaks in DNA irradiated as isolated chromatin compared with chromatin irradiated in whole cells before isolation. For isolated chromatin, values of 6 dV per break have been calculated compared with about 80 eV per break for chromatin irradiated in whole cells, which suggest a large contribution from indirect action by aqueous radicals in isolated chromatin.     

RSC facilitates Rad59-dependent homologous recombination between sister chromatids by promoting cohesin loading at DNA double-strand breaks

Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.     

Analysis of DNA double- and single-strand breaks by two dimensional electrophoresis: action of micrococcal nuclease on chromatin and DNA, and degradation in vivo of lens fiber chromatin.

We describe a novel system for two dimensional electrophoresis at neutral and alkaline pH for determining the double-stranded and single-stranded lengths of DNA. With this system we analysed the mode of micrococcal nuclease digestion of DNA in cellular and SV40 viral chromatin and of supercoiled SV40 DNA. The enzyme reaction occurred in two steps : the enzyme first introduced single-strand breaks, then converted these to double-strand breaks by an adjacent cleavage on the opposite strand. Digestion of cellular chromatin DNA occurred by a similar mechanism. Chromatin fragments produced by limited micrococcal nuclease action contained many single-strand breaks, which may be important when this method is used to prepare chromatin fragments for biochemical and biophysical studies. Nucleosome monomer to tetramer produced at later stages of digestion contained few if any single-strand breaks.     

Phosphorylation of H2AX histone as indirect evidence for double-stranded DNA breaks related to the exchange of nuclear proteins and chromatin remodeling in Chara vulgaris spermiogenesis

Phosphorylation of H2AX histone results not only from DNA damage (caused by ionizing radiation, UV or chemical substances, e.g. hydroxyurea), but also regularly takes place during spermiogenesis, enabling correct chromatin remodeling. Immunocytochemical analysis using antibodies against H2AX histone phosphorylated at serine 139 indirectly revealed endogenous double-stranded DNA breaks in Chara vulgaris spermatids in mid-spermiogenesis (stages V, VI and VII), when protamine-type proteins appear in the nucleus. Fluorescent foci were not observed in early (stages I-IV) and late (VIII-X) spermiogenesis, after replacement of histones by protamine-type proteins was finished. A similar phenomenon exists in animals. Determination of the localization of fluorescent foci and the ultrastructure of nuclei led to the hypothesis that DNA breaks at stage V, when condensed chromatin adheres to the nuclear envelope. This is transformed into a net-like structure during stage VI, probably allowing chromosome repositioning to specific regions in the mature spermatozoid. However, at stages VI and VII, DNA breaks are necessary for transformation of the nucleosomal structure into a fibrillar and finally the extremely condensed status of sleeping genes at stage X.     

Fluorescent analysis of irradiation-induced changes in chromatin state

A fluorescent technique with Hoechst-33258 and acridine orange staining was used to assess changes in chromatin state induced by radiation. Fluorochromes with different modes of binding to DNA were chosen. In T lymphocytes chronic irradiation caused a rearrangement of binding between nonhiston proteins and lipids accompanied by conformational changes in DNA, resulting in chromatin condensation. The decrease in fluorescence probably resulted from a reduction in the number of sites accessible for dye binding. After acute irradiation, the fluorescence intensity decreases predominantly due to double-strand breaks.     

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells

Pluripotent mouse embryonic stem cells (mES cells) exhibit ∼ 100 large γH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (> 10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PK_cs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous γH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of γH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, γH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive γH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.     

In vivo mapping of DNA topoisomerase II-specific cleavage sites on SV40 chromatin

The antitumor drug, m-AMSA (4'-(9-acridinylamino)-methanesulfon-m-anisidide), is known to interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by blocking the enzyme-DNA complex in its putative cleavable state. Treatment of SV40 virus infected monkey cells with m-AMSA resulted in both single- and double-stranded breaks on SV40 viral chromatin. These strand breaks are unusual because they are covalently associated with protein. Immunoprecipitation results suggest that the covalently linked protein is DNA topoisomerase II. These results are consistent with the proposal that the drug action in vivo involves the stabilization of a cleavable complex between topoisomerase II and DNA in chromatin. Mapping of these double-stranded breaks on SV40 viral DNA revealed multiple topoisomerase II cleavage sites. A major topoisomerase II cleavage site was preferentially induced during late infection and was mapped in the DNAase I hypersensitive region of SV40 chromatin.     

Apoptotic cell nuclei favour aggregation and fluorescence quenching of DNA dyes

 Apoptotic cell nuclei are known to stain hyperchromatically with absorption dyes and dimly with many DNA fluorochromes. We hypothesised that both optical phenomena have the same cause - the ability of apoptotic chromatin to aggregate cationic dyes. This hypothesis was tested using prednisolone-primed rat thymus, which is known to contain apoptotic cells. The apoptotic cells were classified as early and late, based on their morphology, in thin and semithin sections and in thymus imprints on slides. Direct reaction for DNA strand breaks (TUNEL) indicated the presence of breaks in both categories of cells, with more intense labelling in late apoptosis. The chromatin ultrastructure of early apoptotic cells initially retained the supranucleosomal order of packaging which characterises control cells, whereas the dense chromatin of late apoptotic cells possessed the degraded structure. Absorption spectra of the toluidine blue-stained early apoptotic cell chromatin revealed a metachromatic shift, indicating a change of DNA conformation and polymerisation of the dye. When the staining was performed by acridine orange (preceded by a short acid treatment), a paradoxical several-fold increase of fluorescence intensity at a several-fold dilution of the dye was found. The simultaneous reduction of the ratio of red to green components of fluorescence confirmed that the concentration-dependent fluorescence quenching was due to aggregation of the dye. The results suggest that the enhanced affinity of the chromatin of early apoptotic cells for cationic dyes is associated with conformational relaxation rather than degradation of DNA. In late apoptotic cells, the very dense packaging of degraded DNA promotes further aggregation of dyes. The results suggest alternative methods for detection and discrimination of early and late apoptotic cells. Accepted: 12 February 1997     

Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA

The occurrence and spatial distribution of intracellular DNA fragmentation was investigated by in situ 3 end labelling of DNA breaks in K562 cells treated in such a way to cause either apoptotic or necrotic cell death. The localisation of DNA breaks was examined by confocal laser microscopy and compared with the electron-microscopic appearance of the cells. In addition, the number of cells with fragmented DNA was counted and compared with the number of dead cells, as determined by the nigrosin dye exclusion test. Apoptosis was induced by cultivation of the cells in the presence of actinomycin D. Cells undergoing apoptosis were characterised by massive intracellular DNA fragmentation that was highly ordered into successive steps. Cells in early stages of the apoptotic process had DNA breaks diffusely distributed in the entire nucleus, except the nucleolus, with crescent-like accumulations beyond the nuclear membrane. In the more advanced stages, the nucleus was transformed into many round bodies with intense labelling. Intracellular accumulations of fragmented DNA corresponded exactly to electron-dense chromatin seen in the electron microscope, whereas diffuse DNA breaks had no morphological correlate at the ultrastructural level. In necrosis induced by ionomycin, NaN₃, or rapid freezing combined with thawing, no DNA fragmentation occurred at the onset of cell death, but appeared 24 h later. This fragmentation was not characterised by a unique morphology, but represented the breakdown of the chromatin in the configuration remaining after cell death. Therefore, apoptosis is characterised by DNA fragmentation that proceeds in a regular orderly sequence at the beginning of cell death, and can be detected by in situ 3end labelling of DNA breaks.     

Modifications of the chromatin arrangement induced by ethidium bromide in isolated nuclei, analyzed by electron microscopy and flow cytometry

Ethidium bromide (EB) is widely used for investigating the DNA conformation in chromatin both with conventional and cytofluorimetric techniques. Since the interaction of the dye with DNA should result in structural deformations which can be different in isolated or in situ chromatin, a study has been performed on the effects caused by different amounts of EB and the analogous propidium iodide on isolated nuclei, in which chromatin maintains its native relationships with the other nuclear structures (envelope, nucleolus, interchromatin RNP, nuclear matrix). The results obtained by comparing ultrastructural observations in thin sections and in freeze-fracturing with conformational analysis in multiparameter flow cytometry indicate that the phenanthridinic fluorochromes, especially at the high concentrations used for cytofluorimetric analyses, cause deep rearrangements of the chromatin in situ. These effects consist both in aggregation and condensation of the fibers into the dense chromatin domains, and in an increase of the supernucleosomal configuration associated with an enlargement of interchromatin spaces in which the RNP particles appear particularly evident. These results, discussed with those available on isolated chromatin, suggest that any unwinding effect of the intercalating dyes on the DNA cause a general condensation of chromatin as a consequence of the constraints which characterize the organization of the chromatin inside the nucleus.