Postirradiation changes in the chromatin structure of thymocytes in irradiated rats

In this work the antibodies were obtained against chromatin isolated from thymocytes of intact and irradiated rats (2 h after exposing to 10 Gy) and against polydeoxyribonucleotides (PDN) extracted from thymus nuclei 6 h following irradiation. All the antibodies under study reacted more readily with the chromatin obtained from the thymus of exposed rats than with the control chromatin. The complexes of DNA with the most firmly bound non-histone proteins, obtained from the three objects under study, reacted with the antibodies with equal efficiency. Thus, a higher reactivity of PDN and chromatin from thymocytes of exposed rats was associated with the decondensation of the latter leading to an increase in availability of a part of antigenic determinants. Using the immunoblotting method we failed to discover any qualitative differences in the protein composition of the chromatin from control and exposed rats.     

A novel protein related to cell cycle-dependent alterations of chromatin structure

We have detected a novel nuclear antigen, AF-2, which appears to be involved in cell cycle-dependent alterations of chromatin structure. Specific monoclonal antibodies detect the antigen spread over the whole cell during mitosis and in islet-like structures in the nuclei of a subpopulation of cells in interphase. Upon nucleolytic digestion of fixed cells, the antigen becomes available to the antibodies in all cells, indicating that AF-2 antigen is present during the whole cell cycle but differentially accessible. Digestion with the single strand specific S1 nuclease reveals that the alteration of chromatin structure induced by the introduction of nicks into the DNA rather than the digestion of DNA bound to the immunogenic epitope accounts for the change in accessibility of AF-2 antigen in interphase nuclei. The epitope recognized by the antibody in human cells is present in two polypeptides of 65 and 36 kDa, respectively, which are tightly bound to chromatin and cross-linkable to the nuclear matrix. The proteins also occur in the midbody during cytokinesis. The immunogenic epitope is conserved between man and fission yeast.     

Migration cues induce chromatin alterations

Directed cell migration is a property central to multiple basic biological processes. Here, we show that directed cell migration is associated with global changes in the chromatin fiber. Polarized posttranslational changes in histone H1 along with a transient decrease in H1 mobility were detected in cells facing the scratch in a wound healing assay. In parallel to the changes in H1, the levels of the heterochromatin marker histone H3 lysine 9 tri-methylation were elevated. Interestingly, reduction of the chromatin-binding affinity of H1 altered the cell migration rates. Moreover, migration-associated changes in histone H1 were observed during nuclear motility in the simple multicellular organism Neurospora crassa . Our studies suggest that dynamic reorganization of the chromatin fiber is an early event in the cellular response to migration cues.     

Early alterations in rat liver chromatin structure after a single dose of diethylnitrosamine

In the chromatin of 24-h regenerating rat livers, derivative melting profiles are characterized by a high proportion of transitions above 90°C. After the injection of diethylnitrosamine there is a rapid shift to lower melting temperatures. This is due to a rearrangement of the chromatin to higher amounts of nucleosomal components but possibly also a consequence of chemical modifications and conformational alterations of the DNA. In the nonregenerating liver essentially the same observations can be made, although reactions proceed significantly slower. These results are in good agreement with the observation that carcinogens are more active in tissues stimulated to rapid proliferation as compared to resting tissues.     

Cytophotometric analysis of T-2 toxin induced alterations in chromatin condensation and neuronal nuclear volume of rat supraoptic-magnocellular neurons

Quantitative cytophotometry and ocular filar micrometry were used to monitor T-2 toxin induced alterations in chromatin and neuronal nuclear volume in supraoptic-magnocellular neurons of rat hypo-thalami. Thirty male Sprague-Dawley rats (200-220g) were given a single i.p. injection of T-2 toxin (0.5, 0.75, 1.0 and 1.5 X LD50), a trichothecene mycotoxin; rats were decapitated 8 hours post-dosing. After stoichiometric Feulgen-DNA staining of brain sections, scanning-integrating microdensitometry was used to quantify changes in the susceptibility of chromatin to Feulgen acid hydrolysis. Changes in neuronal nuclear volumes were also determined histometrically. Within the magnocellular neurons of the supraoptic nuclei, significant reductions in F-DNA reactivity were observed in the 0.5, 0.75, and 1.0 X LD50 groups (i.e. 3.7%, 4.4% and 2.5%, respectively); however, rats receiving 1.5 X LD50 T-2 toxin showed no difference in F-DNA reactivity compared to controls. In addition, ocular filar micrometry demonstrated increased neuronal nuclear volumes in all groups receiving T-2 toxin, and following an inverse trend to that seen with F-DNA stainability. Additional observations included pronounced polydipsia, polyphagia and horripilation in the experimental groups, independent of the dosages employed; these changes were evident within 1 hour post-injection. It is postulated that the T-2 toxin induced reduction in the susceptibility of chromatin to Feulgen acid hydrolysis and concomitant increases in neuronal nuclear volumes represent an early indication of impaired metabolic activity. Since these neurons are important sites of vasopressin (antidiuretic hormone) synthesis, these data suggest an impaired osmoregulatory ability. The pronounced polydipsia which occurred shortly after intoxication is further evidence of this impairment. Although these findings do not provide insight relating to the mechanism of osmoregulatory disruption, it is evident that an impaired ability to osmoregulate is among the earliest indications of acute T-2 toxin mycotoxicosis.     

Alterations of neuronal nuclear matrix and chromatin structure after irradiation under aerobic and anoxic conditions

This study was undertaken to determine if structural alterations of the bulk chromatin and the amount of protein associated with the nuclear matrix in cerebellar neurons depend on radiation dose and a cell's state of oxygenation. After irradiation with 2.5 to 25.0 Gy under both aerobic and anoxic conditions, the sensitivity of the neuronal chromatin to m. nuclease digestion increase linearly with dose up to about 5 Gy, beyond which there was no further increase. The same increase in accessibility of chromatin to micrococcal nuclease digestion was observed when neuronal nuclei were irradiated at 4 degrees C. Neuronal nuclei were stained with propidium iodide (PI) for DNA and with fluorescein isothiocyanate (FITC) for protein, both before and after complete digestion with DNase I, and analyzed by flow cytometry. There was no change in either the PI (P greater than 0.4) or the FITC (P greater than 0.9) fluorescence of undigested nuclei after irradiation. For the DNase I digested nuclei, the PI fluorescence was unchanged after irradiation (P greater than 0.4), but the FITC fluorescence increased significantly (P less than 0.02). This increase in the FITC fluorescence was linear with dose up to about 5 Gy, beyond which there was no further increase. The flow cytometry results from DNase I digested nuclei were identical for neurons irradiated under aerobic or anoxic conditions, indicating that this phenomenon is oxygen independent. This increase in FITC fluorescence after irradiation was inhibited at ice-cold temperatures and probably reflects an increase in protein content at the nuclear matrix that requires metabolism. This may explain our previously observed resistance of nuclear matrix-associated DNA to digestion by DNase I. This protein increase at the nuclear matrix appears to follow "saturation" kinetics identical to that previously reported for repair of DNA strand breaks in cerebellar neurons. However, the exact molecular nature of this process and its role in DNA repair or cell survival remains to be determined.     

Telomeric chromatin structure

Eukaryotic DNA is packaged into nucleosomes, which further condenses into chromosomes. The telomeres, which form the protective end-capping of chromosomes, play a pivotal role in ageing and cancer. Recently, significant advances have been made in understanding the nucleosomal and telomeric chromatin structure at the molecular level. In addition, recent studies shed light on the nucleosomal organisation at telomeres revealing its ultrastructural organisation, the atomic structure at the nucleosome level, its dynamic properties, and higher-order packaging of telomeric chromatin. Considerable advances have furthermore been made in understanding the structure, function and organisation of shelterin, telomerase and CST complexes. Here we discuss these recent advances in the organisation of telomeric nucleosomes and chromatin and highlight progress in the structural understanding of shelterin, telomerase and CST complexes.     

Supranucleosomal structure of chromatin

Rat liver chromatin was moderately digested by micrococcal nuclease and analysed by centrifugation in isokinetic sucrose gradients and electron microscopy. Two classes of particles sedimenting with about 33S and 60S were characterized. Kinetics of their appearance and disappearance during progressive digestion suggests that they represent monomers and dimers cleaved from a higher order (supranucleosomal) structure of chromatin. Biochemical and electron microscopical results suggest that the monomers and dimers contain eight and sixteen nucleosomes, respectively, which are densely packed into 23 nm (monomer) and 29 nm (dimer) globules.     

Sperm chromatin alterations in fertile and subfertile bulls

Alterations in sperm chromatin have been related with subfertility in several mammals. In this study, chromatin alteration types (Base, Basal half, Central axis, Dispersed, and Whole) were assessed by toluidine blue (TB) staining, 6-diamidino-2-fenilindole (DAPI) and anti-protamine 1 antibody (anti-PR1) labeling in sperm samples of fertile and subfertile bulls. Semen samples were obtained from bulls kept in Artificial Insemination Center (fertile bulls) or from bulls subjected to scrotal insulation (subfertile bulls). The percentage of chromatin alterations identified by TB was similar (P?>?0.05) in semen samples of fertile and subfertile bulls. In contrast, a greater (P?<?0.01) chromatin decondensation and heterogeneity were recorded in semen samples of subfertile bulls. In DAPI and anti-PR1 methods, the subfertile bulls samples had a higher (P?<?0.05) percentage of alteration in the base as well as overall chromatin alterations (P?<?0.05). Moreover, the chromatin alterations recorded with TB, DAPI, and anti-PR1 were compared in semen samples of fertile and subfertile bulls. In fertile bulls, the overall chromatin alterations were similar (P?>?0.05) among the methods In contrast, semen samples of subfertile bulls had a higher (P?<?0.05) percentage of overall chromatin alterations when labeled with DAPI. In conclusion, our findings shown that all dye tested had specific sperm stainability and can be feasible to monitor subfertility condition in bulls. Also, different chromatin alteration types in sperm samples of fertile and suberftile bulls were recorded.     

Kinases and chromatin structure

Chromatin structure is regulated by families of proteins that are able to covalently modify the histones and the DNA, as well as to regulate the spacing of nucleosomes along the DNA. Over the years, these chromatin remodeling factors have been proven to be essential to a variety of processes, including gene expression, DNA replication, and chromosome cohesion. The function of these remodeling factors is regulated by a number of chemical and developmental signals and, in turn, changes in the chromatin structure eventually contribute to the response to changes in the cellular environment. Exciting new research findings by the laboratories of Sharon Dent and Steve Jackson indicate, in two different contexts, that changes in the chromatin structure may, in reverse, signal to intracellular signaling pathways to regulate cell fate. The discoveries clearly challenge our traditional view of ‘epigenetics’, and may have important implications in human health.