Postirradiation alterations of neuronal chromatin structure

Previous work from our laboratory suggested that neuronal chromatin structure may be altered immediately after exposure to ionizing radiation. In the present study, whole brains of 4-month-old male Fisher 344 rats were irradiated with a dose of 25 Gy. The kinetics of restoring the chromatin structure to its unirradiated state was investigated in rat cerebellar neurons using three different approaches: (1) measurement of changes in the DNA superhelical structure by the fluorescent halo assay, (2) measurement of changes in chromatin accessibility to digestion by micrococcal nuclease, and (3) measurement of changes in the accessibility of the nuclear-matrix-associated DNA to digestion by DNase I. Immediately after irradiation, the topological constraints on the DNA loops were altered, the chromatin was more accessible to m. nuclease digestion, and the DNA associated with the nuclear matrix was more resistant to digestion by DNase I. Return of the chromatin structure to its unirradiated state as measured by each of the three methods followed biphasic kinetics with the fast phase having a half-time of several minutes and the slow phase having a half-time of several hours. The kinetics are similar to that previously reported for repair of radiation-induced DNA damage in mammalian cells. Although the independent assays used in this study seemed to follow the same kinetics, their relationship at the molecular level remains to be determined.     

Flow cytometric studies of the nuclear matrix

We have devised a method to measure the protein and nucleic acid content of the nuclear matrix using flow cytometry. Nuclear matrices were prepared from nuclei by DNase I digestion followed by 3 M NaCl extraction. The resulting particles were stained with fluorescein isothiocyanate (FITC) for protein and propidium iodide (PI) for double-stranded nucleic acids, and fluorescence as well as forward angle light scatter was detected. The matrices were also subjected to additional chemical or enzymatic perturbations, and changes in the above parameters were measured. Results showed that matrices from heat-shocked cells not only retained the majority of heat-induced excess nuclear protein, but also exhibited higher PI signals than controls after RNase A digestion. This observation did not hold if RNase A digestion preceded high-salt extraction, suggesting that a salt-extractable moiety had been replaced or altered by heat so that double-stranded RNA was protected from the nucleolytic attack. The residual PI fluorescence in matrices from heated cells bore a linear relationship to the increased protein content in those matrices, indicating that the excess protein sequesters matrix-associated RNA. Polyacrylamide gel electrophoresis of matrix polypeptides revealed increased amounts of many proteins as a result of heat as well as the appearance of several new proteins, one of which comigrates with the HSP72/73 heat-shock proteins. The results of these studies show that flow cytometry can be used to study the nuclear matrix and is capable of detecting changes that result from alterations in its protein composition.     

DNase I sensitivity of nuclear DNA measured by flow cytometry

The DNase I digestion kinetics of DNA in isolated nuclei (from HeLa or murine mammary carcinoma, 67 cells) were assayed flow cytometrically by measuring the changes in ethidium bromide (EtBr) fluorescence following various digestion time intervals. The DNase I digestion curve was characterized by an initial 25-30% increase in fluorescence upon addition of the enzyme, a rapid reduction in fluorescence to approximately 50-55% in 30 minutes, and a limit digest of 45-50% beyond 45 minutes. Throughout digestion, the DNA histogram retained its characteristic bimodal shape, showing that histogram rearrangement was not responsible for the changes in EtBr fluorescence. Irradiation with 5 X 10(6) rads (137Cs-gamma-rays) or exposure to 50 mM EDTA caused an increase in EtBr fluorescence similar to that caused by DNase I, suggesting that DNA nicking and/or chromatin loosening were responsible for this increase. Residual DNA assayed by the solubilization of 14C-TdR (thymidine)-labeled DNA indicated a similar kinetic pattern without the initial increase. However, at the limit digest, the fraction of DNA remaining trichloroacetic acid (TCA) insoluble (10%) was smaller than that measured by loss of EtBr fluorescence (50% of initial, 40% of maximum). Part of this difference was due to the presence of TCA soluble DNA trapped within the nuclear matrix (15-20%). This trapped DNA was released when the digested nuclei were exposed to 0.5-1.0 M NaCl just prior to EtBr staining. Exposure of HeLa cells to three agents that are believed to cause changes in chromatin structure resulted in alterations in the DNase I digestion kinetics measured flow cytometrically.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expansion of chicken erythrocyte nuclei upon limited micrococcal nuclease digestion. Correlation with higher order chromatin structure

A sensitive method for measuring nuclear volumes with a Coulter counter is described. It has been applied to the digestion of chicken erythrocyte nuclei by micrococcal nuclease and DNase I. Early in digestion, micrococcal nuclease induced a 20% increase in the effective spherical volume of the nuclei, followed by a gradual reduction. At the peak of nuclear swelling, about 17% of the chromatin was soluble after lysis and its average chain length was about 18 kilobase pairs (kb). DNase I digestion did not give rise to a corresponding expansion of the nuclei. Several preparation conditions, including the treatment of nuclei with 0.2% Triton X-100, led to a loss of the expansion effect upon subsequent micrococcal nuclease digestion. The results support the domain theory of higher order chromatin structure. In the context of this model, the observed maximum nuclear expansion correlates with an average of one nuclease scission per domain.     

Propidium iodide staining correlates with the extent of DNA degradation in isolated nuclei.

Gradual degradation of internucleosomal DNA is a hallmark of apoptosis and can be simulated by incubating isolated thymocyte nuclei in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37 degrees C. Staining of nuclei with the DNA binding fluorescent dye propidium iodide (PI) showed that intensity of fluorescence correlated with the extent of DNA degradation. PI fluorescence was increased in the presence of DNase I. Thus it seems that the cleavage of chromatin DNA by DNase 1 or by the endogenous enzyme increases the accessibility of DNA for the dye. No increase of fluorescence was observed in the presence of the known inhibitors of the endogenous endonuclease: Zn2+ and EGTA. However, the presence of Zn2+ led to decreased staining of the nuclei by PI and caused a shift in the scatter profile of the nuclei, suggesting that a conformational change of chromatin is induced by this ion. This correlation between intensity of PI staining and DNA degradation should be useful to compare endogenous nuclease levels in lymphocyte populations.     

Nuclear protein content and cell progression kinetics following X irradiation

After irradiation with 4 Gy of X rays the nuclear protein and DNA contents (to determine cell-cycle position) of HeLa cells were determined by isolating nuclei and staining them with the fluorescent dyes fluorescein isothiocyanate (FITC) for protein and propidium iodide (PI) for DNA. Immediately following irradiation there was no change in the shape of the bivariate (FITC-PI) histogram. At 3 and 4 h after irradiation the region of the histogram which corresponds to mitotic cells had disappeared. At 6 h nuclei reappeared in this region. The maximum rearrangement of the histogram (i.e., maximum accumulation of cells in G2 with minimum cells in G1) occurred at 10.5 h after irradiation, which is later than the time required for mitotic recovery. No change in nuclear protein content of cells in G1 and S was observed. However, beginning at 4 h after irradiation and continuing throughout the period of observation, a small (10-20%) but significant increase in nuclear protein content was observed for nuclei isolated from cells in G2. The increase in nuclear protein content may be part of the mechanism of G2 arrest and/or may reflect unbalanced growth.     

Response of isolated nuclei to phospholipid vesicles: analysis of chromatin sensitivity to DNase I and micrococcal nuclease

Phosphatidylserine (PS) and phosphatidylcholine (PC) multilamellar vesicles (MLV) affect chromatin structure as analysed by DNase I sensitivity. The kinetics of DNA solubilisation during the digestion of nuclei indicates that phosphatidylserine causes an increase in DNase accessibility while phosphatidylcholine slightly reduces this accessibility. The effect of phosphatidylserine has also been analysed by means of isokinetic sucrose gradients and agarose gel electrophoresis of nuclear DNA solubilised by micrococcal nuclease. This analysis indicates that phosphatidylserine induces a very rapid production of mononucleosome subunits as compared with untreated nuclei.     

Kinetics of nuclease digestion of Physarum polycephalum nuclei at different stages of the cell cycle

The kinetics of nuclease digestion of Physarum polycephalum nuclei by staphylococcal nuclease and DNase I has been studied at different stages of the cell cycle. Significant differences in the digestion behaviour of nuclei from metaphase and interphase have been detected with DNase I but not with staphylococcal nuclease. Furthermore the structure of newly replicated DNA in S phase differs from the bulk in that it is more easily degraded to acid-soluble products by either staphylococcal nuclease or by DNAase I. At least four types of chromatin structure can be distinguished by our digestion kinetics experiments.     

Structure of rat liver nuclei after depletion and reassociation of histone H1

Chromatin in isolated rat liver nuclei was compared with chromatin in (i) nuclei depleted of H1 by acid extraction; (ii) nuclei treated at pH 3.2 (without removal of H1), and (iii) depleted nuclei following reassociation of H1. Electron microscopy and digestion by DNase I, micrococcal nuclease and endogenous Ca/Mg endonuclease were used for this comparative examination. Electron micrographs of H1-depleted nuclei showed a dispersed and finely granular appearance. The rate of DNA cleavage by micrococcal nuclease or DNase I was increased several-fold after H1 removal. Discretely sized intermediate particles produced by Ca/Mg endonuclease in native nuclei were not observed in digests of depleted nuclei. Digestion by micrococcal nuclease to chromatin particles soluble in 60 mM NaCl buffer appeared not to be affected in depleted nuclei. When nuclei were treated at pH 3.2, neither the appearance of chromatin in electron micrographs nor the mode or rate of nuclease digestion changed appreciably. Following reassociation of H1 to depleted nuclei, electron micrographs demonstrated the reformation of compacted chromatin, but the lower rate of DNA cleavage in native nuclei was not restored. Further, H1 reassociation produced a significant decrease in the solubility of nuclear chromatin cleaved by micrococcal nuclease or Ca/Mg endonuclease. In order to evaluate critically the reconstitution of native chromatin from H1-depleted chromatin we propose the use of digestion by a variety of nucleases in addition to an electron microscopic examination.     

Digestion of insect chromatin with micrococcal nuclease, DNase I and DNase I combined with single-strand specific nuclease S1.

The chromatin of the lepidopteran Ephestia kuehniella was digested by micrococcal nuclease, DNase I and S1-nuclease combined with DNase I pretreatment. The resulting DNA fragments were analyzed by gel electrophoresis and compared with the DNA fragments of rat liver nuclei obtained by the same process. Extensive homology was revealed between insect and mammalian chromatin structure. The combined DNase I- S1-nuclease digestion yields double-stranded DNA fragments of lengths from 30 to 110 base-pairs. These DNA fragments are not obtained from nuclei predigested extensively with micrococcal nuclease. The results are discussed with respect to the internal structure of the chromatin subunit.     

Intranuclear distribution of the human myeloid cell nuclear differentiation antigen in HL-60 cells

Based on solubility properties, the human myeloid cell nuclear differentiation antigen exists as at least two distinct populations. Most is easily extracted from isolated nuclei in 0.35 M NaCl, while 20 percent resists such treatment. Compared to undigested nuclei, both the amount of myeloid cell nuclear differentiation antigen (MNDA) released from nuclei after DNase I treatment and the amount resisting further extraction in 0.35 M NaCl increased after DNA was digested with DNase I. Under these conditions, there was a concomitant decrease in the amount of MNDA that was extractable with 0.35 M NaCl. Mixing nuclear protein extracts that contain MNDA with nuclei from cells that do not express this protein demonstrated that the MNDA redistributes from the freely soluble form to the nuclear residual fraction as a consequence of DNase I digestion. These data are consistent with a model in which the amount of MNDA that is tightly bound to salt-washed nuclei is held constant in the presence of an excess of unassociated MNDA in the nucleus, and that the level of MNDA binding to this nuclear fraction increases in proportion to the extent of DNA damage resulting from DNase I digestion.     

The involvement of nuclear nucleases in rat thymocyte DNA degradation after γ-irradiation

Possible mechanisms of internucleosomal DNA fragmentation in thymocytes of irradiated rats were studied. It was shown that thymocyte nuclei contain at least two nucleases that cleave DNA between nucleosomes — a Ca²⁺/Mg²⁺-dependent nuclease and an acidic one which does not depend on bivalent ions. 2 and 3 h after irradiation at a dose of 10 Gy the initial rate of DNA cleavage by Ca²⁺/Mg²⁺-dependent nuclease in isolated nuclei increased three and seven times, respectively, but the kinetics of DNA digestion by acidic nuclease did not change. The experiments with cycloheximide indicated that Ca²⁺/Mg²⁺-dependent endonuclease turns over at a high rate. The activity of the cytoplasmic acidic and Mg²⁺-dependent nucleases was shown to increase (by 40 and 50%, respectively) 3 h after irradiation. The effect is caused by the de novo synthesis of the nucleases. At the same time the activity of nuclear nucleases did not essentially change. The chromatin isolated from rat thymocytes 3 h after irradiation did not differ in its sensitivity to some exogenic nucleases (DNAase I, micrococcal nuclease and nuclease from Serratia marcescens) from the control. Thus, Ca²⁺/Mg²⁺-dependent endonuclease seems to be responsible for the postirradiation internucleosomal DNA fragmentation in dying thymocytes.     

Calmodulin and calmodulin-binding proteins in liver cell nuclei

Three nuclear subfractions were prepared from isolated hepatocytes nuclei. The calmodulin content in whole nuclei was 79 ng/mg of protein. The soluble fraction obtained after digestion of the nuclei with DNase I and RNase A (S1 fraction) contained 252 ng of calmodulin/mg of protein. The pellet obtained after the digestion with nucleases was treated with 1.6 M NaCl, and the soluble fraction and the residual structures obtained after the treatment were called S2 fraction and nuclear matrix, respectively. The calmodulin contents of the S2 fraction and of the nuclear matrix were 68 and 190 ng/mg of protein, respectively. If nuclei were digested only with DNase I, the calmodulin content in the soluble fraction increased to 703 ng/mg of protein, indicating that part of the nuclear calmodulin is associated with active DNA. Five nuclear calmodulin-binding proteins were identified. Two, having apparent molecular masses of 240 and 150 kDa were only found in the nuclear matrix, whereas the other three, having molecular masses of 120, 65, and 40 kDa were found in different proportions in all nuclear subfractions. A calmodulin-dependent inhibition of protein phosphorylation in the S1 fraction was discovered. Purification attempts on the calmodulin-binding proteins of the S1 subfraction by calmodulin affinity chromatography yielded four major polypeptides with apparent molecular masses of about 41, 46, and 120 (two products) kDa. These polypeptides retained the ability to inhibit protein phosphorylation but not the sensitivity to calmodulin.     

The reactive SH1 and SH2 cysteines in myosin subfragment 1 are cross-linked at similar rates with reagents of different length

Nuclei prepared from confluent and mitotically arrested populations of human diploid fibroblast-like cells of different $\text{in}\text{vitro}$ ages were subjected to digestion by micrococcal nuclease and DNase I. There was no age or culture state variation in the susceptibility of DNA to micrococcal nuclease digestion. There was, however, an age related inhibition of DNA digestion by DNase I in nuclei from older confluent but not older arrested cells. It is suggested that this is the result of an age related masking by nucleosome core histones which limits the accessibility of DNA to enzymatic activities in older confluent cells.     

Sea urchin sperm chromatin structure as probed by pancreatic DNase I: Evidence for a novel cutting periodicity

Chromatin from mature sea urchin spermatozoa is highly compacted and composed almost entirely of DNA and the five histones, although sperm H1, H2A, and H2b histones differ from those found in embryo or somatic cell nuclei. Release of acid-soluble DNA during pancreatic DNase I digestion is 20-fold slower from sperm nuclei than from embryonic nuclei. Following DNase I digestion, most sperm nuclear DNA remains at high molecular weight, although there appears to be some release of 10 base oligomer fragments. Size analysis of the higher molecular weight DNA reveals a series of fragments that indicate a cutting periodicity of approximately 500 base pairs. This pattern remains when electrophoretic separation is carried out under denaturing conditions. The 500 base pair cleavage pattern was not detected in digestions of embryonic nuclei. Nucleosomes reconstituted with fractionated core histones from sperm gave, upon digestion, a characteristic 10 base “ladder,” with no resistant high molecular weight DNA. Micrococcal nuclease and DNase II digested sperm nuclei to produce DNA fragments with a calculated repeat length of 248 ± 3 and 246 ± 6 base pairs, respectively. The structural basis for the 500 base pair cutting periodicity in sperm nuclei may reside in the unique sperm H1 histone.     

Nuclease-induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide

A flow cytometric analysis of DNA structural changes induced by cleavage with nucleases was performed on isolated HeLa nuclei by assessing changes in stainability with the DNA-specific fluorochrome propidium iodide (PI). After mild digestion with DNAse I, micrococcal nuclease, or with the single-strand-specific S1 and Neurospora crassa nucleases, fluorescence intensity of nuclei stained with PI increased by about 15-30% above the value of undigested control samples. No significant modifications were observed with the restriction enzymes Eco RI, Alu I, and Not I. The DNAse I-induced increase in fluorescence intensity was also observed with the non-intercalating dye Hoechst 33258, but not with mithramycin. Nuclease-induced fluorescence intensity changes as determined with PI were found to be dependent on the dye concentration. A constant increase (about 20%) was measured at dye/DNA-P ratios greater than 0.11. Below this value (2 micrograms/ml PI), the fluorescence intensity of digested samples was 15-30% lower than that of undigested controls. This behaviour towards intercalating dyes is similar to that of the relaxed (nicked) vs. the supercoiled (intact) form of circular DNA. These results suggest that conformation- but not sequence-specific nucleases induce a relaxation of DNA supercoils.     

Nuclear interactions of wild type and variant glucocorticoid receptors

Nuclease digestion of nuclei from glucocorticoid sensitive and resistant lymphoma cell lines was used to study the nuclear compartmentalization of wild type and variant glucocorticoid receptors. In comparison with wild type, the variant line (S49 143r) had an increased capacity to translocate to the nucleus (nti), but was more completely released from nuclei by nuclease digestion. Approximately 20% of the receptor in wild type nuclei was resistant to release by DNase I digestion, while only less than 5% of the receptor from nti nuclei was retained under the same conditions. Studies with wild type nuclei show that the nuclease resistant portion of receptors was also more resistant to release by increased ionic strength.