Structure of interphase nuclei in relation to the cell cycle. Chromatin organization in mouse L cells temperature-sensitive for DNA replication

Mutant lines of mouse L cells, TS A1S9, and TS C1, show temperature- sensitive (TS) DNA synthesis and cell division when shifted from 34 degrees to 38.5 degrees C. With TS A1S9 the decline in DNA synthesis begins after 6-8 h at 38.5 degrees C and is most marked at about 24 h. Most cells in S, G2, or M at temperature upshift complete one mitosis and accumulate in the subsequent interphase at G1 or early S as a result of expression of a primary defect, failure of elongation of newly made small DNA fragments. Heat inactivation of TS C1 cells is more rapid; they fail to complete the interphase in progress at temperature upshift and accumulate at late S or G2. Inhibition of both cell types is reversible on return to 34 degrees C. Cell and nuclear growth continues during inhibition of replication. Expression of both TS mutations leads to a marked change in gross organization of chromatin as revealed by electron microscopy. Nuclei of wild-type cells at 34 degrees and 38.5 degrees C and mutant cells at 34 degrees C show a range of aggregation of condensed chromatin from small dispersed bodies to large discrete clumps, with the majority in an intermediate state. In TS cells at 38.5 degrees C, condensed chromatin bodies in the central nuclear region become disaggregated into small clumps dispersed through the nucleus. Morphometric estimation of volume of condensed chromatin indicates that this process is not due to complete decondensation of chromatin fibrils, but rather involves dispersal of large condensed chromatin bodies into finer aggregates and loosening of fibrils within the aggregates. The dispersed condition is reversed in nuclei which resume DNA synthesis when TS cells are downshifted from 38.5 degrees to 34 degrees C. The morphological observations are consistent with the hypothesis that condensed chromatin normally undergoes an ordered cycle of transient, localized disaggregation and reaggregation associated with replication. In temperature-inactivated mutants, normal progressive disaggregation presumably occurs, but subsequent lack of chromatin replication prevents reaggregation.     

Replication of heterochromatin: insights into mechanisms of epigenetic inheritance

Heterochromatin is composed of tightly condensed chromatin in which the histones are deacetylated and methylated, and specific nonhistone proteins are bound. Additionally, in vertebrates and plants, the DNA within heterochromatin is methylated. As the heterochromatic state is stably inherited, replication of heterochromatin requires not only duplication of the DNA but also a reinstallment of the appropriate protein and DNA modifications. Thus replication of heterochromatin provides a framework for understanding mechanisms of epigenetic inheritance. In recent studies, roles have been identified for replication factors in reinstating heterochromatin, particularly functions for origin recognition complex, proliferating cell nuclear antigen, and chromatin-assembly factor 1 in recruiting the heterochromatin binding protein HP1, a histone methyltransferase, a DNA methyltransferase, and a chromatin remodeling complex. Potential mechanistic links between these factors are discussed. In some cells, replication of the heterochromatin is blocked, and in Drosophila this inhibition is mediated by a chromatin binding protein SuUR.     

The murine heterochromatin protein M31 is associated with the chromocenter in round spermatids and Is a component of mature spermatozoa

In mature sperm the normal nucleosomal packaging of DNA found in somatic and meiotic cells is transformed into a highly condensed form of chromatin which consists mostly of nucleoprotamines. Although sperm DNA is highly condensed it is nevertheless packaged into a highly defined nuclear architecture which may be organized by the heterochromatic chromocenter. One major component of heterochromatin is the heterochromatin protein 1 which is involved in epigenetic gene silencing. In order to investigate the possible involvement of heterochromatin protein in higher order organization of sperm DNA we studied the localization of the murine homologue of heterochromatin protein 1, M31, during chromatin reorganization in male germ cell differentiation. Each cell type in the testis showed a unique distribution pattern of M31. Colocalization to the heterochromatic regions were found in Sertoli cells, in midstage pachytene spermatocytes, and in round spermatids in which M31 localizes to the centromeric chromocenter. M31 cannot be detected in elongated spermatids or mature spermatozoa immunocytologically, but could be detected in mature spermatozoa by Western blotting. We suggest that M31, a nuclear protein involved in the organization of chromatin architecture, is involved in higher order organization of sperm DNA.     

Correlation between nuclear morphology and rate of deoxyribonucleic acid synthesis in a normal cell line.

Chinese hamster fibroblasts were investigated for the existence of correlations between proliferative activity and nuclear morphology. As a proliferative parameter, the rate of DNA synthesis of individual cells was determined by quantitative 14C-autoradiography. In a second step the images of the Feulgen-stained nuclei were digitized for extraction of features of morphology and texture. These features were correlated with the corresponding DNA synthesis rate values. The following relationships were found: Round nuclei have higher rates of DNA synthesis than flat ones. The more chromatin is packed at the nuclear rim, possibly representing heterochromatin, the lower the rate of DNA synthesis. The DNA synthesis rate also correlates with the graininess of chromatin. Larger areas of condensed chromatin are associated with lower rate values. A fine and irregular network of chromatin, as is typical of immature cell types, is associated with a high rate of DNA synthesis. Although these results are presently confined to the cell line investigated, parallels seem to exist to other cell types, such as erythropoietic cells, which await further investigation.     

THE SYNTHESIS OF DNA, RNA, AND NUCLEAR PROTEIN IN NORMAL AND TUMOR STRAIN CELLS : IV. HeLa Tumor Strain Cells

Interferometric and photometric measurements have been made on HeLa cells, a strain of cells originally derived from a human carcinoma. From a study of the relations between successive physical measurements on individual cells, it was confirmed that, whereas the net syntheses of nuclear RNA and nuclear protein are closely associated during interphase, they are dissociated from DNA replication to a significant extent. These results on nuclear metabolism agree with others previously reported in cell strains derived from tumors; they contrast with results from freshly prepared normal cells, where the net syntheses of DNA, nuclear RNA, and protein are closely associated during interphase. Cytoplasmic measurements on HeLa cells showed that much of the net synthesis of cytoplasmic RNA is associated with DNA replication as in normal cells, and they failed to detect transfer from the nucleus of a stable RNA component synthesized independently from DNA replication. In auxiliary experiments, an inhibition of the onset of DNA synthesis was produced by a dose of X-rays; under these conditions it was shown that the major part of the accumulation of nuclear protein was independent of DNA replication and that the accumulation of nuclear RNA was equivalent to or slightly less than that of nuclear protein. About half the accumulation of cytoplasmic RNA was inhibited when DNA synthesis was blocked.     

Characteristics of DNA replication of the chromosomes of the bone marrow cells in patients with acute leukemia]

A study of the late DNA replication pattern in chromosomes of human acute leucaemia cells revealed a significant diffrence from control. Chromosomes, 2,3 and 4-5 of the acute leucaemia cells finish their DNA replication earlier, and chromosomes 1, 13-15 and 16 later, compared to the control chromosomes. The difference in the pattern of DNA replication between analogous chromosomes of acute leucaemia and donor cells was associated with the discovery of large late-replicating chromatin blocks in the pericentromeric regions of leucaemia cell chromosomes. Some relationship is suggested between the pattern of pericentromeric heterochromatin DNA replication and cell differentiation.     

Protein Tpr is required for establishing nuclear pore‐associated zones of heterochromatin exclusion

Amassments of heterochromatin in somatic cells occur in close contact with the nuclear envelope (NE) but are gapped by channel‐ and cone‐like zones that appear largely free of heterochromatin and associated with the nuclear pore complexes (NPCs). To identify proteins involved in forming such heterochromatin exclusion zones (HEZs), we used a cell culture model in which chromatin condensation induced by poliovirus (PV) infection revealed HEZs resembling those in normal tissue cells. HEZ occurrence depended on the NPC‐associated protein Tpr and its large coiled coil‐forming domain. RNAi‐mediated loss of Tpr allowed condensing chromatin to occur all along the NE's nuclear surface, resulting in HEZs no longer being established and NPCs covered by heterochromatin. These results assign a central function to Tpr as a determinant of perinuclear organization, with a direct role in forming a morphologically distinct nuclear sub‐compartment and delimiting heterochromatin distribution.     

THE EFFECT OF HYDROXYUREA AND FLUORODEOXYURIDINE ON CELL CYCLE EVENTS IN THE CHLOROCOCCAL ALGA SCENEDESMUS QUADRICAUDA (CHLOROPHYTA)1

The effect of hydroxyurea and 5-fluorodeoxyuridine (FdUrd) on the course of growth (RNA and protein synthesis) and reproductive (DNA replication and nuclear and cellular division) processes was studied in synchronous cultures of the chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. The presence of hydroxyurea (5 mg·L^?1)from the beginning of the cell cycle prevented growth and further development of the cells because of complete inhibition of RNA synthesis. In cells treated later in the cell cycle at the time when the cells were committed to division, hydroxyurea present in light affected the cells in the same way as a dark treatment without hydroxyurea; i. e. RNA synthesis was immediately inhibited followed after a short time period by cessation of protein synthesis. Reproductive processes including DNA replication to which the commitment was attained, however, were initiated and completed. DNA synthesis continued until the constant minimal ratio of RNA to DNA was reached. FdUrd (25 mg·L^?1) added before initiation of DNA replication in control cultures prevented DNA synthesis in treated cells. Addition of FdUrd at any time during the cell cycle prevented or immediately stopped DNA replication. However, by adding excess thymidine (100 mg·L^?1), FdUrd inhibition of DNA replication could be prevented. FdUrd did not affect synthesis of RNA, protein, or starch for at least one cell cycle. After removal of FdUrd, DNA synthesis was reinitiated with about a 2-h delay. The later in the cell cycle FdUrd was removed, the longer it took for DNA synthesis to resume. At exposures to FdUrd longer than two or three control cell cycles, cells in the population were gradually damaged and did not recover at all.     

Nuclear reorganization of mammalian DNA synthesis prior to cell cycle exit

In primary mammalian cells, DNA replication initiates in a small number of perinucleolar, lamin A/C-associated foci. During S-phase progression in proliferating cells, replication foci distribute to hundreds of sites throughout the nucleus. In contrast, we find that the limited perinucleolar replication sites persist throughout S phase as cells prepare to exit the cell cycle in response to contact inhibition, serum starvation, or replicative senescence. Proteins known to be involved in DNA synthesis, such as PCNA and DNA polymerase delta, are concentrated in perinucleolar foci throughout S phase under these conditions. Moreover, chromosomal loci are redirected toward the nucleolus and overlap with the perinucleolar replication foci in cells poised to undergo cell cycle exit. These same loci remain in the periphery of the nucleus during replication under highly proliferative conditions. These results suggest that mammalian cells undergo a large-scale reorganization of chromatin during the rounds of DNA replication that precede cell cycle exit.     

The effect of methylnitronitrosoguanidine on DNA synthesis in human and animal cells. Inhibition of DNA synthesis in asynchronous and synchronous cultured cells

Methylnitronitrosoguanidine (MNNG) is reported to inhibit DNA synthesis in intact human cells, in the cells from patients with ataxia telangiectasia (AT) or the cells from two rodent species. DNA synthesis in different cell lines exhibits varying sensitivity to MNNG inhibitory effect. 4-5-fold higher concentrations of MNNG are required for 50% inhibition of DNA synthesis in AT cells or in field vole cells as compared with the concentration required for human cells or Chinese hamster. The different compactness of two chromatin fractions might possibly result in lower sensitivity of DNA synthesis in heterochromatin to MNNG-induced inhibition as compared with the sensitivity of euchromatin. The genetic expression of AT defect on the cellular level is supposed to be connected with changes in supramolecular packaging of chromatin in interphase nuclei.     

Cytochalasin releases mRNA from the cytoskeletal framework and inhibits protein synthesis.

Cytochalasin D was shown to be a reversible inhibitor of protein synthesis in HeLa cells. The inhibition was detectable at drug levels typically used to perturb cell structure and increased in a dose-dependent manner. The drug also released mRNA from the cytoskeletal framework in direct proportion to the inhibition of protein synthesis. The released mRNA was unaltered in its translatability as measured in vitro but was no longer translated in the cytochalasin-treated HeLa cells. The residual protein synthesis occurred on polyribosomes that were reduced in amount but displayed a normal sedimentation distribution. The results support the hypothesis that mRNA binding to the cytoskeletal framework is necessary although not sufficient for translation. Analysis of the cytoskeletal framework, which binds the polyribosomes, revealed no alterations in composition or amount of protein as a result of treatment with cytochalasin D. Electron microscopy with embedment-free sections shows the framework in great detail. The micrographs revealed the profound reorganization effected by the drug but did not indicate substantial disaggregation of the cytoskeletal elements.     

The intranuclear location of a herpes simplex virus DNA-binding protein is determined by the status of viral DNA replication

The herpes simplex viral DNA-binding protein, ICP8, is targeted to two different locations in the cell nucleus as part of its maturation pathway. Prior to viral DNA synthesis ICP8 was found at discrete pre-replicative sites throughout the nucleus, where it exhibited a high salt-labile association with the nuclear matrix. During viral DNA replication ICP8 was localized in randomly distributed replication compartments, where it is bound to viral DNA. Initiation of viral DNA replication caused the protein to move from the prereplicative sites to the replication compartments, while inhibition of replication caused movement in the opposite direction. In cells where viral DNA synthesis was proceeding, a minor population of ICP8 may also have been associated with the prereplicative sites. The prereplicative sites may serve as a nuclear reservoir for ICP8 not bound to replicating or progeny DNA.     

DNA replication, chromatin structure, and histone phosphorylation altered by theophylline in synchronized HeLa S3 cells

The onset of DNA replication normally is coincident with an increase in histone 1 phosphorylation and a relaxation in chromatin structure. In this paper we show that 5 mM theophylline, added 2 h after selective detachment to synchronized HeLa-S-3 cells, delays the onset and reduces the rate of DNA synthesis while theophylline treatment beginning at 8 h has no effect on subsequent DNA synthesis. These actions of theophylline are accompanied by an inhibition of histone 1 phosphorylation and a prevention of the normal relaxation in chromatin structure between G1 and S phases as revealed by image analysis of Feulgen-stained nuclei. The time courses of intracellular cyclic AMP levels, nonhistone protein phosphorylation, and [3H]lysine incorporation are also compared in the same treated and untreated synchronized HeLa cells. Comparison with experiments using 1-beta-D- arabinofuranosylcytosine (Ara-C) shows that the above phenomena are not a direct result of inhibition of DNA synthesis. We interpret our results as evidence that the associations between histone 1 phosphorylation, chromatin relaxation, and the onset of DNA synthesis are temporally and causally related.     

A nuclear lamina‐chromatin‐Ran GTPase axis modulates nuclear import and DNA damage signaling

The Ran GTPase regulates nuclear import and export by controlling the assembly state of transport complexes. This involves the direct action of RanGTP, which is generated in the nucleus by the chromatin‐associated nucleotide exchange factor, RCC1. Ran interactions with RCC1 contribute to formation of a nuclear:cytoplasmic (N:C) Ran protein gradient in interphase cells. In previous work, we showed that the Ran protein gradient is disrupted in fibroblasts from Hutchinson–Gilford progeria syndrome (HGPS) patients. The Ran gradient disruption in these cells is caused by nuclear membrane association of a mutant form of Lamin A, which induces a global reduction in heterochromatin marked with Histone H3K9me3 and Histone H3K27me3. Here, we have tested the hypothesis that heterochromatin controls the Ran gradient. Chemical inhibition and depletion of the histone methyltransferases (HMTs) G9a and GLP in normal human fibroblasts reduced heterochromatin levels and caused disruption of the Ran gradient, comparable to that observed previously in HGPS fibroblasts. HMT inhibition caused a defect in nuclear localization of TPR, a high molecular weight protein that, owing to its large size, displays a Ran‐dependent import defect in HGPS. We reasoned that pathways dependent on nuclear import of large proteins might be compromised in HGPS. We found that nuclear import of ATM requires the Ran gradient, and disruption of the Ran gradient in HGPS causes a defect in generating nuclear γ‐H2AX in response to ionizing radiation. Our data suggest a lamina–chromatin–Ran axis is important for nuclear transport regulation and contributes to the DNA damage response.     

DNA synthesis in BalB/C-3T3 ts 2 cells is restricted by a temperature-sensitive function of late G1 phase

ts 2 BalB/C-3T3 mouse fibroblasts are cdc mutants, which arrest late in G1, at or near the G1/S traverse, upon full expression of the heat-sensitive lesion. The kinetics of temperature inhibition of DNA synthesis in logarithmically growing cultures reveal three stages of heat inactivation. During the first generation time equivalent, normal semiconservative, semidiscontinuous replication proceeds but is reduced as cells exit and do not reenter S phase. During a second such period, a minimal rate of normal DNA synthesis is maintained. Thereafter, as the cells move into a third aborted cell division cycle, the rate of DNA synthesis increases. However, all semiconservative synthesis is then replaced by DNA repair replication. Temperature inactivation of the ts 2 protein results in shutdown of nuclear DNA synthesis. In contrast, normal replication of mitochondrial DNA proceeds at control rate throughout the first stage of temperature inactivation. Synthesis of this organellar genome is quantitatively reduced as the cells move into the second phase of heat inhibition. Titration of chromatin-bound DNA with ethidium bromide revealed that wild-type cells exhibit a changing DNA topology as the temperature is raised. Temperature-inactivated ts 2 cells behave as though their DNA has been topologically frozen in the configuration of control cells at or near entry into S phase.