Structure of interphase chromosomes in the nuclei of Drosophila cells

Fluorescent images of interphase chromatin structures and chromosome structures isolated from reversibly permeable Drosophila cells were analyzed. Decondensed chromatin in early S phase (2.0-2.5 C-value) consisted of a veil-like fibrillary network. Fibrillar chromatin formed rodlets later in the early S phase (2.5-2.75 C). Drosophila chromosomes contain several smaller subunits called rodlets. Fibrillar chromatin turned to chromatin ribbon and the early mid-S-phase globular chromosomes (2.75-3.0 C), then to opened fibrous globular forms later in the mid-S-phase (3.0-3.25 C), to late-S-phase supercoiled ribbons (3.25-3.5 C), end-S-phase elongated prechromosomes (3.5-3.75 C), bent and linear chromosomes (3.75-4.0 C). Early-S phase chromatin fibrils in the nuclei of Drosophila cells are thinner than the veil-like structures in mammalian cells. The connectivity of chromosomes shows linear arrangement (3, 1, 2, 4), with larger chromosomes (1 and 2) inside and smaller chromosomes (3, 4) at the two ends in the chromosomal chain.     

Condensation of interphase chromosomes in nuclei of synchronized CHO cells

The escape of individual interphase chromosomes from nuclei of reversibly permeabilized Chinese hamster ovary (CHO) cells was utilized for the visualization of condensing interphase chromosomes in a cell cycle-dependent manner in synchronized cells. Major interphase chromosomal forms include: (a) mid-S-phase globular chromosomes at 3.0 C-value, (b) late mid-S-phase fibrous hemicircular forms (3.3 C), (c) late-S-phase supercoiled ribbons (3.7 C), and (d) end-S-phase elongated, bent prechromosomal structures (4.0 C).     

Condensation of interphase chromatin in nuclei of synchronized chinese hamster ovary (CHO-K1) cells

Reversibly permeabilized cells have been used to visualize interphase chromatin structures in the presence and absence of biotinylated nucleotides. By reversing permeabilization, it was possible to confirm the existence of a flexible chromatin folding pattern through a series of transient geometric forms such as supercoiled, circular forms, chromatin bodies, thin and thick fibers, and elongated chromosomes. Our results show that the incorporation of biotin-11-dUTP interferes with chromatin condensation, leading to the accumulation of decondensed chromatin structures. Chromatin condensation without nucleotide incorporation was also studied in cell populations synchronized by centrifugal elutriation. After reversal of permeabilization, nuclei were isolated and chromatin structures were visualized after DAPI staining by fluorescent microscopy. Decondensed veil-like structures were observed in the early S phase (at an average C-value of 2.21), supercoiled chromatin later in the early S (2, 55 C), fibrous structures in the early mid S phase (2, 76 C), ribboned structures in the mid-S phase (2, 98 C), continuous chromatin strings later in the mid-S phase (3,28), elongated prechromosomes in the late S-phase (3, 72 C), precondensed chromosomes at the end and after the S phase (3, 99 C). Fluorescent microscopy revealed that neither interphase nor metaphase chromosomes are separate entities but form a linear array arranged in a semicircle. Linear arrangement was confirmed by computer image analysis.     

Gamma irradiation-induced apoptosis in murine pre-B cells prevents the condensation of fibrillar chromatin in early S phase

Local changes in chromatin structure leading to temporally distinct geometric forms were characterized in nuclei of reversibly permeabilized cells. Reversal of permeabilization was tested by 3H-thymidine incorporation and trypan blue dye exclusion. Apoptotic changes were visualized in a cell cycle dependent manner at the chromatin level by fluorescent microscopy in non-irradiated cells and after 400 rad Co60 irradiation. Fluorescent microscopy of chromatin structures belonging mainly to the interphase of the cell cycle confirmed the existence of specific geometric forms in nuclei of non-irradiated cells. In this control population, the following main transitory forms of condensing chromatin were distinguished: decondensed veil-like structures and fibrous structures in early and mid S phase (2.0-2.5 average C-value), chromatin bodies, semicircles later in mid S phase (3.0-3.5 C), precondensed chromosomes in late S (3.5-3.7 C) and metaphase chromosomes at the end and after S phase (3.7-4.0 C). Our results show that upon gamma-irradiation (a) the cellular and nuclear sizes were increased, (b) the DNA content was lower in each elutriated subpopulation of cells, (c) the progression of the cell cycle was arrested in the early S phase at 2.4 C value, (d) the chromatin condensation was blocked between the fibrillar chromatin and precondensed elongated chromosomal forms, and (e) the number and size of apoptotic bodies were inversely correlated with the progression of the cell cycle, with many small apoptotic bodies in early S phase and less and larger apoptotic bodies in late S phase.     

Common pathway of chromosome condensation in Mammalian cells

Chromatin folding in the interphase nucleus is not known. We compared the pattern of chromatin condensation in Indian muntjac, Chinese hamster ovary, murine pre B, and K562 human erythroleukemia cells during the cell cycle. Fluorescent microscopy showed that chromosome condensation follows a general pathway. Synchronized cells were reversibly permeabilized and used to isolate interphase chromatin structures. Based on their structures two major categories of intermediates were distinguished: (1) decondensed chromatin and (2) condensed chromosomal forms. (1) Chromatin forms were found between the G1 and mid-S phase involving veil-like, supercoiled, fibrous, ribboned structures; (2) condensing chromosomal forms appeared in the late-S, G2, and M phase, including strings, chromatin bodies, elongated pre-chromosomes, pre-condensed chromosomes, and metaphase chromosomes. Results demonstrate that interphase chromosomes are clustered in domains; condensing interphase chromosomes are linearly arranged. Our results raise questions related to telomer sequences and to the chemical nature of chromosome connectivity.     

Transition from chromatin bodies to linear chromosomes in nuclei of murine PreB cells synchronized in S phase

Chromatin structures and individual interphase chromosomes escaping nuclei of reversibly permeabilized cells were analyzed in a cell cycle-dependent manner. Cells were synchronized by counterflow centrifugal elutriation. Individual interphase chromosomes became visible as distinct fibrous chromatin bodies from mid-S-phase, turning to elongated chromosomes by the end of S phase. Major interphase chromosomal forms include (1) mid-S-phase chromatin bodies at 3.0 C-value, (2) elongated chromatin bodies later in mid-S-phase (3.25 C-value), (3) chromatin bodies with head and leg portions later in S phase (3.5 C-value), (4) supercoiled ribbons later in S phase seen as twisted prechromosomes (3.7 C-value), and (5) end-S-phase elongated, bent prechromosomal structures (3.9 C-value). The first karyotype analysis of the earliest forms of chromosomes referred to as chromatin bodies was performed.     

Interphase chromosome structures of human cells

Major intermediates of chromosome condensation in erythroleukemia K562 cells are presented. Interphase chromatin structures became visible after reversal of permeabilization. Large-scale chromatin structures and the development of individual interphase chromosomes were observed by fluorescence microscopy. In the linear arrangement the following major intermediates of K562 chromatin condensation could be distinguished: (1) the most decondensed chromatin veil, (2) chromatin ribbon, (3) chromatin funnel, a new intermediate regarded as the earliest visible form of interphase chromosomes, (4) chromatin body, (5) 300 nm chromatin fiber, (6) u, v, or s forms of chromosomes, and (7) linear chromosomes. The observations made in nuclei of K562 cells conform to the model of helical coil chromosome condensation.     

Incomplete chromatin condensation in enlarged rat myelocytic leukemia cells

The distinguishable morphologic features of nuclei of acute myelogenous leukemia cells with enlarged size and finely distributed nuclear chromatin indicate incomplete chromosome condensation that can be related to elevated gene expression. To confirm this, interphase chromosome structures were studied in exponentially growing rat myelomonocytic leukemia 1 cells isolated at the University of Debrecen (My1/De cells). This cell line was established from primary rat leukemia chemically induced by 7,12-dimethylbenz[a]anthracene treatment. The enlarged nuclei of My1/De cells allowed improved fluorescent visualization of chromosomal structures. Increased resolution revealed major interphase intermediates consisting of (1) veil-like chromatin, (2) chromatin ribbon, (3) chromatin funnel, (4) chromatin bodies, (5) elongated prechromosomes, (6) seal-ring, spiral shaped, and circular chromosomal subunits, (7) elongated, bent, u- and v-shaped prechromosomes, and (8) metaphase chromosomes. Results confirmed the existence of the chromatin funnel, the first visible interphase chromosome generated by the supercoiling of the chromatin ribbon. Other intermediates not seen previously included the spiral subunits that are involved in the chromonemic folding of metaphase chromosomes. The existence of spiral subunits favors the helical coil model of chromosome condensation. Incomplete chromatin condensation in leukemia cells throughout the cell cycle is an indication of euchromatization contributing to enhanced gene expression and is regarded as a leukemic factor.     

Linear connection of condensing chromosomes in nuclei of synchronized CHO cells

Reversibly permeabilized cells allowed the analysis of intermediates of large-scale chomatin condensation in a cell cycle-dependent manner. This paper summarizes major intermediates of chromatin condensation and visualizes connectivity between different forms of chromosomes. At the early S phase the veil-like fibrillary chromatin is supercoiled to form chromatin bodies representing the earliest visible chromosomes. Supercoiling results in a chromatin fiber, which turns to rope (thick fiber) forming loops and chromatin ribbon. The elongated shape of prechromosomes indicates that they are arranged head to tail. Bent forms (loop, c-, and v-shaped structures) of interphase chromosomes open at the end of S phase. Linear arrangement of chromosomes was observed to the end of the condensation process, suggesting that connectivity of chromosomes is maintained throughout the cell cycle.     

Mitotic chromatin condensation in vitro using somatic cell extracts and nuclei with variable levels of endogenous topoisomerase II

We report the development of a new method for producing mitotic extracts from tissue culture cells. These extracts reproducibly promote the condensation of chromatin in vitro when incubated with purified interphase nuclei. This condensation reaction is not species specific, since nuclei from chicken, human, and hamster cell lines all undergo chromatin condensation upon incubation with the extract. We have used this extract to investigate the role of DNA topoisomerase II (topo II) in the chromosome condensation process. Chromatin condensation does not require the presence of soluble topo II in the mitotic extract. However, the extent of formation of discrete chromosome-like structures correlates with the level of endogenous topo II present in the interphase nuclei. Our results further suggest that chromatin condensation in this extract may involve two processes: chromatin compaction and resolution into discrete chromosomes.     

Chromatin fiber structure and plectonemic model of chromosome condensation in Drosophila cells

Reversible permeable cells have been used to isolate chromatin structures during the process of chromosome condensation. Analysis of individual structures slipping out from nuclei after reversal of permeabilization revealed that chromosomes of Drosophila cells consist of small units called rodlets. The fluorescent images of chromatin fibers were subjected to computer analysis allowing the computer-aided visualization of chromatin fibers. The zig-zag array of fibers consisting of 12-15 nucleosomes with a length of 270-330 nm (average 300 nm) showed decondensed extended strings, condensed loops, and coiled condensed loops. Theoretical considerations leading to the plectonemic model of chromatin condensation are based on experimental data, and give an explanation how the 30 chromatin fibers are formed and further condensed to the 300 nm chromatin loops in Drosophila cells.     

The state of the chromosomes in the interphase nucleus

In the living interphase nucleus no chromosomal structures are visible. Yet in the injured cell and after treatment with most histological fixatives chromatin structures become apparent. Under certain conditions this appearance of structure in the living interphase nucleus is reversible. We have found that this change in the interphase nucleus is the result of a change in the state of the chromosomes. In the living nucleus the chromosomes are in a greatly extended state, filling the entire nucleus. Upon injury the chromosomes condense and therefore become visible. At the same time the nuclear volume decreases. This behavior of the chromosomes is connected with their content of desoxyribonucleic acid (DNA). This view is based on the following observations: (a) Distribution of DNA in the Nucleus.-(1) The living interphase nucleus of uninjured cells absorbs diffusely at 2537 A. No chromosomal structures are visible in ultraviolet photographs unless they are also distinct in ordinary light. If the chromosomes are made to condense they become visible and the absorption at 2537 A is now localized in these structures. (2) After fixation with formalin and osmic acid interphase nuclei stain diffusely with Feulgen. These fixatives preserve the extended state of the chromosomes. (3) If nuclei are teased out in non-electrolytes (sucrose, glycerin) the chromosomes are extended. Such nuclei stain homogeneously with methyl green. On adding salts the chromosomes condense and the methyl green is now restricted to the visible structures. (b) Extension and Condensation of Isolated Chromosomes.-When chromosomes isolated from interphase nuclei of calf thymus are suspended in sucrose, their volume is four to five times larger than in saline, but they retain their characteristic shapes. Chromosomes from which DNA and histone have been removed do not show this reversible extension and condensation, neither do lampbrush chromosomes of frog oocytes which contain very little DNA. During mitosis a partial condensation of the DNA occurs in prophase, so that the mitotic chromosomes now occupy a much smaller volume of the nucleus. At telophase the chromosomes swell again to fill the entire nucleus.     

Evidence of activity-specific, radial organization of mitotic chromosomes in Drosophila

The organization and the mechanisms of condensation of mitotic chromosomes remain unsolved despite many decades of efforts. The lack of resolution, tight compaction, and the absence of function-specific chromatin labels have been the key technical obstacles. The correlation between DNA sequence composition and its contribution to the chromosome-scale structure has been suggested before; it is unclear though if all DNA sequences equally participate in intra- or inter-chromatin or DNA-protein interactions that lead to formation of mitotic chromosomes and if their mitotic positions are reproduced radially. Using high-resolution fluorescence microscopy of live or minimally perturbed, fixed chromosomes in Drosophila embryonic cultures or tissues expressing MSL3-GFP fusion protein, we studied positioning of specific MSL3-binding sites. Actively transcribed, dosage compensated Drosophila genes are distributed along the euchromatic arm of the male X chromosome. Several novel features of mitotic chromosomes have been observed. MSL3-GFP is always found at the periphery of mitotic chromosomes, suggesting that active, dosage compensated genes are also found at the periphery of mitotic chromosomes. Furthermore, radial distribution of chromatin loci on mitotic chromosomes was found to be correlated with their functional activity as judged by core histone modifications. Histone modifications specific to active chromatin were found peripheral with respect to silent chromatin. MSL3-GFP-labeled chromatin loci become peripheral starting in late prophase. In early prophase, dosage compensated chromatin regions traverse the entire width of chromosomes. These findings suggest large-scale internal rearrangements within chromosomes during the prophase condensation step, arguing against consecutive coiling models. Our results suggest that the organization of mitotic chromosomes is reproducible not only longitudinally, as demonstrated by chromosome-specific banding patterns, but also radially. Specific MSL3-binding sites, the majority of which have been demonstrated earlier to be dosage compensated DNA sequences, located on the X chromosomes, and actively transcribed in interphase, are positioned at the periphery of mitotic chromosomes. This potentially describes a connection between the DNA/protein content of chromatin loci and their contribution to mitotic chromosome structure. Live high-resolution observations of consecutive condensation states in MSL3-GFP expressing cells could provide additional details regarding the condensation mechanisms.     

Cadmium induced apoptotic changes in chromatin structure and subphases of nuclear growth during the cell cycle in CHO cells

CHO cells were grown in the presence of 1 M CdCl₂ and subjected to ATP-dependent replicative DNA synthesis after permeabilization. By decreasing the density of the cell culture replicative DNA synthesis was diminishing. At higher than 2 × 10⁶ cell/ml concentration Cd had virtually no effect on the rate of DNA replication. Growth at higher cell concentrations could be supressed by increasing Cd concentration. After Cd treatment cells were synchronized by counterflow centrifugal elutriation. Cadmium toxicity on cell growth in early and mid S phase led to the accumulation of enlarged cells in late S phase. Flow cytometry showed increased cellular and nuclear sizes after Cd treatment. As the cells progressed through the S phase, 11 subpopulations of nuclear sizes were distinguished. Apoptotic chromatin changes were visualized by fluorescent microscopy in a cell cycle dependent manner. In the control untreated cells the main transitory forms of chromatin corresponded to those we have published earlier (veil-like, supercoiled chromatin, fibrous, ribboned structures, chromatin strings, elongated prechromosomes, precondensed chromosomes). Cadmium treatment caused: (a) the absence of decondensed veil-like structures and premature chromatin condensation in the form of apoptotic bodies in early S phase (2.2–2.4 average C-value), (b) the absence of fibrous structures, the lack of supercoiled chromatin, the appearance of uncoiled ribboned chromatin and perichromatin semicircles, in early mid S phase (2.5–2.9 C), (c) the presence of perichromatin fibrils and chromatin bodies in mid S phase (2.9–3.2 C), (d) early intra-nuclear inclusions, elongated forms of premature chromosomes, the extrusion and rupture of nuclear membrane later in mid S phase (3.3–3.4 C), (e) the exclusion of chromatin bodies and the formation of clusters of large-sized perichromatin granules in late S phase (3.5–3.8 C) and (f) large extensive disruptions and holes in the nuclear membrane and the clumping of incompletely folded chromosomes (3.8–4. C).     

Chromatin-associated protein phosphatase 1 regulates aurora-B and histone H3 phosphorylation

Proper chromosome condensation requires the phosphorylation of histone and nonhistone chromatin proteins. We have used an in vitro chromosome assembly system based on Xenopus egg cytoplasmic extracts to study mitotic histone H3 phosphorylation. We identified a histone H3 Ser(10) kinase activity associated with isolated mitotic chromosomes. The histone H3 kinase was not affected by inhibitors of cyclin-dependent kinases, DNA-dependent protein kinase, p90(rsk), or cAMP-dependent protein kinase. The activity could be selectively eluted from mitotic chromosomes and immunoprecipitated by specific anti-X aurora-B/AIRK2 antibodies. This activity was regulated by phosphorylation. Treatment of X aurora-B immunoprecipitates with recombinant protein phosphatase 1 (PP1) inhibited kinase activity. The presence of PP1 on chromatin suggested that PP1 might directly regulate the X aurora-B associated kinase activity. Indeed, incubation of isolated interphase chromatin with the PP1-specific inhibitor I2 and ATP generated an H3 kinase activity that was also specifically immunoprecipitated by anti-X aurora-B antibodies. Nonetheless, we found that stimulation of histone H3 phosphorylation in interphase cytosol does not drive chromosome condensation or targeting of 13 S condensin to chromatin. In summary, the chromosome-associated mitotic histone H3 Ser(10) kinase is associated with X aurora-B and is inhibited directly in interphase chromatin by PP1.     

Changes of nuclear structure induced by increasing temperatures

Despite the recent improvement in understanding the higher-order structure of chromatin fibers, the organization of interphase chromosomes in specific nuclear domains emerged only recently and it is still controversial. This study took advantage of an integrated approach using complementary techniques in order to investigate the structure and organization of chromatin in interphase nucleus. Native CHO-K1 cells were progressively heated from 310 K to 410 K and the effects of increasing temperatures on nuclear chromatin were analyzed in situ by means of cytometric and calorimetric techniques. Distribution and organization of chromatin domains were analyzed by Fluorescence microscopy, while the mean condensation of nuclear chromatin was measured by Differential scanning calorimetry. The results show as changes of nuclear structures (envelope and matrix, namely) affect significantly organization and condensation of in situ chromatin. Moreover when volume is modified by an external force (the temperature gradient in our case) we observe significant alterations of chromatin structure. These data are in accordance with the hypothesis of an inverse relationship between nuclear volume and chromatin condensation.     

Involvement of Cenp-F in interphase chromatin organization possibly through association with DNA-dependent protein kinase

Cenp-F (also named mitosin) is a 350-kDa human kinetochore protein important for the mitotic progression. It is also a nuclear matrix protein in interphase cells. Here, we showed that overexpression of N-terminal deletion mutants of Cenp-F containing the C-terminal 112 residues induced chromatin condensation into numerous aggregates of varying sizes in interphase nucleus, colocalizing with the exogenous proteins. In situ hybridization using whole chromosome painting probes indicated that the chromatin aggregates were not prematurely condensed individual chromosomes. Neither were they due to apoptosis. We provided evidence showing association of Cenp-F with certain regions of interphase chromatin fibers. Cenp-F associated with the DNA-dependent protein kinase (DNA-PK), a trimeric protein complex critical for genome homeostasis. Moreover, the DNA-PK association activity of Cenp-F mutants correlated with their ability to induce chromatin aggregation. These results imply a role of Cenp-F in organization of interphase chromatin through association and possibly regulation of DNA-PK.