Condensation of Interphase Chromatin in Nuclei ofSynchronized Chinese Hamster Ovary (CHO-K1) Cells
MARIANN GACSI,1 GABOR NAGY,1 GABOR PINTER,1 ALEXEI G. BASNAKIAN,2
and GASPAR BANFALVI1
ABSTRACT
Reversibly permeabilized cells have been used to visualize interphase chromatin structures in the presenceand absence of biotinylated nucleotides. By reversing permeabilization, it was possible to confirm the exis-tence 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 thatthe incorporation of biotin-11-dUTP interferes with chromatin condensation, leading to the accumulation ofdecondensed chromatin structures. Chromatin condensation without nucleotide incorporation was also stud-ied in cell populations synchronized by centrifugal elutriation. After reversal of permeabilization, nuclei wereisolated and chromatin structures were visualized after DAPI staining by fluorescent microscopy. Decondensedveil-like structures were observed in the early S phase (at an average C-value of 2.21), supercoiled chromatinlater in the early S (2, 55 C), fibrous structures in the early mid S phase (2, 76 C), ribboned structures in themid-S phase (2, 98 C), continuous chromatin strings later in the mid-S phase (3,28), elongated prechromo-somes 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 entitiesbut form a linear array arranged in a semicircle. Linear arrangement was confirmed by computer imageanalysis.
43
INTRODUCTION
RELATIVELY LITTLE is known about intermediates of chro-matin folding in the interphase nucleus, owing to the fact
that individual chromosomes cannot be seen during their repli-cation. Chromosome structures have been studied mainly inmetaphase preparations (Benyajati and Worcel, 1976; Adolphet al., 1977; Paulson and Laemmli, 1977; Cook and Brazell,1980; Earnshow and Laemmli, 1983; Rattner and Lin, 1985;Earnshow, 1988). Studies applying chromatin-removal proto-cols reveal further details of nuclear matrix construction of themetaphase chromosome (Wan et al., 1999). Direct approachesto visualize the topography of chromatin remain limited (Cre-mer et al., 1979; Comings, 1980; Sperling and Luedtke, 1981).Chromatin condensation is associated with transcriptional ac-tivities (deCampos-Vidal et al., 1998), and is correlated withphosphorylation of histone H3 (Juan et al., 1998). Chromatin-
remodeling complexes are known to facilitate transcriptionalactivation by opening chromatin structures (Xue et al., 1998).Reconstruction of the structure of interphase nuclei from elec-tron micrographs of serially sectioned nuclei failed to distin-guish among discrete chromosomes since most of the chromatinforms a continuous sticky network (Heslop-Harrison et al.,1988). Consequently, it has been difficult to study the supra-organization of condensing chromosomes within the nuclearmembrane. The experiments described here address this prob-lem by restoring the impermeability of the nuclear envelope bythe reversal of permeabilization in such a way that its functionis maintained, but allowing its reopening any time during thechromosome replication.
Permeable cells were used to analyze replicative intermedi-ates by incorporating pyrimidine analogues substituted at theC-5 position. Substitution at this position allows the introduc-tion of bulky groups such as Hg, which localize in the major
1Department of Animal Anatomy and Physiology, University of Debrecen, Debrecen, Hungary.2Division of Nephrology, University of Arkansas for Medical Sciences, Little Rock, Arkansas.
groove of DNA duplex without interfering with DNA poly-merase activity (Dale et al., 1973; Langer et al., 1981; Basnakianet al., 1989). Biotinylation at C-5 offers additional advantagesbecause biotinylated pyrimidine nucleotides do not disturb nor-mal replication (Blow and Watson, 1987; Hiriyanna et al., 1988),they can be incorporated into permeable cells (Hunting et al.,1985; Nakayasu and Bereznay, 1989), and biotinylation can beused as a tool for the immunofluorescent visualization of newlyreplicated DNA in nuclei of permeabilized cells (Banfalvi et al.,1989). Permeabilization can be reversed to maintain cell viabil-ity and replicative DNA synthesis (Banfalvi et al., 1984). Repli-cation has been studied in permeable cells as well as chromatincondensation in the interphase nucleus after the reversal of per-meabilization (Banfalvi, 1993). Earlier observations indicatedthat biotinylation attenuated the process of chromatin condensa-tion, and could be exploited for the accumulation of chromatinstructures representing different stages of chromosome conden-sation (Banfalvi et al., 1989; Banfalvi, 1993).
This paper confirms the notion that biotinylation interfereswith chromatin condensation and leads to the accumulation ofearly intermediates in chromatin folding. Reversal of perme-ablization was exploited to observe the nucleus in interphasein the absence of nucleotide incorporation and to visualizechromatin folding intermediates in synchronized cells, pro-viding insight into the successive steps of the condensationprocess.
MATERIALS AND METHODS
Chemicals and reagents
Biotin-11-dUTP, other nucleotides, 1,4-diazobicyclo-(2,2,2)-octane were from Sigma. 2,6-Diamino-2-phenylindole(DAPI) was the product of Braunschweig Chemie (Braun-schweig, Germany). Tween 20 was purchased from Pierce(Rockford, IL). Avidin-FITC and biotinylated goat antiavidin
were obtained from Vector Laboratories (Burlingame, CA).Dextran T-150 was purchased from Pharmacia-Biochemicals(Piscataway, NJ). Colcemid (N-methyl-N-deacetyl-colchicine)was the product of Boehringer (Mannheim, Germany). Growthmedia and sera were obtained from Gibco (Grand Island, NY).
Antifade Medium consisted of 90% glycerol, 2% (w/w) 1,4-diazobicyclo-(2,2,2)-octane, 20 mM Tris-Cl, pH 8.0, 0.02%sodium azide, and 25 ng/ml DAPI for blue fluorescent totalstaining of DNA or of 0.2 �g/ml propidium iodide for red DNAstaining. Hypotonic Buffer for reversible permeabilization con-tained 9 mM HEPES, pH 7.8, 5.8 mM dithiothreitol, 4.5% dex-tran T-150, 1 mM EGTA, and 4.5 mM MgCl2. Swelling Bufferconsisted of 50 mM KCl, 10 mM MgSO4, 3 mM dithiothreitol,and 5 mM NaPO4, pH 8.0.
Cell culture
The epithelial-like Chinese hamster ovary cells (CHO-K1,ATCC, Rockville, MD, #CCL61) were grown in F-12 Ham’smedium supplemented with 10% heat-inactivated bovineserum. The stemline chromosome number was hypodiploidwith a chromosome frequency distribution of 2n-22 in 50 cells.Monolayer cultures were grown in plastic 75 cm2 TC flasks(Becton-Dickinson, Mountain View, CA) containing 20 ml ofmedium to reach about 70% confluency. Exponentially grow-ing cells were washed with PBS and monolayers were detachedwith 1.5 ml of 0.25% trypsin and 0.5 mM EDTA in PBS.
Synchronization
Synchronization was performed by counterflow centrifugalelutriation (Offer et al., 2001). Cells were grown for 15 h to afinal concentration of 2–4 � 105 cells/ml. Cells were harvestedby centrifugation at 600 � g for 5 min at 5°C and resuspendedin F-12 medium containing 1% FBS at 107 cells/ml. Cells (2 �108) were introduced into the elutriator rotor (Beckman J-6 MI)equipped with a JE-5.0 elutriation system including a Sander-
GACSI ET AL.44
TABLE 1. CHARACTERIZATION OF SYNCHRONIZED POPULATIONS OF CELLS
Average AverageFlow rate Elutriated Cell nuclear nuclear
Cells subjected to elutriation: 9.7 � 107 (100%).Number of elutriated cells: 8.6 � 107 (89%).Loss of cells during manipulation: 1.1 � 107 (11%).n.d. � not determined.Cellular and nuclear volumes and nuclear diameters were estimated with the Coulter Channelizer. C-values were estimated as
described in Materials and Methods.
son chamber (Beckman Instruments, Inc., Palo Alto, CA) anda MasterFlex peristaltic pump (Cole-Parmer Instruments,Chicago, IL). Eight cell fractions were collected. Elutriationwas performed at a temperature of 20°C and the cells wereeluted in F-12 medium containing 1% fetal bovine serum. Thefraction collected during loading, which contained unloaded anddead cells, was discarded. Fractions (100 ml each) were col-lected at increasing flow rates (Table 1). The first fraction,which contained primarily G1 cells, was not used for the studyof chromatin condensation. Each fraction was routinely moni-tored by light microscopy and analyzed by FACS. The frac-tions contained unaggregated cells, which increased in size witheach fraction elutriated. Cell number, size, and volume weremonitored with a Coulter Multisizer. Cell number was also as-sessed in a Burker chamber and viability (�98%) was deter-mined by Trypan Blue dye exclusion in each elutriated frac-tion. All experiments were repeated three times with similarresults.
Analysis of cell cycle and apoptosis by FACS
Cells were fixed with 70% methanol at room temperatureand stained with 50 �g/ml propidium iodide (Sigma, St. Louis,MO). The cells were analyzed by a FACSan flow cytometer(Becton-Dickinson) using the CellQest software (Becton-Dick-inson) as described by us (Offer et al., 2001). The nuclear DNAcontent, expressed in C-values (1C corresponds to a haploidDNA content per cell), increased from 2C to 4C, and provideda measure of progress through the S phase. Flow cytometricprofiles giving the distribution of DNA content were used tocalculate the average C-value for each elutriated fraction. Thecells of each fraction were divided into 17 subfractions to coverthe total area of the cytometric profile. C-values were calcu-lated from the appropriate area under the flow cytometric pro-file, and were averaged to yield the DNA content for each frac-tion (Basnakian et al., 1989).
DNA synthesis
In vitro DNA synthesis was carried out in permabilized cellsat 37°C for 30 min in the presence of dATP, dGTP, dCTP, andbiotin-11-dUTP as described earlier (Banfalvi et al., 1989).Pulse labeling with biotin-11 dUTP was done for 10 min andwas followed by a 1-h chase in the presence of the 4 dNTP,with the concentration of dTTP increased to 1 mM.
Reversible permeabilization
This method, originally developed for the reversible perme-abilization of murine lymphocytes (Banfalvi et al., 1984), wasadapted to CHO cells. Briefly, 1 ml of Hypotonic Buffer wasadded to 106 cells in the presence of Dextran T-150 as a mo-lecular coat to prevent cells from disruption. Permeabilizationlasted for 2 min at 0°C. For reversal of permeabilization, thehypotonic solution was replaced by F-12 medium containing10% fetal bovine serum, and the cells were incubated in a CO2
incubator at 37°C and 5% CO2 for 3 h.
Isolation of nuclei
Because of the cyclic character of chromatin unfolding andchromosome condensation, which is limited to a relatively short
time, synchronized cell populations were treated with colcemidto arrest the cycle in metaphase. Cells (106) were resuspendedin growth medium after reversal of permeabilization and treatedwith 0.1 �g/ml colcemid for 2 h at 37°C under 5% CO2. Cellswere detached with trypsin, washed with PBS, and incubatedat 37°C for 10 min in Swelling Buffer, followed by centrifu-gation at 500 � g for 5 min. Nuclei were isolated by the slowaddition of 20 volumes of Fixative (methanol:glacial aceticacid, 3:1) and were then centrifuged at 500 � g for 5 min,washed twice in Fixative, and resuspended in 1 ml of Fixative.Cellular and nuclear volumes and nuclear diameter were deter-mined with the Coulter channelizer.
Spreads of nuclear structures
Preparation of nuclei for spreads of chromatin structures usedthe method developed for metaphase chromosomes. Nucleiwere centrifuged at 500 � g for 5 min, washed twice in Fixa-tive, and resuspended in 1 ml fixative. Nuclei were spread overglass slides dropwise from a height of approximately 30 cm.Slides were air dried, stored at room temperature overnight,rinsed with PBS, and dehydrated using increasing concentra-tions of ethanol (70, 90, 95, and 100%).
Visualization of nascent DNA by immunofluorescent amplification
Immunofluorescent visualization of biotinylated DNA wasdescribed earlier (Banfalvi et al., 1989). Briefly, slides con-taining nuclei were preincubated in 4 � SSC, 0.05% Tween 20,and 5% nonfat dry milk at pH 7.4, followed by incubation inthe presence 5 �g/ml avidin-FITC, three washing steps (4 �SSC, 0.05% Tween 20, pH 7.0) and incubation with biotiny-lated goat antiavidin antibody (5 �g/ml). Immunofluorescentamplification was repeated three times.
Visualization of chromatin structures
Dehydrated slides containing biotinylated or nonbiotinylatedchromatin structures were mounted in 35 �l Antifade Mediumunder 24 � 50 mm coverslips. Blue fluorescence of DAPI wasmonitored with an Olympus AX70 fluorescent microscope.
RESULTS
Biotinylation interferes with chromatin folding
Nascent DNA synthesis was carried out in permeable cellsin the presence of biotin-11-dUTP. Cells were then allowed torecover from permeabilization in serum-enriched medium. Col-cemid treatment was used to block the cell cycle in metaphaseand prevent cells from proceeding to the next cell cycle. Repli-cation sites in nuclei of exponentially growing cells were visu-alized after immunofluorescent amplification. Red propidiumiodide and green FITC signals could be visualized simultane-ously using blue excitation light, but the FITC signals were sig-nificantly masked. Because DAPI is spectrally well separatedfrom FITC fluorescence and different shades of its blue colourfluorescence indicate the degree of chromatin compactness,subsequent experiments employed DAPI for fluorescent stain-ing of total DNA. Regarding chromatin staining, it is important
CHROMATIN CONDENSATION 45
to mention that DAPI binds specifically to A–T-rich sequencesin the minor groove of DNA (Parolin et al., 1995).
Control experiment included the visualization of nuclei be-fore and after permeabilization (Fig. 1A and B). After reversalof permeabilization biotinylated-DNA was expelled from the nu-clei as decondensed fibres (Fig. 1C, D, and E). Of the two nu-clei visible in Figure 1F, one is replicating (lower one) and showsthe spatial distribution of nascent biotin-DNA, whereas the up-per nucleus is silent from the point of view of replication. Aswe could not see the biotin label in metaphase chromosomes,pulse-chase experiments were attempted to drive the biotin la-bel into metaphase chromosomes. However, pulse chase failedto drive biotin-labeled DNA from interphase to metaphase chro-mosomes (Fig. 1E); instead, the formation of big chromatin clus-ters and separation and expulsion of biotinylated DNA was ob-
served by the immunofluorescent amplification of nascent DNAas described earlier (Banfalvi et al., 1989). Nevertheless, bi-otinylation is a potential tool to observe early intermediates ofchromatin condensation and initiated the analysis of further in-termediates in the absence of biotinylated nucleotides.
Decondensed chromatin structures after biotinylation
Two generally known chromatin structures can be visualizedin exponentially growing cells: those characteristic of inter-phase nuclei, and metaphase chromosomes. When the isolationof nuclei from permeable cells was attempted without restor-ing cellular membranes by reversing permeabilization, a stickymass of nuclear material was obtained. These experiments sug-gested that not only the cellular but also the nuclear membranewas affected by permeabilization. Restoration of cellular mem-brane function was measured by 3H-thymidine incorporationand Trypan Blue exclusion as described earlier (Banfalvi et al.,1984). In a control experiment, in which biotinylation was omit-ted, reversal of permeabilization did not lead to the accumula-tion of early chromatin condensation intermediates, indicatingthat biotin-DNA can be used as a tool to slow down the con-densation process, thereby allowing the accumulation of earlyintermediates of chromatin folding.
It was observed repeatedly that chromatin condensation inexponentrially growing CHO cells starts with a polarization ofthe nuclear material. Figure 2 shows the early stage of chro-matin decondensation. Although in these experiments most ofthe cells were in the S phase, they represented different sub-phases and stages of chromatin compaction. The polarizationcauses the extrusion of spherical chromatin (Fig. 2A, B, C, F,
GACSI ET AL.46
FIG. 1. Effect of permeabilization and biotinylation on theorganization of chromatin in CHO cells. (A) DAPI-stained nu-clei of unpermeabilized CHO cells. (B) DAPI-stained nuclei ofCHO cells after reversal of permeabilization. (C) CHO cellswere permeabilized and labeled with biotin-11-dUTP. Perme-abilization was reversed by resuspension in growth medium.After 3 h at 37°C, nuclei were isolated, immunofluorescent vi-sualization of biotinylated DNA was as decribed in the Mate-rials and Methods, and then stained with DAPI. (D), (F) Sameas (C), except that the biotin-labeled nuclei were subjected toimmunofluorescence analysis. (E) Same as (D), except that la-beling with biotin-11-dUTP was followed by a 1-h chase in thepresence of 1 mM dTTP. Bar, 5 �m.
FIG. 2. Polarized condensation of biotinylated interphasechromatin. CHO cells were permeabilized, labeled with biotin-11-dUTP, and permeabilization was reversed by resuspensionin growth medium as in Figure 1C. Nuclei were isolated andstained with DAPI. The images were selected to illustrate po-larization of the nuclear material and are at the same magnifi-cation. Bar, 5 �m.
G, and O). DNA is in its decondensed form and the disruptionof the nuclear membrane revealed extruding chromatin as afuzzy, veil-like structure (Fig. 2D, O, P, and Q), leading to theformation of a nuclear plate (Fig. 2D, J, K, L, M, N, and P).The nuclear material either maintained its round shape or un-folded into a supercoiled ribbon structure (Fig. 2Q). Round-shaped chromatin bodies are regarded as sperical forms of chro-mosomes (chromatin bodies), while supercoiled ribbonstructure is representing the early elongated form of prechro-mosomes.
Globular, supercoiled, fibrous, ribboned structuresafter DNA biotinylation
Condensing chromatin structures gradually changed theirshape from globular and circular forms to elongated chromo-somes, as illustrated in Figure 3. Spherical chromatin bodies,which represent less condensed structures (Fig. 3A–D), turn intomore compact circular and fibrous chromatin (Fig. 3D–E). The
formation of globular structures is probably connected with thesupercoiling of the chromatin veil. Judged by the visible num-ber of supercoils (Fig. 3C), which is at least 20, the supercoilsprobably correspond to chromosomes and to chromatin bodies(decondensed chromosomes) in interphase. Some regions of thenuclear material are still in a decondensed cotton-like state (Fig.3D–F), such as the chromosomes seen at the upper right cor-ner, at the bottom and in the center of Figure 3F. Globular in-termediates turn first into elongated strings (Fig. 3D and E) andthen into elongated chromosomes of different lengths and thick-nesses, which represent the next stage of chromosome conden-sation (Fig. 3 E–G). In addition, several transition forms wereobserved, in which elongated prechromosomes are thickened,
CHROMATIN CONDENSATION 47
FIG. 3. Intermediates in the condensation of biotinylatedchromatin. CHO cells were permeabilized, labeled with biotin-11-dUTP, and permeabilization was reversed by resuspensionin growth medium as in Figure 1C. Nuclei were isolated andstained with DAPI. The images were selected to illustrate fre-quently seen patterns, such as: (A,B) chromatin bodies, whichhave been numbered in (B); (C) supercoiled chromatin; (D,E)chromatin bodies turning to fibers; (E–G) transition to elon-gated chromosomes; (G) lobulate arrangement of elongatedchromosomes; (H) metaphase chromosomes. Bar, 5 �m.
FIG. 4. Flow cytometric analysis of CHO cells synchronizedby centrifugal elutriation. CHO cells at different stages in theS phase were obtained by centrifugal elutriation as describedin Materials and Methods. The eight elutriated fractions (E1through E8) that were collected are described in Table 1. (A)Flow cytometric profiles of the elutriated fractions. (B) Nuclearsize of the elutriated fractions measured in a Coulter Channel-izer. (C) Relationship between cell volume and diameter. (D)A sample of each elutriated fraction was subjected to perme-abilization and its reversal and then analyzed by flow cytome-try, showing that reversible permeabilization caused less than5% apoptosis in each fraction. Ap, apoptotic cells; PI, propid-ium iodide.
probably by additional folding and protein binding. In severalinstances, chromosomes were found to be arranged in two lobes,which join at the upper stalk region (Fig. 3C, E, and G). At thefinal stage of their replication condensing chromosomes arealigned in an arc (Fig. 3E). In the lower portion of the nucleusshown in Figure 3F the chromosomes form a condensed clus-ter while in its upper region consists of a the looser structurecomposed of decondensed fibrous chromatin and the elongatedforms of condensing chromosomes. The side-by-side occurenceof two types of chromosomal arrangement in the same nucleussuggests that these represent consecutive stages in chromosomecondensation. The shortening and thickening of elongated pre-condensed forms (Fig. 3G) finally lead to metaphase chromo-somes (Fig. 3H).
Chromatin condensation of nonbiotinylated DNAstudied in synchronized cells
Since biotinylation leads to the accumulation of intermedi-ates in chromatin condensation and interferes with the pro-gression of the condensation process, we examined the inter-mediates in chromatin condensation in the absence ofbiotinylated nucleotides using cells synchronized at differentstages of the cell cycle. Regular chromatin structures were iden-tified as similar forms that recurred in the same synchronizedpopulation of cells. Our analysis focused only on those regu-
larly occurring chromatin structures observed under the fluo-rescent microscope, in which the chromatin network showedcontinuity and the structure was not deformed by physicalforces. At this stage of the work no statistical analysis of chro-matin images is available. Centrifugal elutriation of an expo-nential cell population yielded eight cell fractions, each ofwhich was characterized by the flow rate at which cells wereelutriated and by the average cellular and nuclear volume, nu-clear diameter, and C-value, which define their cell cycle sta-tus (Table 1). The cell cycle parameters were estimated by flowcytometry, which also provided an assessment of the degree ofsynchrony of cells in each fraction (Fig. 4A–C). Flow cytom-etry was also used to assess whether cell permeabilization andits reversal led to apoptosis. This analysis showed less than 5%apoptotic cells after permeabilization and its reversal in eachelutriated fraction (Fig. 4D), which is similar to that seen incontrol cultures not subjected to elutriation and in cultures thatwere not permeabilized (results not shown).
Decondensed chromatin structures in cellssynchronized in the early S phase (2.0–2.5 C)
The cells in Elutriation Fraction 2 were in the early S phase(2.21 C value) and DNA was in a highly decondensed state asshown in Figure 5 (left panels). The chromatin in the fixed nu-clei appeared as a fuzzy, veil-like structure (Fig. 5, left panels).
GACSI ET AL.48
FIG. 5. Decondensed chromatin structures in cells synchronized at 2.0–2.5 C. CHO cells at different stages in the S phase wereobtained by centrifugal elutriation as described in Materials and Methods and Table 1. (Left panels) Elutriation Fraction 2. (Rightpanels) Elutriation Fraction 3. Bar, 5 �m.
In Elutriation Fraction 3 (2.55 C) the nuclear material eithermaintained its round shape with a polarized chromatin plateemerging from the nucleus (Fig. 5, right panels A–D), similarto biotinylated DNA seen in Fig. 2, or the chromatin began tounfold and turn into supercoiled structures (Fig. 5, right panelsE–H). The ribboned chromatin was seen to be twisted in themiddle stalk portion (e.g., Fig. 5, right panel E and G) and grad-ually turned into supercoiled chromatin structures (Fig. 5, rightpanel F and H).
Transition from veiled to ribboned structures in cellssynchronized in the early mid-S phase (2.5–3.0 C)
Elutriation Fraction 4 represented cells in the early mid-Sphase (2.76 C). In the fixed nuclei from these cells, the veiledchromatin gradually developed into supercoiled loops (Fig. 6,left panels A–E), sometimes consisting of thin fibers (Fig. 6,left panels G–H) with a diameter of about 300 nm, which weresimilar to those of euchromatin fibers. Elutriation Fraction 5(2.98 C) revealed further condensation of the fibrous structures,leading to continuous ribbon structures (Fig. 6, right panelsA–F). Presumably due to further supercoiling, the thin chro-
matin string formed a thicker string with a diameter, estimatedat the right end of the chromatin fiber in Figure 6G (right pan-els) of about 700 nm. The thin chromatin string shown in Fig-ure 6H (left panels) is shown magnified for closer scrutiny inFigure 7. The segment of the chromatin string shown in Figure7a includes supercoiled stretches that were more condensed thanlinear intervening stretches, as indicated by fluorescent inten-sities. At the right end of the chromatin string (close to arrow1), chromatin condensation resulted in a thicker precondensedchromosome, representing probably the most condensed part ofthe chromatin string. In Figure 7b condensing supercoiled andless condensed regions appeared to alternate. In the region ofFigure 7c close to arrow 4, chromatin bodies were in a decon-densed cotton-like state.
Linear connection of chromosomes in condensing interphase chromatin in cells synchronized at 3.0–3.5 C
Further evidence for a continuous, linear chromatin structurewas provided by the examination of Elutriation Fraction 6 (Fig.8). Figure 8A and D show decondensed chromosomes that are
CHROMATIN CONDENSATION 49
FIG. 6. Transition to ribboned chromatin structures in cells synchronized at 2.5–3.0 C. CHO cells at different stages in the Sphase were obtained by centrifugal elutriation as described in Materials and Methods and Table 1. (Left panels) Elutriation Frac-tion 4. Fraction 4H was subjected to further analysis shown in Figure 7. (Right panels) Elutriation Fraction 5. Bar, 5 �m.
wrapped around a long chromatin stem or are unfolding likenew leaves. The ends of the chromatin structure appeared to besupercoiled (Fig. 8A), suggesting that condensation from thethin (300 nm) to thick chromatin fibres (700 nm) seen in Fig-ure 6G (right panels) involved supercoiling. In Figure 8B thechromatin is seen as a long string (300 nm) without distin-guishable chromosomes, presumably corresponding to chro-matin loops. The backbone of this chromatin string is tracedwith a black line and the two ends of the continuous structure,about 60 �m in length, are indicated by two small circles (Fig.8C). Continuity seems to be maintained during the formationof elongated prechromosomal forms visualized in Figure 8E.
Distinct forms of early chromosomes in cellssynchronized at 3.5–4.0 C
In the late S phase (Elutriation Fraction 7; average C-valueof 3.72) chromosomes became visible and were arranged in arcsor circles (Fig. 9, left panels). In some cases individual chro-mosomes were clearly distinguishable (Fig. 9, left panels E, G,and H). In the last synchronized population (Elutriation Frac-tion 8, C-value of 3.98), which approached metaphase, chro-mosomes were compact but not yet completely condensed (Fig.9, right panels), but in a few cases (Fig. 9, right panels E–G)began to resemble metaphase chromosomes (Fig. 9, right panelH). Linear connection was maintained in condensed chromo-somes approaching metaphase (Fig. 9, bottom panel J). Thestring of DNA (Fig. 9J) is strengthening the view that the chro-mosomes are arranged head to tail.
Chromatin image analysis
The fluorescent image of a stained nucleus from ElutriationFraction 7 (3.72 C) was subjected to further analysis (Fig. 10).A precalculated intensity mask was fitted to the chromatin im-
age to yield a color-coded intensity map, which is shown inblack and white in Fig. 10B–F, and allowed the intensity as-signments shown diagrammatically in Fig. 10G. From the esti-mated intensities of the 11 chromosomes, it was possible to ten-tatively correlate each of these with the 11 known chromosomesof CHO cells based on chromosome size, as indicated by thenumbers in Fig. 10F. Although, the chromosome assignment istentative at best, it serves to show that individual chromosomescan be visualized in the late S-phase, and are arranged in cir-cular patterns, suggesting some sort of physical association ofneighboring chromosomes.
DISCUSSION
Earlier observations (Banfalvi et al., 1989; Banfalvi, 1993)suggested that biotinylation of DNA interferes with chromatincondensation. Fully condensed metaphase chomosomes did not
GACSI ET AL.50
FIG. 7. Analysis of fibrous chromatin. The chromatin stringfrom Elutriation Fraction 4, shown in Figure 6H (left panels),at higher magnification. The fiber is broken into three segments,with the numbered arrows corresponding to those seen in Fig-ure 6H (left panels). Bar, 5 �m.
FIG. 8. Interconnected chromatin structures in cells synchro-nized at 3.0–3.5 C. CHO cells at different stages in the S phasewere obtained by centrifugal elutriation as described in Mate-rials and Methods and Table 1. The panels show examples ofchromosomes seen in Elutriation Fraction 6. (C) is identical to(B) except that the backbone of the continuous chromosomestring has been traced with a dotted black line and its ends areindicated by small black circles. Bar, 5 �m.
contain FITC foci, suggesting that biotinylation prevents chro-matin folding and leads to decondensed chromatin structures.Our aim was to build on these observations by examining in-termediates in chromatin condensation, taking advantage of thefact that permeabilization allows labeling with biotinylated nu-cleotides at any time during chromosome replication. Expo-nentially growing CHO cells were used in these experiments toassure that most of the cells are in the S phase, where DNA isunfolded and chromatin structures are in a decondensed state.The resulting decondensed chromatin structures could then bevisualized upon reversal of permeabilization.
Results support our earlier notion that the incorporation ofbiotin-labeled nucleotides into nascent DNA prevented chro-matin folding (Banfalvi et al., 1989; Banfalvi, 1993) and thusprovided a means of visualizing early intermediates in chro-mosome replication. Fluorescent images of chromatin structuresconfirm the existence of a flexible folding pattern including sev-
eral transitional forms. Among these forms, it was possible todiscern decondensed veil-like structures turning into ribboned,globular, and fibrous structures. The existence of globular andfibrous structures had been shown in interphase chromatin byhigh-resolution scanning electron microscopy (Iwano et al.,1997).
Biotinylation of DNA appears to interfere with chromatinfolding by blocking chromatin condensation at the fibrous stage.One would therefore expect that the omission of biotinylationmight reveal further intermediates in the condensation process.Experiments carried out with synchronized populations of cellsin the absence of biotinylation not only confirmed the existenceof globular and fibrous structures but revealed further forms.These transitions involved the supercoiling of the chromatinveil, which seemed to separate decondensed chromosomes aschromatin bodies and chromatin loops. The spherical bodies ob-served may correspond to the globular forms of interphase chro-
CHROMATIN CONDENSATION 51
FIG. 9. Circular arrangement of prechromosomes in cells synchronized at 3.5–4.0 C. CHO cells at different stages in the Sphase were obtained by centrifugal elutriation as described in Materials and Methods and Table 1. (Left panels) Elutriation Frac-tion 7. (Right panels) Elutriation Fraction 8. Bar, 5 �m.
mosomes. These gradually changed their shape to chromatinloops, then to semicircular chromatin structures resemblinghorseshoe-like arrays. Finally, the precondensed folded-backstructures of semicircles turned into elongated linear chromo-some forms, which may be the immediate precursors of shortermetaphase chromosomes.
We described earlier that there is a correlation between thenumber of subphases of DNA replication and the number ofchromosomes in CHO and in Drosophila cells (Banfalvi et al.,1997; Rehak et al., 2000). Although, the chromatin folding pat-tern is not completely understood, the linear arrangement ofchromosomes suggests that chromosome replication is relatedto a defined temporal order of chromosome condensation.
Computer image analysis confirmed that condensed chro-mosomes seem to be organized in a circular arrangement, sug-gesting connectivity. An interesting question concerns the roleof telomers in the linkage between chromosomes and their repli-cation. Experiments are under way to identify the chemical ba-sis of chromosome connectivity, whether the linkage of chro-mosomes plays a role in chromosome replication and whether
it is related to the temporal order of chromosome replication.Evidence for a string would be provided by DNA FISH tech-nique with chromsome-specific subtelomere probes, and woulddefinitively prove the linear connection of chromosomes. Un-fortunately, such probes are not available for Chinese hamsterovary cells. Recently, we used other cells (human ery-throleukemia K-562, Drosophila, Indian muntjac) where con-nectivity is already convincing for us, and the liquid hy-bridization FISH technique is under establisment to prove it forothers. In murine preB cells we have compared the chromatinstructures of normal and gamma-irradiated cells, and havefound in nonirradiated cells similar transitory forms. Theseforms include decondensed veil-like structures, fibrous struc-tures in the early and mid-S phase, chromatin bodies, semicir-cles later in the mid-S phase, precondensed chromosomes inthe late S, and metaphase chromosomes at the end and after theS phase (Nagy et al., 2004).
ACKNOWLEDGMENTS
This work was supported by the grant of the Hungarian National Science and Research Foundation to G.B. (OTKAT42762 grant). The reading of the manuscript by Dr. HenryPaulus is gratefully acknowledged.
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FIG. 10. Chromatin image analysis of precondensed chro-mosomes. (A) A nucleus from Elutriation Fraction 7 stainedwith DAPI. (B–F) Image analysis of the nucleus shown in (A)effected by fitting a precalculated intensity mask to the chro-matin image. (C) Chromosome bodies are numbered consecu-tively by the white numbers on the outside of the circulararrangement and (F) in order of size. (G) The chromosomes arepresented schematically in a linear array, the length of the loopscorresponding to the intensity of the corresponding chromatinbody in (F). The top row of numbers correspond to the num-bers in (C), the bottom row to the numbers in (F), which orderthe chromosomes by size. Bar, 5 �m.
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Address reprint requests to:Prof. Gaspar Banfalvi
Department of Animal Anatomy and PhysiologyUniversity of Debrecen
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