©2004, European Journal of Histochemistry

Histone modifications represent an important epigeneticmechanism for the organization of higher order chromatinstructure and gene regulation. Methylation of position-spe-cific lysine residues in the histone H3 and H4 amino terminihas been linked with the formation of constitutive and facul-tative heterochromatin as well as with specifically repressedsingle gene loci. Using an antibody, directed against di-methylated lysine 9 of histone H3 and several other lysinemethylation sites, we visualized the nuclear distribution pat-tern of chromatin flagged by these methylated lysines in 3Dpreserved nuclei of normal and malignant cell types. Opticalconfocal serial sections were used for a quantitative evalua-tion. We demonstrate distinct differences of these histonemethylation patterns among nuclei of different cell typesafter exit of the cell cycle. Changes in the pattern formationwere also observed during the cell cycle. Our data suggestan important role of methylated histones in the reestablish-ment of higher order chromatin arrangements duringtelophase/early G1. Cell type specific histone methylationpatterns are possibly causally involved in the formation ofcell type specific heterochromatin compartments, composedof (peri)centromeric regions and chromosomal subregionsfrom neighboring chromosome territories, which containsilent genes.

Key words: heterochromatin, histone methylation,nuclear architecture, cell cycle, MCF-7 cells.

Correspondence: Marion Cremer, Department of Biology II,Human Genetics, University of Munich, Richard Wagner-Str.10, 80333 Munich, Germany. Phone: international+49.89.21806713. Fax: inernational +49.89.21806719E-mail: marion.cremer@lrz.uni-muenchen.de

Paper accepted February 9, 2004

European Journal of Histochemistry2004; vol. 48

Three dimensional analysis of histone methylation patterns in normaland tumor cell nuclei M. Cremer,*° R. Zinner,* S. Stein,# H. Albiez,* B. Wagler,*° C. Cremer,# T. Cremer**Department of Biology II, Ludwig Maximilians University, Munich; °Institute of Human Genetics,Technical University and GSF; #Kirchhoff Institute of Physics, University of Heidelberg, Germany

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Cell type specific differences of nuclear size,shape and chromatin texture, a parameter forDNA heterogeneity, have been reported for a

long time, but the underlying genetic and epigeneticcauses of these differences and their functionalimplications have remained a puzzle. In 1928 theconcept of heterochromatin was first described byHeitz (Heitz, 1928), based on his light-microscopicstudies of mosses. Heitz named intensely stainednuclear areas heterochromatin and less intenselystained areas euchromatin and predicted that hete-rochromatic areas were genetically passive andwould not contain genes, whereas euchromatinimplied active or potentially active gene-containingregions.

To date, heterochromatin is conventionally classi-fied into two groups: constitutive and facultativeheterochromatin. Constitutive heterochromatin ismostly found around (peri)centromeric regions andconsists of large (> 1 Mb) arrays of tandemlyrepeated sequences. Facultative heterochromatin,containing single copy sequences as well as a varietyof repetitive sequences, cannot be defined at thelevel of characteristic DNA sequences, but itreflects an epigenetic state of chromatin. The best-known example is the inactive X in nuclei of femalemammals. Euchromatin can be transformed intofacultative heterochromatin by epigenetic modifica-tions of DNA and histones.These epigenetic mecha-nisms include methylation of cytosins at CpG sites,as well as methylation and acetylation at specificsites of histone tails (for review see (Bird, 2002)and play an important part in the activation orrepression of gene activity. DNA methylation, his-tone hypoacetylation and histone methylationinvolved in the formation of facultative heterochro-matin are in principle identical to the epigeneticmodifications seen in constitutive heterochromatin(for reviews see (Bird, 2002; Elgin and Grewal,2003; Jones and Baylin, 2002; Lachner et al.,2003; Sims et al., 2003).

ORIGINAL PAPER

Nuclei of tumor cells differ in the amount and dis-tribution of heterochromatin from their normalprogenitor cells. This fact has widely been used inhistopathological diagnosis of tumor cells.Numerous studies of morphometric, densitometricand textural chromatin features were performedwith Feulgen stained nuclei to quantitate cell typespecific features of chromatin texture and ofchanges that occur during tumor progression (Diazet al., 1997; Gil et al., 2002; Mairinger et al.,1999; Mulder et al., 1992; Weyn et al., 2000 b).During the last few years a wealth of informationhas accumulated for many tumor types on specificmutations, chromosomal translocations, gains andlosses of chromosomal subsegments and on grossgenomic instabilities. The application of cDNA-array techniques has made it possible to also ana-lyze in detail changes of the expression patterns intumor cells in comparison to normal cells. In spiteof these important advancements, we still know lit-tle about the putative functional links between theobserved differences of chromatin patterns in nor-mal and malignant cell types and changes of geneexpression patterns that occur during tumor pro-gression at the molecular and cytogenetic level.

Antibodies specifically directed against epigenet-ic markers offer new tools to compare in differentcell types the spatial arrangement of chromatin ona basis that correlates functional aspects with chro-matin topology. Histone tail methylation can belinked to both active chromatin (such as metH3-K4, metH3-K36 and metH3-K79) or repressedchromatin (such as metH3-K9, metH3-K27 andmetH4-K20) (for review see (Lachner et al.,2003). MetH3-K9 flags specifically repressed geneloci as well as constitutive heterochromatin andcreates a binding site for the histone-binding hete-rochromatin-associated protein 1 (HP1) (Lachneret al., 2001), which is essential for establishingconstitutive heterochromatic domains. MetH3-K27especially marks the inactive X (Plath et al.,2003). The organization of such bulk chromosomalregions can be considered as the chromosome levelfunction of histone methylation. Mechanisms bywhich active genes in euchromatic domains aresilenced by lysine methylation mediated processesthat have not yet been fully disclosed can be con-sidered as the gene level function of histone methy-lation (for reviews see (Elgin and Grewal, 2003;Lachner and Jenuwein, 2002; Lachner et al., 2003;Sims et al., 2003). In this paper we report patterns

of (hetero)chromatin marked by methylated lysinesat different positions of H3 and H4 in different nor-mal and tumor cells and during defined cell cyclestages. We used an antibody primarily directedagainst metH3-K9 but it was found that it alsobroadly reacts with other methylated lysines inde-pendent of the respective lysine position (Perez-Burgos et al., 2004 in press), further referred to asmulti-methyl lysine (MML) antibody. We demon-strate distinct differences of these patterns amongcell types and during cell cycle progression. Wepresent and discuss approaches towards a quantita-tive 3D evaluation of these patterns.

Materials and Methods

Cell material and slide preparationHuman lymphocytes and monocytes from a

healthy donor were isolated from peripheral bloodby a Ficoll gradient.These cells were either direct-ly mounted on a cover slip beforefurther processing(see below), or cultivated for 60 hours after stimu-lation with phyto-hemagglutinine in order to obtaincycling lymphocytes. The cell line Jurkat, estab-lished from a patient with an acute T cell leukemia(Schneider et al., 1977), has a stable karyotypewith an X/18 translocation and a trisomy 20(Muller et al., 2004). Primary female humandiploid fibroblasts were obtained from early pas-sages. Paraffine embedded sections of normalhuman colon and colon carcinoma tissue were kind-ly provided by J. Diebold, University of Munich.Thebreast cancer cell line MCF-7 was kindly providedby P. Meltzer (NIH, Bethesda, MD, USA). M-FISHanalysis of the cell passage used for our analysiswas kindly performed by A. Jauch, University ofHeidelberg and revealed a hypertriploid karyotype(n = 76) with numerous chromosomal rearrange-ments.

Adherently growing fibroblasts and MCF-7 cellswere cultivated on coverslips in the appropriate cul-ture medium until subconfluency.Thirty min beforefixation cells were pulse-labeled with BrdU (5 µMfinal concentration) for the later identification of Sphase nuclei. Cells growing in suspension (Jurkatcells, lymphocytes and monocytes) were allowed toattach on polylysine–coated slides for 30 minbefore fixation. For the preparation of 3D pre-served nuclei cells were fixed in 4% paraformalde-hyde/PBS and permeabilized in 0.5% Triton X-100/PBS for 5 min. Slides were stored in 1× PBS

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at 4°C up to a few days until use. Tissue sectionsmounted on coverslips were deparaffinized by twosuccessive 30min incubations in xylole, rehydratedin a descending series of ethanol and rinsed inddH2O. Epitope retrieval was performed bymicrowaving the tissue for 6 × 5 min at 700 W in10 mM Na-Citrate pH 6.0. After cooling down toroom temperature the sections were transferred toPBS and used for immunostaining.

Assessment of the cell cycle stage Detection of BrdU was performed as described by

(Tashiro et al., 2000). Briefly, after blocking in 5%bovine serum albumine/PBS for 15 min, cells wereincubated with mouse-anti-BrdU antibodies(Roche) in 1% BSA, 0.5 × PBS, 30 mM Tris (pH= 7.4), 0.3 mM MgCl2, 0.5 mM 2-mercap-toethanol and 10 µg/ml DNAse I (Roche). As a sec-ondary antibody goat-anti-mouse Alexa 488 orgoat-anti-mouse Alexa 350 (Molecular probes)was applied. A monoclonal antibody against the cellcycle related nuclear protein pKi67 (mouse-anti-pKi67, Dianova) was used for the identification ofcells in G0 and early G1 phase and visualized withthe same secondary antibody as used for the BrdUdetection.

Visualization of heterochromatin byimmunodetection of histone methylation with theMML antibody

An antibody, named multi-methyl lysine (MML),raised in rabbit against 4x branched histone 3 pep-tide, dimethylated at lysine 9 (K9) position wasused. In addition to metH3-K9 this antibody reactsalso with tri-methylated H3-K27 and several othermethylated lysines independent of their respectivepositions (Perez-Burgos et al., 2004 in press).Detection was performed by a goat-anti-rabbit anti-body conjugated either with Cy3 or Alexa 488(Molecular probes).

Visualization of centromeres by immunostainingwith a CREST- antiserum

For the delineation of centromeric regions ahuman antibody directed against centromere spe-cific proteins (Euroimmun) was used and detectedwith a Cy3- conjugated anti-human antibody.

DNA counterstainNuclear DNA was counterstained with 1 µM TO-

PRO-3 (Molecular Probes) for 5 min.

Confocal microscopy and 3D reconstruction ofimage stacks

Nuclei were scanned with an axial distance of250 nm between light optical sections using athree-channel laser scanning confocal microscope(Zeiss LSM 410). For each optical section imageswere collected sequentially for all three fluo-rochromes. Stacks of 8-bit gray scale 2D imageswere obtained with a pixel size of 66 or 88 nm.Confocal images were processed with ImageJ(http://rsb.info.nih.gov/ij/).

Quantitative 3D evaluationThreshold assessment

If not stated otherwise a fixed threshold was setmanually for each nucleus after visual inspection inan attempt to segment specific signals from appar-ent background. All voxels (volume based elements)with intensity values below the threshold were set to0 and were not counted in the respective evaluations.

Evaluation of the radial distribution of MMLlabeled chromatin

A detailed description of the quantitative 3Devaluation of the radial distribution i.e the distancefrom the nuclear center to the nuclear periphery offluorochrome labeled targets in 3D image stacks oflight optical serial sections was published elsewhere(Cremer et al., 2003). As a first step the geometri-cal center and the border of each nucleus weredetermined using the 3D data set of the DNA-coun-terstain fluorescence.The segmented nuclear spacewas divided into 25 equidistant shells with a thick-ness of Dr = 1/25 r. In the second step, chromatindelineated after MML immunostaining was evalu-ated in the 3D stack representing the respectivecolor channel. For each voxel located in the seg-mented nucleus the relative distance rx from thenuclear center was calculated as a fraction of r. Ashell at a given rx contains all nuclear voxels with adistance between rx - Dr/2 and rx + Dr/2. For eachshell all voxels assigned to the MML immunostain-ing were identified and the fluorescence intensitiesderived from the respective emission spectrum weresummed up. This procedure yielded for each shellthe individual relative content of MML stainedchromatin. This relative content within a nuclearshell was plotted as a function of the relative dis-tance r from the 3D center in the entire set of eval-uated nuclei. The cumulative frequencies of voxelintensities plotted against the relative radius were

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tested for significant differences (p ≤ 0.01) with atwo-sample Kolmogorov-Smirnov test.

Definition of objects stained with MML antibodies The 8 bit image stack visualizing immunostained

metH3-K9 was used for the definition of objects inthe following ways: An object was defined by thefact that it contained at least ten voxels with a con-tingent neighborship. Neighborship was defined forall voxels above threshold sharing at least one com-mon side by the software Volocity (Improvision,http://www.improvision.com/), see Figures 5 and 6.For a program written by S. Stein neighborship wasdefined for all voxels sharing at least one commoncorner, edge or side (see Figures 4 and 7).The firstdefinition allows for six neighbors/voxel, the seconddefinition for 26 neighbors/voxel. Object countingwas done for different threshold settings in intervalsof five gray levels until the maximal threshold of255 was reached. 3D image reconstructions ofobjects were done with the software Volocity.

Assessment of the density of MML labeled chromatinThe average density of MML labeled objects for

a given set of nuclei was estimated in two ways: Forthe first approach the sum of voxel intensitiesassigned to an object at a given threshold was divid-ed by the sum of the respective voxels (I/vox). Forthe second approach the sum of voxel intensitiesassigned to an object at a given threshold was divid-ed by the total surface area of all objects (I/surf).The surface of an object is defined by all voxel sidesnot attached to other object voxels. This value(1/µm2) was taken as a parameter of the compact-ness of objects.To test for significant differences (p≤ 0.05) the Mann-Whitney-U test, a distribution-independent rank test for independent samples wasapplied.

Results

MML stained patterns are distinctly different indifferent cell types

The characteristic nuclear pattern of chromatinvisualized after immunostaining using the MMLantibody (further referred to as MML chromatinpattern) and after DNA counterstaining with TO-PRO-3 in different normal and malignant humancell types is shown in Figure 1. The upper row dis-plays the DNA counterstain of a typical confocalmid-section from nuclei of a monocyte (A), a lym-

phocyte (B), a leukemic T-cell line Jurkat (C), anormal fibroblast (D), a normal colon epithelial cell(E) and a colon adenocarcinoma cell (F). The midrow represents the MML pattern and the bottomrow the merged image of the respective nuclei. Allnuclei were in a postmitotic state (further referredto as G0), as revealed by their lack of pKi67 stain-ing (Gerdes et al., 1984).These examples illustratethe variability of histone methylation patterns,ranging from a fairly homogeneous pattern with anadumbrated peripheral accumulation in G0 lympho-cytes, over a granular pattern seen in fibroblasts, topatterns, which exhibit few large and distinct clus-ters in monocytes and normal colon epithelial cells.The clusters of intensely stained MML chromatinare not in all cases reflected by a correspondinghigh staining intensity of the nuclear counterstain(e.g. compare panels A and C). The comparison ofMML patterns between representative normal lym-phocyte nuclei and nuclei from the T-cell derivedleukemic cell line Jurkat revealed distinct differ-ences. In contrast to the commonly observed pat-tern of a rather homogeneous distribution of MMLchromatin in normal G0 lymphocytes, numerousdistinct clusters of MML chromatin were observedin nuclei of the Jurkat cell line. A more peripheralaccumulation of MML chromatin is seen in thenucleus of the colon carcinoma compared to nucleiof normal colon epithelial cells.

MML chromatin patterns change during the cellcycle

The MCF-7 cell line was analyzed for changes ofthe MML chromatin pattern during the cell cycle:We assessed this pattern for nuclei in G0 as definedby a negative staining of pKi67, for nuclei duringearly G1, identified by a speckled pattern of pKi67and a faint outlining of the nucleolar space (Gerdeset al., 1984), and for nuclei in early and mid Sphase defined by their typical patterns of replica-tion foci visualized by immunostaining of incorpo-rated BrdU. Replication foci of early replicatinggene rich chromatin are distributed throughout thenucleus sparing the utmost nuclear periphery andthe perinucleolar regions (Figure 2, panel III B),whereas the gene poor mid replicating chromatinforms a typical layer both at the nuclear peripheryand around nucleoli (Figure 2, panel III C),(O'Keefe et al., 1992).

Typical examples for each cell cycle stage aredepicted in Figure 2. Panel I outlines one confocal

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Figure 1. Chromatin pattern visualized by MML immunostaining in confocal sections from nuclei of different cell types. For each celltype one nucleus is displayed after DNA counterstain with TO-PRO-3 (upper row), the corresponding MML pattern (mid row) and themerged image (bottom row). Bars indicate 5 µmm.

Figure 2. Chromatin patternvisualized by MML immunostain-ing in nuclei of the MCF-7 cellline during different cell cyclestages. Each panel (A – D)shows a confocal mid-section ofa nucleus and represents a dif-ferent cell cycle stage. Panel Idisplays the nuclear counter-stain by TO-PRO-3, panel II thecorresponding MML pattern,panel III either pKi67 or BrdUpattern and panel IV the mergedimage (MML chromatin ingreen, pKi67, BrdU or counter-stain respectively in red). For adetailed description see results.Bar indicates 5 µmm.

mid-section of the nucleus after DNA counterstain,panel II shows the MML chromatin pattern of thecorresponding section, panel III the pKi67 or BrdUpatterns, respectively, and panel IV the mergedimage of MML chromatin with either pKi67 (panelIV A), BrdU (B and C) or with the nuclear counter-stain (D). Visual inspection of nuclei from all cellcycle stages showed a fine granular or reticulatedMML staining with low signal intensity expandingthroughout the entire nucleus.The bulk MML chro-matin with high signal intensity, however, revealeddistinct differences during the cell cycle. In early G1(panel II A) numerous diffusely shaped MML chro-matin clusters are scattered over the entire nucleus.An overlay of the pKi67 and MML chromatin pat-terns shows a high degree of colocalization of thepKi67 signals with these histone methylation sites(IV A). During early S-phase (panel B) the MMLchromatin is preferentially located close to thenuclear periphery and around nucleoli.This space islargely devoid of early replicating chromatin as seenin the merged image by non-overlapping signals (IVB). Notably, the MML chromatin pattern seen inearly S-phase is characteristic for chromatin repli-cating during mid S-phase (O'Keefe et al., 1992).This observation suggests that the MML chromatin

pattern in early S-phase anticipates the pattern ofmid-replicating chromatin. The preferential local-ization around nucleoli and at the nuclear peripheryis maintained during mid-S phase resulting in a par-tial colocalization or association of MML chro-matin and mid-replicating chromatin (IV C). Afterexit of the cell cycle (G0) MML labeled chromatin isconcentrated in a few large, distinctly shaped com-pact clusters of high signal intensity in close associ-ation to the nucleoli and as a few smaller clusters atthe nuclear periphery (IV D).

Topological relationship between MML chromatinpatterns and centromeres

In addition to the antibody against MML chro-matin, MCF-7 cells were co- immunostained with aCREST antiserum that specifically binds to cen-tromeric proteins such as CENP-A and CENP-B(Moroi et al., 1980) and thus delineates the kinteo-chores of centromeres (Figure 3). Panel I shows thenuclear DNA counterstain of a confocal mid-sec-tion, panel II the corresponding centromeric regionsvisualized by the CREST antiserum, panel III theMML chromatin pattern and panel IV the mergedimages of a G0 nucleus and an early S phase nucle-us. Nuclei in G0 were identified by the lack of

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Figure 3. Simultaneous immunostaining of MML chromatin and centromeric regions in a confocal mid-section of a G0 nucleus (upperrow) and an early S-phase nucleus (bottom row) of MCF-7 cells. Panel I: nuclear counterstain with TO-PRO-3, panel II: centromeres,panel III: heterochromatin visualized by metH3-K9, panel IV: merged image of MML chromatin in green, centromeres in red. Arrowsindicate perinucleolar areas of MML chromatin not directly associated to centromeres.

immunostaining with pKi67 antibodies and nucleiin early S-phase by the typical pattern of incorpo-rated BrdU (not shown). The nuclei revealed thetypical MML chromatin patterns, described abovefor G0 and early S-phase (compare Figure 2). In G0

nuclei (Figure 3A), all clusters of centromeric kine-tochores were closely associated to large MMLchromatin clusters, which were located eitheraround nucleoli or at the nuclear periphery. AllMML labeled chromatin that was not associatedwith a centromere was seen as a fine granular pat-tern in the nucleus. In early S-phase, all cen-tromeres were again found either at the nucleoli orat the nuclear periphery, within MML labeled chro-matin compartments. However, a large fraction ofintensely stained MML chromatin, not directlyassociated with centromeres was found in a ringshaped manner around the nucleoli and less dis-tinctly at the nuclear periphery (Figure 3B,arrows).This suggests that the bulk of perinucleolarMML stained chromatin observed in early S-phasecorresponds only to a minor fraction to the consti-tutive heterochromatin of centromeres.

Approaches towards a quantitative 3D evaluationof the MML chromatin pattern

In an attempt to establish a quantitative 3D eval-uation procedure to discriminate the differentMML chromatin patterns observed during differentcell cycle stages, the following parameters wereanalyzed in 3D images: (i) the radial distribution ofMML chromatin clusters, e.g. their distribution as

a function of their distance from the nuclear center,(ii) the number of MML chromatin clusters (fur-ther referred to as objects) at different thresholdlevels, (iii) the density of MML chromatin objectsexpressed either by the mean ratio of the intensityper voxel from all voxels assigned to an object or bythe mean ratio of the intensity per surface assignedto an object. These parameters are exemplifiedbelow for MCF-7 nuclei.

i) Radial distribution of MML chromatin usingfixed threshold levels

Prior to the evaluation of the radial distribution athreshold level of gray value was set manually foreach individual nucleus with the objective to definechromatin that was unequivocally assigned toMML chromatin clusters. The threshold for differ-ent nuclei varied between 90 and 120. Figure 4shows the curves for the radial distribution of MMLlabeled chromatin for the different cell cycle stagesof the MCF-7 cell line. For each cell cycle stage 11to 20 nuclei were evaluated. In each graph the nor-malized chromatin content, represented by itsvoxel-intensity weighted fluorochromes is plottedagainst the relative radius of the nucleus (fordetails see methods).These curves thus explore thepositioning of MML chromatin with regard to thedistance from the geometrical center of the nucle-us.The MML chromatin pattern in G1 nuclei close-ly resemble a Gaussian distribution, with its maxi-mum at around 65% of the relative radius, where-as the pattern in G0 nuclei shows two small peaks,the first around 62% and the second around 86%of the relative radius. With a maximum at around52% of the relative radius, S-phase nuclei show themost internal position of MML chromatin, reflect-ing the accumulation around internally locatednucleoli. Despite the remarkable differences ofthese patterns observed in different cell cycle stagesby visual inspection, the two-sided Kolmogorov-Smirnov did not reveal significant differences.

(ii) Number of MML chromatin objects defined atdifferent threshold levels

In addition we assessed the number of MMLstained objects. In such an analysis, one has to keepin mind that the threshold level for complex struc-tures with a wide range of intensities is inevitablychosen arbitrarily and that different settings of thethreshold level strongly influence the outcome ofsuch an analysis. Figure 5 exemplifies this for one

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Figure 4. Quantitative 3D evaluation of radial distribution ofMML labeled chromatin in 25 concentric shells after immunos-taining in MCF-7 G0 nuclei (red), MCF-7 G1 nuclei (green) andMCF-7 S-phase nuclei (blue). The abscissa denotes the relativeradius r of the nuclear shells, the ordinate the normalized sumof intensities in the voxels assigned to the color channel forMML chromatin. For normalization the area underlying eachcurve for the different series (total relative content of MMLstained chromatin) was set to 100. Bars indicate standard devi-ations of the mean.

nucleus. Figure 5 A shows a mid-section of the rawimages of a G0 MCF-7 nucleus after immunostain-ing with the MML antibody. As typically seen in thiscell type, besides a number of sharply outlined largeclusters with high signal intensity, a network of lowintensity signals, sometimes forming ring like struc-tures expands throughout the nucleus. These lowintensity signals may reflect background, but theymay also represent specific signals possibly reflect-ing small regions containing genes that are con-trolled by histone methylation. In panels 5 B-F thesame nucleus is displayed with different thresholdlevels, marking voxels above the respective thresh-old in red (left-hand row).The resulting 3D objectsafter applying the object definition rules (see meth-ods) are shown in the right-hand row. Note that notevery voxel above the threshold contributes to anobject.While the number of marked voxels decreas-es constantly with increasing threshold levels, thenumber of objects initially increases at very lowthreshold levels until a maximum is reached (B-C).Thereafter the number of objects gradually startsto decrease with increasing threshold levels (C-E).Single objects, which consist of areas with hetero-geneous intensities, can fall apart into severalobjects with increasing threshold levels, if they con-sist of voxels with heterogeneous intensities (com-pare objects marked with an arrow or an arrow-head in panels E and F of Figure 5). Figure 6 illus-trates for three individual nuclei taken from cells atdifferent cell cycle stages how the number ofobjects depends on the threshold value. The curveprogression for the G0 and the S-phase nucleus ismuch steeper compared to the G1 nucleus.This indi-cates that these mainly large objects of G0 and S-phase nuclei had a high degree of homogeneity,which decreased steadily in size over a wide rangeof thresholds and only at very high levels in number.In contrast, the more heterogeneous objects of G1

nuclei tended to fall apart with higher thresholdscompensating for a decrease of object numbers atthese higher thresholds.

(iii) Density of chromatin clusters visualized byMML immunostaining

In a further attempt to establish quantitativeapproaches that do not depend on one arbitrarilyfixed threshold level, the average density of all seg-mented object voxels, either expressed by the inten-sity/voxel or by the intensity/surface (see methodspart) was calculated over a wide range of threshold

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Figure 5. Segmentation of objects at different threshold levels.(A) mid-section of a raw image from confocal serial sections ofa MCF-7 G0 nucleus. (B-F) the same nucleus is displayed withdifferent threshold levels. Voxels above the respective thresholdare marked in red on the left side images, the correspondingobjects in varying colors on the right side images. Note that notall voxels above threshold contribute to an object due to theobject definition as described in the methods part. For this par-ticular nucleus an object maximum is obtained at a thresholdaround 50. The object number is the same at threshold levels110 and 140 (E and F). This is due to a splitting of one largerobject into several ones (marked by arrows) and the disappear-ance of objects between threshold levels 110 and 140 (markedby arrowheads).

levels. This procedure was performed for each cellcycle stage of MCF-7 cells using same set of nucleidescribed above for the evaluation of the radial dis-tribution of MML chromatin (compare Figure 4).As shown in Figure 7 A the average intensity/voxelincluding all voxels assigned to an object was large-ly identical for all cell cycle stages over the entirerange of threshold levels, demonstrating that themean voxel intensity remains constant during the

cell cycle. However, when the intensity/surface wasplotted against threshold levels, (Figure 7 B), dis-tinct differences between the cell cycle stages werefound. The different values for G0 nuclei comparedto S and G1 nuclei mirror the different degree ofcompactness in MML chromatin clusters during therespective cell cycle stages.The higher values for G0

compared to S phase and G1 nuclei indicate thatobjects in G0 have the highest and in G1 the lowest

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Figure 6. Number of objects counted along threshold levels (gray values 15 to 255) of an MCF-7 G0 nucleus (A), an early G1 nucleus(B) and an early S-phase nucleus (C). Panel I shows a projection of 4 confocal mid-sections from each nucleus, encompassing 1µm ofits central part, panel II the number of objects in each nucleus plotted against the threshold levels. In panel III the objects of the entirenucleus are displayed in 3D at threshold levels marked by an asterisk in the graphs.

compactness. Using the Mann-Whitney-U test thedistribution curve for each cell cycle stage was sig-nificantly different (p = 0.01).

Discussion

The observed MML chromatin pattern in G0

nuclei of several normal and malignant cell typesranged from a fairly homogeneous distribution innormal lymphocytes to distinct cluster formation inmonocytes, MCF-7 cells and Jurkat cells. In thesecell types a dimorphic pattern of low intensitystructures and of larger clusters with high signalintensity was observed.The latter were most promi-nent in the MCF-7 G0 nuclei.

The transition from a homogeneous pattern in nor-mal lymphocytes to the pronounced cluster forma-tion observed in nuclei of the Jurkat cell line, whichwas established from a T-lymphocyte leukemia isstriking. It remains speculative to what extent dif-ferences of MML chromatin patterns reflect thechromosome level or gene level function of histone

methylation (see introduction). High-intensity MMLpositive chromatin clusters, which are in addition tothe di-methylation of H3-K9 probably also the resultof a strong tri-methylation of H3-K27 and H4-K20(Peters et al., 2003) may comprise both centromer-ic heterochromatin and surrounding facultative het-erochromatin. The low intensity signals may repre-sent a large number of smaller chromatin regionscontaining gene loci that are controlled by histonemethylation of different lysines.

The distinct differences of MML chromatin pat-terns observed in G0 nuclei from different cell typesraise the question, whether these differences reflectcell type specific arrangements of chromosome ter-ritories (CTs). Present studies of CT arrangementsin various normal and malignant cell types indicatehighly variable neighborhoods of homologous andheterologous chromosomes (Bolzer et al., 2003;Cremer et al., 2001; Lesko et al., 1995; Sun andYokota, 1999). In the light of these findings we con-sider it unlikely that cell type characteristic MMLchromatin patterns can be explained as a conse-quence of cell type specific CT neighborhoods. Incontrast to the high variability of CT neighbor-hoods, radial arrangements of CTs, defined as thedistance from the nuclear center are highly non-random.These radial arrangements of CTs are size-correlated in fibroblast nuclei (Bolzer et al., 2003;Sun et al., 2000), but gene density correlated innuclei such as lymphocytes and numerous othersincluding MCF-7 cells (Boyle et al., 2001; Cremeret al., 2003; Kozubek et al., 2002) with a prefer-ential location of gene poor CTs at the nuclearperiphery and a preferential location of gene denseCTs in the nuclear interior.The quantitative evalua-tion of MML marked chromatin in MCF-7 cells,however, did not reveal a pronounced peripheralposition. This was due to the major fraction ofMML label found in large clusters around thenucleoli. These clusters contain also centromericheterochromatin. Since centromeric regions arehighly methylated at H3-K9 (Lachner andJenuwein, 2002), an essential fraction of the MMLchromatin pattern is reflected by the positions ofcentromeres. In G0 lymphocytes the MML stainingpattern was more intense in the nuclear periphery,which is in agreement with the peripheral locationof centromeric heterochromatin in lymphocytenuclei (Weierich et al., 2003). Possibly, major clus-ters of MML chromatin comprise higher order het-erochromatin compartments built up from cen-

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Figure 7. Evaluation of the density of objects. (A) the meanintensity/voxel averaged over all nuclei of a series is plottedagainst the threshold level. The ratio intensity/voxel increaseswith higher threshold levels since objects with a low intensityare eliminated. The decline of the curve at very high thresholdlevels is due to the fact that values are averaged over all nucleiwithin one series. At very high threshold levels some of thenuclei do not more display any objects, reducing the mean inten-sity/voxel. (B) The mean intensity/ surface averaged over allnuclei of a series is plotted against the threshold level.

tromeric heterochromatin and facultative hete-rochromatic chromosomal subregions from neigh-boring CTs.

Little attention has been paid so far to changes ofpattern formation of MML chromatin during andupon exit of the cell cycle. Our study demonstratesa profound change of the MML chromatin patternin MCF-7 cells between early G1 and S phase andafter exit of the cell cycle. These changes wereremarkable by visual inspection and confirmed byquantitative evaluation. The result raises severalquestions:

1. Do the observed cell cycle changes result fromlocal changes in the level of methylation of H3-K9and other methylated lysines in chromatin configu-rations that are stably positioned during inter-phase? Direct evidence for this assumption is lack-ing. Histone methylation is considered to be a verystable modification and potentially an irreversibleepigenetic mark since histone demethylases havenot been discovered so far. However, stably methy-lated histones might be removed by histoneexchange eliminating their epigenetic potential(Sims et al., 2003).

2. Are cell cycle changes of MML patterns causedby large-scale changes of higher order chromatinarrangements during the cell cycle? Live cell obser-vations of HeLa cells encompassing an entire cellcycle revealed, that - with the exception of the earlyG1-phase (see below) - the position of chromosometerritories and most chromosome subdomains areconstrained within a range of about 1 µm from lateG1 until the end of G2 (Walter et al., 2003). Furtherstudies are indicated to test whether the large-scalestability of CT arrangements during the cell cycle isa general feature of mammalian cell nuclei. If thisshould be the case, the observed changes of theMML chromatin patterns during interphase inMCF-7 cells cannot be explained as a consequenceof major changes of CT arrangements.Furthermore, studies of DNA-replication during S-phase provided evidence that replication foci do notundergo large-scale movements during S-phase.Instead replication factors are recruited to ratherstably positioned chromatin foci (Leonhardt et al.,2000). However, while these observations argueagainst large scale movements of most chromatinfrom mid G1 to late G2, they do not exclude moreexpanded and possibly directed movements of asubset of defined subchromosomal regions.Centromeres provide a case in point. It has recent-

ly been shown for a number of cell types, such asstimulated lymphocytes, lymphoblastoid cells,fibroblasts and a neuroblastoma cell line, that cen-tromeres undergo major movements during the cellcycle and upon exit of the cell cycle (G0 transition)(Solovei et al., 2004). In all cell types studied, cen-tromeres were mostly found in the nuclear interiorand partly at the nuclear periphery during early G1,while in late G1 most centromeres were seen asclusters at the periphery. This location was main-tained through S-phase. Upon exit of the cell cycle(G0 cells) centromeres were found in clusters at thenuclear periphery. A systematic quantitative analy-sis of centromere movements during the cell cyclein MCF-7 cells is still lacking. Since we found cen-tromeric chromatin always in close association withMML labeled chromatin as demonstrated for a G0and an early S-phase nucleus, changes of MMLchromatin patterns during the cell cycle may beexplained in part by the dynamic behavior of cen-tromeric chromatin.

3.The observed changes of MML chromatin pat-terns during the cell cycle lead to the question of apossible functional relevance.The remarkable asso-ciation of MML chromatin sites and pKi67 duringearly G1 may reflect the necessity of chromosometerritories to home in to their final nuclear locationand to establish a polarized orientation of CTs toexpose chromosomal subregions with differenttranscriptional activityin a spatially correct manner(Sadoni et al., 1999), Walter et al., 2003). It hasbeen shown that pKi67 interacts with HP-1, a pro-tein that is recruited at metH3-K9 heterochromatinsites. A transient colocalization of HP-1 and pKi67in heterochromatin foci was observed duringtelophase and early G1 (Kametaka et al., 2002;Scholzen et al., 2002) suggesting a possible dock-ing mechanism for heterochromatin in the newlydeveloping nucleus. On the other side HP-1 inter-acts with the lamin B receptor, an integral compo-nent of the inner nuclear membrane (for review see(Lachner and Jenuwein, 2002). During mitosis,HP-1 largely dissociates from chromosomes andreassembles at the polar surfaces of anaphase chro-mosomes (Kourmouli et al., 2000), whereas p-Ki-67 remains closely associated around chromosomesduring this period. It is tempting to speculate thatthe pKi67/HP-1/metH3-K9 complex plays a role inreestablishing heterochromatin domains duringtelophase/early G1 in a coordinated way with theHP-1 mediated binding of metH3-K9 marked hete-

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rochromatin to the newly formed lamin B at thenuclear envelope (Schermelleh, 2003).

The present data emphasize the importance offurther studies of nuclear architecture changes cor-related with specific epigenetic chromatin modifi-cations such as histone methylations at definedlysine positions. So far studies are still based to alarge part on the qualitative assessment of thestaining patterns observed in light optical nuclearsections. To overcome the subjectivity involved insuch studies, the development of bioimagingapproaches, which guarantee an impartial quanti-tative analysis of chromatin pattern formation withspecific epigenetic marks in space and time is ademanding but requisite task. From the technicalpoint of view a major obstacle for quantitative eval-uations of fluorescently labeled objects in a nucleusis the need of setting a threshold in order to dis-criminate specific, segmented signals from unspe-cific background. This problem becomes crucialwhen numerous targets with complex structuresand a wide range of signal intensities have to besegmented in the same nucleus. Both, automaticand interactive approaches for segmentation havetheir drawbacks (for review see (Gil et al., 2002).A high threshold level may assure that no unspecif-ic background signal is included in the evaluation.On the other hand, low intensity signals, whichwould be eliminated by a high threshold setting,may in fact be specific signals, which should not beignored since they may have important functionalimplications. The importance of utilizing the fullrange of intensities has been shown by (Fay et al.,1997) for an accurate quantitative analysis of thenuclear distribution of nascent RNA and the splic-ing factor SC-35. Approaches that are not limitedto a fixed threshold and allow analyses along alllevels of threshold settings should therefore prefer-ably be implemented.

AcknowledgementsThe authors are most grateful to T. Jenuwein and

A. Peters (Research Institute of MolecularPathology, Vienna, Austria), both for the generousgift of the MML antibody and their helpful com-ments that helped to improve the manuscript. Wealso thank A. Jauch (University of Heidelberg,Germany) for providing the M-FISH analysis ofthe MCF-7 cell line and L. Schermelleh (Universityof Munich) for stimulating discussions and carefulreading of the manuscript.

FundingThis work was supported by grants to T.Cremer

from the Wilhelm Sander Stiftung (2001.079.1)and from the Deutsche Forschungsgemeinschaft(Schwerpunktprogramm “Funktionelle Architekturdes Zellkerns”, CR 59/21-1).

References

Bird A. DNA methylation patterns and epigenetic memory. Genes Dev2002;16:6-21.

Bolzer A, Kreth G, Solovei I, Saracoglu K, Fauth C, Müller S, et al.Complete 3D maps of chromosome positions in human male fibrob-last nuclei and prometaphase rosettes demonstrate a size depend-ent, probabilistic arrangement. submitted 2003.

Boyle S, Gilchrist S, Bridger JM, Mahy NL, Ellis JA, Bickmore WA.The spatial organization of human chromosomes within the nuclei ofnormal and emerin-mutant cells. Hum Mol Genet 2001;10:211-9.

Cremer M, Kupper K, Wagler B, Wizelman L, Hase Jv J, Weiland Y, etal. Inheritance of gene density-related higher order chromatinarrangements in normal and tumor cell nuclei. J Cell Biol 2003;162:809-20.

Cremer M, von Hase J,Volm T, Brero A, Kreth G,Walter J, et al. Non-random radial higher-order chromatin arrangements in nuclei ofdiploid human cells. Chromosome Res 2001;9:541-67.

Diaz G, Zucca A, Setzu MD, Cappai C. Chromatin pattern by variogramanalysis. Microsc Res Tech 1997;39:305-11.

Elgin SC, Grewal SI. Heterochromatin: silence is golden. Curr Biol2003;13:R895-8.

Fay FS,Taneja KL, Shenoy S, Lifshitz L, Singer RH. Quantitative dig-ital analysis of diffuse and concentrated nuclear distributions ofnascent transcripts, SC35 and poly(A). Exp Cell Res 1997;231:27-37.

Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cellcycle analysis of a cell proliferation-associated human nuclear anti-gen defined by the monoclonal antibody Ki-67. J Immunol 1984;133:1710-5.

Gil J, Wu H, Wang BY. Image analysis and morphometry in the diag-nosis of breast cancer. Microsc Res Tech 2002;59:109-18.

Heitz E. Das Heterochromatin der Moose. I. Jahrb. Wiss Botanik1928;69:762-818.

Jones PA, Baylin SB.The fundamental role of epigenetic events in can-cer. Nat Rev Genet 2002;3:415-28.

Kametaka A, Takagi M, Hayakawa T, Haraguchi T, Hiraoka Y, YonedaY. Interaction of the chromatin compaction-inducing domain (LRdomain) of Ki-67 antigen with HP1 proteins. Genes Cells 2002;7:1231-42.

Kourmouli N, Theodoropoulos PA, Dialynas G, Bakou A, Politou AS,Cowell IG, et al. Dynamic associations of heterochromatin protein 1with the nuclear envelope. Embo J 2000;19:6558-68.

Kozubek S, Lukasova E, Jirsova P, Koutna II, Kozubek M, Ganova A,et al. 3D Structure of the human genome: order in randomness.Chromosoma 2002;111:321-31.

Lachner M, Jenuwein T.The many faces of histone lysine methylation.Curr Opin Cell Biol 2002;14:286-98.

Lachner M, O'Carroll D, Rea S, Mechtler K, Jenuwein T. Methylationof histone H3 lysine 9 creates a binding site for HP1 proteins.Nature 2001;410:116-20.

Lachner M, O'Sullivan RJ, Jenuwein T. An epigenetic road map for his-tone lysine methylation. J Cell Sci 2003;116:2117-24.

Leonhardt H, Rahn HP, Weinzierl P, Sporbert A, Cremer T, Zink D, etal. Dynamics of DNA replication factories in living cells. J Cell Biol2000;149:271-80.

Lesko SA, Callahan DE, LaVilla ME, Wang ZP, Ts'o PO. The experi-mental homologous and heterologous separation distance his-tograms for the centromeres of chromosomes 7, 11, and 17 in inter-phase human T-lymphocytes. Exp Cell Res 1995;219:499-506.

Mairinger T, Mikuz G, Gschwendtner A. Nuclear chromatin textureanalysis of nonmalignant tissue can detect adjacent prostatic ade-

22

M. Cremer et al.

nocarcinoma. Prostate 1999;41:12-9.Moroi Y, Peebles C, Fritzler MJ, Steigerwald J,Tan EM. Autoantibody

to centromere (kinetochore) in scleroderma sera. Proc Natl AcadSci USA 1980;77:1627-31.

Mulder JW, Offerhaus GJ, de Feyter EP, Floyd JJ, Kern SE,VogelsteinB, et al. The relationship of quantitative nuclear morphology tomolecular genetic alterations in the adenoma-carcinoma sequenceof the large bowel. Am J Pathol 1992;141:797-804.

Muller S, Eder V, Wienberg J. A nonredundant multicolor bar code asa screening tool for rearrangements in neoplasia. Genes Chrom-osomes Cancer 2004;39:59-70.

O'Keefe RT, Henderson SC, Spector DL. Dynamic organization of DNAreplication in mammalian cell nuclei: spatially and temporallydefined replication of chromosome-specific α-satellite DNAsequences. J. Cell Biol 1992;116:1095-110.

Perez-Burgos L, Peters A, Opravil S, Kauer M, Mechtler K, JenuweinT. Generation and characterization of methyl-lysine antibodies.Methods Enzymol 2004 (in press).

Peters AH, Kubicek S, Mechtler K, O'Sullivan RJ, Derijck AA, Perez-Burgos L, et al. Partitioning and plasticity of repressive histonemethylation states in mammalian chromatin. Mol Cell 2003;12:1577-89.

Plath K, Fang J, Mlynarczyk-Evans SK, Cao R, Worringer KA, WangH, et al. Role of histone H3 lysine 27 methylation in X inactivation.Science 2003;300:131-5.

Sadoni N, Langer S, Fauth C, Bernardi G, Cremer T,Turner BM, et al.Nuclear organization of mammalian genomes. Polar chromosometerritories build up functionally distinct higher order compartments.J Cell Biol 1999;146:1211-26.

Schermelleh L. Dynamic organization of chromosomes in the mam-malian cell nucleus. (Dissertation, Univ. of Munich 2003).

Schneider U, Schwenk HU, Bornkamm G. Characterization of EBV-genome negative "null" and "T" cell lines derived from children withacute lymphoblastic leukemia and leukemic transformed non-Hodgkin’s lymphoma. Int J Cancer 1977;19:621-6.

Scholzen T, Endl E,Wohlenberg C, van der Sar S, Cowell IG, Gerdes J,et al. The Ki-67 protein interacts with members of the heterochro-matin protein 1 (HP1) family: a potential role in the regulation ofhigher-order chromatin structure. J Pathol 2002;196:135-44.

Sims RJ 3rd, Nishioka K, Reinberg D. Histone lysine methylation: a sig-nature for chromatin function.Trends Genet 2003;19:629-39.

Solovei I, Schermelleh L, Düring K, Engelhardt A, Stein S, Von HaseJ, et al. Differences in centromere position during cell cycle and atGo in four different types of human cells. (submitted 2004).

Sun HB, Shen J,Yokota H. Size-dependent positioning of human chro-mosomes in interphase nuclei. Biophys J 2000;79:184-90.

Sun HB, Yokota H. Correlated positioning of homologous chromo-somes in daughter fibroblast cells. Chromosome Res 1999;7:603-10.

Tashiro S, Walter J, Shinohara A, Kamada N, Cremer T. Rad51 accu-mulation at sites of DNA damage and in postreplicative chromatin.J Cell Biol 2000;150:283-91.

Walter J, Schermelleh L, Cremer M,Tashiro S, Cremer T. Chromosomeorder in HeLa cells changes during mitosis and early G1, but is sta-bly maintained during subsequent interphase stages. J Cell Biol2003;160:685-97.

Weierich C, Brero A, Stein S, von Hase J, Cremer C, Cremer T, et al.Three-dimensional arrangements of centromeres and telomeres innuclei of human and murine lymphocytes. Chromosome Res 2003;11:485-502.

Weyn B, Jacob W, da Silva VD, Montironi R, Hamilton PW,ThompsonD, et al. Data representation and reduction for chromatin texture innuclei from premalignant prostatic, esophageal, and colonic lesions.Cytometry 2000;41:133-8.

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