Nuclear structure and gene activity in human differentiated cells

Journal of

Structural Biology Journal of Structural Biology 139 (2002) 76–89 www.academicpress.com

Nuclear structure and gene activity in human differentiated cells
,a Michal Kozubek,b Hana Gajov
a,c Eva B
artov
a,a Stanislav Kozubek,a,* Pavla Jirsova a b Emilie Luk
asov
a, Magdalena Skaln
ıkov
a, Alena Ga nov
a,a Irena Koutn
a,b and Michael Hausmannd a

c

Institute of Biophysics, Academy of Sciences of the Czech Republic, Kr
alovopolsk
a 135, 612 65 Brno, Czech Republic b Faculty of Informatics, Masaryk University, Botanick
a 68 a, 602 00 Brno, Czech Republic Department of Ophthalmology, Children’s Hospital, Faculty of Medicine, Masaryk University, Cernopoln
ı 9, Brno, Czech Republic d € berle-Strasse 3-5, D-69120 Heidelberg, Germany Kirchhoff Institute of Physics, University of Heidelberg, Albert-U Received 21 September 2001, and in revised form 16 February 2002

Abstract The nuclear arrangement of the ABL, c-MYC, and RB1 genes was quantitatively investigated in human undifferentiated HL-60 cells and in a terminally differentiated population of human granulocytes. The ABL gene was expressed in both cell types, the c-MYC gene was active in HL-60 cells and down-regulated in granulocytes, and expression of the RB1 gene was undetectable in HL-60 cells but up-regulated in granulocytes. The distances of these genes to the nuclear center (membrane), to the center of the corresponding chromosome territory, and to the nearest centromere were determined. During granulopoesis, the majority of selected genetic structures were repositioned closer to the nuclear periphery. The nuclear reposition of the genes studied did not correlate with the changes of their expression. In both cell types, the c-MYC and RB1 genes were located at the periphery of the chromosome territories regardless of their activity. The centromeres of chromosomes 8 and 13 were always positioned more centrally within the chromosome territory than the studied genes. Close spatial proximity of the c-MYC and RB1 genes with centromeric heterochromatin, forming the chromocenters, correlated with gene activity, although the nearest chromocenter of the silenced RB1 gene did not involve centromeric heterochromatin of chromosome 13 where the given gene is localized. In addition, the role of heterochromatin in gene silencing was studied in retinoblastoma cells. In these differentiated tumor cells, one copy of the RB1 gene was positioned near the heterochromatic chromosome X, and reduced RB1 gene activity was observed. In the experiments presented here, we provide evidence that the regulation of gene activity during important cellular processes such as differentiation or carcinogenesis may be realized through heterochromatin-mediated gene silencing. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Nuclear compartments; Gene expression; FISH; Centromeric heterochromatin

1. Introduction Cell differentiation is often accompanied by changes in nuclear organization that are related to rearrangement of chromosome territories (Cremer and Cremer, 2001; Leitch et al., 1994; Manuelidis, 1990). A shift of some genetic elements towards nuclear periphery was determined during myogenesis (Chaly and Munro, 1996) and granulopoesis (B
artov
a et al., 2000a, 2001). Each pathway of cellular maturation is characterized by specific patterns of gene expression that is modulated by *

Corresponding author. Fax: +420-5-41240498. E-mail address: [email protected] (S. Kozubek).

epigenetic factors including spatial organization of the cell nucleus (Cockell and Gasser, 1999; Francastel et al., 2000). The coordination of gene activity is related to the accessibility of DNA to regulatory factors (Brown et al., 1997, 1999; Bulger and Groudine, 1999; Francastel et al., 1999; Hendrich and Willard, 1995) and to transcriptional machinery (Cook, 1994, 1995). Nuclear functions such as replication, transcription, splicing, and repair are organized in distinct compartments within the cell nucleus (Chevret et al., 2000; Cremer et al., 1993, 2000; Ferreira et al., 1997; Spector, 1993; Schul et al., 1998; Verschure et al., 1999). The compartmentalization of transcriptionally active and inactive chromatin was described by Kurz et al.

1047-8477/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 7 - 8 4 7 7 ( 0 2 ) 0 0 5 6 0 - 9

E. B
artov
a et al. / Journal of Structural Biology 139 (2002) 76–89

(1996). In these experiments genes were found preferentially at the periphery of individual chromosome territories. This arrangement of chromosome territories was independent of transcriptional activity of studied gene loci, although nonexpressed anonymous fragments were found randomly distributed or preferentially localized in the interior of the corresponding chromosome territory. On the other hand, analyses of active and inactive X chromosomes revealed that transcriptionally active ANT2 (Xq24–q25) and ANT3 (Xp22.3) genes were positioned more peripherally in their chromosomal territory and the inactive ANT2 gene was located more centrally in the X inactive region (Dietzel et al., 1999). Nuclear positioning of expressed and nonexpressed DNA sequences within their respective chromosome territories was also studied in the experiments of Volpi et al. (2000). All genetic regions analyzed by these authors in B-cells were located predominantly at the periphery of the chromosomal territory. Active genes were observed more often on chromatin loops extending outward from the periphery of the chromosome territory as compared with fibroblasts where the studied locus was silent. In contrast, centromeric regions never appeared on a large external chromatin loop (Volpi et al., 2000). Considerable evidence that transcriptionally active chromatin is compartmentalized was published by Verschure et al. (1999). Newly synthesized and nascent RNA was presented in the interchromatin areas that were positioned throughout chromosome territories. Thus, the transcriptionally active loci are not necessarily located on the periphery of the territories, but are found predominantly at or near the surface of compact chromatin domains, which are territorial subunits. This interchromatin compartment starts at nuclear pores, expands between chromosome territories, extends into their interior (Solovei et al., 2000; Verschure et al., 1999), and possibly ends with its smallest branches between
100 kb and
1 Mb chromatin loop domains (Cremer and Cremer, 2001). Such invaginating interchromatin channels form a structural compartment that provides a functionally relevant barrier playing an important role in transcriptional regulation (Cremer and Cremer, 2001). Several reports have suggested that spatial arrangements of chromatin in the nucleus have a relationship not only with the functional state of the nucleus, but also cellular functions such as cell cycle progression (Weimer et al., 1992) and differentiation (B
artov
a et al., 2000b, 2001; Manuelidis, 1990). It is postulated that cellular differentiation may be related to nuclear differentiation (Leitch et al., 1994) accompanied by chromatin condensation (see Summary Francastel et al., 2000) and repositioning of certain centromeres closer to the nuclear periphery (B
artov
a et al., 2001; Chaly and Munro, 1996). In addition, the distances between genes and centromeres play an important role in the regulation of specific gene activity in differentiating cells (Cockell and Gasser,

77

1999; Francastel et al., 2000). All these observations lead to the question of how higher order organization regulates cell differentiation and activation of specific genes. In the present study, we investigated the changes of chromatin organization during granulocytic cell maturation. The ascertained structural changes of the ABL, c-MYC, and RB1 genes were compared with the transcriptional activity of studied gene loci. The spatial arrangement of selected active and inactive genes was determined with respect to the whole nucleus and in relationship to corresponding chromosome territories. In addition, simultaneous visualization of studied genes and all centromeric heterochromatin revealed differences in the distances of active and inactive loci from the nearest chromocenter (cluster of centromeres; Alcobia et al., 2000). Our experiments showed that gene repositioning in the cell nucleus, which represents an important feature of granulopoesis, does not correlate with the activity of studied loci. The RB1 and c-MYC genes were found on the periphery of chromosome territories independently of their activity, irrespectively of G0 =G1 as well as in G2 stages of the cell cycle and independently of the level of differentiation. Gene silencing during granulocytic differentiation correlates with the distance to centromeric heterochromatin; however, the nearest chromocenter regulating the RB1 (13q14) gene activity does not involve centromeric heterochromatin of chromosome 13 where the RB1 gene is localized. The potential influence of heterochromatin on RB1 gene silencing was also investigated in differentiated human retinoblastoma tumor cells with an (X;13) translocation. Spreading or proximity of the heterochromatic and methylated chromosome X to the q arm of chromosome 13 is suspected to induce functional monosomy of the RB1 gene.

2. Materials and methods 2.1. Cell preparation and fixation HL-60 cells. Cells of HL-60, a human leukemic promyelocytic cell line, were cultivated in IMDM medium as recommended by the American Tissue Culture Collection (Manassas, MD), and 10% fetal calf serum (PAN, Germany) was added to the medium. The cells grew to 25 passages at 37 °C in humidified atmosphere containing 5% CO2 . Cell viability was tested by 15% eosin staining (Sigma). Exponentially proliferating HL-60 cells at an initial cell density of 2  105 cells/ml were treated with all trans-retinoic acid (RA 10 lM), a differentiating agent. Granulocytic cell maturation was verified by the analysis of surface antigen expression using FITC-labeled CD33 and CD11b antibodies. Isotypic control

78

E. B
artov
a et al. / Journal of Structural Biology 139 (2002) 76–89

staining was performed with FITC-conjugated mouse monoclonal antibodies of the IgG1 isotype. CD33 and CD 11b expression in differentiated cells was measured by a FACSCalibur flow cytometer (Becton Dickinson), equipped with an argon ion laser (the 488 nm laser line was used for excitation) and CellQuest software running on the Apple Macintosh computer connected to the flow cytometer was used. Peripheral blood cells. Dextran sulfate solution was added to heparinized human peripheral blood. Samples were kept at room temperature (RT) for 20–60 min to allow the sedimentation of red cells. The leukocyte-rich plasma on the top of the red cell fraction was carefully collected and centrifuged at 300g for 8 min at RT. The supernatant was discarded and the cell pellet was resuspended in 1 ml of phosphate buffer saline (PBS). A layer of Ficoll-Hypaque solution (5 ml) was carefully put on the cell suspension. The cells were then centrifuged at 400g for 30 min at 4 °C. A buffy coat containing lymphocytes and monocytes appeared as a white, cloudy band between the plasma and the Ficoll-Hypaque and was collected. The granulocytic fraction under the Ficoll-Hypaque layer was fixed. Cell fixation. Cytological preparations were made using the cell fixation either by formaldehyde or by a methanol/acetic acid mix. For formaldehyde fixation, dehydration of the cells was avoided to preserve their native three-dimensional (3D) structure as much as possible. The suspension of cells was washed in PBS and spread onto a microscope slide. After 30 s of air drying, the cells were fixed in 4% formaldehyde in PBS for 5 min, washed thoroughly in PBS (3  4 min), permeabilized in 0.2% saponine in PBS followed by 0.2% Triton X-100 in PBS for 10 min each, washed in 0.1 M Tris– HCl (pH 7.2) for 2 min, equilibrated in 20% glycerol in PBS for 20 min, and treated with liquid nitrogen. An alternative method of cell fixation was hypotonic shock in 75 mM KCl and fixation in methanol:acetic acid solution (3:1). This cell preparation was used in the experiments analyzing the locus distance to the nearest point on the nuclear membrane (see Section 2.7). 2.2. Retinoblastoma tumor cell preparation and fixation A differentiated, endophytic tumor sample of an unilateral retinoblastoma was cytogenetically analyzed. Cut fragments of fresh tissue were treated in suspension with 75 mM KCl for 20 min at 37 °C and fixed in a 3:1 methanol:acetic acid. The cells were centrifuged at 200g for 5 min and then postfixed with 4% formaldehyde. In this state of fixation the tumor cells were placed onto a slide and again fixed with 4% formaldehyde for 5 min at RT. Three-dimensional fixation was performed according to Francastel et al. (1999) and was selected only for the retinoblastoma tumor cells analyzed using fluorescence in situ hybridization (FISH).

2.3. Fluorescence in situ hybridization For FISH techniques we used the following biotinylated or digoxigenin-labeled DNA probes (Oncor, USA): unique sequences of the ABL, c-MYC, and RB1 genes, a pan-centromeric probe, and painting probes for whole chromosomes 8 and 13. The hybridization procedure and posthybridization washing were performed according to the instructions of the manufacturer. In brief, after fixation and cell permeabilization the chromosomal target DNA was denatured at 70 °C in 70% formamide in 2X SSC (pH 7.0) for 3 min. Unique sequence probe (8–10 ll) was denatured at 72 °C for 10 min and applied on the slide with fixed cells. The whole chromosome painting probes (10 ll) were denatured at 70 °C for 10 min, incubated at 37 °C for 1 h to preanneal, and then applied to the chromatin target. Hybridization was performed overnight at 37 °C in a humidified chamber in the horizontal position. Posthybridization washing was performed in 50% formamide in 2X SSC, pH 7.0, at 43 °C for 15 min, in 2X SSC with 0.1% Tween 20, pH 7.0, at 37 °C for 8 min and then in 4X SSC with 0.2% Igepal, at RT, for 4 min. Rhodamin-antidigoxigenin or FITC-avidin (37 ll) was applied to the slides. Incubation under a plastic cover slip was at 37 °C for 15 min at which time the cover slip was removed and the slides were washed in 4X SSC with 0.2 % Igepal at 37 °C for 4 min each. Counterstaining was performed with DAPI (40 ,6-diamidine-20 -phenylindole) (0.2 lg=ml). The c-MYC and RB1 gene visualization was performed simultaneously with the corresponding chromosomal territory, using classical multilabeling FISH techniques. In these experiments the chromosomal and unique sequence DNA probes were denatured separately and their mixture was applied to the target cells on microscope slides. Bearing in mind that the FISH technique can modify the nuclear structure, we used optimized protocols suggested by Kurz et al. (1996) and Verschure et al. (1999). These authors performed experiments with the same nuclei before and after FISH. Spatial distribution of PML bodies and centromeres was analyzed. The FISH techniques suggested by the authors only resulted in small changes in the distributions of PML bodies and centromeres that were detected using antibody staining. After repeated FISH, used in some of our experiments, the modification of the nuclear structure can easily be verified using the same DNA probes in subsequent hybridizations. According to our experience, the positions of genetic elements remain the same. 2.4. Prime in situ labeling (PRINS) Alpha satellite centromeric regions of chromosomes 1, 8, 9, 10, and 13 were detected using PRINS techniques. Slides with fixed interphase cells were placed on

E. B
artov
a et al. / Journal of Structural Biology 139 (2002) 76–89

a heating block of the PCR thermal cycler (PTC 100, MJ Research, USA) and incubated with 30 ll PRINS reaction mixture: 3 ll 10X PRINS reaction buffer (Roche, Germany), 3 ll Rhodamine PRINS labeling mix (Roche, Germany) containing 500 lM dATP, 500 lM dCTP, 500 lM dGTP, 50 lM dTTP, and l0 lM rhodamine-dUTP in 50% glycerol. One hundred to 150 pmol of primers and 2.0 U Taq DNA polymerase Unis (TopBio, Czech Republic) were used. The final volume of 30 ll was obtained by adding deionized water. The following primers were used: centromere 1, 50 -ATT CCA TTA GAT GAT GAC CCC TTT CAT-30 ; centromere 8, 50 -CTA TCA AT A GAA ATG TTC AGC ACA GTT-30 ; centromere 9, 50 -TAT CTG CAA GCG GAC GTT TTA-30 ; centromere 10, 50 -ACTGGAACGCACA GATGACAAAGC-30 ; and centromere 13, 50 -TGATGT GTGTACCCAGCT-30 . After application of the PRINS mixture the specimen was denatured at 94 °C for 3 min, followed by an incubation at the appropriate primer annealing temperature for 40 min. After gentle removal of the coverslip, a stop buffer (50 mM NaCl and 50 mM EDTA, pH 8.0) was added and the incubation continued at 60 °C for 4 min. Washing was performed at RT for 5 min three times in PBS containing 0.2% Tween 20 and then at RT for 5 min three times in PBS. DAPI staining (0.2 lg=ml) was used to visualize the cell nuclei. Overnight incubation of slides at 4 °C improved the fluorescence signal to noise ratio. 2.5. Simultaneous visualization of centromeric regions and chromosome territories A combination of PRINS and FISH techniques was used for simultaneous visualization of centromeres and whole chromosome territories. After the PRINS reaction, the denatured and annealed chromosome probe was applied to the denatured target DNA of the cells with nuclei stained by PRINS. FISH was performed overnight. Standard washing and immunochemical detection was done according to the protocol of classical FISH (see Section 2.3). The PRINS signals were analyzed simultaneously with the FISH signals representing compact chromosomal territories. 2.6. Reverse transcription polymerase chain reaction (RT-PCR) RNA was extracted from cells using an RNA-blue isolation kit (Top-Bio). The RNA content was determined spectrophotometrically and visualized in a 1% formaldehyde gel, stained with ethidium bromide. A 5X Mops (3-[N-morpholino]propanesulfonic acid) (Sigma) was used as the running buffer. The RNA (0.5 lg) was reverse-transcribed using 25 U/ll moloney-murine leukemia virus reverse transcriptase (M-MLV); the reaction was performed in a solution containing: 1.9 ll DEPC

79

(diethyl pyrocarbonate) water, 2 ll reaction buffer for reverse transcriptase [250 mM Tris–Cl, pH 8.3, 375 mM KCl, 15 mM MgCl2 , 5 mM dithiotreitol (DTT)], 0.5 ll dNTP (deoxynucleoside triphosphates, 10 mM of each), 0.3 ll DTT (dithiotreitol, 100 mM), and 38.7 pmol of 30 (antisense) primer in 0.3 ll. The reaction mixture was incubated in a thermal cycler (PTC 100, MJ Research) for 1 h at 37 °C. The reaction was stopped at 99 °C for 5 min and the mixture then cooled on ice. The ABL fragment of the length of 195 bp, the c-MYC fragment of the length of 345 bp, and the RB1 gene fragment of the length of 135 bp were amplified. The reaction mixture with cDNA was diluted with 40 ll PCR mix containing 4 ll PCR buffer (10 mM Tris–Cl, pH 8.3, 50 mM KCl, 1.2 mM MgCl2 ), 1 ll dNTP (10 mM of each), and 25–38 pmol of primers per reaction. Taq polymerase (1.5 U) (Top-Bio) was used for PCR amplification. The primers for the ABL gene detection were selected according to Hughes et al. (1991): forward primer, 50 TTCAGCGGCCAGTAGTCTGACTT-30 ; and reverse primer, 50 -TGTGATTATAGCCTAAGACCCGGAGC TTTT-30 . Forward primer for the c-MYC gene, 50 -CCA CCAGCAGCGACTCTG-30 , and reverse primer for the c-MYC gene, 50 -CCAAGACGTTGTGTGTTC-30 , were selected and purchased from Stratagene (USA) and sequences compared with those of Bernard et al. (1983). RT-PCR analysis of the RB1 gene was performed according to Antosz et al. (1997), who used the following forward primer, 50 -GGAGTTCGCTTGTATTAC-30 , and reverse primer, 50 -AACCTCAAGA GCGCACGC30 . The PCR for the ABL, c-MYC, and RB1 genes, respectively, was performed in a thermal cycler in the following steps: single predenaturation step at 94 °C for 5 min (ABL, c-MYC) and 3 min (RB1); 70 °C for Taq polymerase addition was used as a hot start; 35 cycles with denaturation at 94 °C for 45 s for (ABL, c-MYC) and for 30 s (RB1); annealing at 63 °C for 45 s (ABL), at 61 °C for 45 s (c-MYC), and at 56.6 °C for 30 s (RB1). Elongation was at 72 °C for 1 min (ABL), for 1.5 min (c-MYC), and for 45 s (RB1). Final elongation at 72 °C for 10 min and cooling at 4 °C was used. One sample without M-MLV reverse transcriptase was used as a negative control in reverse transcription and one sample without cDNA was used as a negative control in the PCR. Eight microliters of the PCR products was applied on 2% agarose gel. 1X TBE (45 mM Tris–Cl, 45 mM H3 BO3 , 1 mM EDTA, pH 8.0) was used as a running buffer. Electrophoresis was performed at 10 V/cm for 3 h. RT-PCR analyses were repeated three times. 2.7. Image acquisition and analysis A high-resolution cytometer based on a Leica DMRXA microscope (Leica, Germany), with a Quantix CCD camera (Photometrix, Tuscon, AZ) and a 2X Pentium II 266 MHz computer (256 MB ram and

80

E. B
artov
a et al. / Journal of Structural Biology 139 (2002) 76–89

2  30 GB HDD) was preferentially used for 2D image acquisition. In addition, 3D image acquisition and analysis were performed using a Zeiss Axiovert 100 microscope (Carl Zeiss Jena, Germany) equipped with a confocal unit Carv (Atto Instruments, USA). These images of 40 optical sections in axial steps of 0.3 lm were acquired with a Micromax fully programmable digital CCD camera (Princeton Instruments). Both cytometers were computer-controlled. Image acquisition and analysis were performed using the FISH 2.0 software (Kozubek et al., 1997, 1999a,b). Usually around 500–1000 images were stored overnight in the computer memory and subsequently analyzed. The algorithms used for automatic findings of nuclei and fluorescent signals inside the cell nuclei were described by Kozubek et al., 1999a,b and the algorithms used for lobular nuclei of granulocytes were described by B
artov
a et al. (2001). Special topographic parameters of genes (centromeres and chromosomes) were computed, namely the membrane to gene (centromere, chromosome) distances (MG; MN; MHÞ. These distances were measured as the Euclidean distance between the given gene (centromere, chromosome) and the nearest point on the nuclear boundary (defined by FISH 2.0 software, developed in our laboratory). Such analysis is appropriate for pyknotic nuclei with an irregular shape such as in neutrophils. The same topographic parameter was used for both granulocytes and undifferentiated HL-60 cells. Image segmentation was carried out as follows. The 3D positions of chromosomes, centromeres, and genes were calculated relative to the bary center (fluorescence intensity weight center) of the nucleus obtained from the DAPI counterstain. Chromosome positions were represented either by the points of their maximum intensity or by their intensity bary centers. The information relating to the signal coordinates was written into text files and further analyzed in Sigma Plot (Jandel Scientific, CA). The threshold for segmentation is determined from the histogram of the image where two peaks are found. The first peak corresponds to the background level of

the color intensity; the second peak is formed by the objects (nuclei, chromosomes). The value of intensity for the threshold is taken from the local minimum between the two peaks. This is the point of the maximal gradient of the intensity of a given color. Using 3D analyses, nuclear bary (weight) center to gene distances were measured (CG  SE) and normalized to the nuclear area. The distances between the ABL (c-MYC, RB1) gene signals and the nearest cluster of centromeric regions were measured in nuclei of 3D fixed cells. The method published by Sch€ ubeler et al. (2000) describing the analysis of gene distances from the nearest centromere was used. The average distances between the centromere (gene) and the fluorescence intensity bary (weight) center of a chromosome (RGav) were determined. Minimal distances between genetic loci (gene to nearest cluster of centromeres) (RGmin) were computed. In addition, the nuclear area (A), nuclear radius (R), and chromosome territory area were also determined. The results were statistically analyzed using Student’s t test.

3. Results 3.1. Expression of the ABL, c-MYC, and RB1 genes during granulocytic differentiation Semiquantitative RT-PCR analysis was employed to compare the levels of ABL (9q34.1), c-MYC (8q24.12– q24.13), and RB1 (13q14.2) transcripts in human undifferentiated HL-60 cells, after all-trans-retinoic-induced differentiation of HL-60 cells, and in terminally differentiated human peripheral blood granulocytes. In these experiments, a stable level of ABL gene expression was found in undifferentiated HL-60 cells as well as differentiated granulocytes (Fig. 1A). The c-MYC gene down-regulation (Fig. 1B) and RB1 gene up-regulation (Fig. 1C) during RA-induced granulopoiesis as well as in mature granulocytes were confirmed.

Fig. 1. Changes in gene expression were detected using the RT-PCR. (A) Stable ABL gene activity was found in undifferentiated HL-60 cells (HL), during in vitro (RA) induced granulopoiesis (4 days), as well as in terminally differentiated human peripheral blood granulocytes (GR). (B) The c-MYC gene was down-regulated in granulocytes (RA and GR) as compared with HL-60 cells (HL). On the other hand, the RB1 gene was upregulated during the studied cell maturation (C). A DNA marker (M) was used for the verification of the RT-PCR product length. One sample without M-MLV reverse transcriptase was used as a negative control in reverse transcription and one sample without cDNA was used as an negative control in PCR (not shown).

E. B
artov
a et al. / Journal of Structural Biology 139 (2002) 76–89

3.2. Changes of the location of selected genetic elements in the cell during granulocytic differentiation do not correlate with changes of gene expression Three-dimensional distances between the center of nucleus and the genetic element as well as 2D distances between the nearest membrane point and the genetic element were measured for the ABL, c-MYC, and RB1 genes and corresponding centromeres of chromosomes 8, 9, and 13 in the nuclei of human promyeloid (undifferentiated) HL-60 cells as well as in the nuclei of human granulocytic (terminally differentiated) cells. The detected changes of the nuclear topography in cells fixed by the standard methanol/acetic acid procedure did not differ significantly from those obtained from samples fixed by formaldehyde.

81

The center of nucleus to element distance distributions determined for 3D fixed cells are shown for the genes studied in Fig. 2, together with dashed lines that correspond to theoretical random distributions. From the comparison, it is evident that the experimental distributions are different from the theoretical random ones. Particularly for the ABL gene, the distribution is substantially shifted to the center of the nucleus. Average 2D distances of genetic elements from the nearest point on the nuclear membrane analyzed in fixed cells at different stages of granulocytic differentiation (in vitro and in vivo granulopoiesis) are shown in Table 1. Due to similar topography of granulocytes obtained in vitro and in vivo, the majority of experiments were based on the comparison of nuclear parameters between progenitor HL-60 cells and peripheral blood granulocytes (the

Fig. 2. Three-dimensional distributions of the nuclear fluorescence bary center to gene distances (CG) determined for the ABL (9q34.1), c-MYC (8q24.12–q24.13), and RB1 (13q14.2) loci in undifferentiated HL-60 cells, as well as in terminally differentiated granulocytes. The distances were normalized to the nuclear area (A) (see Section 2). Mean values of the distributions (CG/A) and their standard errors are given in each panel. The center of nucleus to element distance distributions determined for 3D fixed cells are shown together with theoretical dashed lines correspond to random distributions.

Table 1 Peripheral relocation of genetic loci during human granulocytic cell differentiationa Control undifferentiated HL-60 cells ABL Centromere 9 c-MYC Centromere 8 RB1 Centromere 13 a *

MG ¼ 54:1  1:7% MC ¼ 37:6  0:6% MG ¼ 34:3  1:2% MC ¼ 33:9  1:2% MG ¼ 36:9  1:4% MC ¼ 28:1  1:5%

RA differentiated HL-60 cells 

MG ¼ 47:1  1:6% MC ¼ 36:3  1:1% MG ¼ 24:6  1:0% MC ¼ 19:8  1:0% MG ¼ 35:9  1:6% MC ¼ 27:6  0:6%

Peripheral blood granulocytes MG ¼ 38:1  1:1% MC ¼ 30:5  0:7% MG ¼ 26:2  0:9% MC ¼ 24:8  1:4% MG ¼ 37:1  1:0% MC ¼ 28:8  1:3%

Average 2D distances of genetic loci positioning from the nearest point on the nuclear membrane related to the nuclear area. Student’s t test was applied for statistical analysis with statistical significance at P < 0:05.

82

E. B
artov
a et al. / Journal of Structural Biology 139 (2002) 76–89

in vivo system). Analyzing both 2D and 3D results, the selected genetic elements of chromosomes 8 and 9 were found to be shifted to the nuclear membrane in granulocytes, although to a different extent. On the other hand, the RB1 gene and the centromere of chromosome 13 were not repositioned (Table 1 and Fig. 2). The range of 3D positions of the c-MYC and RB1 genes was between 70 and 80% of the nuclear radius for both HL-60 cells and granulocytes. A similar value was also obtained for the ABL gene in granulocytes in contrast to a much more central location of this gene (55.3%) in undifferentiated 3D fixed HL-60 cells. The centromeres of chromosomes 8, 9, and 13 were located closer to the nuclear membrane than the corresponding c-MYC, ABL, and RB1 genes in both promyelocytic cells and granulocytes (Table 1). Changes in the distance distributions of the center of nucleus (membrane) to gene for the ABL, c-MYC, and RB1 loci did not correlate with the changes of the given gene expression observed during granulocytic differentiation. 3.3. Topography of chromosome territories does not influence gene expression The distributions of measured distances between the genetic element and the fluorescence intensity bary center of the corresponding chromosome territory were calculated for the c-MYC, and the RB1 genes (along with corresponding centromeres) in the nuclei of HL-60 cells as well as in the nuclei of granulocytes. The downregulated c-MYC gene and the up-regulated RB1 gene as well as centromeres 8 and 13 were located closer to the center of the corresponding chromosome territory in granulocytes as compared with HL-60 cells (Tables 2 and 3). The centromeres of chromosomes 8 and 13 were always positioned more centrally within the chromo-

some territory than the c-MYC and the RB1 genes. The genetic locus to territory center distances (in lm) were reduced in granulocytes for a number of genetic elements (see Table 3), which seems to reflect the condensation of chromosome territories. We found that centromeres 1, 8, 10, and 13 are repositioned closer to the fluorescence intensity bary center of the corresponding chromosomes in differentiated cells as compared with HL-60 cells. Visual observation revealed that both active and inactive c-MYC and RB1 genes were located at the periphery of chromosome territories (Fig. 3). Examples for RB1 locations in the G1 (A) and G2 (B) stages of undifferentiated HL-60 cells and in terminally differentiated human peripheral blood granulocytes (C and D) are shown. In all cases the RB1 gene is located on the periphery of the chromosome territories. In some granulocytic nuclei the active RB1 gene appeared to be located on loops extended outward of the chromosome territory. A similar phenomenon was observed in the population of promyeloid HL-60 cells with undetectable RB1 expression. The frequency of these extensions was similar for the c-MYC and RB1 genes in all cell types. These observations imply that active and inactive c-MYC and RB1 genes are, in the majority of cell nuclei, positioned on the periphery of chromosome territory regardless of their activity and independently of the cell cycle (in G0 =G1 as well as in G2 stages) or stage of differentiation. 3.4. Heterochromatin-mediated gene silencing in undifferentiated HL-60 cells and terminally differentiated granulocytes Using FISH techniques and high-resolution cytometry, the influence of centromeric heterochromatin on the gene expression that changed during granulopoiesis of

Table 2 Nuclear positioning of the c-MYC and RB1 genes relative to the bary center of the corresponding chromosome territories

Undifferentiated promyeloid HL-60 cells Peripheral blood granulocytes

c-MYC to chromosome territory center distances

RB1 to chromosome territory center distances

RGav ¼ 2:73  0:08 lm RGav ¼ 1:12  0:03 lm

RGav ¼ 1:48  0:04 lm RGav ¼ 1:17  0:04 lm

*

Student’s t test was applied for statistical analysis with statistical significance at P 6 0:05. The average distances of gene to fluorescence bary center of chromosome territory (RGav) were measured in lm and normalized to the relative size of territory.

Table 3 Average nuclear location of selected centromeres in corresponding chromosomal territory of undifferentiated HL60 cells and peripheral blood granulocytes (GR)a Centromere/chromosome (R/G)

1R/1G

8R/8G

9R/9G

10R/10G

13R/13G

RGav (lm) HL-60 RGav (lm) GR

1:65  0:09 lm 1:21  0:1 lm

1:44  0:06 lm 0:87  0:07 lm

0:98  0:07 lm 0:91  0:03 lm

1:59  0:1 lm 0:92  0:06 lm

1:28  0:09 lm 0:88  0:05 lm

a

The average distances of centromere to fluorescence intensity bary center of chromosome territory (RGav) were detected and normalized to the relative size of territory. * Student’s t test was applied for statistical analysis with statistical significance at P 6 0:05.

E. B
artov
a et al. / Journal of Structural Biology 139 (2002) 76–89

83

Fig. 3. The location of the RB1 gene (13q14.2) relative to the corresponding chromosome 13 territory visualized in the nuclei of undifferentiated HL-60 cells in the G1 (A) and G2 (B) stages of the cell cycle is shown as examples. The G2 stage is characterized by double dots of the fluorescence signals (B) though with similar center of territory to gene distance distributions. For the purposes of illustration, the RB1 gene is also shown in the chromosome 13 territory of human peripheral blood granulocytes (G0 stage of the cell cycle) (C and D). Bar 2 lm.

Fig. 5. Three-dimensional illustration of the nuclear topography of the RB1 gene and its relationship to the clusters of centromeres. In undifferentiated HL-60 cells, the inactive RB1 genes were located very close to the nearest centromeric regions (A). In peripheral blood granulocytes, much longer distances between the RB1 genes and the centromeric heterochromatin were observed in most cases (B). The lateral maximum intensity projection image obtained from 40 optical sections is shown (a; a0 ), as well as the y–z (b; b0 ) and x–z (c; c0 ) projections. The crosses indicate the position of analyzed RB1 gene. Bar 2 lm.

84

E. B
artov
a et al. / Journal of Structural Biology 139 (2002) 76–89

HL-60 cells and in terminally differentiated human peripheral blood granulocytes was studied. The centromeres, forming the chromocenters, were visualized using pan-centromeric DNA probe and the distance of the investigated gene to the nearest centromere signal was measured for each cell nucleus. The average minimal ABL to chromocenter distance was constant for both cell types (Fig. 4A), which correlates with the fact that its expression measured by the RT-PCR technique is also constant (Fig. 1A). The c-MYC gene, when inactive

(Fig. 1B) in peripheral blood granulocytes, was found in proximity to the nearest chromocenter (Fig. 4B). The active RB1 (Fig. 1C) gene was positioned outside all chromocenters in granulocytes, while the inactive RB1 gene was located in proximity to the nearest chromocenter in HL-60 cells (Fig. 4C). For the purposes of illustration, the chromocenters are shown together with the RB1 gene in Fig. 5. The inactive gene is located very close to the centromeric heterochromatin of undifferentiated HL-60 cells (Fig. 5A). In the population of

Fig. 4. Distributions of the gene to nearest cluster of centromere distances. Simultaneous visualization of genes and all centromeric regions forming the chromocenters was used to determine distributions for the ABL (A), c-MYC (B), and RB1 (C) genes in undifferentiated cells (HL-60), as well as in terminally differentiated granulocytes (GR). Mean values of the distributions (RGmin) normalized to the nuclear area and their standard errors are given in each panel.

E. B
artov
a et al. / Journal of Structural Biology 139 (2002) 76–89

peripheral blood granulocytes, the active RB1 gene is located outside the centromeric regions forming the chromocenters (Fig. 5B). Furthermore, the mutual distance between the RB1 gene and its own centromeric region (centromere of chromosome 13) was measured. The average RB1 to centromere 13 distance relative to the nuclear radius was 21:9  0:8% in undifferentiated HL-60 cells and 20:0  1:0% in peripheral blood granulocytes. Similar distributions of the RB1 to centromere 13 distances indicate that the centromeric region corresponding to the silenced gene is probably not involved in heterochromatin-mediated gene down-regulation.

85

3.5. One copy of the RB1 gene might be silenced by its positioning close to chromosome X heterochromatin in differentiated retinoblastoma tumor cells The reason for the cytogenetic analyzes of retinoblastoma tumor cells was our study of the RB1 gene silencing. These tumor cells are a completely different cell type (in comparison with granulocytes) but changes in RB1 (13q14) gene expression play an important role in the progression of this tumor. Visualization of the chromosome territory in male retinoblastoma tumor cells revealed a close proximity of chromosome X and one of the chromosome 13 territories (Figs. 6A–C). The

Fig. 6. Topography of the RB1 gene, chromosome X and 13 territories in the nuclei of male differentiated retinoblastoma tumor cells. Chromosome X (green) and 13 (red) territories were visualized in the tumor cells carrying the (X;13) translocation (A–C). Chromosome territory boundaries were determined using FISH 2.0 software; black points indicate the chromosome territory fluorescence intensity bary centers and the yellow region is the area of chromosome X and 13 possible translocation (B). In the second fluorescence in situ hybridization, the two copies of the RB1 gene were visualized (D). Their positions were determined in the nuclear weight center coordinates. After the analysis and superposition of the images obtained from both hybridizations, one copy of the RB1 gene was frequently found in the boundary region between the chromosome X and 13 territories (E). In the overlaid figure (E) the red diamonds (green square) show the positioning of the fluorescence intensity bary center of chromosome 13 (X) territories and the pink triangles indicate the location of the RB1 genes. Bar represents 2 lm. Using RT-PCR analysis, reduced expression of the RB1 gene in retinoblastoma tumor cells was found, in comparison with the human peripheral blood granulocytes (GR), (F). M represents the DNA marker. One sample without M-MLV reverse transcriptase was used as a negative control in reverse transcription and one sample without cDNA was used as an negative control in PCR (not shown).

86

E. B
artov
a et al. / Journal of Structural Biology 139 (2002) 76–89

minimum HSA X to HSA 13 distances determined in 2D were lower than 40% of the nuclear radius in approximately 60% of the tumor cell nuclei. For comparison, only 30% of peripheral lymphocytes of the same patient showed such small distances. Such a high proportion of nuclei with close proximity of chromosome X and 13 territories strongly suggests the existence of a t(X;13) translocation, confirmed by metaphase spreads from lymphocytes of this patient. Using repeated FISH (Kozubek et al., 1999a,b), the territories of both chromosomes, as well as two copies of the RB1 gene, were visualized for the same cell nuclei (Figs. 6C,D). The presence of two signals of the RB1 gene in tumor cells indicates that the primary sequences of both copies of the gene remained intact and that the translocation did not destroy the RB1 locus because in this case three signals would be observed. Of course, we cannot exclude point mutations or some deletions; however, it would be the second aberration in the chromosome 13 territory (in addition to the translocation), which is less probable. The positions of the RB1 genes were determined using FISH 2.0 software (Kozubek et al., 1999a,b), replaced by symbols and overlaid with chromosome territories (Fig. 6E). One copy of the RB1 gene was always located in the fusion area between chromosomes 13 and X (Fig. 6E) and may have been silenced by the proximity or spreading of methylated (Jones et al., 1997) and heterochromatic (Tajbakhsh et al., 2000) chromosome X into the neighborhood of the translocated region of chromosome 13. To support this hypothesis, the RB1 gene expression was determined in retinoblastoma tumor cells and compared with other cell types with normal RB1 gene expression (granulocytes and lymphocytes). A partial RB1 gene inactivation was observed in retinoblastoma tumor cells (Fig. 6F), which could indicate functional monosomy of the RB1 gene. For completeness, the position of the RB1 gene in the corresponding chromosome territory was investigated for normal and translocated chromosomes. The average distances of the RB1 gene to the fluorescence intensity bary center of the nontranslocated (translocated) chromosome 13 territory were 1:1  0:06 lm (1:2  0:08 lm). The average 3D distance of the center of nucleus to RB1 gene for the nontranslocated chromosome 13 territories was 72:8  1:7% and for translocated territories was 71:3  1:9%. These nuclear parameters were very similar for both active and inactive RB1 genes in retinoblastoma cells and corresponded to the parameters obtained for 3D fixed blood cells (Fig. 2).

4. Discussion In experiments presented in this paper the spatial and functional dynamics of selected genetic elements such as the ABL, c-MYC, and RB1 genes were studied during

human granulocytic cell differentiation. Changes in the activity of studied genes were described in the given maturation process (B
artov
a et al., 2000a; Lachman and Skoultchi, 1984; Li et al., 1998; Matikainen and Hurme, 1994; Perego et al., 1998; Ramsay et al., 1986; Savoysky et al., 1996). The role of chromatin structure in regulation of studied gene expression was tested using the following three hypotheses: (i) active (inactive) gene location in the whole nucleus, (ii) gene location within the corresponding chromosome territory, and (iii) association of genes with centromeric heterochromatin. Testing the first hypothesis, we have found that the c-MYC and ABL genes as well as related chromosomes 8 and 9 were shifted to the nuclear membrane in human granulocytes as compared with human undifferentiated HL-60 cells, though to a different extent. On the other hand, the RB1 gene and the centromere of acrocentric chromosome 13 were not repositioned. These results confirm that structural changes in cell nuclei occur during cellular differentiation (Manuelidis, 1990; Chaly and Munro, 1996; B
artov
a et al., 2001); however, they do not correlate with changes in gene expression. Experiments testing the gene location within a given chromosome territory showed that a down-regulated c-MYC gene and an up-regulated RB1 gene were located closer to the corresponding fluorescence intensity bary center of chromosome territory in human granulocytes as compared to undifferentiated HL-60 cells. A similar shift to the center of the territory was observed for the centromeric regions of chromosomes 1, 8, 10, and 13 studied during cell maturation. Presumably the repositioning of genetic elements to the center of the corresponding chromosomal territory reflects the effect of chromosome condensation that is extensively induced during granulopoiesis. Our visual observation showed that both the active and the inactive c-MYC and RB1 genes were located at the periphery of chromosomal territory (see Fig. 3). On the other hand, we found that the centromeric regions are located closer to the chromosomal bary center than the studied active or inactive gene loci. These findings support the observation of Kurz et al. (1996) whose analyses revealed that the studied genes are preferentially located at the periphery of chromosome territories, independently of their activity. A peripheral location within the chromosome 17 territory for the TP53 gene was also revealed in our experiments analyzing human T-lymphocytes. Visually, in many lymphocytes, the TP53 gene was positioned on the loops extending outward in the given territory. Such loops were observed more frequently for TP53 in human lymphocytes as compared with the active c-MYC or RB1 genes which could indicate a specificity in gene location. Similar positioning of active genes on the chromatin loops was described by Volpi et al. (2000) for the loci of major histocompatibility complex (MHC).

E. B
artov
a et al. / Journal of Structural Biology 139 (2002) 76–89

The third hypothesis that we tested relates to the influence of centromeric heterochromatin on gene silencing. In the corresponding experiments, association of the ABL, c-MYC, and RB1 genes with the centromeric heterochromatin was analyzed. We determined the distributions of distances between the given genes and the nearest centromeric region. We observed a substantial reduction of given distances for both the inactive c-MYC gene in granulocytes as compared with HL-60 cells and the inactive RB1 gene in HL-60 cells as compared with granulocytes. The ABL gene had a stable position to the nearest chromocenter which correlated with its stable expression. The dynamics in localization of genes is often linked with cis-acting elements. It has been found that tissuespecific enhancers and locus control regions (LCRs) prevent active genes from being included in a region of transcriptional inactive condensed chromatin (heterochromatin) that forms during cell maturation (Bulger and Groudine, 1999; Francastel et al., 2000). Localization away from centromeric heterochromatin is required to achieve general hyperacetylation and an open chromatin structure of the locus, whereas mechanism involving LCR/promoter histone H3 hyperacetylation is required for higher level transcription of b-globin genes (Sch€ ubeler et al., 2000). Contributing to these studies is our observation of centromeric heterochromatin-mediated gene silencing during granulocytic differentiation. In addition, we found that the nearest chromocenter regulating the RB1 (13q14) gene activity did not involve centromeric heterochromatin of chromosome 13 where the RB1 gene is localized. Several mechanisms were considered as an explanation of the structural modification of gene activity. For example, Ikaros, a DNA-binding protein localized in discrete foci in the nuclei of murine B-lymphocytes, is in close association with the centromeric heterochromatin. Strong correlation was found between these foci and the location of transcriptionally inactive genes (Brown et al., 1997, 1999). In addition, during differentiation of human lymphocytes it was found that promoter-specific binding factor Ikaros mediates the association of celltype-specific genes with centromeric heterochromatin. Ikaros regulates the gene movement to centromeric heterochromatin, whereas activated genes are released (Cockell and Gasser, 1999). Thus, gene positioning on the periphery of chromosome territory could facilitate not only access to the transcriptional machinery (enabling gene activation), but also the access to the factors inhibiting gene expression (e.g., clusters of centromeric heterochromatin). We also investigated the role of heterochromatin in the regulation of gene expression in differentiated retinoblastoma tumor cells. Despite the fact that two copies of the RB1 gene were documented in the tumor cells, reduced expression of the gene was detected. This

87

observation indicates that some epigenetic mechanism might regulate RB1 gene activity. We cannot exclude a second aberration directly in the RB1 gene. However, the appearance of two coincidental mutations in the same chromosome is less probable. A simple explanation of the reduced activity of the RB1 gene is offered by the following observation. In the majority of retinoblastoma tumor cells, one copy of the RB1 gene was found in the area of fusion of chromosomes X and 13, suggesting that heterochromatin and/or methylation of chromosome X might spread to chromosome 13 and produce functional monosomy of the RB1 gene located on proximal 13q. A similar phenomenon was observed in a male patient suffering from retinoblastoma (Jones et al., 1997). According to some authors, chromosome X is one of the gene-poor and heterochromatic chromosomes (Deloukas et al., 1998; Tajbakhsh et al., 2000). Several links of evidence suggest an important role of methylated heterochromatin in transcriptional inactivation of genes. In approximately 15% of retinoblastoma tumors, the RB1 CpG islands are methylated (Sakai et al., 1991). Methylation of the RB1 promoter reduces the gene activity characteristic for a retinoblastoma tumor (Greger et al., 1994). Constitutional hypermethylation of the RB1 gene may, therefore, be expected to occur in translocations involving chromosome 13 and X or in imprinted autosomal chromosome territories (Jones et al., 1997). Since our observations are only circumstantial, further studies are needed to explore the mechanisms of the RB1 gene silencing. It seems that methylation may play an important role not only in RB1 gene silencing in the retinoblastoma tumor, but also be an important factor in centromere-mediated gene silencing. Such considerations are supported by the observation that the centromeric regions of many chromosomes are heavily methylated (Barbin et al., 1994). A global view of gene regulation leads to the conclusion that gene activation is a multilevel process influenced by many factors. The first condition for active gene expression is transcriptional competence. Most of genes are predetermined to transcriptional competence by chromosome and nuclear compartmentalization (Cremer et al., 1993, 2000; Cremer and Cremer, 2001; Ferreira et al., 1997; Lichter et al., 1988; Pinkel et al., 1988; Sadoni et al., 1999; Spector, 1993; Verschure et al., 1999). However, this compartmentalization itself does not explain hereditary changes of gene expression that appear during differentiation. Changes of gene expression accompanying cell maturation are obviously conditioned by changes of transcriptional competence due to some epigenetic regulatory mechanism. The regulation of gene expression is ascribed to different epigenetic processes such as heterochromatinization (see Summary, Hendrich and Willard, 1995), modification of

88

E. B
artov
a et al. / Journal of Structural Biology 139 (2002) 76–89

chromatin structure (see Minireview, Chevret et al., 2000), DNA methylation (Jones, 1999; Singal and Ginder, 1999), or chromatin-mediated silencing (Francastel et al., 1999, 2000). The results presented here suggest that such an epigenetic mechanism may be related to association (disassociation) of an inactive (active) gene to centromeric heterochromatin forming chromocenters. We also showed that the nearest chromocenter of the silenced gene need not involve centromeric heterochromatin of the related chromosome where the given gene is localized. Thus, clustering of heterochromatic centromeres into the chromocenters and the process of heterochromatinization itself could be an important mechanism influencing the regulation of tissue-specific gene expression.

Acknowledgments This work was supported in part by the Academy of Sciences of the Czech Republic (Grants S50004010 and B5004102), the Grant Agency of Czech Republic (301/ 01/0186), the Volkswagen Stiftung (I/75946), Hanover, Germany, and by Grant MSM 143300002 (Ministry of Education of the Czech Republic). We thank RNDr. H. Konecn
a, Ph.D., from the Laboratory of Plant Molecular Physiology, Faculty of Sciences, Masaryk University Brno (Czech Republic) for oligonucleotide synthesis, and Dr. Denise Kozikowski for critical reading of this manuscript. The work of Michal Hausmann was partly supported by a NCI/CCR Intramural Research Award.

References Alcobia, I., Dilao, R., Parreira, L., 2000. Spatial associations of centromeres in the nuclei of haematopoietic cells: evidence for celltype-specific organisational patterns. Blood 95, 1608–1615. Antosz, H., Kitlinska, J., Kwiatkowska-Drabik, B., Koczkodaj, D., Wach, M., Antosz, M., Rudzki, S., Wojcierowski, J., 1997. RB1 gene expression in B-cell lymphocytic leukaemia cases with deletion in the 13q14 region. Cytobios 92, 111–121. Barbin, A., Montpellier, C., Kokalj-Vokac, N., Gibaud, A., Niveleau, A., Malfoy, B., Dutrillaux, B., Bourgeois, C.A., 1994. New sites of methylcytosine-rich DNA detected on metaphase chromosomes. Hum. Genet. 94, 684–692. B
artov
a, E., Kozubek, S., Jirsov
a, P., Kozubek, M., Luk
asov
a, E., Skaln
ıkov
a, M., Cafourkov
a, A., Koutn
a, I., Pasekov
a, R., 2001. Higher-order chromatin structure of human granulocytes. Chromosoma 110, 360–370. B
artov
a, E., Kozubek, S., Kozubek, M., Jirsov
a, P., Luk
asov
a, E., Skaln
ıkov
a, M., Cafourkov
a, A., Koutn
a, I., 2000a. Nuclear topography of the c-MYC gene. Gene 244, 1–11. B
artov
a, E., Kozubek, S., Kozubek, M., Jirsov
a, P., Luk
asov
a, E., Skaln
ıkov
a, M., Buchn
ıckov
a, K., 2000b. The influence of the cell cycle, differentiation and irradiation on the nuclear location of the ABL, BCR, and c-MYC genes in human leukemic cells. Leukocyte Res. 24, 233–241.

Bernard, O., Cory, S., Gerondakis, S., Webb, E., Adams, J.M., 1983. Sequence of the murine and human cellular myc oncogenes and two modes of MYC transcription regulating from chromosome translocation in B lymphoid tumors. EMBO J. 2, 2375–2383. Brown, K.E., Baxter, J., Graf, D., Merkenschlager, M., Fisher, A.G., 1999. Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division. Mol. Cell 3, 207–217. Brown, K.E., Guest, S.S., Smale, S.T., Hahm, K., Merkenschlager, M., Fisher, A.G., 1997. Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin. Cell 91, 845–854. Bulger, M., Groudine, M., 1999. Looping versus linking: toward a model for long-distance gene activation. Genes Dev. 13, 2465– 2477. Chaly, N., Munro, S.B., 1996. Centromeres reposition to the nuclear periphery during L6E9 myogenesis in vitro. Exp. Cell Res. 223, 274–278. Chevret, E., Volpi, E.V., Sheer, D., 2000. Mini review: form and function in the human interphase chromosome. Cytogenet. Cell Genet. 90, 13–21. Cockell, M., Gasser, S.M., 1999. Nuclear compartments and gene regulation. Curr. Opin. Genet. Dev. 9, 199–205. Cook, P.R., 1994. RNA polymerase: structural determinant of the chromatin loop and chromosome. BioEssays 16, 425–430. Cook, P.R., 1995. A chromometric model for nuclear and chromosome structure. J. Cell Sci. 108, 2927–2935. Cremer, T., Cremer, C., 2001. Chromosome domains, nuclear architecture and gene regulation in mammalian cells. Nature Rev. Genet. 2, 292–301. Cremer, T., Kreth, G., Koester, H., Fink, R.H.A., Heintzmann, R., Cremer, M., Solovei, I., Zink, D., Cremer, C., 2000. Chromosome domains, interchromatin domain compartment, and nuclear matrix: an integrated view of the functional nuclear architecture. Crit. Rev. Eukaryotes Gene Expression 12, 179–212. Cremer, T., Kurz, A., Zirbel, R., Dietzel, S., Rinke, B., Schrock, E., Speicher, M.R., Mathieu, U., Jauch, A., Emmerich, P., Scherthan, H., Ried, T., Cremer, C., Lichter, P., 1993. Role of chromosome territories in the functional compartmentalisation of the cell nucleus. Cold Spring Harbor Symp. Quant. Biol. 58, 777–792. Deloukas, P., Schuler, G.D., Gyapay, G., Beasley, E.M., Soderlund, C., et al., 1998. A physical map of 30,000 human genes. Science 282, 744–746. Dietzel, S., Schiebel, K., Little, G., Edelmann, P., Rappold, G.A., Eils, R., Cremer, C., Cremer, T., 1999. The 3D positioning of ANT2 and ANT3 genes within female X chromosome domains correlates with gene activity. Exp. Cell Res. 252, 363–375. Ferreira, J., Paolella, G., Ramos, C., Lamond, A.I., 1997. Spatial organization of large-scale chromatin domains in the nucleus: a magnified view of single chromosome domains. J. Cell. Biol. 139, 1597–1610. Francastel, C., Sch€ ubeler, D., Martin, D.I.K., Groudine, M., 2000. Nuclear compartmentalisation and gene activity. Nature Rev. Mol. Cell. Biol. 1, 137–143. Francastel, C., Walters, M.C., Groudine, M., Martin, D.I.K., 1999. A functional enhancer suppresses silencing of a transgene and prevents its localisation close to centromeric heterochromatin. Cell 99, 259–269. Greger, V., Debus, N., Lohman, D., Hopping, W., Passarge, E., Horsthemke, B., 1994. Frequency and parental origin of hypermethylated RB1 alleles in retinoblastoma. Hum. Genet. 94, 491–496. Hendrich, B.D., Willard, H.F., 1995. Epigenetic regulation of gene expression: the effect of altered chromatin structure from yeast to mammals. Hum. Mol. Genet. 4, 1765–1777. Hughes, T.P., Morgan, G.J., Martiat, P., Goldman, J.M., 1991. Detection of residual leukemia after bone-marrow transplant for chronic myeloid leukemia: role of polymerase chain reaction in predicting relapses. Blood 77, 874–878.

E. B
artov
a et al. / Journal of Structural Biology 139 (2002) 76–89 Jones, C., Booth, C., Rita, D., Jazmines, L., Brandt, B., Newlan, A., Horsthemke, B., 1997. Bilateral retinoblastoma in male patient with an X;13 translocation: evidence for silencing of the RB1 gene by the spreading of X inactivation. Am. J. Hum. Genet. 60, 1558– 1562. Jones, P.A., 1999. The DNA methylation paradox. Trends Genet. 15, 34–37. Kozubek, M., Kozubek, S., Luk
asov
a, E., Mareckov
a, A., B
artov
a, E., Skaln
ıkov, M., Jergov
a, A., 1999a. High-resolution cytometry of FISH dots in interphase cell nuclei. Cytometry 36, 279–293. Kozubek, S., Luk
asov
a, E., Mareckov
a, A., Skaln
ıkov, M., Kozubek, M., B
artov
a, E., Kroha, V., Krahulcov
a, E., 1999b. The topological organisation of chromosomes 9 and 22 in cell nuclei has a determinative role in the induction of t(9,22) translocations and in the pathogenesis of t(9,22) leukemias. Chromosoma 108, 426–435. Kozubek, S., Luk
asov
a, E., R
yznar, L., Kozubek, M., Liskov
a, A., Govorun, R.D., Krasavin, E.A., Homeck, G., 1997. Distribution of ABL and BCR genes in cell nuclei of normal and irradiated lymphocytes. Blood 89, 4537–4545. Kurz, A., Lampel, S., Nickolenko, J.E., Bradl, J., Benner, A., Zirbel, R.M., Cremer, T., Lichter, P., 1996. Active and inactive genes localise preferentially in the periphery of chromosome domains. J. Cell Biol. 135, 1195–1205. Lachman, H., Skoultchi, A.I., 1984. Expression of c-MYC changes during differentiation of mouse erythroleukaemia cells. Nature 310, 592–594. Leitch, A.R., Brown, J.K.M., Mosg€ oller, W., Schwarzacher, T., Heslop-Harrison, J.S., 1994. The spatial localisation of homologous chromosomes in human fibroblasts at mitosis. Hum. Genet. 93, 275–280. Li, Z.R., Hromchak, R., Mudipalli, A., Bloch, A., 1998. Tumor suppressor proteins as regulators of cell differentiation. Cancer Res. 58, 4282–4287. Lichter, P., Cremer, C., Borden, J., Mauelidis, L., Ward, D.C., 1988. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80, 224–234. Manuelidis, L., 1990. A view of interphase chromosomes. Science 250, 1533–1540. Matikainen, S., Hurme, M., 1994. Comparison of retinoic acid and phorbol-myristate acetate as inducers of monocytic differentiation. Int. J. Cancer 57, 98–103. Perego, R.A., Bianchi, C., Brando, B., Urbano, M., Del Monte, U., 1998. Increment of nonreceptor tyrosine kinase Arg RNA as evaluated by semiquantitative RT-PCR in granulocyte and macrophage-like differentiation of HL-60 cells. Exp. Cell Res. 245, 146– 154. Pinkel, D., Landegent, J., Collins, C., Fuscoe, J., Segraves, R., Lucas, J., Gray, J., 1988. Fluorescence in situ hybridisation with human chromosome-specific libraries: detection of trisomy 21 and trans-

89

location of chromosome 4. Proc. Natl. Acad. Sci. USA 85, 9138– 9142. Ramsay, R.G., Ikeda, K., Rifkind, R.A., Marks, P.A., 1986. Changes in gene expression associated with induced differentiation of erythroleukemia: protooncogenes, globin genes, and cell division. Proc. Natl. Acad. Sci. USA 83, 6849–6853. Sadoni, N., Langer, S., Frauth, C., Bernardi, G., Cremer, T., Turner, B.M., Zink, D., 1999. Nuclear organisation of mammalian genome: polar chromosome domains build up functionally distinct higher order compartments. J. Cell Biol. 146, 1211–1226. Sakai, T., Toguchida, J., Ohtani, N., Yandell, D.W., Rapaport, J.M., Dryja, T.P., 1991. Allele-specific hypermethylation of the retinoblastoma tumor-suppressor gene. Am. J. Hum. Genet. 48, 880–888. Savoysky, E., Yoshida, K., Ohtomo, T., Yamaguchi, Y., Akamatsu, K.I., Yamazaki, T., Yoshida, S., Tsuchiya, M., 1996. Downregulation of telomerase activity is an early event in the differentiation of HL60 cells. Biochem. Biophys. Res. Commun. 226, 329– 334. Sch€ ubeler, D., Francastel, C., Cimbora, D.M., Reik, A., Martin, D.I.K., Groudine, M., 2000. Nuclear localisation and histone acetylation: a pathway for chromatin opening and transcriptional activation of the human b-globin locus. Genes Dev. 14, 940–950. Schul, W., de Jong, L., van Driel, R., 1998. Nuclear neighbours: the spatial and functional organization of genes and nuclear domains. J. Cell. Biochem. 70, 159–171. Singal, R., Ginder, G.D., 1999. DNA methylation. Blood 93, 4059– 4070. Solovei, I., Kienle, D., Little, G., Eils, R., Savelyeva, L., Schwab, M., Jager, W., Cremer, C., Cremer, T., 2000. Topology of double minutes (dmins) and homogenously staining regions (HSRs) in nuclei of human neuroblastoma cell lines. Genes Chromosomes Cancer 29, 297–308. Spector, D.L., 1993. Macromolecular domains within the cell nucleus. Annu. Rev. Cell Biol. 9, 265–315. Tajbakhsh, J., Bornfleth, H., Lampel, S., Cremer, C., Lichter, P., 2000. Spatial distribution of GC-rich and AT-rich DNA sequences within human chromosome territories. Exp. Cell Res. 255, 229–237. Verschure, P.J., van der Kraan, I., Manders, E.M.M., van Driel, R., 1999. Spatial relationship between transcription sites and chromosome domains. J. Cell Biol. 147, 13–24. Volpi, E.V., Chevret, E., Jones, T., Vatcheva, R., Williamson, J., Beck, S., Campbell, R.D., Goldsworthy, M., Powis, S.H., Ragoussis, J., Trowsdale, J., Sheer, D., 2000. Large-scale chromatin organization of the major histocompatibility complex and other regions of human chromosome 6 and its response to interferon in interphase nuclei. J. Cell Sci. 113, 1565–1576. Weimer, R., Kr€ uger, J., Poot, M., Schmid, M., 1992. Characterisation of centromere arrangements and test for random distribution in G0 , G1 , S, G2 , G1 , and early S phase in human lymphocytes. Hum. Genet. 88, 673–682.