Telomere-surrounding regions are transcription-permissive 3D nuclear compartments in human cells

Experimental Cell Research 307 (2005) 52 – 64 www.elsevier.com/locate/yexcr

Telomere-surrounding regions are transcription-permissive 3D nuclear compartments in human cells Ana Sofia Quinaa,b, Leonor Parreiraa,b,T a

Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Av. Professor Egas Moniz, 1649-028 Lisboa, Portugal b Instituto Gulbenkian de Cieˆncia, 2781-901 Oeiras, Portugal Received 9 December 2004, revised version received 12 February 2005 Available online 29 March 2005

Abstract Positioning of genes relative to nuclear heterochromatic compartments is thought to help regulate their transcriptional activity. Given that human subtelomeric regions are rich in highly expressed genes, we asked whether human telomeres are related to transcription-permissive nuclear compartments. To address this question, we investigated in the nuclei of normal human lymphocytes the spatial relations of two constitutively expressed genes (ACTB and RARA) and three nuclear transcripts (ACTB, IL2RA and TCRB) to telomeres and centromeres, as a function of gene activity and transcription levels. We observed that genes and gene transcripts locate close to telomere clusters and away from chromocenters upon activation of transcription. These findings, together with the observation that SC35 domains, which are enriched in premRNA processing factors, are in close proximity to telomeres, indicate that telomere-neighboring regions are permissive to gene expression in human cells. Therefore, the associations of telomeres observed in the interphase nucleus might contribute, as opposed to chromocenters, for the establishment of transcription-permissive 3D nuclear compartments. D 2005 Elsevier Inc. All rights reserved. Keywords: Telomeres; Centromeres; Active genes; Gene transcripts; Transcription; Nuclear organization

Introduction Permissive and non-permissive environments for gene transcription are known to exist in the interphase nucleus of mammalian cells. Well known repressive domains are those formed by pericentromeric heterochromatic regions, which are capable of inducing the stable repression of spatially juxtaposed genes (reviewed in [1]). Transcription repression mediated by pericentromeric chromatin can occur in cis, akin to the position-effect-variegation (PEV) earlier described in Drosophila cells, or in trans, in which case the euchromatic gene becomes spatially juxtaposed with

* Corresponding author. Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Av. Professor Egas Moniz, 1649-028 Lisboa, Portugal. Fax: +351 21 799 95 27. E-mail addresses: [email protected], [email protected] (L. Parreira). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.02.025

centromeric heterochromatin through higher-order levels of chromatin organization (reviewed in [2]). Importantly, repressive effects in trans can be transient in nature as suggested by recent reports indicating that genes may dynamically relocate towards, or from, repressive environments, in apparent relation with their transcriptional activity (reviewed in [3]). Therefore, centromeres, whether in isolation or clustered in chromocenters, as it is the rule in differentiated mammalian cells, appear to behave as the heterochromatic-silencing core of a broader domain, in which the coordinated sequestering, or release, of surrounding euchromatic genes may be facilitated during cell differentiation [4]. Previous observations that the chromocenters present in different types of hematopoietic cells correspond to cell-type specific and ontogenically determined spatial patterns of centromere association [4,5] further suggest that these heterochromatic compartments may act as important epigenetic regulators of tissue-specific gene expression.

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Other heterochromatic repetitive DNA sequences, such as the telomere repeats at the end of chromosomal arms, have been shown to also induce silencing of adjacent genes in the budding yeast Saccharomyces cerevisiae, a phenomenon that has been called Telomere Position Effect (TPE) (reviewed in [6]). Whether similar effects do occur in human or other mammalian cells, in which btelomericQ genes locate tens to thousands of kilobases away from the telomeres, is still unclear. Experiments in which reporter genes were integrated at close proximity to telomeric repeats in different human cell lines showed that the integrated genes are expressed at reduced levels [7–9]. However, in these experiments, exogenous genes were artificially inserted in the vicinity of telomeric repeats, in the absence of an intact adjacent subtelomeric region in between, what might have had a negative effect on transgene transcription (see also [10]). In fact, studies of TPE in human cells, in the presence of natural subtelomeric regions, failed to conclusively demonstrate a direct influence of telomeres on the activity of genes located in cis [11–14]. Furthermore, expression analysis of 34 natural telomeric genes (located 13 to 282 kb from the telomere) in young and senescent human fibroblasts showed that telomere length was not sufficient to determine the expression status of the nearby genes, and analysis of 8 tandem telomeric genes on 16 q (ranging from 170 to 640 kb from the telomere) revealed a discontinuous pattern of expression independent of telomere proximity and length [15]. On the other hand, it has been shown that many subtelomeric regions of the human genome are enriched in expressed genes, corresponding, at the cytogenetic level, to G-light bands, which are early-replicating, gene-rich and with the highest transcriptional and recombination activity of the genome [16–19]. In addition, gene expression profiles of normal and pathological human tissues revealed that 40 to 50% of highly expressed genes are preferentially clustered near telomeres [20], the same happening to transcription-coupled repair sites (related to a nucleotide excision repair pathway that depends on ongoing transcription) on human cells [21]. It is, thus, conceivable that (sub)telomeric regions might somehow be related to (or help establishing) transcription-permissive nuclear environments in human cells. If this so, it would be expected that the spatial relations between active (or inactive) genes and telomeres should follow an opposite dynamics to that observed between genes and chromocenters. To specifically address this question, we investigated in 3D preserved nuclei of normal human lymphocytes, the spatial relations of 2 bhousekeepingQ genes (ACTB and RARA) and 3 specific nuclear transcripts (ACTB, IL2RA and TCRB) to telomeres and centromeres, as a function of gene activity or transcription levels. The data show that active genes and gene transcripts locate close to telomeres, and away from centromeres. The additional observation that SC35 (splicing factor SC35) domains, which are enriched in pre-mRNA processing factors, are preferen-

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tially positioned close to telomeres further strengths the hypothesis that telomeres and/or telomere-neighboring regions might contribute to the establishment of permissive environments for gene expression in the interphase nucleus of human cells.

Materials and methods Cells Peripheral blood lymphocytes (PBL) from healthy donors were used and resting lymphocytes isolated as described [5]. Bromodeoxyuridine (BrdU; Roche Diagnostics GmbH, Mannhein, Germany) incorporation confirmed that the isolated lymphocytes were mainly in the G0 phase of the cell cycle (fewer than 5% of the cells were BrdU positive). For mitogenic stimulation experiments, PBL were stimulated by the addition of phytohemagglutinin (PHA; Seromed, Biochrom KG, Berlin, Germany) [5]. In order to distinguish cell cycle phases, BrdU was added to the PHA-stimulated cell cultures prior to harvesting [22], and different stages within the S-phase (BrdU positive cells; ~35% of PHA-stimulated lymphocytes) were identified based on the morphological patterns of BrdU incorporation [23]. To discriminate cells in the G2 phase of the cell cycle, a BrdU pulse and chase strategy was used as described [22]. In the serum stimulation experiment, resting lymphocytes were left for 24 h in 5% FCS medium, followed by stimulation with 20% FCS during 20 and 45 min (based on [24,25]). In situ hybridization and signal detection in 3D preserved cells Fluorescent DNA in situ hybridization experiments were done as described [5,22]. For in situ detection of RNA, cells were fixed with 3.7% paraformaldehyde/PBS, permeabilized as for DNA hybridization and incubated in 50% formamide in 2 SSC for 5 min at room temperature, followed by hybridization without previous DNA denaturation. RNA probes (see below) were heated at 908C for 3 min, and the hybridization mixture added to a final concentration of 10 ng/Al RNA probe, 50% formamide, 0.02% BSA, 10% dextran sulphate, 2 mM vanadyl– ribonucleoside complex and 2 SSC. Hybridization was performed for 3 h to overnight at 378C in a moist chamber. Post-hybridization washes were done in 50% formamide/2 SSC at 458C (2 for 30 min). All solutions and materials were RNase-free. Specificity of RNA signals was confirmed by treating samples with RNase. For the simultaneous detection of RNA and DNA, RNA was first hybridized for 3 h, followed by post-hybridization washes as above, detection of RNA signal, post-fixation with 3.7% paraformaldehyde/PBS, DNA denaturation and DNA hybridization (omitting the RNase treatment step). Indirect

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immunofluorescence detection of centromeres (human antikinetochore autoimmune serum K55, kindly provided by Dr. W. van Venrooij, University of Nijmegen, The Netherlands), nuclear envelope (rabbit anti-lamin-B antibody, kindly provided by Dr. S. Georgatos, The University of Crete, Greece) and SC35 domains (mouse monoclonal serum against the splicing factor SC35) [26] was done immediately after post-hybridization washes. Probes for in situ hybridization Telomeres were detected with a plasmid probe specific for telomeric repeats present in all human telomeres (pHUTel) [27] kindly provided by Dr. M. Rochi (DAPEG, Bari, Italy). The ACTB gene was detected with a 20 Kb genomic clone (MA161) spanning the entire gene (provided by Dr. M. Antoniou, King’s College London, UK) and the RARA gene with the genomic PAC clone RP5-1112G21 (~150 Kb, covering the entire gene) [28] purchased from BACPAC Resources (CHORI, Oakland, USA). DNA probes were labeled with DNP, digoxigenin or biotin by nick-translation [22]. For the detection of RNA transcripts, specific RNA probes were generated. The ACTB riboprobe was generated from I.M.A.G.E. Consortium [LLNL] cDNA CloneID 611151 (~2 Kb), purchased from the Mammalian Gene Collection (UK HGMP Resource Centre, Cambridge, UK) [29]. The IL2RA riboprobe was generated from a ~2.3 Kb cDNA clone kindly provided from Dr. W. Leonard (National Heart, Lung and Blood Institute, Bethesda, MD, USA) [30]. The TCRB riboprobe clone was generated from a ~0.4 Kb genomic clone encompassing TCR Ch1, and cross-hybridizing to Ch2, gene segments [31]. Generation of RNA probes was as follows: plasmids were linearized by restriction enzyme digestion, phenol/chloroform extracted, labeled with digoxigenin-11-UTP (DIG RNA Labeling Mix; Roche Diagnostics GmbH, Penzberg, Germany) when transcribed from the promoters of T7 or T3 RNA polymerases (Stratagene, Amsterdam, The Netherlands) in order to give antisense probes and incubated in Carbonate buffer (60 mM Na2CO3/40 mM NaHCO3, pH 10.2) to regulate probe size. Prior to hybridization, 20 Ag tRNA E. coli was added to 50 ng of the RNA probe, ethanol precipitated and re-suspended in 90% formamide/2 SSC.

nuclear volume (average of 24 and 31 sections for G0 and PHA-stimulated lymphocytes, respectively, corresponding to 0.3 Am increments in the z axis). A minimum of 30 nuclei were analyzed in each experiment. Three-dimensional image stacks of entire nuclei (at the resolution of 512  512 pixels) were recorded for analysis by Metamorph Imaging Series v.4.5 (Universal Imaging Corporation, USA) and ImageJ 1.29 (NIH, Springfield, VA, USA) softwares. In order to avoid ambiguous determination of the limits of hybridization/ immunodetection signals, distance measurements were done from the centroid of each signal. For each signal, the absolute X and Y coordinates of the signal’s intensity center within a certain optical section were determined (in almost all cells, the signals could be seen in several sequential planes), based on the information given by LSM 510 software (version 2.8) on the increment between sections along the z axis (0.3 Am) and on the x/y scaling (pixels/Am) of each stack. The combined weighted intensity center of each signal corresponded to the centroid (x, y and z coordinates of the signal’s maximum intensity) of the complete hybridization/immunodetection signal. An extension of the Pythagoras theorem to the 3-dimensional space returned, for each cell, the Euclidian distances between the centroids of the alleles/transcripts and the centroids of the closest heterochromatic domains. Printing-quality images were prepared with Adobe Photoshop 7.0. Statistical analysis The Kolmogorov–Smirnov goodness-of-fit test was used to determine how well the statistical distribution of the allelic/transcript minimal distances (the sample data) conformed to a normal distribution. To determine if two sample data came from the same normal population, the non-paired Student’s t test was used. If comparison was made in more than two samples, the analysis of variance (ANOVA) was used instead. The Chi-square test of homogeneity was used to determine if the sampled cells were drawn from homogeneous lymphocyte populations with respect to the distribution among 2 or more distance patterns (e.g. Cen N Tel, Cen  Tel, Cen b Tel, see below). Regression analysis was used to test for a linear relationship between the number of heterochromatic domains and the absolute distances. The level of significance chosen for all the tests was P = 0.05.

Confocal microscopy and image processing Analysis of gene expression by semi-quantitative RT-PCR Analysis of hybridization signals was performed with the confocal microscope Zeiss LSM-510 (Carl Zeiss, Jena, Germany), with the Planapochromat 63/1.4 objective. Excitation lasers of 488, 543 and 630 nm were used. Only 3D preserved nuclei were selected for analysis [22]. Depending on the size of the nucleus (the average diameter of human lymphocyte nuclei is 6 Am and 9 Am for G0 and PHAstimulated cells, respectively), a variable number of equidistant optical sections were obtained covering the entire

In three independent experiments, total RNA was extracted from 0.5–1  107 resting and PHA-stimulated lymphocytes using the TriZol reagent (Gibco-BRL) according to the instructions of the manufacturer. Approximately 1.1 Ag and 4.2 Ag of total RNA were recovered per 106 resting and stimulated cells, respectively. Of these, 1 Ag of total RNA was reverse-transcribed to cDNA as described [32], followed by semi-quantitative PCR analysis (performed in the linear

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range of amplification) of ACTB and RARA cDNA, obtained from ~4  104 cells. A primer-pair specific for ACTB exons 3 and 4 was used which gives a PCR product of 446 bp [33]. Amplification conditions for ACTB were as follows: initial denaturation at 958C for 3 min, followed by 948C for 1 min, 578C for 1 min, 728C for 2 min, for a total of 16 to 30 cycles and a final extension at 728C for 10 min. Primers R2 (specific for RARA exon 2) and R5 (specific for RARA exon 3) were used for RARA amplification (PCR products of 185 bp) [32]. Amplification conditions for these primers were as follows: initial denaturation at 958C for 3 min, followed by 958C for 50 s, 588C for 1 min, 728C for 1 min, for a total of 23 to 32 cycles, and a final extension at 728C for 10 min. Relative quantification of transcripts (arbitrary units) was done by image profiling of pixel intensity along ethidium bromide-stained PCR bands (ImageJ 1.29 software).

Results The 3-dimensional organization of telomeres and centromeres in the nucleus of peripheral blood lymphocytes We began by characterizing the spatial organization of human telomeres and centromeres in the nuclei of

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quiescent and PHA-stimulated peripheral blood lymphocytes. As seen in Fig. 1A, telomeres tend to associate with each other forming conspicuous structures of different sizes (hereafter called Telomere Associations or TAs) [34– 36]. These are more evident in quiescent (G0) nuclei (average number of 25 telomere signals, for a theoretical number of 92 per nucleus) than in stimulated G1 cells (BrdU negative; average number of 35). The data corroborate the findings of Nagele et al. [35] and the recent observation that telomeres cluster together in a dynamic way in the nucleus of human cells [37]. Similarly, centromeric associations, or chromocenters [5,36], increase in number upon cell stimulation (mean of 13 and 24 per nucleus in G0 and G1, respectively) (Fig. 1B). The simultaneous visualization of TAs and chromocenters revealed that these two heterochromatic domains occupy distinct compartments in lymphocyte nuclei (Fig. 1C). In resting lymphocytes (G0), centromeres locate preferentially at the periphery of the nuclei while telomeres occupy more central positions [5,36,38]. Upon PHA stimulation, some centromeres, as well as telomeres, are also found within the nuclear interior. Nonetheless, associations between TAs and chromocenters were infrequent (average of 2 and 5 per quiescent and stimulated G1 nuclei, respectively).

Fig. 1. The 3-dimensional organization of telomeres in the nucleus of human peripheral blood lymphocytes. (A) Average number of telomere signals per nucleus in sequential stages of the cell cycle. (B) Average number of chromocenters and telomeric associations (TAs) in resting and G1 lymphocytes. (C) Single confocal sections of resting (upper panel) and G1 lymphocytes (lower panel) showing that TAs (green) and chromocenters (red) occupy distinct nuclear compartments. Nuclear lamina detected with anti-lamin B antibody (blue). Scale bar, 5 Am.

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In resting lymphocytes, the positioning of the ACTB alleles in relation to TAs and chromocenters differs between donors and different time points, whereas RARA alleles are consistently closer to TAs We next investigated how the alleles of transcriptionally active genes would spatially relate to these two types of heterochromatic domains. For this purpose, two bhousekeepingQ genes residing in distinct chromosomal locations and chromatin contexts were chosen: the ACTB gene, which is located in a light G-band at a subtelomeric position in chromosome 7 (p22.1), and the RARA gene, located in a dark G-band (17q12) cytogenetically closer to the centromere of chromosome 17 than to either of the telomeres. Given that the cells under analysis are normal primary cells rather than clonal and immortalized, cells from different donors (A and B), at different time points for each donor (A1, A2, B1, B2, etc) were investigated. The distance of the 2 alleles of each gene to the closest TA and chromocenter was analyzed in the nuclei of resting and stimulated lymphocytes in each donor (see Materials and methods). This approach returns concrete physical distances for the most probable localization of a gene, as assessed by the distances between the centroids of immunofluorescence/hybridization signals. It is of note that distances between centroids of V0.5 Am often correspond to associated or overlapping signals, reflecting a close proximity in the 3-dimensional nucleus (average nuclear diameter of 6 Am and 9 Am for G0 and PHA-stimulated lymphocytes, respectively) (see also [39]). The minimal allelic distances of ACTB (considering the 2 alleles together) to the closest chromocenters and TAs in the nuclei of resting lymphocytes from 2 donors (A and B), in 2 different time points, are shown in Fig. 2A. Significant

variations were observed between donors and within the same donor at different time points (P b 0.0001 for both comparisons). Average minimal distances to the closest chromocenter varied from ~1 Am, in donor A, time point 1 (A1) and donor B, time point 2 (B2), to ~1.8 Am, in donor B, time point 1 (B1). In relation to TAs, allelic distances varied, again, between donors (P = 0.0001) and within donor B in different time points (B1 versus B2) (donor A not investigated in different time points). Distance analysis of the RARA alleles in relation to the closest chromocenters and TAs was done in the resting lymphocytes from donors A and B (2 time points and 1 time point analyzed, respectively). As shown in Fig. 2B, the RARA gene is consistently closer to TAs than to chromocenters (average distances 0.9–1 Am and 1.9 Am, P = 0.25 and P = 0.7, respectively), a pattern which, contrary to that observed for ACTB, did not differ between donors or with time. To simultaneously analyze the spatial relationship of each ACTB and RARA allele with chromocenters and TAs, cells were classified into 3 populations according to the allelic distances to both domains in each nucleus: Cen N Tel, where the allelic distances of both alleles to the closest chromocenters are higher than the allelic distances of both alleles to the closest TAs; Cen b Tel, where both alleles are located closer to chromocenters than to TAs; and Cen  Tel, where one allele is closer to a chromocenter than to a TA, while the other is closer to a TA than to a chromocenter. As depicted in Fig. 3A, in donor A (only one time point analyzed), the two ACTB alleles are either close to TAs (41%) or differ in relation to the proximity to either domain (44%). As to donor B, the distribution patterns varied in different time points: in B2 cells, 23% of the nuclei had both alleles close to TAs, the reverse being observed for 33% of

Fig. 2. Minimal distances of ACTB and RARA genes to chromocenters and TAs in resting lymphocytes. Box-plots depict: (A) the minimal distances of ACTB alleles to the closest chromocenters (Chrom) and TAs in the nuclei from 2 donors (A and B) in two different time points (1 and 2); (B) the minimal distances of RARA alleles to the closest chromocenters (Chrom) and TAs in the nuclei of resting lymphocytes from 2 donors (A and B) and in two different time points for donor A (A2 and A3). Box range: percentiles 25–75%; 5 mean; 1% and 99% percentiles; -min and max values. *P b 0.05.

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particularly in genes related to the cytoskeleton machinery, such as actin genes [40]. Indeed, the transcription activity of these genes was shown to respond to serum induction within minutes [24,25]. Accordingly, we reasoned that physiological variations in individual micro-environmental factors (such as subtle differences in concentration of blood serum factors) could help explain the observed variations in the ACTB gene positions among donors. To address this question, an in vitro serum stimulation assay of resting lymphocytes from donor A, time point 2, was performed, followed by analysis of allelic distances to chromocenters and TAs. As shown in Fig. 4A, the greatest variability in distances of ACTB alleles (considering the 2 alleles simultaneously) to the closest chromocenters and TAs was observed in resting lymphocytes and in lymphocytes cultured for 24 h with 5% FCS. This variation progressively diminished with serum stimulation, with the gene eventually becoming equidistant from chromocenters and TAs (average distances of ~1.4 Am

Fig. 3. Distance patterns of ACTB and RARA alleles, in relation to chromocenters and TAs, when simultaneously considered at the cell population level (resting lymphocytes). Distribution of cells among the three distances patterns (please see Results for pattern description) of ACTB (A) and RARA (B) alleles in different donors and time points. *P b 0.0001.

the nuclei, the remaining 43% corresponding to pattern Cen  Tel; in contrast, the majority of B1 nuclei (83%) had the two ACTB alleles closer to TAs than to chromocenters. Strikingly, the latter pattern was observed for the two RARA alleles, in all three donors investigated (Fig. 3B). In summary, the positioning of the ACTB alleles in relation to chromocenters and TAs in resting lymphocytes may differ between donors and, within the same donor, in different time points. At the single cell level, the two ACTB alleles may be found closer to chromocenters than to telomeres, closer to telomeres than to chromocenters or equidistant in relation to these heterochromatic domains. In contrast, the RARA gene is consistently found closer to TAs than to chromocenters in the nuclei of resting lymphocytes of different donors and in different time points. This indicates that the distance between a particular gene and the centromere, or the nearest telomere, of the respective chromosome, when assessed at the cytogenetic level, does not necessarily translate into 3dimensionally distances in the interphase nucleus. The ACTB alleles localize close to telomeres in conditions of increased gene transcription Upon serum stimulation It has been shown that variation in the expression levels of human genes among individuals is a frequent phenomenon,

Fig. 4. Localization of ACTB gene in the nuclei of serum-stimulated lymphocytes. (A) Box-plot depicting the minimal distance of ACTB alleles to the closest chromocenters (Chrom) and TAs in the nuclei of resting lymphocytes from donor A, time 2, cultured for 24 h in medium supplied with 5% FCS, and subsequently stimulated with 20% FCS during 20 or 45 min. Box range: percentiles 25–75%; 5 mean; 1% and 99% percentiles; -min and max values. (B) Cell distribution among the three distance patterns of ACTB alleles.

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and ~1.3 Am, respectively, at 45 min of serum stimulation). When the allelic distances to both domains were simultaneously considered at the cell population level (Fig. 4B), the overall distribution among the three cell populations (Cen N Tel, Cen  Tel and Cen b Tel) was found to differ, significantly, between cells cultured with 5% FCS and serum-stimulated lymphocytes (with 20% FCS for 45 min; P b 0.001). In contrast to resting cells, which are equally distributed between the Cen N Tel (41%) and Cen  Tel (44%) populations, most lymphocytes cultured with 5% FCS (73%) have now one ACTB allele closer to a chromocenter, while the other allele is closer to a TA (Cen  Tel pattern). After serum stimulation (45 min in the presence of 20% FCS), the number of cells in the Cen  Tel population diminishes to 30% while the proportion of cells with both alleles either closer to TAs or to chromocenters increases (39% and 30%, respectively). Therefore, under conditions that do not promote ACTB transcription (5% FCS medium) [24,25], the two ACTB alleles tend to position differently in relation to chromocenters and to TAs, one locating closer to a chromocenter and farther from a TA, the other showing the reciprocal positioning. In conditions that promote ACTB gene transcription (20% FCS medium) [24,25], the two alleles become equidistant to both domains. Thus, both conditions of serum deprivation and serum stimulation appear to induce a rapid relocalization of ACTB within the interphase nucleus. Upon PHA stimulation We then asked if the observed dynamic relocalization of ACTB also occurred in the nuclei of cycling lymphocytes, in which overall transcriptional levels are increased (see Materials and methods), and locus repositioning has been described for some tissue-specific genes [41]. To answer this question, ACTB minimal allelic distances to the closest chromocenters and TAs were determined in the nuclei of PHA-stimulated lymphocytes (72 h) and compared with quiescent lymphocytes. Similarly to that observed under serum stimulation, gene distances to both structures diminished with PHA stimulation (Fig. 5A, donor A). The ACTB gene is now located significantly closer to TAs than to chromocenters (average minimal distances of ~1.1 Am and ~1.4 Am, respectively). The consistency of these results was confirmed by analyzing lymphocytes from a different donor. As depicted in Fig. 5A (donor C), upon stimulation with PHA, the ACTB gene relocates, again, closer to TAs (average minimal distances of ~1.2 Am versus ~1.5 Am in resting cells) than to chromocenters (average minimal distances of ~1.5 Am in both stimulated and non-stimulated lymphocytes), indicating that the ACTB gene tends to locate close to TAs in conditions of increased gene activity (see below). This is in contrast with the RARA gene, which maintains its preferential position closer to TAs than to chromocenters (average distances of ~0.9 Am and ~1.7 Am, respectively) in stimulated cells, akin to what is observed in resting cells (Fig. 5B).

Allelic distances are not related to the number of domains in the nuclei of resting and stimulated lymphocytes Theoretically, the number of chromocenters or TAs in the nucleus could have influenced gene distances to these structures. Two observations indicate that this is not the case. First, the variability in ACTB distances, either to chromocenters or TAs, between donors and in different time points for the same donor, observed in resting lymphocytes, shows that a wide range of allelic distances can be found in nuclei in which the average number of domains is relatively stable. Secondly, in the nuclei of stimulated lymphocytes, where both centromeric and telomeric domains increase in number, a similar variation of distances is observed, irrespective of the number of domains (P N 0.05; unpublished data). The same behavior is observed for the RARA gene, both in the nuclei of resting and stimulated lymphocytes (unpublished data). Therefore, a higher number of domains does not imply a decrease in the minimal allelic distances, suggesting that the spatial positioning of a gene in relation to chromocenters and telomeric associations might depend, at least in part, on functional rather than on structural reasons. Transcriptional activity of ACTB and RARA upon PHA stimulation The observation that PHA stimulation leads to a repositioning of ACTB but not of RARA prompted us to see whether this was related to differences in transcriptional activity of any of these genes upon mitogenic stimulation. To address this point, semi-quantitative RT-PCR for each gene was performed from cDNA derived from equivalent number of cells in three independent PHA stimulation experiments. As shown in Fig. 5C, the transcriptional activity of ACTB is markedly increased upon PHA stimulation whereas the effect on RARA transcription is, comparatively, much subtler. The data, therefore, suggest that the presence (or absence) of spatial repositioning of these housekeeping genes, in relation to chromocenters and TAs, might be correlated with the occurrence of (or absence of) changes in their transcriptional activity in different metabolic/cell-cycle stage contexts. Specific nuclear RNA signals are found close to telomeric associations and far from chromocenters in the nuclei of PHA-stimulated lymphocytes To support the latter possibility, the minimal distances between the ACTB nuclear RNA signals and the closest chromocenters and TAs were determined in PHA-stimulated nuclei. RNA-FISH performed in both resting and PHAstimulated lymphocytes showed that ACTB RNA signals were barely detectable in resting cells. In contrast, 61% of the stimulated lymphocytes had visible foci of ACTB RNA signals (co-localizing with the respective gene; unpublished

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Fig. 5. Nuclear positioning, in relation to TAs and chromocenters, and transcriptional activity of ACTB and RARA in PHA-stimulated lymphocytes. Box-plots showing the minimal distances of ACTB (A) and RARA (B) alleles to the closest chromocenters (Chrom) and TAs, in resting and 72 h PHA-stimulated lymphocytes from different donors. Box range: percentiles 25–75%; 5 mean; 1% and 99% percentiles; -min and max values. *P b 0.005. (C) Semiquantitative RT-PCR assay of ACTB (upper panel) and RARA (lower panel) transcripts in resting (R) and PHA-stimulated (S) lymphocytes in 3 independent PHA stimulation experiments (1, 2, 3). Plot profiles show the pixel intensity along ethidium bromide-stained PCR bands (please see Materials and methods). Number of cycles corresponds to linear amplification of PCR products. W—water.

data). Two signals were visible in 56% of the nuclei, one signal in the remaining 44%. Such variability in ACTB nuclear RNA signals has been previously reported and considered as reflecting gene activity at the allelic level [25]. As depicted in Figs. 6A and D, ACTB nuclear transcripts were positioned significantly closer to TAs than to chromocenters (average distance of 0.9 Am and 1.6 Am, respectively). To assess if this positioning was also observed for other active genes, in situ detection of RNA from a gene highly expressed upon mitogenic stimulation (the alpha subunit of the IL2 receptor gene, IL2RA) and a gene

specifically expressed in T-lymphocytes (T-cell receptor beta locus, TRCB) was performed in PHA-stimulated lymphocytes from the same donor. Both the transcripts from TCRB and IL2RA genes were visualized as single dots in the nucleus of 93% and 73% of stimulated lymphocytes, respectively. These observations could be explained by monoallelic expression of both genes. Allelic exclusion is known to occur for TCRB transcription (reviewed in [42]), but, as to IL2RA, this is, to our knowledge, the first observation showing that this gene might be preferentially expressed from one allele at the single cell level.

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Fig. 6. Positioning of nuclear RNA signals from ACTB, IL2RA and TCRB genes in relation to TAs and chromocenters. (A–C) Representative triple-color confocal sections of PHA-stimulated lymphocytes with simultaneous visualization of centromeres (blue), telomeres (green) and specific RNA signals for ACTB (A), IL2RA (B) and TCRB (C) (white arrows). Dashed circles indicate the approximate contours of the nuclear sections. Scale bar, 5 Am. (D) Box-plot depicting the distances of the ACTB, the IL2RA and the TCRB nuclear transcripts to the closest chromocenters (Chrom) and TAs in the nuclei of PHA-stimulated lymphocytes from donor B. Box range: percentiles 25–75%; 5 mean; 1% and 99% percentiles; -min and max values. *P b 0.0001.

Strikingly, and similar to ACTB gene transcripts, IL2RA and TCRB nuclear RNA signals are consistently positioned closer to TAs than to chromocenters (Fig. 6). When statistically compared, the three nuclear transcripts do not differ in relation to the minimal distances to the closest chromocenters (average distance of ~1.6 Am; P = 0.9). However, they are differently located as to the minimal distances to the closest TAs: the transcripts of TCRB are found closer to TAs (average distance of 0.6 Am) in comparison to the transcripts of the housekeeping ACTB gene ( P b 0.0001). Moreover, distance variances of IL2RA and TRCB transcripts, but not of the ACTB transcripts, in relation to the closest TAs, are lower than those to the closest chromocenters ( P b 0.02). The distances of TCRB transcripts to the closest TAs were, indeed, the least variant of all the observed distances to a heterochromatic domain. The latter finding was further observed in stimulated lymphocytes from one other donor (unpublished data). SC35 domains are found close to telomeric associations and far from chromocenters in the nuclei of PHA-stimulated lymphocytes To investigate whether TAs might associate with nuclear domains related to transcriptional activity, the spatial relationship to SC35 domains, enriched in splicing and mRNA processing factors and found close to active genes and early-replicating bands [43], was determined. For that, telomeres, centromeres and SC35 domains were simultaneously detected in PHA-stimulated lymphocytes. Within each nucleus, SC35 domains were found to vary in size and

number (range of 6 to 25 domains per lymphocyte nucleus). Strikingly, recurrent associations of SC35 domains with TAs, but not with chromocenters, were observed (Fig. 7A). Frequently, more than one TA was found surrounding SC35 domains. Analysis of the minimal distances between the 3–4 most prominent SC35 domains and the closest chromocenters and TAs confirmed that SC35 domains are located significantly closer to TAs than to chromocenters (average distances of 0.8 Am and 1.7 Am, respectively) (Fig. 7B). Thus, and similar to that observed with active genes, a nuclear domain enriched in pre-mRNA processing factors (SC35 domains) also tends to locate near telomeres in the nuclei of human lymphocytes.

Discussion In this work, we have analyzed the nuclear positioning of constitutively expressed genes (ACTB and RARA) and gene transcripts (from ACTB, IL2RA and TCRB) in relation to centromeric and telomeric domains, in resting and stimulated human lymphocytes. The data show that active genes and transcripts are consistently located close to telomeric domains and away from centromeres. Furthermore, ACTB alleles are rapidly relocated near telomeres upon upregulation of gene transcription, suggesting a cause/effect relationship between gene activity and proximity to telomeric domains. To investigate how genes or gene transcripts are spatially related to chromocenters and telomeres, we measured, in the same nucleus, the distance of the 2 alleles of each gene, and

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Fig. 7. Positioning of SC35 domains in relation to TAs and chromocenters. (A) Triple-color single confocal section representative of PHA-stimulated lymphocytes hybridized with the telomeric probe (green) and immunolabeled for centromeres (blue) and SC35 domains (red). White arrows point to close proximity between SC35 domains and telomeres. Dashed circle indicates the approximate contour of the nuclear section. Scale bar, 5 Am. (B) Box-plot depicting the minimal distances of the 3–4 most prominent SC35 domains in PHA-stimulated nuclei to the closest chromocenters (Chrom) and TAs. Box range: percentiles 25–75%; 5 mean; 1% and 99% percentiles; -min and max values. *P b 0.0001.

of each transcript signal, to the closest TA and chromocenter. Consistent with in vivo observations showing that DNA sequences move inside the nucleus by a random diffusion process within a constrained subvolume [44,45], we observed that all the minimal distances (absolute values) of each allele/transcript to either heterochromatic domain behaved as normal distributions. Therefore, our findings, obtained in a random sample of cells, can be interpreted as reflecting the probabilistic allelic position in relation to a particular domain, in a given population of cells. Firstly, we observed that in resting cells the ACTB and RARA genes behave differently as to their localization in relation to TAs and chromocenters. While distances of ACTB to both domains vary among donors and with time in the same donor, the RARA gene was consistently closer to TAs than to chromocenters in different donors and time points. Two previously reported findings made us hypothesize that such a discrepancy could somehow be related to differences in the dynamics of transcription between the two genes. One is that genes coding for cytoskeleton proteins (as it is the case of ACTB) are among those with the highest variation in transcription levels in normal individuals [40]. The other is that the ACTB, but not the RARA gene, contains a serum responsive element in its regulatory region [46], indicating that it is capable of rapidly responding to different serum concentrations [24,25]. Indeed, a rapid relocalization of the two ACTB alleles in relation to both domains was observed upon serum stimulation, a finding reminiscent of those previously reported for other gene loci, which were shown to loop out from the respective chromosome territories upon upregulation of their transcriptional activity [47–49]. It is, therefore, plausible that circumstantial

variations in individual physiological factors, such as subtle differences in the concentration of serum factors, might explain the observed heterogeneity in ACTB gene positioning among donors and time points. The RARA gene, in contrast, was invariably close to telomeres in resting cells, irrespective of donor and time point. In fact, contrary to the ACTB gene, whose repositioning close to telomeres upon PHA stimulation paralleled an increase in transcription, the non-variable position of RARA correlated with constant transcriptional activity (which did not change upon mitogenic stimulation). It has been shown in human lymphocytes that RARA and other constitutively expressed regions of chromosome 17 locate towards the nuclear center within the territory of the chromosome, in an opposite position to that of the respective centromere [50]. By contrast, a region with low expression levels localized towards the nuclear membrane and near to the centromere [50]. Although positioning of chromosome 17 telomeres had not been investigated, other reports have shown that telomeres and centromeres of several human chromosomes locate at opposite regions in the respective chromosome territory [38,51]. A possibility, then, is that the RARA gene and other active loci in chromosome 17, but not inactive ones, locate close to the telomeres of their own territory, within a more euchromatic compartment [52]. The in situ visualization of nuclear transcripts from the ACTB, IL2RA (both upregulated upon mitogenic stimulation) and TCRB (expression restricted to T-lymphocytes) genes showed that the three transcripts were consistently close to telomeric domains and away from chromocenters, further confirming the relationship between transcriptional permissiveness and spatial proximity to telomeres.

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Two intriguing observations argue in favor that proximity of active genes to telomeres, rather than coincidental, might be of functional importance. One is that, even though ACTB and TCRB genes reside in the same chromosome (in opposite arms), the former being genetically closer to 7 p telomere (~5 Mb) than the latter to 7 q telomere (~17 Mb), TCRB transcripts were located closer to TAs than ACTB transcripts. Second, the distances between TCRB transcripts and the closest TAs were the least variant of all the measured distances to a heterochromatic domain. Notably, TCRB transcripts and telomere signals were frequently touching each other. These findings are compatible with an active role for the 3D surroundings of telomeres in the maintenance of gene activity. Accordingly, the strict positioning of TCRB transcripts near a telomeric domain would help maintain the (very active) transcriptional status of this allelic-excluded Tcell-specific gene. Conversely, genes with variable transcriptional activity (such as ACTB) [25] would locate at distances compatible with their bjumpingQ between positions close to telomeres or to chromocenters in order to transiently regulate transcription. Indeed, in serum-deprived cells, we observed that the two ACTB alleles were differentially located in relation to TAs and chromocenters, one being always closer to the former than the other. Therefore, an unequal allelic positioning may reflect unequal transcription activities at the allelic level. If so, the differential and dynamic positioning of the ACTB alleles in relation to TAs and chromocenters could reflect a previously unforeseen way for timely regulating gene transcription at the allelic level, perhaps in response to variable time point requirements of the respective product by the cell. Finally, we observed that, in the nuclei of stimulated lymphocytes, SC35 domains are in close proximity to telomeres and far from chromocenters. Also, and similarly to what was very recently observed in some human flattened cell types [52], we detected recurrent associations between SC35 domains and telomeres. It is known that highly acetylated chromatin [53], several active genes (including ACTB) and early-replicating gene-rich R bands cluster in a non-random fashion around SC35 domains in human cells [43,54]. In addition, telomeres have been shown to dynamically associate with one other class of nuclear bodies, the PML bodies, which are also frequently surrounded by transcriptionally active regions of the human genome [37,39]. Taken together, the present data show that the 3D environment of telomeres is not repressive but rather permissive to gene transcription in human cells. This is in contrast with the telomere-mediated silencing effects observed in budding yeast cells (reviewed in [6]). Such a discrepancy is not surprising, however, if one considers that human and yeast telomeres differ in several fundamental aspects among which their spatial and functional relations to distinct nuclear compartments. In fact, whereas yeast telomeres cluster in a zone near the nuclear envelope, where they are thought to contribute for the establishment of a

repressive environment through the action of telomereassociated Sir proteins [55], in human cells, the nuclear periphery is almost devoid of telomeres (which are centrally located) and mostly occupied by (transcription-repressive) pericentromeric heterochromatin (our data and [34,36, 38,56]). Whether telomeres are directly involved in the establishment of a permissive compartment for gene expression in the nuclear interior (as it is the case for centromeres in the peripheral repressive compartment) is an open question. Noteworthy in this respect, however, is that many human subtelomeric regions are highly enriched in active genes (see Introduction). A reasonable assumption, therefore, is that the spatial relation between telomere clusters and gene activity reported here might reflect the presence of high local concentrations of positive factors for gene transcription. Our observation that the alleles of a housekeeping gene locate close to telomeres upon activation of transcription is reminiscent, yet with putative opposing effects, of previous correlative evidence for closeness of specific genes to centromeres and gene-silencing, in murine lymphoid cells [41]. Hence, a likely possibility is that, in mammalian cells, chromocenters and telomeric domains may contribute in opposite ways for the regulation of gene transcription.

Acknowledgments The authors are grateful to Professor Joa˜o Ferreira (Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa) for discussion and suggestions of technical procedures, to Drs. Jorge Carneiro (Instituto Gulbenkian de Cieˆncia, Oeiras) for help with statistical analysis, Jose´ Braga and Jose´ Rino (Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa) for help with digital imaging. The work was supported by a grant from Fundac¸a˜o para a Cieˆncia e Tecnologia (FCT) POCTI/37953/2001 and ASQ by a FCT fellowship.

References [1] S.M. Gasser, Positions of potential: nuclear organization and gene expression, Cell 104 (2001) 639 – 642. [2] N. Dillon, R. Festenstein, Unravelling heterochromatin: competition between positive and negative factors regulates accessibility, Trends Genet. 18 (2002) 252 – 258. [3] J. Baxter, M. Merkenschlager, A.G. Fisher, Nuclear organisation and gene expression, Curr. Opin. Cell Biol. 14 (2002) 372 – 376. [4] I. Alcobia, A.S. Quina, H. Neves, N. Clode, L. Parreira, The spatial organization of centromeric heterochromatin during normal human lymphopoiesis: evidence for ontogenically determined spatial patterns, Exp. Cell Res. 290 (2003) 358 – 369. [5] I. Alcobia, R. Dila˜o, L. Parreira, Spatial associations of centromeres in the nuclei of hematopoietic cells: evidence for cell-type-specific organizational patterns, Blood 95 (2000) 1608 – 1615. [6] W.-H. Tham, V.A. Zakian, Transcriptional silencing at Saccharomyces telomeres: implications for other organisms, Oncogene 21 (2002) 512 – 521.

A.S. Quina, L. Parreira / Experimental Cell Research 307 (2005) 52–64 [7] C.N. Sprung, L. Sabatier, J.P. Murnane, Effect of telomere length on telomeric gene expression, Nucleic Acids Res. 24 (1996) 4336 – 4340. [8] J.A. Baur, Y. Zou, J.W. Shay, W.E. Wright, Telomere position effect in human cells, Science 292 (2001) 2075 – 2077. [9] C.E. Koering, A. Pollice, M.P. Zibella, S. Bauwens, A. Puisieux, M. Brunori, C. Brun, L. Martins, L. Sabatier, J.F. Pulitzer, E. Gilson, Human telomeric position effect is determined by chromosomal context and telomeric chromatin integrity, EMBO Rep. 3 (2002) 1055 – 1061. [10] Z.E. Smith, D.R. Higgs, The pattern of replication at a human telomeric region (16p13.3): its relationship to chromosome structure and gene expression, Hum. Mol. Genet. 8 (1999) 1373 – 1386. [11] G. Jiang, F. Yang, P.G.M. van Overveld, V. Vedanarayanan, S.M. van der Maarel, M. Ehrlich, Testing the position-effect variegation hypothesis for fascioscapulohumeral muscular dystrophy by analysis of histone modification and gene expression in subtelomeric 4q, Hum. Mol. Genet. 12 (2003) 2909 – 2921. [12] S.T. Winokur, Y.-W. Chen, P.S. Masny, J.H. Martin, J.T. Ehmsen, S.J. Tapscott, S.M. van der Maarel, Y. Hayashi, K.M. Flanigan, Expression profiling of FSHD muscle supports a defect in specific stages of myogenic differentiation, Hum. Mol. Genet. 12 (2003) 2895 – 2907. [13] H. Zhang, K.-H. Pan, S.N. Cohen, Senescence-specific gene expression fingerprints reveal cell-type-dependent physical clustering of up-regulated chromosomal loci, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 3251 – 3256. [14] H.-L. Chen, C.-Y. Lu, Y.-H. Hsu, J.-J. Lin, Chromosome positional effects of gene expressions after cellular senescence, Biochem. Biophys. Res. Commun. 313 (2004) 576 – 586. [15] Y. Ning, J.-F. Xu, Y. Li, L. Chavez, H.C. Riethman, P.M. Lansdorp, N.-P. Weng, Telomere length and the expression of natural telomeric genes in human fibroblasts, Hum. Mol. Genet. 12 (2003) 1329 – 1336. [16] S. Saccone, A. De Sario, G.D. Valle, G. Bernardi, The highest gene concentrations in the human genome are in telomeric bands of metaphase chromosomes, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 4913 – 4917. [17] J.M. Craig, W.A. Bickmore, The distribution of CpG islands in mammalian chromosomes, Nat. Genet. 7 (1994) 376 – 382. [18] H.C. Riethman, Z. Xiang, S. Paul, E. Morse, X.-L. Hu, J. Flint, H.-C. Chi, D.L. Grady, R.K. Moyzis, Integration of telomere sequences with the draft human genome sequence, Nature 409 (2001) 948 – 951. [19] M.J. Lercher, A.O. Urrutia, A. Pavle´cˇk, L.D. Hurst, A unification of mosaic structures in the human genome, Hum. Mol. Genet. 12 (2003) 2411 – 2415. [20] H. Caron, B. van Schaik, M. van der Mee, F. Baas, G. Riggins, P. van Sluis, M.-C. Hermus, R. van Asperen, K. Boon, P.A. Vouˆte, S. Heisterkamp, A. van Kampen, R. Versteeg, The human transcriptome map: clustering of highly expressed genes in chromosomal domains, Science 291 (2001) 1289 – 1292. [21] J. Surralle´s, M.J. Ramirez, R. Marcos, A.T. Natarajan, L.H.F. Mullenders, Clusters of transcription-coupled repair in the human genome, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 10571 – 10574. [22] H. Neves, C. Ramos, M.G. Silva, A. Parreira, L. Parreira, The nuclear topography of ABL, BCR, PML, and RARa genes: evidence for gene proximity in specific phases of the cell cycle and stages of hematopoietic differentiation, Blood 93 (1999) 1197 – 1207. [23] H. Nakayasu, R. Berezney, Mapping replicational sites in the eukaryotic cell nucleus, J. Cell Biol. 108 (1989) 1 – 11. [24] A.M. Femino, F.S. Fay, K. Fogarty, R.H. Singer, Visualization of single RNA transcripts in situ, Science 280 (1998) 585 – 590. [25] J.M. Levsky, S.M. Shenoy, R.C. Pezo, R.H. Singer, Single-cell gene expression profiling, Science 297 (2002) 836 – 840. [26] X.-D. Fu, T. Maniatis, Factor required for mammalian assembly is localized to discrete regions in the nucleus, Nature 343 (1990) 437 – 441. [27] E. Gilson, T. Muller, J. Sogo, T. Laroche, S.M. Gasser, RAP1 stimulates single- to double-strand association of yeast telomeric

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42] [43]

[44]

[45]

[46]

[47]

63

DNA: implications for telomere–telomere interactions, Nucleic Acids Res. 22 (1994) 5310 – 5320. G. Specchia, F. Albano, C.T. Storlazzi, L. Anelli, A. Zagaria, V. Liso, M. Rocchi, t(15;17) in acute promyelocytic leukemia is not associated with submicroscopic deletions on der(17), Haematologica 87 (2002) 775 – 777. G. Lennon, C. Auffray, M. Polymeropoulos, M.B. Soares, The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression, Genomics 33 (1996) 151 – 152. W.J. Leonard, J.M. Depper, G.R. Crabtree, S. Rudikoff, J. Pumphrey, R.J. Robb, M. Kro¨nke, P.B. Svetlik, N.J. Peffer, T.A. Waldmann, W.C. Greene, Molecular cloning and expression of cDNAs for the human interleukin-2 receptor, Nature 311 (1984) 626 – 631. J.J.M. Van Dongen, I.L.M. Wolvers-Tettero, Analysis of immunoglobulin and T cell receptor genes: Part I. Basic and technical aspects, Clin. Chim. Acta 198 (1991) 1 – 91. A.S. Quina, P. Gameiro, M.S. Costa, M. Telhada, L. Parreira, PMLRARA fusion transcripts in irradiated and normal hematopoietic cells, Genes Chromosomes Cancer 29 (2000) 266 – 275. E. Porfiri, L.M. Secker-Walker, A.V. Hoffbrand, J.F. Hancock, DCC tumor suppressor gene is inactivated in hematologic malignancies showing monosomy 18, Blood 81 (1993) 2696 – 2701. M.E. Lude´rus, B. van Steensel, L. Chong, O.C. Sibon, F.F. Cremers, T. de Lange, Structure, subnuclear distribution, and nuclear matrix association of the mammalian telomeric complex, J. Cell Biol. 135 (1996) 867 – 881. R.G. Nagele, A.Q. Velasco, W.J. Anderson, D.J. McMahon, Z. Thomson, J. Fazekas, K. Wind, H.-Y. Lee, Telomere associations in interphase nuclei: possible role in maintenance of interphase chromosome topology, J. Cell Sci. 114 (2000) 377 – 388. C. Weierich, A. Brero, S. Stein, J. von Hase, C. Cremer, T. Cremer, I. Solovei, Three-dimensional arrangements of centromeres and telomeres in nuclei of human and murine lymphocytes, Chromosome Res. 11 (2003) 485 – 502. C. Molenaar, K. Wiesmeijer, C. Verwoerd, S. Khazen, R. Eils, H.J. Tanke, R.W. Dirks, Visualizing telomeres dynamics in living mammalian cells using PNA probes, EMBO J. 22 (2003) 6631 – 6641. J. Amrichova´, E. Luka´sˇova´, S. Kozubek, M. Kozubek, Nuclear and territorial topography of chromosome telomeres in human lymphocytes, Exp. Cell Res. 289 (2003) 11 – 26. J. Wang, C. Shiels, P. Sasieni, P.J. Wu, S.A. Islam, P.S. Freemont, D. Sheer, Promyelocytic leukaemia nuclear bodies associate with transcriptionally active genomic regions, J. Cell Biol. 164 (2004) 515 – 526. V.G. Cheung, L.K. Conlin, T.M. Weber, M. Arcaro, K.-Y. Jen, M. Morley, R.S. Spielman, Natural variation in human gene expression assessed in lymphoblastoid cells, Nat. Genet. 33 (2003) 422 – 425. K.E. Brown, J. Baxter, D. Graf, M. Merkenschlager, A.G. Fisher, Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division, Mol. Cell 3 (1999) 207 – 217. B. Khor, B.P. Sleckman, Allelic exclusion at the TCRbeta locus, Curr. Opin. Immunol. 14 (2002) 230 – 234. L.S. Shopland, C.V. Johnson, M. Byron, J. McNeil, J.B. Lawrence, Clustering of multiple specific genes and gene-rich R-bands around SC-35 domains: evidence for local euchromatic neighbourhoods, J. Cell Biol. 162 (2003) 981 – 990. P. Heun, A. Taddei, S.M. Gasser, From snapshots to moving pictures: new perspectives on nuclear organization, Trends Cell Biol. 11 (2001) 519 – 525. J.R. Chubb, S. Boyle, P. Perry, W.A. Bickmore, Chromatin motion is constrained by association with nuclear compartments in human cells, Curr. Biol. 12 (2002) 439 – 445. S.-Y. Ng, P. Gunning, S.-H. Liu, J. Leavitt, L. Kedes, Regulation of the human h-actin promoter by upstream and intron domains, Nucleic Acids Res. 17 (1989) 601 – 615. E.V. Volpi, E. Chevret, T. Jones, R. Vatcheva, J. Williamson, S. Beck, R.D. Campbell, M. Goldsworthy, S.H. Powis, J. Ragoussis,

64

[48]

[49]

[50]

[51]

A.S. Quina, L. Parreira / Experimental Cell Research 307 (2005) 52–64 J. Trowsdale, D. Sheer, 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 (2000) 1565 – 1576. R.R.E. Williams, S. Broad, D. Sheer, J. Ragoussis, Subchromosomal positioning of the epidermal differentiation complex (EDC) in keratinocyte and lymphoblast interphase nuclei, Exp. Cell Res. 272 (2002) 163 – 175. S. Chambeyron, W.A. Bickmore, Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription, Genes Dev. 18 (2004) 1119 – 1130. E. Luka´sˇova´, S. Kozubek, M. Kozubek, M. Falk, J. Amrichova´, The 3D structure of human chromosomes in cell nuclei, Chromosome Res. 10 (2002) 535 – 548. M. Nogami, O. Nogami, K. Kagotani, M. Okumura, H. Taguchi, T. Ikemura, K. Okumura, Intranuclear arrangement of human chromosome 12 correlates to large-scale replication domains, Chromosoma 108 (2000) 514 – 522.

[52] R. Tam, K.P. Smith, J.B. Lawrence, The 4q subtelomere harboring the FSHD locus is specifically anchored with peripheral heterochromatin unlike most human telomeres, J. Cell Biol. 167 (2004) 269 – 279. [53] M.J. Hendzel, M.J. Kruhlak, D.P. Bazett-Jones, Organization of highly acetylated chromatin around sites of heterogeneous nuclear RNA accumulation, Mol. Biol. Cell 9 (1998) 2491 – 2507. [54] Y. Xing, C.V. Johnson, P.T. Moen Jr., J.A. McNeil, J. Lawrence, Nonrandom gene organization: structural arrangements of specific pre-mRNA transcription and splicing with SC-35 domains, J. Cell Biol. 131 (1995) 1635 – 1647. [55] F. Hediger, F.R. Neumann, G. Van Houwe, K. Dubrana, S.M. Gasser, Live imaging of telomeres: yKu and Sir proteins define redundant telomere-anchoring pathways in yeast, Curr. Biol. 12 (2002) 2076 – 2089. [56] M. Ferguson, D.C. Ward, Cell cycle dependent chromosomal movement in pre-mitotic human T-lymphocyte nuclei, Chromosoma 101 (1992) 557 – 565.