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Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells

Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human... Research Article Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells 1 2 2 1, Anne E. Wiblin , Wei Cui , A. John Clark and Wendy A. Bickmore * MRC Human Genetics Unit, Crewe Road, Edinburgh, EH4 2XU, UK Roslin Institute, Roslin BioCentre, Midlothian, EH25 9PS, UK *Author for correspondence (e-mail: [email protected]) Accepted 13 May 2005 Journal of Cell Science 118, 3861-3868 Published by The Company of Biologists 2005 doi:10.1242/jcs.02500 Summary Nuclear organisation is thought to be important in but instead we detect a relocalisation of the OCT4 locus, to regulating gene expression. Here we investigate whether a position outside its chromosome territory. There is also a human embryonic stem cells (hES) have a particular smaller proportion of centromeres located close to the nuclear organisation, which could be important for nuclear periphery in hES cells compared to differentiated maintaining their pluripotent state. We found that whereas cells. We conclude that hES cell nuclei have a distinct the nuclei of hES cells have a general gene-density-related nuclear architecture, especially at loci involved in radial organisation of chromosomes, as is seen in maintaining pluripotency. Understanding this level of hES differentiated cells, there are also distinctive localisations cell biology provides a framework within which other for chromosome regions and gene loci with a role in large-scale chromatin changes that may accompany pluripotency. Chromosome 12p, a region of the human differentiation can be considered. genome that contains clustered pluripotency genes including NANOG, has a more central nuclear localisation in ES cells than in differentiated cells. On chromosome 6p Key words: Centromere, Chromosome territory, Embryonic stem we find no overall change in nuclear chromosome position, cell, NANOG, Nucleus, OCT4 Introduction spatial and radial distribution of chromosomes have been documented in different tissues of the animal (Parada et al., The human genome is spatially organised within the nuclei of 2004) as well as during the differentiation of T cells (Kim et differentiated cells. There is a radial arrangement of al., 2004). However, to date no significant change in radial chromosome territories (CTs): gene-rich chromosomes such as position of a human chromosome within the nucleus has been chromosome 19 (HSA19) concentrate in the centre of the documented during differentiation, although there may be nucleus and more gene-poor chromosomes (e.g. chromosome changes in chromosome associations (Kuroda et al., 2004). 18) localise toward the nuclear periphery (Croft et al., 1999; Within CTs themselves, the position of gene clusters is Boyle et al., 2001; Cremer et al., 2001; Cremer et al., 2003). altered in different differentiated human cell types (Volpi et al., Centromeres are also generally found at the nuclear periphery, 2000; Williams et al., 2002). This aspect of nuclear or around nucleoli (Carvalho et al., 2001; Weierich et al., 2003; organisation has not been studied in human stem cells, but in Gilchrist et al., 2004), whereas telomeres are mainly found in the mouse, movement of specific genes out of CTs has been the nuclear interior (Weierich et al., 2003). Gene clusters, and seen upon the differentiation of ES cells (Chambeyron and individual chromosomal domains also have distinctive Bickmore, 2004). Human centromeres are localised close to localisations within respect to their CTs (Volpi et al., 2000; either the nuclear periphery or the nucleolus (Carvalho et al., Williams et al., 2002; Mahy et al., 2002a). In model organisms it is clear that nuclear organisation can 2001; Weierich et al., 2003). However, changes of centromere regulate gene expression (Spector, 2003). Data are consistent distribution in relation to cell cycle, physiological or differentiation state have been reported (reviewed by Gilchrist with nuclear organisation also being a determinant of gene et al., 2004). In addition, lineage-specific centromere expression for the human genome. Therefore, there may be associations into chromocentres have been reported during differences in the nuclear organisation of different cell types. lymphoid and myeloid differentiation, with an overall increase Indeed, in some human cell types (amniocytes and fibroblasts) in centromere clustering towards later stages of differentiation with flat/ellipsoid-shaped nuclei, HSA18 can be found toward the nuclear centre rather than at the nuclear periphery, as is (Beil et al., 2002; Alcobia et al., 2003). typical in cells with more spherical nuclei (lymphocytes, If nuclear organisation regulates gene expression, then it keratinocytes, colon and cervix epithelial cells) (Cremer et al., may have a key role in restricting it, as cells become more 2001; Cremer et al., 2003). In the mouse, differences in the committed to a differentiation pathway. Therefore it is Journal of Cell Science 3862 Journal of Cell Science 118 (17) important to determine how the genome is organised in the For 2D analysis, cells were swollen in 75 mM KCl before fixation in 3:1 methanol:acetic acid. Hybridisation was as described previously nucleus of pluripotent cells, and particularly in stem cells (Croft et al., 1999) but with the denaturing time reduced to 1.15 (Fisher and Merkenschlager, 2002). The organisation of human minutes for hES cells. For 3D analysis, hES cells were trypsinised and chromosomes and centromeres has been studied in washed twice in PBS before permeabilisation in CSK buffer (100 mM haemopoietic progenitor cells (Cremer et al., 2003) and in NaCl, 300 mM sucrose, 3 mM MgCl , 10 mM PIPES, pH 6.8, 0.5% + 2 CD34 stem cells from umbilical cord blood (Alcobia et al., Triton X-100) for 5 minutes on ice. After washing in PBS, cells were 2003). However, there have been no studies of nuclear fixed with 4% paraformaldehyde/PBS for 10 minutes, washed again organisation in hES cells. in PBS and cytospun onto slides at 11 g (Shandon, Cytospin3) for 5 Human ES cells have been derived from the inner cell mass minutes. Slides were then subjected to freeze-thaw in 20% of blastocysts, and as well as being able to self-renew, they glycerol/PBS and FISH was carried out as described previously (Croft have the ability to differentiate into all three embryonic germ et al., 1999). To check the preservation of nuclear structure after cytospinning, we compared the nuclear organisation of centromeres layers when injected into severe combined immunodeficient in primary fibroblasts grown on slides to that of primary fibroblasts mice (Thomson et al., 1998). It is anticipated that hES cells cytospun onto slides. will be an important tool for understanding early human After hybridisation, biotinylated probes were detected using development, with the hope that they may also have therapeutic fluorochrome-conjugated avidin (FITC or Texas Red) (Vector potential. Although they share many features with mouse ES Laboratories) followed by biotinylated anti-avidin (Vector (mES) cells, including the expression of common genes Laboratories) and a final layer of fluorochrome-conjugated avidin. important for pluripotency, there are also key differences Digoxigenin-labelled probes were detected with sequential layers of between mES and hES cells (Pera and Trounson, 2004; Ginis FITC-conjugated antidigoxigenin (BCL) and FITC-conjugated anti- et al., 2004). Moreover, there are fundamental differences in sheep antibody (Vector Laboratories). Slides were counterstained with the organisation of chromosomes between the human and 0.5 g/ml DAPI. Telomere FISH was carried out using a telomere PNA FISH Kit (DAKO). mouse genomes. Therefore, mES cells cannot serve as a suitable model for studying the nuclear organisation of human stem cells and an investigation of hES cell nuclei is required. Immunofluorescence Here we compared the nuclear organisation of differentiated Centromeres were detected by immunofluorescence using either a human cells with hES cells. We show that hES cells have a CENP-C antibody (gift of W. Earnshaw, Wellcome Trust Centre for radial organisation of chromosomes in the nucleus that relates Cell Biology, University of Edinburgh, UK) and FITC-conjugated to gene density and that is typical of many differentiated cell anti-rabbit secondary antibody, or CREST serum and a Texas Red types. However, we find differences in the localisation of anti-human secondary antibody. PML bodies were detected using chromosomes and gene loci with known roles in pluripotency. 5S10 monoclonal antibody and Texas Red anti-mouse secondary We also describe differences in centromere position in hES cell antibody. Nucleoli were detected using a Ki67 antibody and FITC- conjugated anti-rabbit secondary antibody. All secondary antibodies nuclei. were supplied by Jackson ImmunoResearch Laboratories. Materials and Methods Human ES cell culture and analysis Image capture and image analysis Human ES cell lines H1 (46XY), H7 and H9 (46XX) (Thomson et 2D slides were examined using a Zeiss Axioplan fluorescence al., 1998) were grown as previously described, with minor microscope fitted with a triple band-pass filter (Chroma #83000). modification (Xu et al., 2001). Briefly, the cells were cultured on Grey-scale images were captured with a cooled CCD camera Matrigel-coated culture dishes with mouse embryonic fibroblast (Princeton Instruments Pentamax) and analysed using custom IPLab conditioned medium supplemented with 8 ng/ml basic fibroblast scripts. For 3D analysis, a focus motor was used to collect images at growth factor. Cells were routinely split 1:3 with collagenase. H7 cells 0.25m intervals in the z-plane using a Xillig CCD camera. 3D image were passage (p)55. H1 cells were used at p42-65 and H9 cells were stacks were analysed using IPLab and deconvolved using Hazebuster at p39-55. (Vaytek). The cells were analysed by flow cytometry for the hES cell surface The radial distribution of CTs was determined in 2D specimens by antigens SSEA4 and Tra-1-60 using a FACScan (BD Biosciences). an erosion script, as previously described (Croft et al., 1999). The Briefly, hES cells were harvested by trypsin/EDTA and washed with radial distribution of specific gene loci was assessed manually across phosphate-buffered saline (PBS). After treatment with 10% goat the five erosion shells from the edge (shell 1) to the centre (shell 5) of serum to block non-specific binding, the cells were incubated with the nucleus. These distributions were normalised to the proportion of monoclonal antibodies against SSEA4 (1:5, DSHB, IA) or Tra-1-60 the total DAPI signal present in each shell. 3D chromosome position (1:12, Chemicon) on ice for 30 minutes. The cells were then treated was determined as previously described (Bridger et al., 2000). with goat anti-mouse IgG3-FITC or goat anti-mouse IgM-PE (both at Analysis of probe position relative to the surface of CTs, and 1:100, Southern Biotechnologies). Finally, 10 cells were acquired for interphase separation (d) were as previously described (Mahy et al., each sample and analysed with CELLQUEST software. 2002a; Mahy et al., 2002b; Chambeyron and Bickmore, 2004). Human (46XY) 1HD primary fibroblasts and FATO LCLs (46XY) Differences in the nuclear position of CTs and gene loci were tested were grown as described previously (Croft et al., 1999). for statistical significance using a Mann-Whitney U test in Minitab 13. This is a nonparametric test of the hypothesis that two groups come from the same distribution, without assuming that the data are Fluorescence in situ hybridisation normally distributed. Chromosome paints were labelled with biotin-16-dUTP by nick 3D analysis of centromeres and telomeres in the z-plane was translation or by PCR amplification (Croft et al., 1999) or obtained performed using a custom IPLab script. Briefly, the script defines the commercially (Cambio). BACs were labelled by nick translation with outline of the DAPI nucleus in each frame of the z-stack, calculates digoxigenin-11-dUTP. 200 ng paint and 70 ng BAC were used per the highest level of intensity for each fluorescent spot and locates slide, with 6 g human Cot1 DNA (GIBCO BRL) as competitor. which frame the spot is positioned in. Journal of Cell Science Human ES cell nuclear organisation 3863 Results An altered nuclear distribution of 12p in human ES cells HSA18 and 19 have a radial distribution in the nuclei of The data in Fig. 1 suggest that CTs in hES cell nuclei have a human ES cells gene-density-related radial organisation similar to that seen in many differentiated human cell types. (Cremer et al., 2001; The radial distribution of CTs in the nucleus, related to their Cremer et al., 2003). To determine whether there might be gene density, was first described for HSA18 and 19 (Croft et changes in the nuclear distribution of specific CTs in hES cells, al., 1999). These chromosomes are of approximately the same we examined the radial position of the CTs that carry genes with size (76 and 63 Mb, respectively) but HSA18 is very gene- a known role in maintaining the undifferentiated state. OCT4 poor, harbouring an estimated 449 genes, whereas HSA19 is (POU5F1) is located within a cluster of non-class I genes very gene-rich with 1528 genes (http://www.ensembl.org/ embedded within the MHC class I region on HSA6p21.33. Homo_sapiens/). HSA18 is found towards the nuclear OCT4 expression is essential to maintain the undifferentiated periphery in a variety of differentiated cells and HSA19 is in phenotype of hES cells (Matin et al., 2004). NANOG (12p13.31) the centre of the nucleus (Croft et al., 1999; Cremer et al., expression is also required to maintain the undifferentiated state 2003). This radial distribution is conserved amongst primates of hES cells (Zaehres et al., 2005). We hybridised chromosome (Tanabe et al., 2002) and it is also applicable to other human paints for 6p and 12p, together with BACs for OCT4 and chromosomes (Cremer et al., 2001; Boyle et al., 2001). NANOG, to nuclei from hES cells and lymphoblastoid cells We investigated the radial position of HSA18 and HSA19 in (LCLs) (Fig. 2A). The radial position of the CTs was the nuclei of H1 (XY) and H9 (XX) hES cells. The cells were established using the same erosion analysis as used in Fig. 1. cultured on Matrigel and the expression of cell-surface antigens We have previously reported that human chromosomes 6 and SSEA-4 and Tra-1-60 cells was analysed by flow cytometry. Of 12 have nuclear distributions in LCLs and fibroblasts that are the H1 cells, 70% were SSEA-4 positive and 55% were positive intermediate between those of HSA18 and HSA19, i.e. they are for Tra-I-60, indicating that most of the cells in the culture were located neither at the nuclear periphery, nor in the nuclear centre undifferentiated (Draper et al., 2002; Carpenter et al., 2004). (Boyle et al., 2001). This was confirmed here for 6p and 12p in Chromosome position was first established using fluorescence LCLs (Fig. 2B). There was no significant difference in the radial in situ hybridisation (FISH) with chromosome paints for position of 6p between LCLs and hES cells. However, 12p was HSA18 and 19 in 2D preparations (Fig. 1A). Although this located significantly closer to the nuclear centre (shell 5) in hES flattens nuclear morphology, it does not alter the measured cells compared to LCLs (P=0.04) (Fig. 2B). radial distribution of chromosomes (Croft et al., 1999), and it allows for rapid and automated analysis of large numbers of nuclei. The radial position of each CT was established from the Nuclear organisation of pluripotency genes in ES and distribution of hybridisation signal, relative to that of total DNA, differentiated cells in five erosion shells (Croft et al., 1999; Boyle et al., 2001). In both cell lines, HSA19 has a more central nuclear location than If CT radial position differs between ES cells and differentiated HSA18 (P0.001), and data for H1 cells is shown in Fig. 1B. cell types, then it might be expected that the radial position of This was confirmed by 3D analysis of H1 cells (Fig. 1C). specific gene loci on these chromosomes follow that of their HSA18 is significantly closer to the nuclear periphery than host chromosome. Consistent with this, NANOG (12p), but not HSA19 in the x and y-axes (P0.001), though differences OCT4 (6p) was located closer to the nuclear centre in hES through the z-axis were not significant (P=0.68). compared with LCLs (Fig. 2C). Fig. 1. The radial distribution of HSA18 and 19 in hES cells. (A) hES cell nuclei, counterstained with DAPI (blue) and hybridised with chromosome paints for HSA18 or 19. (B) Distribution of HSA18 and 19 hybridisation signals within the nucleus of H1 ES cells analysed by erosion of 2D images into five concentric shells from the edge (1) to the centre (5) of the nucleus. The mean (±s.e.m.) proportion of hybridisation signal, normalised to the amount of DAPI signal, is shown for each shell (n=50). (C) Analysis of HSA18 and 19 hybridisation signals within 3D-preserved hES cell nuclei. Graphs are the distributions of the centres of the HSA18 and 19 territories, along the fractional radius of each nucleus, along the x, y and z-axes (n=20). Bar, 5 m. Journal of Cell Science 3864 Journal of Cell Science 118 (17) As well as having a radial organisation within the nucleus, CTs also have a distinctive architecture themselves. In differentiated cells, gene-rich domains and regions of coordinately regulated gene expression, loop out from CTs (Volpi et al., 2000; Mahy et al., 2002a). One of the gene-rich domains of the human genome that we have previously shown to loop out from its CT in LCLs is the distal part of 11p15.5 (Mahy et al., 2002a). We found that loci from 11p15.5 (positions 0.25-2.1 Mb, NCBI build 35, http:// www.ensembl.org/Homo_sapiens) are also located outside the 11p territory in hES cell nuclei, even though this region of the genome does not contain any genes with a known role in maintaining pluripotency (Table 1). In contrast, RCN,which is expressed in both LCLs (Mahy et al., 2002b) and hES cells (Ramalho-Santos et al., 2002), but which is located in a low gene-density region at 11p13 (32Mb), remains inside the CT (Table 1). Therefore, CT architecture is well developed in hES cells, and is organised in a similar manner to differentiated cells, with regions of generally high gene density located out side of CTs. To determine whether a specific CT architecture could be detected at pluripotency genes expressed in ES cells, we analysed the intra-CT position of NANOG and OCT4. We measured the distances between hybridisation signals for BACs for the specific loci, and the visible edge of the hybridisation signal for the corresponding CT (Mahy et al., 2002). We found that NANOG is located well within the 12p CT in both LCL and ES cells (Table 1, Fig. 3A). However, the intra-CT behaviour of NANOG contrasts with that of OCT4, which is a non-class I gene, embedded within the MHC Class I region (Fig. 3A). Classical class I region genes are expressed constitutively in human LCLs and fibroblasts. Unlike mES cells, hES cells also express class I Fig. 2. Radial distribution of 6p, 12p, OCT4 and NANOG in ES genes (Tian et al., 1997; Drukker et al., 2002; Draper et al., cells. (A) Interphase hybridisation of BAC probes containing OCT4 2002; Carpenter et al., 2004). The Class I and Class III regions or NANOG (red), and chromosome paints for either 6p or 12p (green), within the nuclei of human ES cells counterstained with have been found outside CTs in LCLs (Volpi et al., 2000). We DAPI (blue). (B) Distribution of HSA6p and 12p hybridisation confirmed this using BACs that flank OCT4 and that contain signals within the nucleus of ES cells, by erosion of 2D images into either another non-class I gene (FLOT1), or the most 5 concentric shells from the edge (1) to the centre (5) of the centromeric genes of the class I region (MICB). We found that nucleus. The mean (±s.e.m.) proportion of hybridisation signal, these regions were located, on average, outside the 6p CT in normalised to the amount of DAPI signal, is shown for each shell hES cells (Table 1 and Fig. 3A). However, the mean position (n=50). C) Distribution of hybridisation signals from OCT4 or of the intervening OCT4 locus differed between ES and LCL NANOG-containing BACs within the nucleus of ES cells, by cells. On average, OCT4 was just inside the CT in LCLs, but erosion of 2D images into five concentric shells from the edge (1) outside the CT in ES cells (Table 1 and Fig. 3A). This to the centre (5) of the nucleus. The proportion of hybridisation difference was small, but statistically significant (P=0.041). signals, normalised to the amount of DAPI signal, is shown for each shell (n=50). Bar, 5 m. Analysing the distribution of distances revealed that this Table 1. Intra-CT position of loci in hES cells and LCLs Position relative to CT edge Cytogenetic Genomic position Locus position (Mb) Probe name LCL (m) ES (m) IFITM3 11p15.5 0.2 D11S483 –1.4±0.3 –0.70±1.13 INS 11p15.5 2.1 cINS/IGF2 –0.6±0.2 –0.55±0.15 RCN 11p13 32 cH11148 0.6±0.2 0.26±0.04 NANOG 12p13.31 7.8 RP11-358I17 0.23±0.06 0.32±0.04 FLOT1 6p21.33 30.8 RP11-324F19 –0.07±0.07 –0.11±0.08 OCT4 6p21.33 31.2 RP11-1058J10 0.03±0.06 –0.15±0.09 MICB 6p21.33 31.6 RP11-184F16 –0.25±0.09 –0.31±0.16 The cytogenetic position and genome position (from NCBI build 35, http://www.ensembl.org/Homo_sapiens) of each locus is indicated, together with the name of the cosmid or BAC probe used in FISH. Mean (±s.e.m.) position, in m, of specific loci relative to the edge of CTs in nuclei from hES cells and from LCLs. Negative values indicate positions outside the visible limits of the CT. LCL data for 11p15.5 loci is taken from Mahy et al., 2002a. Journal of Cell Science Human ES cell nuclear organisation 3865 chromatin structure (s.d.=0.52-0.6; median/mean ~1.0) (Sachs et al., 1995; Chambeyron and Bickmore, 2004). There was no -0.5 ES significant difference in the mean-squared interphase distance outside LCL -0.4 (<d >) between OCT4 and MICB BACS (genomic distance, 350 2 2 kb) for hES cells and LCLs (<d >=0.5±0.06 and 0.41±0.04 m -0.3 respectively, P=0.41). However, there was a significantly larger interphase separation between OCT4 and FLOT1 (genomic -0.2 2 2 distance, 400 kb) in LCLs (<d >=0.33±0.04 m ) compared to -0.1 2 hES cells (0.24±0.03 m ), P=0.04. In both LCLs and hES cells edge the large sizes of the d values measured around OCT4, are consistent with the presence of a generally open chromatin fibre structure, rather than a compact one (Gilbert et al., 2004). 0.1 These data suggest that both the intra-CT architecture and 0.2 the long-range chromatin configuration around the OCT4 locus inside differ between hES cells and a differentiated cell type that does 0.3 not express this marker of pluripotency. 7.7 7.9 30.6 30.8 31.0 31.2 31.4 31.6 12p 6p GDF3 GTF2H4 HLA-B MICB TNF DDR1 FLOT1 OCT-4 NANOG HLA-E DHX16 STELLA SLC2A14 HLA-C MICA BAT1BAT2 CLEC7 Localisation and clustering of centromeres in human ES Class I Class I Class III APOBEC1 cells RP11-324F19 RP11-1058J10 RP11-184F16 RP11-358I17 We detected distinctive nuclear organisation of chromosome arms and specific gene loci in hES cells. To investigate other non-genic regions we compared the position and number of centromere clusters in hES cells with that in two diploid OCT4 B 40 differentiated cell types: LCLs and primary fibroblasts. outside territory inside 35 ES territory edge territory Centromeres were detected in paraformaldehyde-fixed cells LCL using antibodies that recognise CENP-C or CREST serum. 25 There were no significant differences in the extent of centromere clustering between hES cells and these two differentiated cell types. The average number of centromere signals scored per cell was 34, 36 and 38 for ES, LCL and proliferating fibroblasts, respectively (n=20). Centromere position was analysed with respect to the nuclear periphery, or <-1.0 -1 to -0.6 -0.6 to -0.2 -0.2 to 0.2 0.2 to 0.6 0.6 to 1 1.0 to the nucleolus (detected with antibody that recognises pKi67) distance between probe and territory edge (μm) (Fig. 4A). A significantly lower proportion of centromeres was associated with the nuclear periphery of hES cells in Fig. 3. Intrachromosome territory organisation of NANOG and comparison with LCLs (P<0.04) or fibroblasts (P<0.001) (Fig. OCT4. (A) Position (mean±s.e.m.) in m, relative to the inside, edge 4B). Similar proportions of centromeres were associated with or outside CTs, for loci including NANOG and OCT4, as well as loci nucleoli in hES cells and fibroblasts (P>0.39). In hES cells a flanking OCT4, in the nuclei of hES cells () and LCLs () (n=100). Negative values indicate localisation outside the CT. The significantly higher proportion of centromeres were not map of the genomic regions around OCT4 and NANOG (according to associated with either the nuclear periphery or the nucleolus NCBI build 35) is shown below. Genes present in the BACs used are than either differentiated cell type (P<0.004). These highlighted in bold. (B) Histogram of the distribution of FISH differences were confirmed by examination of centromere signals from a BAC containing OCT4, relative to the edge of the distribution through the z-axis of nuclei (Fig. 4C). Centromeres chromosome 6p CT, in nuclei from hES cells (open bars) and LCLs have a normal distribution along the z-axis of ES cell nuclei, (filled bars). Negative distance indicates localisation outside the in contrast with a bimodal distribution towards the top and visible limits of the CT (n=100). bottom surface of the nucleus of fibroblasts. Telomeres are dispersed throughout the nucleoplasm of differentiated cells (Weierich et al., 2003). Most primary change in mean intra-CT position represented, not a change in diploid somatic cells, including fibroblasts, do not have active the overall percentage of OCT4 loci found well (>0.2 m) telomerase activity, and so are subject to progressive telomere outside the CT (36% for both LCLs and ES cells), but a shortening. Germ cells and stem cells in contrast have active reduction in the number of loci found deep within the CT (>0.6 telomerase, and robust telomerase activity is detected in hES m), and a consequent increase in the OCT4 loci positioned at cells (Thomson et al., 1998). We found that telomeres had a the CT edge (Fig. 3B). near-normal distribution in the centre of the nucleus of both During the differentiation of mouse ES cells, the movement hES cells and LCLs, though this is skewed towards the bottom of loci relative to the surface of CTs is generally accompanied of the nucleus in fibroblasts (Fig. 4D). by cytologically detectable changes in chromatin condensation Lastly, we also analysed the nuclear distribution of PML (Chambeyron and Bickmore, 2004). To investigate this further, bodies. The function of these nuclear bodies remains unknown, we measured the interphase distance (d) between OCT4 and the though they have been implicated in transcriptional regulation, flanking BAC clones. In all cases, the distribution of d values apoptosis, and DNA damage and stress sensing (Dellaire and conformed to that expected of a random-walk model of Journal of Cell Science mean distance from territory edge (μm) % of signals < 3866 Journal of Cell Science 118 (17) the radial organisation of CTs, is already established in hES cells. Gene-poor chromosome 18 is located toward the nuclear periphery of ES cells, whereas gene-rich HSA19 is more internal (Fig. 1). HSA18 is seen towards the nuclear periphery of a variety of differentiated cell types including lymphocytes (Croft et al., 1999), keratinocytes, and leukaemic and cancer cell lines (Cremer et al., 2003). However, a peripheral localisation of HSA18 was not seen in the very flat nuclei of amniotic fluid cells and quiescent fibroblasts (Bridger et al., 2000; Cremer et al., 2001). The nuclei of H1 ES cells are quite spherical (average height:length ratio=1.02±0.1), more similar to the shape of lymphocyte nuclei (ratio=1.00±0.1), than to those of fibroblasts (ratio=0.25±0.4). A differential localisation of HSA18 and 19 was also reported for granulocyte- macrophage colony-forming cells (GM-CFCs) and it has been suggested that radial distribution is also present in the pluripotent haematopoietic progenitor cells (Cremer et al., 2003). As we show that this radial distribution is already present in hES cells, we think it highly likely that a similar nuclear organisation will be present in most, if not all, foetal and adult stem cells. Chromosome 12p is located in the centre of the nucleus in ES cells Differences in the radial distribution of mouse chromosomes have been documented in different tissues and during T-cell differentiation (Parada et al., 2004; Kim et al., 2004). However, to date no significant change in radial position of a human chromosome within the nucleus had been documented during differentiation, although there may be changes in chromosome associations (Kuroda et al., 2004). Here we have detected a significantly more central nuclear Fig. 4. Centromere and telomere localisation in hES cells. localisation for the short arm of human chromosome 12 in ES (A) Localisation of centromeres (CREST, red), and nucleoli (Ki67, cells. It is interesting to note that recurrent gains of green) in single image frames, taken at 0.75 m intervals, through chromosome 12, including iso12p, have been found in human the z-axis of hES cell nuclei counterstained with DAPI (blue). Note ES cells (Draper et al., 2004). It has been suggested that the absence of centromeres from the nuclear periphery. (B) Mean increased dosage of genes on chromosome 12 (and therefore (±s.e.m.) proportion of centromeres per cell that are associated with presumably increased gene expression levels) is the nuclear periphery (left), the nucleolus (middle), or neither of advantageous to the propagation of undifferentiated ES cells. these nuclear compartments (right), in H1 ES cells (open bars), LCLs Although the functional significance of positioning in the (filled bars) and fibroblasts (hatched bars) (n=20). The mean nuclear centre of mammalian cells is unknown, the presence (±s.e.m.) distribution of (C) centromeres and (D) telomeres through the z-plane from the top (0) to the bottom (1) of nuclei from ES, LCL in this zone of the nucleus of the most gene-dense human and fibroblast cells (n=20). Bar, 5 m. chromosomes (Boyle et al., 2001) suggests that it may confer some transcriptional advantage. Chromosome 12p contains a cluster of genes whose expression is linked to the Bazett-Jones, 2004). Their nuclear distribution has not been maintenance of pluripotency. NANOG expression is required extensively studied, but many transcriptionally active genomic to maintain ES cells in an undifferentiated state (Zaehres et regions, including parts of the major histocompatibility al., 2005). It is located just proximal of two other genes, complex (MHC) at 6p, are reported to be associated with them STELLA and GDF3, which are also expressed in ES cells and (Wang et al., 2004). The average number of PML bodies scored downregulated upon differentiation (Clark et al., 2004). A in hES cells (11), is lower than that seen in LCLs (15) or BAC that covers this gene cluster also shows a more central fibroblasts (27), but despite the differences in abundance of nuclear position in hES cells when compared with LCLs (Fig. PML bodies between cell types, their intranuclear distribution 2C). Is it possible that it is the transcriptional activity of this towards the central mid-plane of the nucleus was the same in gene cluster that is driving the nuclear localisation of 12p in all three cell types (data not shown). hES cells? Preferential association of inactive genes with the nuclear Discussion periphery has been reported in differentiated cells, compared The radial distribution of chromosome territories is with their position in expressing cell types (Zink et al., 2004). present in hES cells However, we detect no association of either NANOG or OCT4 with the nuclear periphery in differentiated cells (Fig. 2C). We found that a major organisational feature of human nuclei, Journal of Cell Science Human ES cell nuclear organisation 3867 A relocalisation of OCT4, with respect to its Epigenome (LSHG-CT-2004-503433). We thank W. Earnshaw (University of Edinburgh) for the gift of anti-CenpC antibody. chromosome territory, in hES cells In contrast to the central nuclear localisation of 12p and NANOG in human ES cells, we detected no significant References difference in the radial nuclear position of 6p or OCT4 between Alcobia, I., Quina, A. S., Neves, H., Clode, N. and Parreira, L. (2003). The ES and differentiated cells. Both gene and chromosome spatial organization of centromeric heterochromatin during normal human lymphopoiesis: evidence for ontogenically determined spatial patterns. Exp. territory remain in an intermediate nuclear position (Fig. 2). Cell Res. 290, 358-369. However, we found that compared with LCLs, OCT4 is located Beil, M., Durschmied, D., Paschke, S., Schreiner, B., Nolte, U., Bruel, A. significantly closer to, or just beyond the CT edge in hES cells and Irinopoulou, T. (2002). Spatial distribution patterns of interphase (Fig. 3). In both cell types, the flanking Class I and Class III centromeres during retinoic acid-induced differentiation of promyelocytic leukemia cells. Cytometry 47, 217-225. MHC regions were located outside the 6p CT, consistent with Boyle, S., Gilchrist, S., Bridger, J. M., Mahy, N. L., Ellis, J. A. and other results (Volpi et al., 2000). The local chromatin structure Bickmore, W. A. (2001). The spatial organization of human chromosomes of OCT4 has not been studied in hES cells but, in the mouse, within the nuclei of normal and emerin-mutant cells. Hum. Mol. Genet. 10, increased DNA methylation and histone deacetylation of the 211-219. Oct4 enhancer/promoter are seen in trophoblast cells compared Bridger, J. M., Boyle, S., Kill, I. R. and Bickmore, W. A. (2000). Re- modelling of nuclear architecture in quiescent and senescent human with ES cells (Hattori et al., 2004). The data we have presented fibroblasts. Curr. Biol. 10, 149-152. here would be consistent with a more long-range remodelling Carpenter, M. K., Rosler, E. S., Fisk, G. J., Brandenberger, R., Ares, X., of chromatin architecture around OCT4, which might also Miura, T., Lucero, M. and Rao, M. S. (2004). Properties of four human contribute to its transcriptional regulation. embryonic stem cell lines maintained in a feeder-free culture system. Dev. Dyn. 229, 243-258. Therefore, for both of the best-studied genes involved in Carvalho, C., Pereira, H. M., Ferreira, J., Pina, C., Mendonca, D., Rosa, pluripotency, we find a distinctive nuclear organisation in A. C. and Carmo-Fonseca, M. (2001). Chromosomal G-dark bands human ES cells. In the case of NANOG, the whole chromosome determine the spatial organization of centromeric heterochromatin in the arm is localised towards the nuclear centre, whereas for OCT4 nucleus. Mol. Biol. Cell 12, 3563-3572. Chambeyron, S. and Bickmore, W. A. (2004). 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Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells

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Abstract

Research Article Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells 1 2 2 1, Anne E. Wiblin , Wei Cui , A. John Clark and Wendy A. Bickmore * MRC Human Genetics Unit, Crewe Road, Edinburgh, EH4 2XU, UK Roslin Institute, Roslin BioCentre, Midlothian, EH25 9PS, UK *Author for correspondence (e-mail: [email protected]) Accepted 13 May 2005 Journal of Cell Science 118, 3861-3868 Published by The Company of Biologists 2005 doi:10.1242/jcs.02500 Summary Nuclear organisation is thought to be important in but instead we detect a relocalisation of the OCT4 locus, to regulating gene expression. Here we investigate whether a position outside its chromosome territory. There is also a human embryonic stem cells (hES) have a particular smaller proportion of centromeres located close to the nuclear organisation, which could be important for nuclear periphery in hES cells compared to differentiated maintaining their pluripotent state. We found that whereas cells. We conclude that hES cell nuclei have a distinct the nuclei of hES cells have a general gene-density-related nuclear architecture, especially at loci involved in radial organisation of chromosomes, as is seen in maintaining pluripotency. Understanding this level of hES differentiated cells, there are also distinctive localisations cell biology provides a framework within which other for chromosome regions and gene loci with a role in large-scale chromatin changes that may accompany pluripotency. Chromosome 12p, a region of the human differentiation can be considered. genome that contains clustered pluripotency genes including NANOG, has a more central nuclear localisation in ES cells than in differentiated cells. On chromosome 6p Key words: Centromere, Chromosome territory, Embryonic stem we find no overall change in nuclear chromosome position, cell, NANOG, Nucleus, OCT4 Introduction spatial and radial distribution of chromosomes have been documented in different tissues of the animal (Parada et al., The human genome is spatially organised within the nuclei of 2004) as well as during the differentiation of T cells (Kim et differentiated cells. There is a radial arrangement of al., 2004). However, to date no significant change in radial chromosome territories (CTs): gene-rich chromosomes such as position of a human chromosome within the nucleus has been chromosome 19 (HSA19) concentrate in the centre of the documented during differentiation, although there may be nucleus and more gene-poor chromosomes (e.g. chromosome changes in chromosome associations (Kuroda et al., 2004). 18) localise toward the nuclear periphery (Croft et al., 1999; Within CTs themselves, the position of gene clusters is Boyle et al., 2001; Cremer et al., 2001; Cremer et al., 2003). altered in different differentiated human cell types (Volpi et al., Centromeres are also generally found at the nuclear periphery, 2000; Williams et al., 2002). This aspect of nuclear or around nucleoli (Carvalho et al., 2001; Weierich et al., 2003; organisation has not been studied in human stem cells, but in Gilchrist et al., 2004), whereas telomeres are mainly found in the mouse, movement of specific genes out of CTs has been the nuclear interior (Weierich et al., 2003). Gene clusters, and seen upon the differentiation of ES cells (Chambeyron and individual chromosomal domains also have distinctive Bickmore, 2004). Human centromeres are localised close to localisations within respect to their CTs (Volpi et al., 2000; either the nuclear periphery or the nucleolus (Carvalho et al., Williams et al., 2002; Mahy et al., 2002a). In model organisms it is clear that nuclear organisation can 2001; Weierich et al., 2003). However, changes of centromere regulate gene expression (Spector, 2003). Data are consistent distribution in relation to cell cycle, physiological or differentiation state have been reported (reviewed by Gilchrist with nuclear organisation also being a determinant of gene et al., 2004). In addition, lineage-specific centromere expression for the human genome. Therefore, there may be associations into chromocentres have been reported during differences in the nuclear organisation of different cell types. lymphoid and myeloid differentiation, with an overall increase Indeed, in some human cell types (amniocytes and fibroblasts) in centromere clustering towards later stages of differentiation with flat/ellipsoid-shaped nuclei, HSA18 can be found toward the nuclear centre rather than at the nuclear periphery, as is (Beil et al., 2002; Alcobia et al., 2003). typical in cells with more spherical nuclei (lymphocytes, If nuclear organisation regulates gene expression, then it keratinocytes, colon and cervix epithelial cells) (Cremer et al., may have a key role in restricting it, as cells become more 2001; Cremer et al., 2003). In the mouse, differences in the committed to a differentiation pathway. Therefore it is Journal of Cell Science 3862 Journal of Cell Science 118 (17) important to determine how the genome is organised in the For 2D analysis, cells were swollen in 75 mM KCl before fixation in 3:1 methanol:acetic acid. Hybridisation was as described previously nucleus of pluripotent cells, and particularly in stem cells (Croft et al., 1999) but with the denaturing time reduced to 1.15 (Fisher and Merkenschlager, 2002). The organisation of human minutes for hES cells. For 3D analysis, hES cells were trypsinised and chromosomes and centromeres has been studied in washed twice in PBS before permeabilisation in CSK buffer (100 mM haemopoietic progenitor cells (Cremer et al., 2003) and in NaCl, 300 mM sucrose, 3 mM MgCl , 10 mM PIPES, pH 6.8, 0.5% + 2 CD34 stem cells from umbilical cord blood (Alcobia et al., Triton X-100) for 5 minutes on ice. After washing in PBS, cells were 2003). However, there have been no studies of nuclear fixed with 4% paraformaldehyde/PBS for 10 minutes, washed again organisation in hES cells. in PBS and cytospun onto slides at 11 g (Shandon, Cytospin3) for 5 Human ES cells have been derived from the inner cell mass minutes. Slides were then subjected to freeze-thaw in 20% of blastocysts, and as well as being able to self-renew, they glycerol/PBS and FISH was carried out as described previously (Croft have the ability to differentiate into all three embryonic germ et al., 1999). To check the preservation of nuclear structure after cytospinning, we compared the nuclear organisation of centromeres layers when injected into severe combined immunodeficient in primary fibroblasts grown on slides to that of primary fibroblasts mice (Thomson et al., 1998). It is anticipated that hES cells cytospun onto slides. will be an important tool for understanding early human After hybridisation, biotinylated probes were detected using development, with the hope that they may also have therapeutic fluorochrome-conjugated avidin (FITC or Texas Red) (Vector potential. Although they share many features with mouse ES Laboratories) followed by biotinylated anti-avidin (Vector (mES) cells, including the expression of common genes Laboratories) and a final layer of fluorochrome-conjugated avidin. important for pluripotency, there are also key differences Digoxigenin-labelled probes were detected with sequential layers of between mES and hES cells (Pera and Trounson, 2004; Ginis FITC-conjugated antidigoxigenin (BCL) and FITC-conjugated anti- et al., 2004). Moreover, there are fundamental differences in sheep antibody (Vector Laboratories). Slides were counterstained with the organisation of chromosomes between the human and 0.5 g/ml DAPI. Telomere FISH was carried out using a telomere PNA FISH Kit (DAKO). mouse genomes. Therefore, mES cells cannot serve as a suitable model for studying the nuclear organisation of human stem cells and an investigation of hES cell nuclei is required. Immunofluorescence Here we compared the nuclear organisation of differentiated Centromeres were detected by immunofluorescence using either a human cells with hES cells. We show that hES cells have a CENP-C antibody (gift of W. Earnshaw, Wellcome Trust Centre for radial organisation of chromosomes in the nucleus that relates Cell Biology, University of Edinburgh, UK) and FITC-conjugated to gene density and that is typical of many differentiated cell anti-rabbit secondary antibody, or CREST serum and a Texas Red types. However, we find differences in the localisation of anti-human secondary antibody. PML bodies were detected using chromosomes and gene loci with known roles in pluripotency. 5S10 monoclonal antibody and Texas Red anti-mouse secondary We also describe differences in centromere position in hES cell antibody. Nucleoli were detected using a Ki67 antibody and FITC- conjugated anti-rabbit secondary antibody. All secondary antibodies nuclei. were supplied by Jackson ImmunoResearch Laboratories. Materials and Methods Human ES cell culture and analysis Image capture and image analysis Human ES cell lines H1 (46XY), H7 and H9 (46XX) (Thomson et 2D slides were examined using a Zeiss Axioplan fluorescence al., 1998) were grown as previously described, with minor microscope fitted with a triple band-pass filter (Chroma #83000). modification (Xu et al., 2001). Briefly, the cells were cultured on Grey-scale images were captured with a cooled CCD camera Matrigel-coated culture dishes with mouse embryonic fibroblast (Princeton Instruments Pentamax) and analysed using custom IPLab conditioned medium supplemented with 8 ng/ml basic fibroblast scripts. For 3D analysis, a focus motor was used to collect images at growth factor. Cells were routinely split 1:3 with collagenase. H7 cells 0.25m intervals in the z-plane using a Xillig CCD camera. 3D image were passage (p)55. H1 cells were used at p42-65 and H9 cells were stacks were analysed using IPLab and deconvolved using Hazebuster at p39-55. (Vaytek). The cells were analysed by flow cytometry for the hES cell surface The radial distribution of CTs was determined in 2D specimens by antigens SSEA4 and Tra-1-60 using a FACScan (BD Biosciences). an erosion script, as previously described (Croft et al., 1999). The Briefly, hES cells were harvested by trypsin/EDTA and washed with radial distribution of specific gene loci was assessed manually across phosphate-buffered saline (PBS). After treatment with 10% goat the five erosion shells from the edge (shell 1) to the centre (shell 5) of serum to block non-specific binding, the cells were incubated with the nucleus. These distributions were normalised to the proportion of monoclonal antibodies against SSEA4 (1:5, DSHB, IA) or Tra-1-60 the total DAPI signal present in each shell. 3D chromosome position (1:12, Chemicon) on ice for 30 minutes. The cells were then treated was determined as previously described (Bridger et al., 2000). with goat anti-mouse IgG3-FITC or goat anti-mouse IgM-PE (both at Analysis of probe position relative to the surface of CTs, and 1:100, Southern Biotechnologies). Finally, 10 cells were acquired for interphase separation (d) were as previously described (Mahy et al., each sample and analysed with CELLQUEST software. 2002a; Mahy et al., 2002b; Chambeyron and Bickmore, 2004). Human (46XY) 1HD primary fibroblasts and FATO LCLs (46XY) Differences in the nuclear position of CTs and gene loci were tested were grown as described previously (Croft et al., 1999). for statistical significance using a Mann-Whitney U test in Minitab 13. This is a nonparametric test of the hypothesis that two groups come from the same distribution, without assuming that the data are Fluorescence in situ hybridisation normally distributed. Chromosome paints were labelled with biotin-16-dUTP by nick 3D analysis of centromeres and telomeres in the z-plane was translation or by PCR amplification (Croft et al., 1999) or obtained performed using a custom IPLab script. Briefly, the script defines the commercially (Cambio). BACs were labelled by nick translation with outline of the DAPI nucleus in each frame of the z-stack, calculates digoxigenin-11-dUTP. 200 ng paint and 70 ng BAC were used per the highest level of intensity for each fluorescent spot and locates slide, with 6 g human Cot1 DNA (GIBCO BRL) as competitor. which frame the spot is positioned in. Journal of Cell Science Human ES cell nuclear organisation 3863 Results An altered nuclear distribution of 12p in human ES cells HSA18 and 19 have a radial distribution in the nuclei of The data in Fig. 1 suggest that CTs in hES cell nuclei have a human ES cells gene-density-related radial organisation similar to that seen in many differentiated human cell types. (Cremer et al., 2001; The radial distribution of CTs in the nucleus, related to their Cremer et al., 2003). To determine whether there might be gene density, was first described for HSA18 and 19 (Croft et changes in the nuclear distribution of specific CTs in hES cells, al., 1999). These chromosomes are of approximately the same we examined the radial position of the CTs that carry genes with size (76 and 63 Mb, respectively) but HSA18 is very gene- a known role in maintaining the undifferentiated state. OCT4 poor, harbouring an estimated 449 genes, whereas HSA19 is (POU5F1) is located within a cluster of non-class I genes very gene-rich with 1528 genes (http://www.ensembl.org/ embedded within the MHC class I region on HSA6p21.33. Homo_sapiens/). HSA18 is found towards the nuclear OCT4 expression is essential to maintain the undifferentiated periphery in a variety of differentiated cells and HSA19 is in phenotype of hES cells (Matin et al., 2004). NANOG (12p13.31) the centre of the nucleus (Croft et al., 1999; Cremer et al., expression is also required to maintain the undifferentiated state 2003). This radial distribution is conserved amongst primates of hES cells (Zaehres et al., 2005). We hybridised chromosome (Tanabe et al., 2002) and it is also applicable to other human paints for 6p and 12p, together with BACs for OCT4 and chromosomes (Cremer et al., 2001; Boyle et al., 2001). NANOG, to nuclei from hES cells and lymphoblastoid cells We investigated the radial position of HSA18 and HSA19 in (LCLs) (Fig. 2A). The radial position of the CTs was the nuclei of H1 (XY) and H9 (XX) hES cells. The cells were established using the same erosion analysis as used in Fig. 1. cultured on Matrigel and the expression of cell-surface antigens We have previously reported that human chromosomes 6 and SSEA-4 and Tra-1-60 cells was analysed by flow cytometry. Of 12 have nuclear distributions in LCLs and fibroblasts that are the H1 cells, 70% were SSEA-4 positive and 55% were positive intermediate between those of HSA18 and HSA19, i.e. they are for Tra-I-60, indicating that most of the cells in the culture were located neither at the nuclear periphery, nor in the nuclear centre undifferentiated (Draper et al., 2002; Carpenter et al., 2004). (Boyle et al., 2001). This was confirmed here for 6p and 12p in Chromosome position was first established using fluorescence LCLs (Fig. 2B). There was no significant difference in the radial in situ hybridisation (FISH) with chromosome paints for position of 6p between LCLs and hES cells. However, 12p was HSA18 and 19 in 2D preparations (Fig. 1A). Although this located significantly closer to the nuclear centre (shell 5) in hES flattens nuclear morphology, it does not alter the measured cells compared to LCLs (P=0.04) (Fig. 2B). radial distribution of chromosomes (Croft et al., 1999), and it allows for rapid and automated analysis of large numbers of nuclei. The radial position of each CT was established from the Nuclear organisation of pluripotency genes in ES and distribution of hybridisation signal, relative to that of total DNA, differentiated cells in five erosion shells (Croft et al., 1999; Boyle et al., 2001). In both cell lines, HSA19 has a more central nuclear location than If CT radial position differs between ES cells and differentiated HSA18 (P0.001), and data for H1 cells is shown in Fig. 1B. cell types, then it might be expected that the radial position of This was confirmed by 3D analysis of H1 cells (Fig. 1C). specific gene loci on these chromosomes follow that of their HSA18 is significantly closer to the nuclear periphery than host chromosome. Consistent with this, NANOG (12p), but not HSA19 in the x and y-axes (P0.001), though differences OCT4 (6p) was located closer to the nuclear centre in hES through the z-axis were not significant (P=0.68). compared with LCLs (Fig. 2C). Fig. 1. The radial distribution of HSA18 and 19 in hES cells. (A) hES cell nuclei, counterstained with DAPI (blue) and hybridised with chromosome paints for HSA18 or 19. (B) Distribution of HSA18 and 19 hybridisation signals within the nucleus of H1 ES cells analysed by erosion of 2D images into five concentric shells from the edge (1) to the centre (5) of the nucleus. The mean (±s.e.m.) proportion of hybridisation signal, normalised to the amount of DAPI signal, is shown for each shell (n=50). (C) Analysis of HSA18 and 19 hybridisation signals within 3D-preserved hES cell nuclei. Graphs are the distributions of the centres of the HSA18 and 19 territories, along the fractional radius of each nucleus, along the x, y and z-axes (n=20). Bar, 5 m. Journal of Cell Science 3864 Journal of Cell Science 118 (17) As well as having a radial organisation within the nucleus, CTs also have a distinctive architecture themselves. In differentiated cells, gene-rich domains and regions of coordinately regulated gene expression, loop out from CTs (Volpi et al., 2000; Mahy et al., 2002a). One of the gene-rich domains of the human genome that we have previously shown to loop out from its CT in LCLs is the distal part of 11p15.5 (Mahy et al., 2002a). We found that loci from 11p15.5 (positions 0.25-2.1 Mb, NCBI build 35, http:// www.ensembl.org/Homo_sapiens) are also located outside the 11p territory in hES cell nuclei, even though this region of the genome does not contain any genes with a known role in maintaining pluripotency (Table 1). In contrast, RCN,which is expressed in both LCLs (Mahy et al., 2002b) and hES cells (Ramalho-Santos et al., 2002), but which is located in a low gene-density region at 11p13 (32Mb), remains inside the CT (Table 1). Therefore, CT architecture is well developed in hES cells, and is organised in a similar manner to differentiated cells, with regions of generally high gene density located out side of CTs. To determine whether a specific CT architecture could be detected at pluripotency genes expressed in ES cells, we analysed the intra-CT position of NANOG and OCT4. We measured the distances between hybridisation signals for BACs for the specific loci, and the visible edge of the hybridisation signal for the corresponding CT (Mahy et al., 2002). We found that NANOG is located well within the 12p CT in both LCL and ES cells (Table 1, Fig. 3A). However, the intra-CT behaviour of NANOG contrasts with that of OCT4, which is a non-class I gene, embedded within the MHC Class I region (Fig. 3A). Classical class I region genes are expressed constitutively in human LCLs and fibroblasts. Unlike mES cells, hES cells also express class I Fig. 2. Radial distribution of 6p, 12p, OCT4 and NANOG in ES genes (Tian et al., 1997; Drukker et al., 2002; Draper et al., cells. (A) Interphase hybridisation of BAC probes containing OCT4 2002; Carpenter et al., 2004). The Class I and Class III regions or NANOG (red), and chromosome paints for either 6p or 12p (green), within the nuclei of human ES cells counterstained with have been found outside CTs in LCLs (Volpi et al., 2000). We DAPI (blue). (B) Distribution of HSA6p and 12p hybridisation confirmed this using BACs that flank OCT4 and that contain signals within the nucleus of ES cells, by erosion of 2D images into either another non-class I gene (FLOT1), or the most 5 concentric shells from the edge (1) to the centre (5) of the centromeric genes of the class I region (MICB). We found that nucleus. The mean (±s.e.m.) proportion of hybridisation signal, these regions were located, on average, outside the 6p CT in normalised to the amount of DAPI signal, is shown for each shell hES cells (Table 1 and Fig. 3A). However, the mean position (n=50). C) Distribution of hybridisation signals from OCT4 or of the intervening OCT4 locus differed between ES and LCL NANOG-containing BACs within the nucleus of ES cells, by cells. On average, OCT4 was just inside the CT in LCLs, but erosion of 2D images into five concentric shells from the edge (1) outside the CT in ES cells (Table 1 and Fig. 3A). This to the centre (5) of the nucleus. The proportion of hybridisation difference was small, but statistically significant (P=0.041). signals, normalised to the amount of DAPI signal, is shown for each shell (n=50). Bar, 5 m. Analysing the distribution of distances revealed that this Table 1. Intra-CT position of loci in hES cells and LCLs Position relative to CT edge Cytogenetic Genomic position Locus position (Mb) Probe name LCL (m) ES (m) IFITM3 11p15.5 0.2 D11S483 –1.4±0.3 –0.70±1.13 INS 11p15.5 2.1 cINS/IGF2 –0.6±0.2 –0.55±0.15 RCN 11p13 32 cH11148 0.6±0.2 0.26±0.04 NANOG 12p13.31 7.8 RP11-358I17 0.23±0.06 0.32±0.04 FLOT1 6p21.33 30.8 RP11-324F19 –0.07±0.07 –0.11±0.08 OCT4 6p21.33 31.2 RP11-1058J10 0.03±0.06 –0.15±0.09 MICB 6p21.33 31.6 RP11-184F16 –0.25±0.09 –0.31±0.16 The cytogenetic position and genome position (from NCBI build 35, http://www.ensembl.org/Homo_sapiens) of each locus is indicated, together with the name of the cosmid or BAC probe used in FISH. Mean (±s.e.m.) position, in m, of specific loci relative to the edge of CTs in nuclei from hES cells and from LCLs. Negative values indicate positions outside the visible limits of the CT. LCL data for 11p15.5 loci is taken from Mahy et al., 2002a. Journal of Cell Science Human ES cell nuclear organisation 3865 chromatin structure (s.d.=0.52-0.6; median/mean ~1.0) (Sachs et al., 1995; Chambeyron and Bickmore, 2004). There was no -0.5 ES significant difference in the mean-squared interphase distance outside LCL -0.4 (<d >) between OCT4 and MICB BACS (genomic distance, 350 2 2 kb) for hES cells and LCLs (<d >=0.5±0.06 and 0.41±0.04 m -0.3 respectively, P=0.41). However, there was a significantly larger interphase separation between OCT4 and FLOT1 (genomic -0.2 2 2 distance, 400 kb) in LCLs (<d >=0.33±0.04 m ) compared to -0.1 2 hES cells (0.24±0.03 m ), P=0.04. In both LCLs and hES cells edge the large sizes of the d values measured around OCT4, are consistent with the presence of a generally open chromatin fibre structure, rather than a compact one (Gilbert et al., 2004). 0.1 These data suggest that both the intra-CT architecture and 0.2 the long-range chromatin configuration around the OCT4 locus inside differ between hES cells and a differentiated cell type that does 0.3 not express this marker of pluripotency. 7.7 7.9 30.6 30.8 31.0 31.2 31.4 31.6 12p 6p GDF3 GTF2H4 HLA-B MICB TNF DDR1 FLOT1 OCT-4 NANOG HLA-E DHX16 STELLA SLC2A14 HLA-C MICA BAT1BAT2 CLEC7 Localisation and clustering of centromeres in human ES Class I Class I Class III APOBEC1 cells RP11-324F19 RP11-1058J10 RP11-184F16 RP11-358I17 We detected distinctive nuclear organisation of chromosome arms and specific gene loci in hES cells. To investigate other non-genic regions we compared the position and number of centromere clusters in hES cells with that in two diploid OCT4 B 40 differentiated cell types: LCLs and primary fibroblasts. outside territory inside 35 ES territory edge territory Centromeres were detected in paraformaldehyde-fixed cells LCL using antibodies that recognise CENP-C or CREST serum. 25 There were no significant differences in the extent of centromere clustering between hES cells and these two differentiated cell types. The average number of centromere signals scored per cell was 34, 36 and 38 for ES, LCL and proliferating fibroblasts, respectively (n=20). Centromere position was analysed with respect to the nuclear periphery, or <-1.0 -1 to -0.6 -0.6 to -0.2 -0.2 to 0.2 0.2 to 0.6 0.6 to 1 1.0 to the nucleolus (detected with antibody that recognises pKi67) distance between probe and territory edge (μm) (Fig. 4A). A significantly lower proportion of centromeres was associated with the nuclear periphery of hES cells in Fig. 3. Intrachromosome territory organisation of NANOG and comparison with LCLs (P<0.04) or fibroblasts (P<0.001) (Fig. OCT4. (A) Position (mean±s.e.m.) in m, relative to the inside, edge 4B). Similar proportions of centromeres were associated with or outside CTs, for loci including NANOG and OCT4, as well as loci nucleoli in hES cells and fibroblasts (P>0.39). In hES cells a flanking OCT4, in the nuclei of hES cells () and LCLs () (n=100). Negative values indicate localisation outside the CT. The significantly higher proportion of centromeres were not map of the genomic regions around OCT4 and NANOG (according to associated with either the nuclear periphery or the nucleolus NCBI build 35) is shown below. Genes present in the BACs used are than either differentiated cell type (P<0.004). These highlighted in bold. (B) Histogram of the distribution of FISH differences were confirmed by examination of centromere signals from a BAC containing OCT4, relative to the edge of the distribution through the z-axis of nuclei (Fig. 4C). Centromeres chromosome 6p CT, in nuclei from hES cells (open bars) and LCLs have a normal distribution along the z-axis of ES cell nuclei, (filled bars). Negative distance indicates localisation outside the in contrast with a bimodal distribution towards the top and visible limits of the CT (n=100). bottom surface of the nucleus of fibroblasts. Telomeres are dispersed throughout the nucleoplasm of differentiated cells (Weierich et al., 2003). Most primary change in mean intra-CT position represented, not a change in diploid somatic cells, including fibroblasts, do not have active the overall percentage of OCT4 loci found well (>0.2 m) telomerase activity, and so are subject to progressive telomere outside the CT (36% for both LCLs and ES cells), but a shortening. Germ cells and stem cells in contrast have active reduction in the number of loci found deep within the CT (>0.6 telomerase, and robust telomerase activity is detected in hES m), and a consequent increase in the OCT4 loci positioned at cells (Thomson et al., 1998). We found that telomeres had a the CT edge (Fig. 3B). near-normal distribution in the centre of the nucleus of both During the differentiation of mouse ES cells, the movement hES cells and LCLs, though this is skewed towards the bottom of loci relative to the surface of CTs is generally accompanied of the nucleus in fibroblasts (Fig. 4D). by cytologically detectable changes in chromatin condensation Lastly, we also analysed the nuclear distribution of PML (Chambeyron and Bickmore, 2004). To investigate this further, bodies. The function of these nuclear bodies remains unknown, we measured the interphase distance (d) between OCT4 and the though they have been implicated in transcriptional regulation, flanking BAC clones. In all cases, the distribution of d values apoptosis, and DNA damage and stress sensing (Dellaire and conformed to that expected of a random-walk model of Journal of Cell Science mean distance from territory edge (μm) % of signals < 3866 Journal of Cell Science 118 (17) the radial organisation of CTs, is already established in hES cells. Gene-poor chromosome 18 is located toward the nuclear periphery of ES cells, whereas gene-rich HSA19 is more internal (Fig. 1). HSA18 is seen towards the nuclear periphery of a variety of differentiated cell types including lymphocytes (Croft et al., 1999), keratinocytes, and leukaemic and cancer cell lines (Cremer et al., 2003). However, a peripheral localisation of HSA18 was not seen in the very flat nuclei of amniotic fluid cells and quiescent fibroblasts (Bridger et al., 2000; Cremer et al., 2001). The nuclei of H1 ES cells are quite spherical (average height:length ratio=1.02±0.1), more similar to the shape of lymphocyte nuclei (ratio=1.00±0.1), than to those of fibroblasts (ratio=0.25±0.4). A differential localisation of HSA18 and 19 was also reported for granulocyte- macrophage colony-forming cells (GM-CFCs) and it has been suggested that radial distribution is also present in the pluripotent haematopoietic progenitor cells (Cremer et al., 2003). As we show that this radial distribution is already present in hES cells, we think it highly likely that a similar nuclear organisation will be present in most, if not all, foetal and adult stem cells. Chromosome 12p is located in the centre of the nucleus in ES cells Differences in the radial distribution of mouse chromosomes have been documented in different tissues and during T-cell differentiation (Parada et al., 2004; Kim et al., 2004). However, to date no significant change in radial position of a human chromosome within the nucleus had been documented during differentiation, although there may be changes in chromosome associations (Kuroda et al., 2004). Here we have detected a significantly more central nuclear Fig. 4. Centromere and telomere localisation in hES cells. localisation for the short arm of human chromosome 12 in ES (A) Localisation of centromeres (CREST, red), and nucleoli (Ki67, cells. It is interesting to note that recurrent gains of green) in single image frames, taken at 0.75 m intervals, through chromosome 12, including iso12p, have been found in human the z-axis of hES cell nuclei counterstained with DAPI (blue). Note ES cells (Draper et al., 2004). It has been suggested that the absence of centromeres from the nuclear periphery. (B) Mean increased dosage of genes on chromosome 12 (and therefore (±s.e.m.) proportion of centromeres per cell that are associated with presumably increased gene expression levels) is the nuclear periphery (left), the nucleolus (middle), or neither of advantageous to the propagation of undifferentiated ES cells. these nuclear compartments (right), in H1 ES cells (open bars), LCLs Although the functional significance of positioning in the (filled bars) and fibroblasts (hatched bars) (n=20). The mean nuclear centre of mammalian cells is unknown, the presence (±s.e.m.) distribution of (C) centromeres and (D) telomeres through the z-plane from the top (0) to the bottom (1) of nuclei from ES, LCL in this zone of the nucleus of the most gene-dense human and fibroblast cells (n=20). Bar, 5 m. chromosomes (Boyle et al., 2001) suggests that it may confer some transcriptional advantage. Chromosome 12p contains a cluster of genes whose expression is linked to the Bazett-Jones, 2004). Their nuclear distribution has not been maintenance of pluripotency. NANOG expression is required extensively studied, but many transcriptionally active genomic to maintain ES cells in an undifferentiated state (Zaehres et regions, including parts of the major histocompatibility al., 2005). It is located just proximal of two other genes, complex (MHC) at 6p, are reported to be associated with them STELLA and GDF3, which are also expressed in ES cells and (Wang et al., 2004). The average number of PML bodies scored downregulated upon differentiation (Clark et al., 2004). A in hES cells (11), is lower than that seen in LCLs (15) or BAC that covers this gene cluster also shows a more central fibroblasts (27), but despite the differences in abundance of nuclear position in hES cells when compared with LCLs (Fig. PML bodies between cell types, their intranuclear distribution 2C). Is it possible that it is the transcriptional activity of this towards the central mid-plane of the nucleus was the same in gene cluster that is driving the nuclear localisation of 12p in all three cell types (data not shown). hES cells? Preferential association of inactive genes with the nuclear Discussion periphery has been reported in differentiated cells, compared The radial distribution of chromosome territories is with their position in expressing cell types (Zink et al., 2004). present in hES cells However, we detect no association of either NANOG or OCT4 with the nuclear periphery in differentiated cells (Fig. 2C). We found that a major organisational feature of human nuclei, Journal of Cell Science Human ES cell nuclear organisation 3867 A relocalisation of OCT4, with respect to its Epigenome (LSHG-CT-2004-503433). We thank W. 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Journal of Cell Science – The Company of Biologists

Published: Sep 1, 2005

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