Article |
Address correspondence to W.A. Bickmore, MRC Human Genetics Unit, Crewe Road, Edinburgh, EH4 2XU, UK. Tel.: 44-131-332-2471. Fax: 44-131-343-2620. E-mail: W.Bickmore{at}hgu.mrc.ac.uk
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Abstract |
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To investigate whether localization outside of the visible confines of chromosome territories can also occur for regions that are not coordinately regulated, we have examined the spatial organization of human 11p15.5 and the syntenic region on mouse chromosome 7. This region is gene rich but its genes are not coordinately expressed, rather overall high levels of transcription occur in several cell types. We found that chromatin from 11p15.5 frequently extends away from the chromosome 11 territory. Localization outside of territories was also detected for other regions of high gene density and high levels of transcription. This is shown to be partly dependent on ongoing transcription. We suggest that local gene density and transcription, rather than the activity of individual genes, influences the organization of chromosomes in the nucleus.
Key Words: gene density; chromatin; chromosome territories; nuclear organization; transcription
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Introduction |
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We have previously shown that individual human genes can be transcribed from within the interior of chromosome territories that are not located in the nuclear center (Mahy et al., 2002). This showed that genes do not need to be either at the visible surface of interphase chromosome territories, or at the centre of the nucleus, in order to be transcribed. These genes were located in regions of moderate gene-density (the R-band 11p13). In contrast, the gene-dense major histocompatibilty complex (MHC)* locus is frequently observed on loops of chromatin that extend away from the human chromosome 6 territory that is detected by FISH with a chromosome paint, particularly when transcription of genes from this region is induced (Volpi et al., 2000). Similarly, the epidermal differentiation complex (EDC) at 1q21 is frequently located outside of the chromosome 1 territory in keratinocytes, cells in which the genes of the EDC are highly expressed (Williams et al., 2002). It was not clear whether localization outside of chromosome territories was a particular feature of regions of the genome that contain genes with related functions, and that are coordinately expressed, or whether it might represent a more general facet of genome organization wherever genes are particularly clustered together, or where the overall levels of transcription from a large number of genes across a region is high.
To address this, we have used FISH to examine territory organization of regions of the human genome with high gene densities and generally high levels of transcription. The T-band 11p15.5 contains at least 47 known genes within the most distal 4.5 megabase (Mb) of DNA. We found that many megabases of this chromatin is frequently found outside of the visible confines of the 11p territory. By extending this observation to other gene-dense parts of the human genome including; 11q13 and 16p13.3 and gene-dense regions of chromosomes 21 and 22, we suggest that there is a correlation between domains of high gene density and localization outside of chromosome territories. We show that the frequency of extraterritory localization decreases, but is not eliminated, when transcription is inhibited. This level of higher-order genome organization is conserved in the mouse, indicating that it likely has functional significance. We suggest that the propagation of a decondensed chromatin fibre outside of the confines of chromosome territories creates an environment that is permissive to transcription increasing the overall transcriptional potential of the domain (Tumbar et al., 1999), and that the structure of chromosome territories is, in part, driven by transcription.
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Results |
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To address this, we have analyzed the organization of distal 11p15, including the very gene-rich, subtelomeric T-band 11p15.5. The most distal 4.5 Mb of HSA11p is well characterized due to its association with Beckwith-Wiedemann Syndrome, and because of the cluster of imprinted genes located there. The region contains at least 47 known genes (Redeker et al., 1994; Alders et al., 1997; Hu et al., 1997; Reid et al., 1997; Lee et al., 1999; Engemann et al., 2000; Onyango et al., 2000; Paulsen et al., 2000; Fig. 1). In addition, it has a density of CpG islands that is much higher than that of 11p13 (Craig and Bickmore, 1994).
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It was possible that distal loci appeared outside of the chromosome territory because the HSA11p paint did not include sequences near the end of the chromosome. However, examination of metaphase chromosomes showed that chromosome paint FISH signal extended right to the end of the chromosome arm, ending at a point coincident with a probe to the 11p telomere (unpublished data). Furthermore, the most proximal sequence on HSA11p, the centromere, was positioned at the visible edge (but inside of) the HSA11p territory (Fig. 3 b).
Location of chromatin outside of chromosome territories is not common to telomeric or imprinted regions
11p15.5 may locate outside of the chromosome territory because of its distal position close to the 11p telomere. There is some evidence for associations between different telomeres and gene-rich subtelomeric regions in human nuclei (Stout et al., 1999; Nagele et al., 2001). However, other telomeric sequences that we examined (e.g., 18pter and 18qter) were located at the edge of, but not outside of, their respective chromosome territories (unpublished data).
11p15 contains clusters of imprinted genes. The mechanistic basis of imprinting is yet to be fully defined, but aspects of higher-order chromatin structure have been implicated, and homologous loci of human and mouse genes subject to imprinting have been reported to be transiently associated during late S-phase (LaSalle and Lalande, 1996). We considered it unlikely that the nuclear organization of 11p15.5 was linked to the imprinted state of genes in this region, as both alleles were found outside of chromosome territories in many nuclei. However, we wished to determine whether other imprinted regions of the human genome were also located outside of chromosome territories. There is a large cluster of imprinted genes, associated with Prader-Willi and Angelmann syndromes, located at 15q1113. We found that loci from the imprinted region of 15q1113 are positioned within the HSA15q territory (Fig. 4) indicating that localization outside of chromosome territories is not a common feature of imprinted regions.
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To assess the long-range effects of gene density on intraterritory position, we used the published sequences of HSA21 and HSA22 (The chromosome 21 mapping and sequencing consortium, 2000; Dunham et al., 1999; Saccone et al., 2001) to identify gene-rich (and GC-rich) and gene-poor 1 Mb regions across the long arms of both chromosomes. In two-dimensional FISH to lymphoblast nuclei, the localization of each probe relative to the edge of the chromosome 21 or 22 territories corresponded well to the local estimated gene-density (Fig. 4), except for the gene-rich probe 154H4, which is close to the centromere of chromosome 22. In three-dimensional analysis the borders of the chromosome 21 and 22 territories were too indistinct to be able to reliably measure locus position. Linear regression of the data in Fig. 4 confirms the correlation between gene density and localization relative to the edge of chromosome territories (r2 = 67%).
Transcription has a role in localizing chromatin outside of chromosome territories
Localization of chromatin outside of territories could reflect an "open" chromatin structure across large regions poised for transcription, and/or could be due to the process of transcription itself. Not only are 11p15.5, 11q13, and 16p13 regions of high gene- and CpG island density but they are also domains where there is a high density of transcribed genes and where the levels of expression from many genes is high in many cell types (Caron et al., 2001).
To investigate this we analysed the nuclear organization of 11p15 and 11q13 loci after transcription had been inhibited with Actinomycin D (ActD) or 5,6-dichloro-b-ribofuranosylbenzimidazole (DRB; Chodosh et al., 1989; Croft et al., 1999). The proportion of signals from probe cI-11p15-46 (11p15.5) located >0.2 µm beyond the territory edge decreased from 42% in control cells to 35 and 28% in ActD or DRB-treated cells, respectively (Fig. 5 a). The 72% of signals from 80N22 in 11q13 observed outside of the 11q territory dropped to 60% in ActD treated cells (Fig. 5 b). Even the modest 20% of signals from the ß-globin locus (11p15.4) that could bee seen outside of the 11p territory in control cells was decreased to 15% of treated cells (Fig. 5 c). Hence ongoing transcription likely contributes to localization outside of chromosome territories, but gene dense domains can still locate outside of chromosome territories in the absence of transcription.
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Conservation of chromatin organization in the mouse
We have previously shown that the relative position of loci within chromosome territories is conserved between mouse and human (Mahy et al., 2002). To determine whether conservation of spatial organization extends to include loci that locate outside of chromosome territories, we examined the organization of loci from the region of the mouse chromosome 7 (MMU7) that is in conserved synteny with the BWS-associated region on HSA11p15.5 (Fig. 1). BACs 300P2 (Paulsen et al., 2000) and 245N5 (Engemann et al., 2000) were used in combination with an MMU7 chromosome paint (Jentsch et al., 2001) in two-dimensional FISH to MAA-fixed ES cell nuclei. 40% of 245N5 signals and 29% of 300P2 signals were outside of the MMU7 territory (Table I; Fig. 6). This contrasts with only 3% of signals from the Wt1 gene on MMU2, the region of conserved synteny to human 11p13 (Mahy et al., 2002). We conclude that the localization of gene-rich chromatin external to chromosome territories is conserved between human and mouse cells and that this indicates its functional importance.
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Discussion |
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To establish if localization outside of territories is not uncommon, we have analysed the organization of several gene-dense regions of the human genome. Here we show that one of the sites of highest gene density on chromosome 11p (11p15.5), but where the genes are functionally unrelated and have different patterns of gene expression, can also be found outside of the HSA11p chromosome territory (Table I; Figs. 2 and 3). We saw similar "extra-territory" localizations for two other gene-rich T-bands of the human genome (11q13 and 16p13; Figs. 2 e and 4). A strong correlation (r2 = 67%) between gene-density and chromosome territory organization was confirmed by a systematic analysis along the long arms of chromosomes 21 and 22 (Fig. 4). It is interesting to note that even though the cell types that we examined do not express globin genes, the localization of the ß-globin locus close to the surface of, but within, the chromosome 11 territory (Fig. 2 a; Kurz et al., 1996) contrasts with the localization of the -globin region outside of the chromosome 16 territory (Fig. 4). This adds to the growing list of differences in chromatin structure and nuclear organization that have been described for
-and ß globin genes (Brown et al., 2001).
Transcriptional activity can influence localization outside of territories
Localization of chromatin outside of the confines of chromosome territories could result from the process of transcription itself, or could reflect the structure of the chromatin fibre (e.g., histone modifications) in domains poised for transcription. Extrusion of the MHC and EDC loci from their chromosome territories is clearly related to the levels of transcription from these complexes (Volpi et al., 2000; Williams et al., 2002). Even though the loci that we have identified here as being frequently located outside of territories do not contain genes whose expression is coordinately switched on in the cell types that we studied, there are high levels of gene expression emanating from these regions. Genome-wide expression profiling using human ESTs highlighted the distal part of 11p, and regions likely corresponding to 11q13 and 16p13.3, as large regions of increased gene expression in many different cell and tissue types, including primary fibroblasts used in our study (Caron et al., 2001). Many of the genes in 16p13 are also known to be widely expressed (Daniels et al., 2001). Together with the results of Volpi et al. (2000) and Williams et al. (2002) this suggests that it may indeed be high levels of gene expression over a large genomic region, rather than just the density of genes per se, that determines whether chromatin domains will locate outside of chromosome territories.
To investigate this we examined the localization of loci outside of territories after treatment of cells with agents (ActD and DRB) that inhibit transcription. We did observe a decrease in the number of signals seen outside of chromosome territories in treated cells compared to controls and a concomitant increase in the signals located within the bulk chromosome territory (Fig. 5). Retraction of gene-dense domains into the confines of condensed chromosome territories in the absence of transcription is consistent with the compaction of the territory of (gene-dense) human chromosome 19 after treatment with ActD or DRB (Croft et al., 1999), and with the failure of an mouse mammary tumor virus promoter array to decondense upon steroid hormone addition in DRB-treated cells (Müller et al., 2001).
However, even in DRB or ActD-treated cells most signals from 11p15.5 and 11q13 loci are still outside of territories. ActD had most effect on territories in which the loops of chromatin extending beyond the territory edge contain only some of the loci from the region (Fig. 5 d) i.e., they may not be fully extended. The role of transcription may be during formation of the loops, whereas other factors, e.g., chromatin structure, may maintain them. The visible decondensation of a 90-Mb lacO array can be induced by a transcriptional activator, even in the absence of transcription itself, and is accompanied by increased levels of histone acetylation (Tumbar et al., 1999). 11p15.5, 11q13, and 16p13 are all regions of the human genome with hyperacetylated histones (Jeppesen, 1997), and are identified here as regions that frequently locate outside of chromosome territories.
Reconsidering the concept of chromosome territories
Volpi et al. (2000) suggested that FISH signals located outside of chromosome territories are the visual manifestation of chromatin decondensation over large regions. Here we have shown that this phenomenon is quite widespread, and not limited to clusters of coordinately regulated genes. Previous studies of long-range chromatin decondensation as the result of transcriptional activator binding (Tumbar et al., 1999) or steroid hormone recruitment and transcription (Müller et al., 2001) on artificial reporter arrays have tried to quantify the level of chromatin compaction. In our study of endogenous loci in human cells we observe a maximal distance between an extended 11p15.5 locus and the 11p territory of 2 µm in pFa-fixed cells, the mean distances being 1 µm (Fig. 3 c). Based on the human genome sequence, the genomic distance between 11ptel and IGF2 is
1.5 Mb (Fig. 1). This represents a fourfold higher level of compaction than that seen in the presence of transcription from a reporter array (2 Mb extending over an average of 6 µm; Müller et al., 2001). However, it is a similar level of decondensation to that reported by Tumbar et al. (1999)(90 Mb extended across a 30 µm fiber). Hence, there is still a large degree of higher-order structure, beyond a 30-nm fiber, in regions that extend out from chromosome territories
The data presented here, and previously published (Volpi et al., 2000; Williams et al., 2002) lead us to suggest that the organization of chromosomes within the nucleus is probably somewhere in between the complete decondensation of chromatin fibres like spaghetti on a plate suggested >30 y ago and the model of a discrete territorial organization favored recently (for review see Cremer and Cremer, 2001). Although the chromosome territory is a useful term to describe the appearance of hybridization signals from complex chromosome paints at the light microscope level, does it have any biological significance if there are many genomic regions contained outside of these domains and "invisible" by chromosome painting?
To answer this question it will be important to determine whether extended chromatin fibers are located in a "space" between chromosome territories, or whether they are embedded with the territories of other chromosomes. A light microscopy study of in vivo labelled chromatin has demonstrated that, although in general the borders between chromosome territories are well defined, fiber-like structures can be observed embedded in other chromosome territories (Visser and Aten, 1999).
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Materials and methods |
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FISH performed on MAA-fixed cells (two-dimensional analysis) or on three-dimensional preserved cells fixed with 4% pFa buffered in PBS, probe detection, examination of slides, and image capture, were as previously described (Mahy et al., 2002).
Image analysis
Analysis of probes located within chromosome territories in two-dimensional samples was as previously described (Mahy et al., 2002). Where probe signals appeared outside of the chromosome territory, the following script was used. Nuclear area was calculated from the segmented DAPI image. Locus-specific hybridization signals were segmented and a region of interest was manually defined around them. Hybridization signal from the chromosome territory was then segmented by thresholding, without knowledge of the locus signal, and a region of interest manually defined around the detectable territory. The area of the territory was calculated. A segmentation disc was dilated out from the locus signal centroid, and then eroded until a pixel containing territory signal was found. This was taken to be the nearest edge of the territory to the locus, and the radius of the disc was calculated, representing the distance (µm) from the centre of the locus to the nearest edge of the territory. A similar procedure was used to determine the distance between the territory centroid and territory edge. To verify the reproducibility of this analysis, the localization of 11p15 probes cI-11p15-46 and ß-globin LCR, as well as an 11q13 probe were assessed in lymphoblastoid cells in separate experiments by independent investigators. For the 11p15.5 probe cI-11p15-46, both investigators scored the mean position of the locus as outside of the chromosome 11p territory and >0.7 µm away from the territory edge (-1.0 ± 0.34; -0.70 ± 0.2). ß-Globin was measured within the 11p territory and close to the chromosome territory edge (0.29 ± 0.1; 0.1 ± 0.2).
Because actual territory size varied between chromosomes, between cell types, and between species, the locus to territory edge distance was normalized by dividing it by the radius of a circle of equal area to that of the territory. Thus, a value of 0 denotes a locus at the edge of a territory and negative values describe loci that locate outside of the detectable limits of the chromosome territory. A value of 1.0 denotes a locus at the theoretical territory center, but in practice values of 1.0 are not seen because territories are not circular. On this scale, the actual mean territory centroids were at 0.64±0.02 and 0.63±0.03 (HSA11p and q, respectively), 0.59±0.03 (HSA16p), and 0.66±0.03 (MMU7).
Three-dimensional images were analyzed as previously described (Mahy et al., 2002), using the program MAPaint (Mouse Atlas Project, http://genex.hgu.mrc.ac.uk/).
Statistical analyses of data by linear regression, and by Students t test, were carried out using Microsoft Excel.
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Footnotes |
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Acknowledgments |
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N. Mahy was supported by a studentship from the Medical Research Council, and W.A. Bickmore is a Centennial Fellow of the James S. McDonnell Foundation.
Submitted: 22 July 2002
Revised: 4 November 2002
Accepted: 4 November 2002
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