Nicola L. Mahy, Paul E. Perry, Susan Gilchrist, Richard A. Baldock and Wendy A. Bickmore

Medical Research Council Human Genetics Unit, Edinburgh EH4 2XU, United Kingdom

Address correspondence to Wendy A. Bickmore, MRC Human Genetics Unit, Crewe Rd., Edinburgh, EH4 2XU, UK.    Tel.:   +44-131-332-2471.     Fax:    +44-131-343-2620.
E-mail:    W.Bickmore@hgu.mrc.ac.uk

The position of genes within the nucleus has been correlated with their transcriptional activity. The interchromosome domain model of nuclear organization suggests that genes preferentially locate at the surface of chromosome territories. Conversely, high resolution analysis of chromatin fibers suggests that chromosome territories do not present accessibility barriers to transcription machinery.

To clarify the relationship between the organization of chromosome territories and gene expression, we have used fluorescence in situ hybridization to analyze the spatial organization of a contiguous 1 Mb stretch of the Wilms' tumor, aniridia, genitourinary anomalies, mental retardation syndrome region of the human genome and the syntenic region in the mouse. These regions contain constitutively expressed genes, genes with tissue-restricted patterns of expression, and substantial regions of intergenic DNA. We find that there is a spatial organization within territories that is conserved between mouse and humans: certain sequences do preferentially locate at the periphery of the chromosome territories in both species. However, we do not detect genes necessarily at the periphery of chromosome territories or at the surface of subchromosomal domains. Intraterritory organization is not different among cell types that express different combinations of the genes under study.

Our data demonstrate that transcription of both ubiquitous and tissue-restricted genes is not confined to the periphery of chromosome territories, suggesting that the basal transcription machinery and transcription factors can readily gain access to the chromosome interior.

Introduction:

There is a well-established functional link between chromatin structure at the level of the nucleosome and gene expression (Jenuwein and Allis, 2001). However, the functional significance of higher-order chromatin structures in transcription remains unclear and has been examined at different levels; from large scale structures above the 30-nm fiber to whole chromosome territories (Mahy et al., 2000). It was thought that large scale chromatin organization above the 30-nm fiber might affect transcription by presenting an accessibility barrier to large protein complexes (Zirbel et al., 1993; Kurz et al., 1996). Hence, it has been proposed that transcription and RNA processing might occur in a space between territories called the interchromosome domain (ICD)* compartment (for review see Cremer and Cremer, 2001). In support of this model, specific gene transcripts and components of the splicing machinery have been reported to concentrate at the border of chromosome territories (Zirbel et al., 1993). However, poly(A) RNA is not excluded from chromatin domains, and nascent RNA can be seen deep within chromosome territories (Abranches et al., 1998; Verschure et al., 1999). The idea of a compact chromosome territory that would act as a barrier to the
transcription machinery is contradicted by studies of chromatin fibers at higher resolution by both light microscopy and EM (Belmont et al., 1999).

If transcription does occur close to the surface of chromosome territories, genes should preferentially be found there, and noncoding sequences should be more internal. The intraterritory positions of a small number of individual genes from scattered genomic locations have been examined. In one report, three coding regions of the human genome were all located in the periphery of their respective chromosome territories independent of transcriptional status, whereas a noncoding sequence was not (Kurz et al., 1996). An active gene has also been found in a more peripheral location within the active X chromosome (Xa) territory than its inactive counterpart in the inactive X chromosome (Xi) territory (Dietzel et al., 1999). The imprinted SNRPN genes are found at the periphery of both chromosome 15 homologues (Nogami et al., 2000). Over larger regions, it has been shown that the gene-rich major histocompatibility complex (MHC) lies on large chromatin loops that extend away from the surface of the bulk chromosome 6 territory that is detectable with a
chromosome paint (Volpi et al., 2000). An extreme interpretation of these data is that most (active) genes lie on the surface of chromosome territories. However, with the exception of the Xi, both early and late replicating DNA that are usually equated with gene-rich and gene-poor domains, respectively, appear to be distributed throughout chromosome territories (Visser et al., 1998). Similarly, the most GC-rich fraction of the human genome (which has a high gene density) is also distributed throughout the volume of territories (Tajbakhsh et al., 2000).

Models of higher-order chromatin fiber and chromosome organization have implications for the spatial organization of genomic DNA in the nucleus both at the long range (chromosome band) level and at a more local megabase (Mb) level. To investigate the relationships between the organization of chromosome territories and gene expression, we have used fluorescence in situ hybridization (FISH) on both two-dimensional (2D) and three-dimensional (3D) samples to analyze the spatial organization of a contiguous 1 Mb stretch of the human genome and the syntenic region in the mouse. We have used cell types that express different repertoires of the genes from this region.

Distal human chromosome 11p13 is well mapped and sequenced because of its involvement in the Wilms' tumor, aniridia, genitourinary anomalies, mental retardation (WAGR) syndrome. It is moderately gene rich (Craig and Bickmore, 1994; Fantes et al., 1995; Gawin et al., 1999) and contains both ubiquitously expressed genes and genes whose expression is tissue restricted (Kent et al., 1997; Gawin et al., 1999; Kleinjan et al., 2002). There are also large (~300 kb) intergenic stretches of DNA (Kent et al., 1997; Gawin et al., 1999). Gene order and spacing are conserved at the region of conserved synteny on mouse chromosome 2 (MMU2E) (P. Gautier, personal communication) (http://www.ensembl.org/Mus_musculus/; http://www.ncbi.nlm.nih.gov/Omim/Homology/human11.html). We have compared the organization of these genomic regions to a more distal region on human chromosome 11 (HSA11) (11p15) and its region of conserved synteny in the mouse MMU7.

Our data show that there is spatial organization within chromosome territories. However, genes from 11p13 or MMU2E do not preferentially localize at the periphery of their chromosome territories. All of the 11p13 loci studied, including expressed genes, have a mean position well within the bulk chromosome territory compared with a locus from 11p15 that locates at the territory edge. Tissue-restricted genes are not relocated to the territory periphery in expressing cells.

We conclude that in general there is no gross remodelling of chromosome territory organization to accommodate small changes in gene expression within mammalian cells. Rather, it is more likely that small local changes in large scale chromatin fiber conformation accompany gene expression (Belmont et al., 1999). Our data may be compatible with the modified ICD model that suggests that this compartment penetrates into territories, ending at the surface of compact chromosomal subdomains of 0.3–0.45 µm diameter (Verschure et al., 1999; for review see Cremer and Cremer, 2001). Chromatin fibers containing transcriptionally active DNA may be then be decondensed at the surface of these sub-domains or extend into the interchromatin spaces (Verschure et al., 1999). However, although we have found that a ubiquitously expressed gene is indeed located at the surface of, or outside of, such subdomains and although adjacent noncoding DNA is located within the compact subdomain, we find no clear correlation between chromosome territory subdomains and the expression of tissue-restricted genes.
...

Discussion:

Transcription of genes can occur within the interior of a chromosome territory

Components of the splicing machinery and specific gene transcripts of an integrated human papilloma virus genome are often excluded from the interior of chromosome territories (Zirbel et al., 1993). Furthermore, three coding regions of the human genome have been observed at the periphery of chromosome territories, and a single nontranscribed sequence was randomly distributed or more internally positioned (Kurz et al., 1996). Within the context of the ICD compartment model of nuclear organization (Cremer and Cremer, 2001), an extreme interpretation of these data is that most or all genes lie on the periphery of chromosome territories and that the territory interior is filled with intergenic sequence (Zirbel et al., 1993; Kurz et al., 1996). However, here we have shown that both coding and noncoding sequences from 11p13 are similarly
located inside of the HSA11p territory. Both ubiquitously expressed genes and genes with a tissue-restricted expression pattern can be transcribed from within the territory interior (Figs. 3 and 6). This argues against the concept of a compact chromosome territory and indicates that the basal transcription machinery and transcription factors must be able to readily access chromatin located within the interior of chromosome territories. Our data indicate that large scale chromatin remodelling to position genes on the surface of a territory is not required to facilitate transcription but rather that any changes to the chromatin fiber are likely to be local (Belmont et al., 1999).

Therefore, our data question the extent to which the organization of chromosomes within territories can contribute to control of gene expression. Replication has been shown to take place in foci located throughout the entire chromosome territory volume (Visser et al., 1998). This demonstrates that activity of macromolecular enzyme complexes takes place throughout chromosome territories and is not confined to the territory surface (Zirbel et al., 1993; Kurz et al., 1996). In addition, transcription sites have been visualized throughout the nucleus (Abranches et al., 1998; Verschure et al., 1999), and using FRAP nuclear proteins with diverse functions and distinct distribution patterns have been shown to diffuse rapidly throughout the entire nucleus (Phair and Misteli, 2000). The fact that the human WAGR locus is positioned inside the HSA11p chromosome territory rather than at its periphery is consistent with these observations.

Intraterritory organization is not random and is conserved

Although we did not find genes from 11p13 preferentially at the periphery of the HSA11 territory, we did find that territories are spatially organized. In contrast to 11p13 DNA, sequences from 11p15 were preferentially located at the surface of the HSA11 territory (Fig. 3).

We extended our study of the WAGR locus to the syntenic region in the mouse. This is the first time that the organization of murine chromosome territories has been considered. We found that sequences, including expressed genes, from the murine WAGR locus were located in a similar relative position inside of the territory of MMU2 as the human region is within the HSA11p territory (Fig. 5). Likewise, the region of MMU7 in conserved synteny with HSA11p15 is located at the periphery of the MMU7 territory (Fig. 5). Conservation of sequence organization within chromosome territories suggests that it has functional significance, but it remains to be determined what that is. The conservation of spatial organization within chromosome territories is interesting in light of the conservation of territory position within the nucleus that has been seen in vertebrates (Croft et al., 1999; Boyle et al., 2001; Habermann et al., 2001; Tanabe et al., 2002).

Small scale changes in the transcription profile of a region do not require territory reorganization

Three studies have indicated a role for active transcription in organizing gene position within a chromosome territory (Dietzel et al., 1999; Volpi et al., 2000; Williams et al., 2002). Volpi et al. (2000) noted a difference in the incidence of extrusion of chromatin containing the MHC locus from the surface of HSA6 in cell lines with different expression profiles of MHC genes. Inducing transcription from this region using IFN increased the incidence of chromatin looping (Volpi et al., 2000). A relationship between transcription status and position was observed for a single gene within the Xa and Xi territories. The SLC25A5 (previously ANT2) gene was more peripheral within the Xa chromosome from which it is actively transcribed than within the Xi territory in which it is silent (Dietzel et al., 1999).

Using cell lines that express the tissue-restricted WT1 or PAX6 genes (Fig. 1), we found no significant alteration of the intraterritory position adopted by the WAGR locus that correlated with increased gene expression. Genes that are inactive in lymphoblasts and fibroblasts were not relocated to the HSA11p territory surface when actively transcribed but rather remain within the chromosome territory in a position similar to those of neighboring ubiquitously active genes and noncoding sequences (Fig. 6).

Differences in intraterritory organization dependent on transcription status of associated genes were ascertained previously within the context of chromosome domains subject to large scale transcriptional differences. The MHC locus consists of families of genes with the same or a related function; therefore, all of the genes are subject to the same regulation kinetics (Volpi et al., 2000). Similarly, the human epidermal differentiation complex at 1q21 contains functionally related genes involved in keratinocyte differentiation. This region appears to be extended outside of the HSA1 territory in keratinocytes where the genes are highly expressed but not in lymphoblasts where they are silent (Williams et al., 2002). Transcriptional activation of a 90-Mb heterochromatic region containing 256 copies of the lac operator sequence has also been associated with large scale chromatin decondensation (Tumbar et al., 1999). Lastly, the Xi adopts a unique structure which differs from the Xa territory (Eils et al., 1996; Dietzel et al., 1999) and is generally devoid of sites of transcription (Verschure et al., 1999). It is unclear how widely applicable observations made from study of X chromosome territory organization will be to the rest of the human karyotype.

Do levels of chromatin packaging within territories contribute to control of gene expression?

A high resolution chromosome painting study has suggested that locally compacted and unfolded regions within chromosome territories form distinct subdomains of 0.3–0.45 µm diameter and that chromatin is organized in such a way that transcriptionally active loci are at the surface of large scale chromatin fibers (Verschure et al., 1999). These data imply that limitations on protein accessibility within chromosome territories are likely to map to the periphery of large scale chromatin fibers or larger chromosome subdomains formed by the folding of these fibers. Transmission EM localization of uridine or BrUTP incorporation has shown heavy labeling at the edge of condensed large scale chromatin domains rather than at the surface of chromosomes itself (Fakan and Nobis, 1978; Wansink et al., 1996; Cmarko et al., 1999). Transcripts of 1a1 collagen located within the chromosome 17 territory similarly often coincide within holes in the chromosome paint (Clemson and Lawrence, 1996). Such observations have been used to extend the
concept of the ICD (now interchromatin domain) to include channels penetrating into chromosome territories (Cremer and Cremer, 2001), ending in branches between 1 Mb and 100 kb chromatin loop domains. Transcription apparently occurring within chromosome territories is therefore actually occurring on the "surface" of chromosome subdomains (Verschure et al., 1999). In accordance with this idea, we found that a ubiquitously transcribed gene often colocalized with unlabeled or less intensely labeled areas of the chromosome territory, whereas the linked intergenic locus was positioned frequently in intensely labeled (compact) subdomains of the territory (Fig. 7). The fact that we found gene sequences themselves and not just their transcripts in apparent "holes" in the chromosome territory implies that these are not in fact channels devoid of chromatin but rather that they correspond to areas of decondensed chromatin fibers not detectable by chromosome painting. The positions of genes with a tissue-restricted expression pattern with respect to
chromosome subdomains did not appear to correlate with their expression status. Therefore, the role of subchromosomal packaging in facilitating transcription remains open to debate and further investigation.

...

Footnotes:

* Abbreviations used in this paper: 2D, two-dimensional; 3D, three-dimensional; BAC, bacterial artificial chromosome; ES, embryonic stem; FISH, fluorescence in situ hybridization; ICD, interchromosomal domain/interchromatin domain; MAA, methanol acetic acid (3:1 vol/vol); Mb, megabase; MHC, major histocompatibility complex; pFa, paraformaldehyde; RT, reverse transcriptase; WAGR, Wilms' tumor, aniridia, genitourinary anomalies, mental retardation; Xa, active X chromosome; Xi, inactive X chromosome.

Acknowledgments:

We thank Hongen Zhang, Michael Bittner, and Jeffrey Trent (National Institutes of Health, Bethesda, MD) for providing us with HSA11p paint, Dirk-Jan Kleinjan and Colin Miles for mouse BACs, and Irina Solovei (University of Munich, Munich, Germany) for MMU2 paint. The CD5a cell line was a gift from Alan Prescott (University of Dundee, Dundee, UK). We are indebted to Andrew Carothers for input into data analysis and statistics.

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.

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