Since the advent of FISH (fluorescence in situ hybridization), there have been major advances in our understanding of how the genome is organized in interphase nuclei. Indeed, this organization is found to be non-random and individual chromosomes occupy discrete regions known as territories. Determining the factors that drive the spatial positioning of these territories within nuclei has caused much debate; however, in proliferating cells, there is evidently a correlation between radial positioning and gene density. Indeed, gene-poor chromosomes tend to be located towards the nuclear edge, while those that are more gene-rich are positioned more internally. These observations pose a number of questions: first, what is the function of this global organization and, secondly, is it representative of that occurring at a more local scale? During the course of this review, these questions will be considered, in light of the current literature regarding the role of transcription factories and the nuclear matrix in interphase genome organization.
Over the past decade, much progress has been made in order to elucidate the nuclear organization of the genome. It is now accepted that this organization is in fact non-random and that individual chromosomes occupy discrete regions known as chromosome territories. Interestingly, this spatial arrangement of chromosomes appears to alter both on differentiation  and exit from the cell cycle . Studies have shown that individual chromosomes have preferential neighbours in nuclei ; an organization which appears to be cell-type-specific .
A correlation between gene density and chromosome organization exists; generally, gene-rich chromosomes are positioned within the nuclear centre, whereas gene-poorer chromosomes are located towards the nuclear periphery . Within chromosomes, transcriptionally inactive alleles are embedded in the heart of the territory, while active alleles tend to be positioned either towards the territory edge or outside the territory altogether . Indeed, gene-rich chromatin loops radiating away from chromosome territories have been reported [7,8]. Recent research suggests that both GC content and replication timing could also be important for positioning chromatin domains within nuclear space [9,10].
Nuclear periphery: an area of repression?
Historically, the nuclear edge has been regarded as an area of transcriptional inactivity. Constitutive heterochromatin is consistently found to associate with both the nuclear periphery and nucleoli, while the early replicating, transcriptionally active, euchromatin occupies a more internal position. This observation concurs with the apparent gene-density-dependent radial positioning of chromosomes revealed by FISH (fluorescence in situ hybridization) studies . Unfortunately, however, the picture is not as straightforward as perhaps once imagined; work is continually being published which makes the transcriptional distinction between the nuclear edge and interior increasingly less certain.
Research shows that the nuclear interior is unquestionably a hot bed of transcriptional activity. RIDGEs (regions of increased gene expression) are found located within the nuclear interior, while anti-RIDGEs, transcriptionally inactive genomic regions, are often observed at the periphery [11–14]. Indeed, Kosak et al.  report that the majority of transcribed genes are positioned within the nuclear interior. In line with this, transcriptionally active alleles tend to cluster within the nuclear interior, unlike their inactive counterparts .
Is the nuclear periphery in mammals, however, incompatible with transcription? By tracking nascent RNA production, it is evident that transcription occurs throughout the nucleus, both within the nuclear interior and at the nuclear edge . Work performed using mice demonstrates that the IFNγ (interferon γ) locus is located at the nuclear periphery regardless of whether the gene is active or inactive . A study involving HIV further strengthens the opinion that the nuclear edge is not unsuited to gene expression . Recently, three groups have focused on the transcriptional consequences of artificially tethering regions of the genome to the nuclear edge in mammalian cells. Reddy et al.  reported that while cxcl5 and cxcl1 were significantly down-regulated as a result of such anchorage to the nuclear periphery, no noteworthy changes were observed in any of the other genes examined. Conclusions from the studies of Bickmore and co-workers  and Kumaran and Spector  appear to confirm this, in that targeting to the nuclear edge represses some, but not all, genes. To further complicate matters, it is emerging that NPCs (nuclear pore complexes) are in fact areas of high transcriptional activity; this view is, in part, formed by observing events occurring in both yeast and Drosophila . This chimes with Blobel's early gene-gating hypothesis where he suggested that high levels of gene transcription at NPCs would logically facilitate mRNA export .
What is the functional relevance of gene repositioning?
Specific genes are found to be repositioned from the nuclear periphery to the interior during their activation [25–27]. Other studies demonstrate that when expression is increased, loci move towards the nuclear interior, while when repressed they preferentially associate with the nuclear edge [28–31]. However, it is becoming apparent that rather than nuclear repositioning dictating transcriptional activity, it is expression that drives repositioning. Indeed, purely relocating the Mash1 locus from the periphery to a more interior position was not sufficient to activate transcription . Furthermore, the looping out of a gene from its chromosome territory, into a more interior position, was insufficient to up-regulate gene expression . Taken together, these conclusions infer that an internal nuclear position is more important for attaining high transcriptional activity, than for the actual activation event itself .
Gene positioning and transcription factories
It is generally accepted that transcription occurs in discrete foci known as transcription factories, which are found to be distributed throughout the nucleus, attached to an underlying nucleoskeleton. These structures were originally identified by tracking the incorporation of BrdU (bromodeoxyuridine) into nascent transcripts. The number of these factories per nucleus is still debatable, with reported values ranging from hundreds to thousands. They are also very small; on average, factories are 40–100 nm in diameter. Current models suggest that gene foci migrate towards these protein-rich factories, where they are fed through the immobilized RNA polymerase II complex and transcribed (see Figure 1). Transcription in these foci is catalysed by one of three distinct RNA polymerases; in general, RNA polymerase I is restricted to the nucleolus, while RNA polymerases II and III are nucleoplasmic (for a review, see ).
NM-associated RNA polymerase II transcription factories
Contrary to popular belief, mRNA is generated from active genes during infrequent bursts rather than continual periods of transcription . The majority of active genes reside within transcription factories, while inactive loci are found outside these structures . There is some evidence to indicate that certain genes preferentially co-transcribe. Using RNA FISH, Osborne et al.  demonstrated that in mouse B lymphocytes, the proto-oncogene, Myc, preferentially shares the same transcription factory as the highly transcribed Igh; a significant finding since the two gene loci are frequently involved in lymphoma-related translocation events. Importantly, it appears that functionally related genes come together in order to be transcribed. The mouse β-globin gene co-localizes, at high frequency, to the same transcription factory as other erythroid-specific genes (see Figure 2); an observation that has been proven by both RNA FISH and 4C (chromosome conformation capture-on-chip) studies [37,38]. In yeast, tRNA genes, which are generally scattered throughout the genome, are reported to cluster into nucleolar-based factories when transcribed . Furthermore, the transcription of specific snRNA genes is found to occur at coiled bodies .
The co-transcription of specific genes
Recent work by Peter Cook's group suggests that such factories appear to specialize in the production of certain transcript species. Minichromosomes were introduced into cells and their transcription tracked using FISH. Interestingly, only ~20 transcription factories were employed to transcribe the 8000 minichromosomes present per cell; this indicates: (i) that factory specificity exists and (ii) that one factory can process multiple transcripts simultaneously. The study reported that this selectivity was driven by intronic status and promoter type . This implies that specific sequences are transcribed by selected factories; thus, surely if this is the case, the distribution of different transcription factory types would influence the position of genes within the nucleus. Perhaps some genes are positioned within the vicinity of necessary factories, which contain the required transcription factors, while other genes need to migrate some distance in order to be expressed or repressed. This begs the question: which particular elements target gene sequences to specific transcription factories? Elucidating such factors will also aid in understanding the basis of genome organization.
What role does the NM (nuclear matrix) play in organizing the genome?
The NM or nucleoskeleton is proposed to be a permanent network of core filaments, underlying thicker fibres, present regardless of transcriptional activity. It is found to be both RNA and protein rich; indeed, treatment with RNase A destroys filament integrity. In addition to mediating the organization of entire chromosomes, the NM has also been demonstrated to tether telomeres via their TTAGGG repeats. Numerous proteins are found to associate with and form part of the NM; one such protein family is the lamins. It is apparent that these proteins partially mediate the structure's role in organizing the genome; both A- and B-type lamins bind MARs (matrix-attachment regions) as well as telomeric and repetitive DNA (for review see ).
Importantly, the NM provides the foundation on which transcription factories operate . A fully functional NM is required to support transcription; if lamin proteins are missing or mutant, this process is perturbed. Spann et al.  reported that the presence of mutant lamin A inhibits the activity of RNA polymerase. Furthermore, depleting lamin B1 levels by interference technology significantly disrupts RNA synthesis .
Since the NM has a role to play in mediating the organization of both chromatin and transcription factories, it is logical to hypothesize that the structure is also indirectly responsible for the positioning of genes. It is possible to conceive a situation whereby MARs tether chromatin to the NM at particular points, thus influencing the global organization of the genome. If these anchorage points are disrupted, this could, in theory, perturb both global (chromosome) and local (gene) chromatin structure.
Perturbed genome organization in disease
Within the literature, evidence exists which suggests that genome organization is perturbed in disease. In several cancers, certain human chromosomes are found to be incorrectly positioned [46,47]. This also occurs in laminopathy patients whose cells harbour mutations in LMNA; both HSA (human chromosome) 13 and 18 are found to occupy a more interior position than controls .
However, what effect does this disorganization have on the behaviour of specific genes and how does it contribute to pathology? The repositioning of chromosomes or genes seen in disease could merely reflect the perturbation of nuclear architecture and the inability of certain structures, such as the NM, to organize the genome. Most interestingly, gene positioning could be used for diagnostics since specific genes in breast tumour tissue are consistently mispositioned compared with control tissue .
It is possible that if genes are incorrectly positioned, their normal local environment changes significantly, thus altering the type of transcription factories available in the vicinity. As a result of this, gene expression could be misregulated, with genes either not participating in transcription or being forced to use suboptimal factories. Indeed, it is evident that the relationship between the positioning of certain genes and their transcriptional activity needs to be studied more extensively before being fully understood.
What can evolution tell us?
Perhaps one of the most thought-provoking articles on genome organization in recent years has been that published by Solovei et al. . The group investigated and compared the nuclear architecture of rod photoreceptor cells in both nocturnal and diurnal mammals. Significantly, the results questioned the widely accepted theory; that the heterochromatin-rich regions of the periphery are a staple in nuclear organization. The study discovered that, in rod photoreceptor cells of the mouse, this typical arrangement is in fact reversed; gene-dense regions decorate the nuclear edge, while gene-poor regions localize in the interior. In line with this, the majority of transcription machinery is also found peripherally. Interestingly, all genes, regardless of transcriptional activity, were located towards the nuclear edge. The authors reported that this ‘inverse’ pattern of organization in the retina was indeed associated with nocturnal vision; diurnal mammals have conventionally arranged rod photoreceptor nuclei. This finding has led to many questions; one of which centres around evolution. The demonstration that an ‘inverse’ version of the ‘conventional’ genome organization exists, suggests that each must confer an evolutionary advantage to the corresponding organisms. Solovei et al.  suggest that this inverse arrangement facilitates, in some way, the transmission of photons. Whether or not this is the sole reason, the study reveals the flexibility of the organization and indicates that it remains unchanged through evolution for a purpose. Evidently, this ‘purpose’ will be inextricably linked to transcriptional regulation; however, for the time being, the answer remains elusive. It is extremely important to identify those elements which encode the global organization of the genome.
Over the last decade, there have been major advances in our understanding of how the genome is organized. It is now becoming increasingly apparent that the functional relevance of this organization is somewhat complex; indeed, the transcriptional distinction between the nuclear periphery and nuclear interior is not as straightforward as once imagined. The repositioning of certain genes correlates with changes in expression; however, with other loci, it does not. It is likely that gene repositioning is used in order to regulate the expression of specific genes; it is probably not representative of a blanket genomic response. Furthermore, the significance and role of transcription factories in mediating this organization should not be overlooked. As our understanding of these factories increases, so too will our comprehension of genome organization and the nuclear structures which dictate it.
Nuclear Envelope Disease and Chromatin Organization 2009: Independent Meeting held at College of St Hild and St Bede, University of Durham, Durham, U.K., 22–23 April 2009. Organized and Edited by Chris Hutchison (Durham, U.K.).
L.S.E. is funded through European Union Framework Programme 7 EURO-Laminopathies grant and the Brunel Progeria Research Fund