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Chromosome organization: new facts, new models
Review
TRENDS in Cell Biology
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Chromosome organization: new facts, new models Miguel R. Branco and Ana Pombo MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK
The study of nuclear organization has radically changed the way we envision gene regulation, imposing a paradigm shift from a seemingly featureless nucleus to a highly compartmentalized and complex organelle. The positioning of genes, regulatory sequences and transcription factors in relation to each other and to landmarks in the nucleus, such as nuclear bodies and the lamina, is important in determining which genes are transcribed at any one time. Investigating chromatin organization during interphase is therefore essential to the understanding of gene expression. The recent discovery of interactions between distal chromatin segments that occur within the same chromosome or across different chromosomes, and that have a role in transcription regulation, suggests a re-evaluation of current models of chromosome organization and the development of new ones. Spatial regulation of gene activity The human genome comprises 20 000–25 000 protein-coding genes [1], of which only a fraction is transcribed at any one time in each cell. Transcription is tightly regulated to ensure correct expression of the genes required in each phase of the cell cycle, stage of differentiation and environmental condition that is imposed on the cell. It is therefore not surprising that increasingly complex layers of gene regulation have been discovered over the years. The linear DNA sequence contains regulatory elements, such as promoters and enhancers, which activate genes depending on the availability of particular transcription factors [2]. Furthermore, the accessibility of transcription factors and RNA polymerases (Pols) to DNA is influenced by the condensation state of chromatin, which is in turn regulated by several epigenetic determinants such as histone modifications, binding of heterochromatin protein 1 (HP1) and Polycomb group (PcG) (see Glossary) proteins, and the activity of chromatin remodelling complexes [3–5]. An extra layer of regulation that has been emerging as crucial to gene regulation is the spatial organization of factors and sequences within the nucleus [6–9]. Several nuclear components are found compartmentalized in nuclear bodies that are distinct in their composition, morphology and abundance [10,11]. The nucleolus is the most prominent and well studied nuclear body, and harbours all the machinery essential for the transcription of 45S rRNA genes, including Pol I [6]. This imposes spatial Corresponding author: Branco, M.R. ([email protected]). Available online 2 January 2007. www.sciencedirect.com
restrictions on rRNA gene-containing chromosomes, which have a nucleolar-organizing region (NOR) that promotes their association with nucleoli even when they are inactive [12]. Transcription by Pols II and III has also been shown to occur in sites containing many Pols, termed ‘transcription factories’ (50 nm diameter in HeLa, F9 and ES cells, and amphibian A1 cells), which are evenly distributed throughout the nucleoplasm, and which might similarly restrict the positioning of other genes on a smaller scale [6,13,14]. Other nuclear bodies, such as speckles, promyelocytic leukaemia (PML) bodies and Cajal bodies, have been shown to have roles in mRNA maturation, RNP assembly and other nuclear processes [11]. However, a better understanding of the function of these compartments is still necessary, especially in regard to whether their spatial proximity to certain genes is important for transcription
Glossary Cajal bodies (or coiled bodies): nuclear bodies rich in the protein coilin and other factors involved in the biogenesis of small nuclear ribonucleoprotein particles. Chromosome conformation capture (3C): biochemical technique for the detection of interactions between chromatin segments within the nucleus. It is based on crosslinking of proximal fragments through protein bridges, digestion of chromatin, intramolecular ligation of crosslinked fragments and their detection by PCR. Chromosome conformation capture-on-chip (4C) [29]: genome-wide adaptation of 3C, in which all of the crosslinking events to a fragment of interest can be analysed by microarrays. Heterochromatin protein 1 (HP1): binds chromatin preferentially at heterochromatic regions by interaction of its chromodomain with the repressive mark methylated lysine 9 at histone H3 and recruits the corresponding methyltransferase, Su(var)3-9. Locus control region (LCR): an enhancer element that ensures high expression levels independent of integration site of an associated transgene. Nucleolar organizing region (NOR): chromosomal locus containing ribosomal RNA genes. In humans, they are present in five chromosomes: 13, 14, 15, 21 and 22. Open-ended 3C [28] and circular chromosome conformation capture (also abbreviated 4C) [30]: other genome-wide adaptations of 3C, but in which crosslinked fragments are analysed by sequencing. Polycomb group (PcG): family of proteins involved in repression of homeotic genes, which form multimeric protein complexes. The Polycomb repressive complex 2 (PRC2) has a role in the deposition of repressive marks, whereas PRC1 is involved in the maintenance of silent chromatin. Promyelocytic leukaemia (PML) bodies: nuclear bodies rich in the PML protein. A translocation causing the fusion of PML with retinoic acid receptor (RAR) a leads to acute PML and disrupts the formation of PML bodies. RNA tagging and recovery of associated proteins (RNA-TRAP): biochemical technique for the detection of chromatin fragments interacting with active genes. It involves tagging nascent transcripts, with subsequent purification of the chromatin found in close proximity to the tagged RNA and the detection of specific chromatin regions by PCR. Splicing speckles: nuclear bodies rich in splicing factors and poly(A)+ RNA. Xist: a noncoding RNA that coats one of the female X chromosomes early in the process of X inactivation.
0962-8924/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2006.12.006
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regulation [15]. Association of genes with other nuclear landmarks, such as the lamina [16] or centromeric clusters [17], has been correlated with gene silencing. Spreading of repressive marks in these heterochromatic regions in a nonlinear, three-dimensional way (as opposed to spreading along the linear DNA sequence) to associated genes might have a role in the spatial regulation of transcription. It is in this context that the study of chromosome organization during interphase is essential for understanding gene expression. Each chromosome occupies its own distinct space or territory (Figure 1), and assumes a nonrandom position within the nucleus [18–20]. Despite the apparent simplicity of this arrangement, the initial models of chromosome organization have been challenged by recent observations and technical developments. It seems that interactions between distal segments of chromatin, within the same chromosome or across different chromosomes, are more common than previously thought [13,21–30]. These numerous examples of functionally relevant intra- and interchromosomal associations attribute further importance to the organization of chromatin in the process of gene expression, and move us to revise current models of chromosome organization. Here, we review three models [chromosome territory–interchromatin compartment (CT-IC), lattice and interchromosomal network (ICN) models] that describe different aspects of chromosome structure and inevitably bring about different views on how long-range chromatin interactions might be established. Other aspects of chromatin organization or spatial regulation of gene expression are embedded within the discussion of these three models, or have been omitted owing to limitations of space. Chromosome organization: the early days When Flemming first observed mitotic chromosomes (see [31] for a review), he could only speculate as to what happened to them once cells entered interphase. Given that chromosomes were no longer seen as separate entities within the cell nucleus, the most likely scenario was that they unfolded and became entangled. A more organized view of chromatin came from the studies of Rabl and Boveri, who showed that plant chromosomes maintain a polarized orientation during interphase, with centromeres and telomeres located to opposite sides of the nucleus, reflecting their conformation during mitosis (the so-called Rabl configuration) (see [8] for a review). From this observation, they also suggested that chromosomes might maintain their spatial separation and occupy distinct territories in the nucleus. Evidence for the existence of chromosome territories (CTs) in interphase nuclei was first originated in mammalian cells. In an elegant study, Thomas Cremer et al. [32] microirradiated Chinese Hamster nuclei with a UV laser, and visualized the sites of damage at the following metaphase. Instead of obtaining a scattered signal across several chromosomes, the authors observed discrete labelling in parts of only a few chromosomes, readily indicating that chromatin must be highly compartmentalized within the nucleus. Direct visualization of CTs in mammalian interphase nuclei was only possible after the development of whole chromosome paint probes, in combination with fluorescence in situ hybridization www.sciencedirect.com
Figure 1. Chromosomes occupy discrete territories during interphase. Two chromosome-specific paints (chromosome 1 in green, chromosome 2 in red) were hybridized to a cryosection (100–200 nm thick) of human T lymphocytes by cryo-FISH [46], revealing the territorial organization of chromosomes during interphase.
(FISH) [33,34] (Figure 1). This has since become a standard tool that has enabled extensive study of chromosome organization during interphase, and that in combination with other methods has given rise to the models that we summarize here. The CT-IC model The discovery of a territorial organization of chromosomes during interphase brought about several questions. Namely, how are genes and regulatory regions organized within a CT, and is there a relationship between position and activity? In experiments that aimed to investigate where transcription and splicing occurred in relation to CTs, Zirbel et al. [35] simultaneously labelled CTs and splicing speckles (as labelled by the Sm antigen) or the transcript of an integrated human papilloma virus genome. Both the viral transcripts and the Sm antigen showed a preferential position outside CTs, accumulating in a nuclear compartment distinct from the CTs. The domain that apparently separated CTs was named the interchromosome domain (ICD), and a model emerged in which active genes would be located at the periphery of CTs so that they would be accessible to transcription and splicing factors [36] (Figure 2a). Conversely, inactive genes would be located in the interior of CTs, which limited the accessibility of the transcription machinery. Initial studies on the position of chromatin segments in relation to their CT seemed to support this view because coding sequences often located to the periphery of CTs and noncoding sequences towards the interior [37]. Moreover, a small number of genes were shown to loop out of their own CT following activation [38,39], suggesting a migration into the ICD to facilitate transcription. However, a generalized correlation between position relative to the CT and gene activity has not been found; coding sequences can be seen
Review
TRENDS in Cell Biology
at the interior of CTs, irrespective of their transcriptional status [40], and noncoding sequences are seen at the periphery of CTs [41]. Interestingly, in the case of X inactivation during ES cell differentiation, there is evidence that Xist-mediated gene silencing brings inactive genes to the centre of the inactive X chromosome, whereas genes escaping inactivation are located at the periphery [42] (this conformation seems to change when epigenetic marks take over the silencing role of Xist [43]). Apart from this exception, transcription sites are found evenly scattered across the nucleus, including within CTs [44–46]. To incorporate these observations, an updated CT-IC model was suggested [47], in which CTs are subdivided into ‘1 megabase pair (Mbp) chromatin domains’ that constitute a level of chromatin organization above the 30 nm fibre (Figure 2b). The ICD is then seen as a network of gaps between chromatin domains that enables access of proteins into the CT (the meaning of the acronym ICD was thus changed to ‘interchromatin domain’). Although there are no experimental clues to how chromatin is organized within a 1 Mbp domain, the model suggests that each 1 Mbp domain is built up as a rosette of small loops, termed ‘100 kilobase pair (kb) chromatin domains’, which are in contact with the ICD [7,47]. The model therefore predicts that active genes locate at the periphery of the 100 kbp domains, whereas inactive ones assume a more internal position (Figure 2b, inset). An alternative view on the subchromosomal organization of chromatin is the random-walk/giant-loop model, which suggests that chromatin forms flexible loops of 3 Mbp, with the base of the loops following a random walk [48]. This form of organization is normally invoked to explain the looping of genes out of their own CTs [38,39], although there is no evidence that such loops are made of a single, completely unfolded chromatin fibre. It is conceivable that different subchromosomal regions might fold according to each of these models, depending on their local chromatin properties. According to the CT-IC model, CTs and also chromatin domains (e.g. p and q arms) exhibit little or no intermingling between them [49], as suggested by some studies [50,51]. In support of this, an ultrastructural study by Visser et al. [52] has shown that chromatin labelled by BrdU incorporation forms distinct domains when seen on the electron microscope (EM). By comparing the patterns of labelled chromatin (detected using a gold-conjugated antibody) with that of a total DNA stain (osmium ammine), the authors concluded that chromatin domains are mostly separated by the ICD, with only some exhibiting contacts or a very small extent of intermingling [52]. However, given the discrete character of the gold particles, it is difficult to define boundaries between BrdU-labelled and -unlabelled chromatin, so this study cannot exclude the existence of more extensive interactions between chromatin domains or CTs. Although the CT-IC model is a robust hypothesis that has been well accepted, it remains to be shown whether higher-order structures such as the 100 kbp domain exist, and what are the functional elements that drive such an organization. Furthermore, the CT-IC model does not take into account potential functional interactions between distal chromatin segments and/or attachments to nuclear www.sciencedirect.com
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landmarks, although these interactions could have a role in organizing the 100 kbp and 1 Mbp domains. The lattice model Early investigations into the structure of isolated chromatin using the EM revealed fibres with a diameter of 10 nm [53]. This fibre was later resolved into a beads-on-a-string structure, on account of the evenly spaced wrapping of DNA around core histones, and which was shown to fold further into a 30 nm fibre [53]. However, visualization of such structures in the interphase mammalian nucleus was hampered by the lack of contrast between chromatin and all of the proteins and RNA present in the nucleus. Staining methods were developed to provide such contrast, some of which included a detergent treatment to extract nuclear proteins. These preparations revealed two main classes of fibres during G1, one with a diameter of 100–130 nm and the other 60–80 nm, which further unfolded locally into the 30 nm fibre [54]. However, this would still not provide a global high-resolution picture of chromosome organization because most chromatin in the nucleus exists in a decondensed state that does not provide enough contrast for visualization by conventional transmission EM [53]. One solution to this problem is electron spectroscopic imaging (ESI). This technique explores the differences in energy loss that electrons suffer when they interact with different elements within a sample. To apply this principle to EM, an imaging spectrometer analyses the electrons that have interacted with the sample, providing nanometer-resolution maps of specific elements in the sample. Given the high content of nitrogen in proteins, and that of phosphorus in nucleic acids, this tool has been explored for high-resolution studies of chromatin organization during interphase. In agreement with the early studies, chromatin seems to be organized mainly into 10 nm and 30 nm fibres, with varying local concentrations within the nucleus [53] (Figure 2c). Other higher-order structures, such as the 1 Mbp domain proposed in the CT-IC model, are not supported by ESI images. Instead, they are suggested to be randomly organized high concentrations of 10 nm and 30 nm fibres, which makes them irresolvable by lower-resolution methods [53]. However, the resolving power of ESI is challenged when it comes to heterochromatin, so that it can only be speculated whether heterochromatin reflects a high concentration of disorganized 10 nm and 30 nm fibres, or whether in this case chromatin folds into a particularly organized and compact conformation, forming a higher-order structure. The lattice model of chromosome organization was proposed based on these observations [53] (Figure 2d). It contradicts the CT-IC model because it argues against the presence of large chromatin-free channels within the nucleus, which are not seen by ESI; the ICD becomes simply the space within the lattice of 10 nm and 30 nm fibres. The latter conformation is in agreement with experiments that show that all of the nucleus is highly accessible to macromolecules moving by diffusion [55]. Because no large spaces, other than those occupied by bodies such as nucleoli and PML bodies, are observed, this model proposes that fibres from different chromosomes are able to intermingle to a certain extent at the edges of CTs, so that chromatin fibres form a nearly-continuous lattice across the whole nucleoplasm. A
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Figure 2. Three models of chromosome organization in the mammalian interphase nucleus. (a,b) The CT-IC model was initially proposed as the interchromosome domain (ICD) model (a) (modified, with kind permission of Springer Science and Business Media, from Ref. [35]), in which CTs were separated from each other by an interchromosomal space rich in the transcription machinery and processing factors; active genes were located at the periphery of CTs and inactive ones in the interior. In its current version, the CT-IC model (b) (modified, with kind permission of Springer Science and Business Media, from Ref. [49]) proposes that CTs are composed of ‘1 Mbp www.sciencedirect.com
Review
TRENDS in Cell Biology
caveat in the ESI analysis is that phosphorus maps will have a contribution from the nuclear distribution of RNA, which might fill the gaps between chromatin domains. However, the secondary structure of RNAs and their association with proteins would have to make them indistinguishable from chromatin structures to enable their erroneous classification as chromatin in an interchromatin space. Combination of immunogold labelling with ESI showed that Pol II is found at the surface of chromatin fibres throughout the whole nucleus, with the exception of nucleoli and heterochromatin. In the lattice model, folding into a 30 nm fibre could be sufficient to regulate gene activity by limiting accessibility to its interior, without the need to invoke a more complex or compact structure such as the 100 kbp domain. The ICN model Several long-range interactions have recently been described between distal genomic loci within the same chromosome or in different chromosomes, and which are functionally relevant (see later) [13,21–27]. This suggests an additional layer of positional regulation involving chromosome organization. Intrachromosomal associations, established by means of chromatin looping, had already been suggested as a mechanism of action by which enhancer elements regulate genes located several kb away [56]. After the development of biochemical techniques enabling the detection of chromatin interactions [chromosome conformation capture (3C) and RNA tagging and recovery of associated proteins (RNA-TRAP) [57,58]], looping was first shown to occur in mammalian cells in the b-globin locus, where the locus control region (LCR) physically interacts with active genes lying 50 kb away [26]. Our notion of proximity based on the linear organization of the genome has since been challenged because a growing number of distant interactions are revealed. The mouse bglobin gene Hbb1 was shown to interact in fetal liver cells with other erythroid-specific genes lying several Mbp away, and the simultaneous colocalization with Pol II foci suggested a role of transcription factories as mediators for the interactions [13]. Intrachromosomal associations have also been shown to occur within the TH2 cytokine locus in mouse T cells, where a functional role is suggested by the dependence of these interactions on specific transcription factors [24] and on the presence of a hypersensitive site on the Rad50 gene [59]. Surprisingly, the same authors later reported an interchromosomal association between the TH2 locus on chromosome 11 and the Ifng gene on chromosome 10, which is also dependent on the same hypersensitive site, and is detected in naı¨ve T cells (where the genes
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are inactive) but not in differentiated TH1 and TH2 cells [25]. Several other examples of interactions between genes lying in different chromosomes have been characterized, such as those between a- and b-globin [13,60], Igf2/H19 and Wsb1/Nf1 [22], and both X inactivation centres during X inactivation onset [21,27]. An interesting example was recently reported, in which the enhancer element H interacts in each sensory neuron with only one of 1300 olfactory receptor genes distributed across several different chromosomes [23]. The interaction is in correlation with the gene that is active in each neuron, suggesting a mechanism for the choice of olfactory receptor gene expression that is dependent on positional information of chromatin. Furthermore, new technical developments have extended 3C studies to genome-wide approaches {open-ended 3C [28], chromosome conformation capture-on-chip (4C) [29], and circular chromosome conformation capture (also abbreviated 4C) [30]}, which have revealed extensive intraand interchromosomal interactions across the genome. The degree of genome flexibility and dynamics that is suggested by the detection of interchromosomal associations, points to a higher degree of intermingling between CTs than initially thought. This is in line with studies on chromatin motion, which show that chromatin moves mainly by constrained diffusion within 0.4 mm [61–63] but can exhibit movements up to 1.5 mm [62], and even directional movement [64]. Results from radiation biology have also been suggestive of chromosome intermingling because the frequency and complexity of translocations that are induced after irradiation of cells are not fully compatible with a separation of CTs, at least according to some models [65–67]. Using a novel high-resolution FISH method (cryo-FISH), we have recently shown that intermingling between CTs is significant, and estimated that 20% of the nuclear volume contains areas in which sequences from more than one chromosome are present [46]. Intermingling is confirmed at the ultrastructural level, after immunogold labelling of the painted chromosomes and imaging on the EM [46]. Therefore, a high potential for interchromosomal interactions exists, which will be reflected in the frequency of chromosome rearrangements. In fact, the amount of intermingling between pairs of CTs correlates with their translocation potential [46]. No difference in the average DNA concentration is detected between regions of intermingling and the interior of CTs [46], providing evidence that there is no clear distinction in terms of chromatin organization between the CT interior and its periphery, in agreement with the lattice model. In the absence of an interchromosomal space, genes that loop out from their own CTs [38,39] are likely to be intermingled
chromatin domains’, which are in turn made up by ‘100 kbp chromatin loop domains’; the ICD’’ or compartment extends into the interior of CTs, contacting the surface of the 100 kbp domains, where active genes are located. (c,d) The lattice model stems mainly from observations made using ESI (c), (modified, with kind permission of Springer Science and Business Media, from Ref. [53]), which provides high-resolution maps of particular elements such as phosphorus (enriched in nucleic acids) and nitrogen (enriched in proteins). At low magnification [(c) (i)], the nucleus seems to contain chromatin structures folded above the 30 nm fibre. At high resolution, chromatin is visualized as a network of 10 nm and 30 nm fibres in varying concentrations [(c) (ii) overlay of net phosphorus map in yellow and net nitrogen map in blue; (c) (iii) net phosphorus map]. The lattice model (d) (modified, with kind permission of Springer Science and Business Media, from Ref. [53]) proposes that interphase chromatin is made up solely by the latter fibres. There are no large channels or gaps devoid of chromatin, only the small spaces within the chromatin lattice that provide enough accessibility for macromolecules; chromatin fibres from different CTs are able to intermingle at CT boundaries. (e,f) The ICN model incorporates recent evidence for intraand interchromosomal associations into the context of chromosome organization. Some functional interactions between CTs might be mediated by RNA Pol II factories (e) because simultaneous labelling of chromosome 3 (Chr3, red), the whole genome except chromosome 3 (WG-3, blue) and RNA Pol II (Pol II, green), reveals that areas of intermingling between CTs can contain Pol II foci. The ICN model (f) proposes that functional interactions occurring within a CT and between CTs, and mediated by Pol II or other nuclear components, form a network that contributes to the organization of chromosomes within the nucleus. Together with other functional attachments and the physical properties of chromatin, this cell type-specific network will influence CT position and tissue-specific translocations. www.sciencedirect.com
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with neighbouring CTs. Indeed, the MHC II locus is seen more often within other chromosomes after looping out of its CT, following transcriptional activation [38,39], and interchromosomal interactions with HoxB1 (as detected by open-ended 3C) are more frequent after its differentiation-induced looping out [28]. Foci of active Pol II are detected within areas of intermingling (Figure 2e), and inhibition of transcription changes the pattern of intermingling across specific pairs of chromosomes [46]. If transcription factories stabilize some interchromatin associations, these results suggest that transcription helps to dictate chromosome conformation within the nucleus, and might help to explain why different cell types, with their specific transcriptional programmes, have distinct chromosome arrangements that are conserved in evolution [68,69]. The ICN model (Figure 2f) proposes that the balance between tissue-specific intra- and interchromosomal associations, together with functional tethering to other nuclear landmarks such as the lamina and nucleoli, will define chromosome position and conformation [46]. This will consequently establish which loci are close enough to have the potential to be misrepaired following DNA damage, thus determining tissue-specific translocations. Following exit from mitosis, as the transcription repertoire of the cell is re-established, a network of interactions forms across the genome, imposing spatial restrictions to the formation of other interactions, thereby functioning as a means of epigenetic regulation. Inevitably, the same spatial restrictions will also drive a passive proximity between chromatin segments not involved in functional interactions. Future efforts should aim to distinguish passive proximity from effective functional interactions. The ICN model brings the newly found interchromatin interactions into the context of general chromosome organization, suggesting that particular functional relationships might help to shape the genome. However, unlike the CTIC and lattice models, it incorporates few considerations on differences in structure and folding of active and inactive chromatin. Nevertheless, it suggests that the interaction of chromatin with transcription factories forms one kind of organizational unit, in which active sequences contact Pol II in the interior, and inactive chromatin loops out from the factories [70]. This structure would potentially resemble the 1 Mbp domains suggested in the CT-IC model but with the active genes located at the base of each 100 kbp loop and not at their periphery. Concluding remarks: converging views? Although the models presented here disagree in certain points, they are far from being mutually exclusive. Instead, they tend to describe different aspects of chromosome organization, pointing towards a unification of these views. It is accepted that chromosomes occupy territories and are not fully intertwined within the nucleus. The CT-IC model proposes that genes found within compact chromatin domains are silenced owing to inaccessibility of transcription factors, whereas active genes are in contact with an ICD, which contains the proteins required for transcription. The ICD is seen as a network of reasonably large www.sciencedirect.com
chromatin-free channels, branching off into progressively smaller channels that contact the surface of 100 kbp domains. The lattice model challenges this aspect and focuses on the organization of chromatin at the nanostructural level. By ESI, the majority of nuclear chromatin is found in the form of 10 nm and 30 nm fibres, with no evidence for large chromatin-free channels. However, different concentrations of these fibres are seen across the nucleus. This suggests that the ICD (as defined in the CT-IC model) might not be totally devoid of chromatin but simply contain a lower concentration of DNA fibres, thus appearing as gaps when seen by light microscopy or lower-sensitivity contrasting methods used for transmission EM [53]. Whether devoid of chromatin or not, there is now evidence that regions that are weakly stained by DNA dyes are distributed randomly in the nucleus and in relation to CTs, and do not show a preference for CT edges [46]. Finally, the ICN model mainly explores how functional interactions between chromatin segments across the genome, and with nuclear bodies or the lamina, might influence and be influenced by chromosome organization. It thus aims to add new functional views on the spatial regulation of gene expression to the context of nuclear organization. As predictions from each of these models are tested, new and refined views of chromosome organization will be proposed, helping to unravel the complex relationships between gene regulation and the spatial organization of the genome within the interphase nucleus. Acknowledgements We thank Drs Thomas Cremer and David Bazett-Jones for permission to reproduce published material and for providing us with high-resolution images. We thank the Medical Research Council (UK) and Fundac¸a˜o para a Cieˆncia e Tecnologia (Portugal) for funding.
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chromosome 6 and its response to interferon in interphase nuclei. J. Cell Sci. 113, 1565–1576 Mahy, N.L. et al. (2002) Spatial organization of active and inactive genes and noncoding DNA within chromosome territories. J. Cell Biol. 157, 579–589 Scheuermann, M.O. et al. (2004) Topology of genes and nontranscribed sequences in human interphase nuclei. Exp. Cell Res. 301, 266– 279 Chaumeil, J. et al. (2006) A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced. Genes Dev. 20, 2223–2237 Clemson, C.M. et al. (2006) The X chromosome is organized into a generich outer rim and an internal core containing silenced nongenic sequences. Proc. Natl. Acad. Sci. U. S. A. 103, 7688–7693 Abranches, R. et al. (1998) Transcription sites are not correlated with chromosome territories in wheat nuclei. J. Cell Biol. 143, 5–12 Verschure, P.J. et al. (1999) Spatial relationship between transcription sites and chromosome territories. J. Cell Biol. 147, 13–24 Branco, M.R. and Pombo, A. (2006) Intermingling of chromosome territories in interphase suggests role in translocations and transcription-dependent associations. PLoS Biol. 4, e138 Cremer, T. and Cremer, C. (2001) Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat. Rev. Genet. 2, 292–301 Sachs, R.K. et al. (1995) A random-walk/giant-loop model for interphase chromosomes. Proc. Natl. Acad. Sci. U. S. A. 92, 2710– 2714 Cremer, T. et al. (2000) Chromosome territories, interchromatin domain compartment, and nuclear matrix: an integrated view of the functional nuclear architecture. Crit. Rev. Eukaryot. Gene Expr. 10, 179–212 Munkel, C. et al. (1999) Compartmentalization of interphase chromosomes observed in simulation and experiment. J. Mol. Biol. 285, 1053–1065 Visser, A.E. and Aten, J.A. (1999) Chromosomes as well as chromosomal subdomains constitute distinct units in interphase nuclei. J. Cell Sci. 112, 3353–3360 Visser, A.E. et al. (2000) High resolution analysis of interphase chromosome domains. J. Cell Sci. 113, 2585–2593 Dehghani, H. et al. (2005) Organization of chromatin in the interphase mammalian cell. Micron 36, 95–108 Belmont, A.S. and Bruce, K. (1994) Visualization of G1 chromosomes: a folded, twisted, supercoiled chromonema model of interphase chromatid structure. J. Cell Biol. 127, 287–302 Verschure, P.J. et al. (2003) Condensed chromatin domains in the mammalian nucleus are accessible to large macromolecules. EMBO Rep. 4, 861–866 Bulger, M. and Groudine, M. (1999) Looping versus linking: toward a model for long-distance gene activation. Genes Dev. 13, 2465–2477 Carter, D. et al. (2002) Long-range chromatin regulatory interactions in vivo. Nat. Genet. 32, 623–626 Dekker, J. et al. (2002) Capturing chromosome conformation. Science 295, 1306–1311 Lee, G.R. et al. (2005) Hypersensitive site 7 of the TH2 locus control region is essential for expressing TH2 cytokine genes and for longrange intrachromosomal interactions. Nat. Immunol. 6, 42–48 Brown, J.M. et al. (2006) Coregulated human globin genes are frequently in spatial proximity when active. J. Cell Biol. 172, 177–187 Abney, J.R. et al. (1997) Chromatin dynamics in interphase nuclei and its implications for nuclear structure. J. Cell Biol. 137, 1459– 1468 Chubb, J.R. et al. (2002) Chromatin motion is constrained by association with nuclear compartments in human cells. Curr. Biol. 12, 439–445 Gasser, S.M. (2002) Visualizing chromatin dynamics in interphase nuclei. Science 296, 1412–1416 Chuang, C.H. et al. (2006) Long-range directional movement of an interphase chromosome site. Curr. Biol. 16, 825–831 Hlatky, L. et al. (2002) Radiation-induced chromosome aberrations: insights gained from biophysical modelling. Bioessays 24, 714–723 Holley, W.R. et al. (2002) A model for interphase chromosomes and evaluation of radiation-induced aberrations. Radiat. Res. 158, 568– 580
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67 Sachs, R.K. et al. (2000) Random breakage and reunion chromosome aberration formation model; an interaction-distance version based on chromatin geometry. Int. J. Radiat. Biol. 76, 1579–1588 68 Tanabe, H. et al. (2002) Non-random radial arrangements of interphase chromosome territories: evolutionary considerations and functional implications. Mutat. Res. 504, 37–45
69 Mora, L. et al. (2006) Chromosome territory positioning of conserved homologous chromosomes in different primate species. Chromosoma 115, 367–375 70 Cook, P.R. (2002) Predicting three-dimensional genome structure from transcriptional activity. Nat. Genet. 32, 347–352
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Chromosome organization: new facts, new models Miguel R. Branco and Ana Pombo MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK
The study of nuclear organization has radically changed the way we envision gene regulation, imposing a paradigm shift from a seemingly featureless nucleus to a highly compartmentalized and complex organelle. The positioning of genes, regulatory sequences and transcription factors in relation to each other and to landmarks in the nucleus, such as nuclear bodies and the lamina, is important in determining which genes are transcribed at any one time. Investigating chromatin organization during interphase is therefore essential to the understanding of gene expression. The recent discovery of interactions between distal chromatin segments that occur within the same chromosome or across different chromosomes, and that have a role in transcription regulation, suggests a re-evaluation of current models of chromosome organization and the development of new ones. Spatial regulation of gene activity The human genome comprises 20 000–25 000 protein-coding genes [1], of which only a fraction is transcribed at any one time in each cell. Transcription is tightly regulated to ensure correct expression of the genes required in each phase of the cell cycle, stage of differentiation and environmental condition that is imposed on the cell. It is therefore not surprising that increasingly complex layers of gene regulation have been discovered over the years. The linear DNA sequence contains regulatory elements, such as promoters and enhancers, which activate genes depending on the availability of particular transcription factors [2]. Furthermore, the accessibility of transcription factors and RNA polymerases (Pols) to DNA is influenced by the condensation state of chromatin, which is in turn regulated by several epigenetic determinants such as histone modifications, binding of heterochromatin protein 1 (HP1) and Polycomb group (PcG) (see Glossary) proteins, and the activity of chromatin remodelling complexes [3–5]. An extra layer of regulation that has been emerging as crucial to gene regulation is the spatial organization of factors and sequences within the nucleus [6–9]. Several nuclear components are found compartmentalized in nuclear bodies that are distinct in their composition, morphology and abundance [10,11]. The nucleolus is the most prominent and well studied nuclear body, and harbours all the machinery essential for the transcription of 45S rRNA genes, including Pol I [6]. This imposes spatial Corresponding author: Branco, M.R. ([email protected]). Available online 2 January 2007. www.sciencedirect.com
restrictions on rRNA gene-containing chromosomes, which have a nucleolar-organizing region (NOR) that promotes their association with nucleoli even when they are inactive [12]. Transcription by Pols II and III has also been shown to occur in sites containing many Pols, termed ‘transcription factories’ (50 nm diameter in HeLa, F9 and ES cells, and amphibian A1 cells), which are evenly distributed throughout the nucleoplasm, and which might similarly restrict the positioning of other genes on a smaller scale [6,13,14]. Other nuclear bodies, such as speckles, promyelocytic leukaemia (PML) bodies and Cajal bodies, have been shown to have roles in mRNA maturation, RNP assembly and other nuclear processes [11]. However, a better understanding of the function of these compartments is still necessary, especially in regard to whether their spatial proximity to certain genes is important for transcription
Glossary Cajal bodies (or coiled bodies): nuclear bodies rich in the protein coilin and other factors involved in the biogenesis of small nuclear ribonucleoprotein particles. Chromosome conformation capture (3C): biochemical technique for the detection of interactions between chromatin segments within the nucleus. It is based on crosslinking of proximal fragments through protein bridges, digestion of chromatin, intramolecular ligation of crosslinked fragments and their detection by PCR. Chromosome conformation capture-on-chip (4C) [29]: genome-wide adaptation of 3C, in which all of the crosslinking events to a fragment of interest can be analysed by microarrays. Heterochromatin protein 1 (HP1): binds chromatin preferentially at heterochromatic regions by interaction of its chromodomain with the repressive mark methylated lysine 9 at histone H3 and recruits the corresponding methyltransferase, Su(var)3-9. Locus control region (LCR): an enhancer element that ensures high expression levels independent of integration site of an associated transgene. Nucleolar organizing region (NOR): chromosomal locus containing ribosomal RNA genes. In humans, they are present in five chromosomes: 13, 14, 15, 21 and 22. Open-ended 3C [28] and circular chromosome conformation capture (also abbreviated 4C) [30]: other genome-wide adaptations of 3C, but in which crosslinked fragments are analysed by sequencing. Polycomb group (PcG): family of proteins involved in repression of homeotic genes, which form multimeric protein complexes. The Polycomb repressive complex 2 (PRC2) has a role in the deposition of repressive marks, whereas PRC1 is involved in the maintenance of silent chromatin. Promyelocytic leukaemia (PML) bodies: nuclear bodies rich in the PML protein. A translocation causing the fusion of PML with retinoic acid receptor (RAR) a leads to acute PML and disrupts the formation of PML bodies. RNA tagging and recovery of associated proteins (RNA-TRAP): biochemical technique for the detection of chromatin fragments interacting with active genes. It involves tagging nascent transcripts, with subsequent purification of the chromatin found in close proximity to the tagged RNA and the detection of specific chromatin regions by PCR. Splicing speckles: nuclear bodies rich in splicing factors and poly(A)+ RNA. Xist: a noncoding RNA that coats one of the female X chromosomes early in the process of X inactivation.
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regulation [15]. Association of genes with other nuclear landmarks, such as the lamina [16] or centromeric clusters [17], has been correlated with gene silencing. Spreading of repressive marks in these heterochromatic regions in a nonlinear, three-dimensional way (as opposed to spreading along the linear DNA sequence) to associated genes might have a role in the spatial regulation of transcription. It is in this context that the study of chromosome organization during interphase is essential for understanding gene expression. Each chromosome occupies its own distinct space or territory (Figure 1), and assumes a nonrandom position within the nucleus [18–20]. Despite the apparent simplicity of this arrangement, the initial models of chromosome organization have been challenged by recent observations and technical developments. It seems that interactions between distal segments of chromatin, within the same chromosome or across different chromosomes, are more common than previously thought [13,21–30]. These numerous examples of functionally relevant intra- and interchromosomal associations attribute further importance to the organization of chromatin in the process of gene expression, and move us to revise current models of chromosome organization. Here, we review three models [chromosome territory–interchromatin compartment (CT-IC), lattice and interchromosomal network (ICN) models] that describe different aspects of chromosome structure and inevitably bring about different views on how long-range chromatin interactions might be established. Other aspects of chromatin organization or spatial regulation of gene expression are embedded within the discussion of these three models, or have been omitted owing to limitations of space. Chromosome organization: the early days When Flemming first observed mitotic chromosomes (see [31] for a review), he could only speculate as to what happened to them once cells entered interphase. Given that chromosomes were no longer seen as separate entities within the cell nucleus, the most likely scenario was that they unfolded and became entangled. A more organized view of chromatin came from the studies of Rabl and Boveri, who showed that plant chromosomes maintain a polarized orientation during interphase, with centromeres and telomeres located to opposite sides of the nucleus, reflecting their conformation during mitosis (the so-called Rabl configuration) (see [8] for a review). From this observation, they also suggested that chromosomes might maintain their spatial separation and occupy distinct territories in the nucleus. Evidence for the existence of chromosome territories (CTs) in interphase nuclei was first originated in mammalian cells. In an elegant study, Thomas Cremer et al. [32] microirradiated Chinese Hamster nuclei with a UV laser, and visualized the sites of damage at the following metaphase. Instead of obtaining a scattered signal across several chromosomes, the authors observed discrete labelling in parts of only a few chromosomes, readily indicating that chromatin must be highly compartmentalized within the nucleus. Direct visualization of CTs in mammalian interphase nuclei was only possible after the development of whole chromosome paint probes, in combination with fluorescence in situ hybridization www.sciencedirect.com
Figure 1. Chromosomes occupy discrete territories during interphase. Two chromosome-specific paints (chromosome 1 in green, chromosome 2 in red) were hybridized to a cryosection (100–200 nm thick) of human T lymphocytes by cryo-FISH [46], revealing the territorial organization of chromosomes during interphase.
(FISH) [33,34] (Figure 1). This has since become a standard tool that has enabled extensive study of chromosome organization during interphase, and that in combination with other methods has given rise to the models that we summarize here. The CT-IC model The discovery of a territorial organization of chromosomes during interphase brought about several questions. Namely, how are genes and regulatory regions organized within a CT, and is there a relationship between position and activity? In experiments that aimed to investigate where transcription and splicing occurred in relation to CTs, Zirbel et al. [35] simultaneously labelled CTs and splicing speckles (as labelled by the Sm antigen) or the transcript of an integrated human papilloma virus genome. Both the viral transcripts and the Sm antigen showed a preferential position outside CTs, accumulating in a nuclear compartment distinct from the CTs. The domain that apparently separated CTs was named the interchromosome domain (ICD), and a model emerged in which active genes would be located at the periphery of CTs so that they would be accessible to transcription and splicing factors [36] (Figure 2a). Conversely, inactive genes would be located in the interior of CTs, which limited the accessibility of the transcription machinery. Initial studies on the position of chromatin segments in relation to their CT seemed to support this view because coding sequences often located to the periphery of CTs and noncoding sequences towards the interior [37]. Moreover, a small number of genes were shown to loop out of their own CT following activation [38,39], suggesting a migration into the ICD to facilitate transcription. However, a generalized correlation between position relative to the CT and gene activity has not been found; coding sequences can be seen
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at the interior of CTs, irrespective of their transcriptional status [40], and noncoding sequences are seen at the periphery of CTs [41]. Interestingly, in the case of X inactivation during ES cell differentiation, there is evidence that Xist-mediated gene silencing brings inactive genes to the centre of the inactive X chromosome, whereas genes escaping inactivation are located at the periphery [42] (this conformation seems to change when epigenetic marks take over the silencing role of Xist [43]). Apart from this exception, transcription sites are found evenly scattered across the nucleus, including within CTs [44–46]. To incorporate these observations, an updated CT-IC model was suggested [47], in which CTs are subdivided into ‘1 megabase pair (Mbp) chromatin domains’ that constitute a level of chromatin organization above the 30 nm fibre (Figure 2b). The ICD is then seen as a network of gaps between chromatin domains that enables access of proteins into the CT (the meaning of the acronym ICD was thus changed to ‘interchromatin domain’). Although there are no experimental clues to how chromatin is organized within a 1 Mbp domain, the model suggests that each 1 Mbp domain is built up as a rosette of small loops, termed ‘100 kilobase pair (kb) chromatin domains’, which are in contact with the ICD [7,47]. The model therefore predicts that active genes locate at the periphery of the 100 kbp domains, whereas inactive ones assume a more internal position (Figure 2b, inset). An alternative view on the subchromosomal organization of chromatin is the random-walk/giant-loop model, which suggests that chromatin forms flexible loops of 3 Mbp, with the base of the loops following a random walk [48]. This form of organization is normally invoked to explain the looping of genes out of their own CTs [38,39], although there is no evidence that such loops are made of a single, completely unfolded chromatin fibre. It is conceivable that different subchromosomal regions might fold according to each of these models, depending on their local chromatin properties. According to the CT-IC model, CTs and also chromatin domains (e.g. p and q arms) exhibit little or no intermingling between them [49], as suggested by some studies [50,51]. In support of this, an ultrastructural study by Visser et al. [52] has shown that chromatin labelled by BrdU incorporation forms distinct domains when seen on the electron microscope (EM). By comparing the patterns of labelled chromatin (detected using a gold-conjugated antibody) with that of a total DNA stain (osmium ammine), the authors concluded that chromatin domains are mostly separated by the ICD, with only some exhibiting contacts or a very small extent of intermingling [52]. However, given the discrete character of the gold particles, it is difficult to define boundaries between BrdU-labelled and -unlabelled chromatin, so this study cannot exclude the existence of more extensive interactions between chromatin domains or CTs. Although the CT-IC model is a robust hypothesis that has been well accepted, it remains to be shown whether higher-order structures such as the 100 kbp domain exist, and what are the functional elements that drive such an organization. Furthermore, the CT-IC model does not take into account potential functional interactions between distal chromatin segments and/or attachments to nuclear www.sciencedirect.com
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landmarks, although these interactions could have a role in organizing the 100 kbp and 1 Mbp domains. The lattice model Early investigations into the structure of isolated chromatin using the EM revealed fibres with a diameter of 10 nm [53]. This fibre was later resolved into a beads-on-a-string structure, on account of the evenly spaced wrapping of DNA around core histones, and which was shown to fold further into a 30 nm fibre [53]. However, visualization of such structures in the interphase mammalian nucleus was hampered by the lack of contrast between chromatin and all of the proteins and RNA present in the nucleus. Staining methods were developed to provide such contrast, some of which included a detergent treatment to extract nuclear proteins. These preparations revealed two main classes of fibres during G1, one with a diameter of 100–130 nm and the other 60–80 nm, which further unfolded locally into the 30 nm fibre [54]. However, this would still not provide a global high-resolution picture of chromosome organization because most chromatin in the nucleus exists in a decondensed state that does not provide enough contrast for visualization by conventional transmission EM [53]. One solution to this problem is electron spectroscopic imaging (ESI). This technique explores the differences in energy loss that electrons suffer when they interact with different elements within a sample. To apply this principle to EM, an imaging spectrometer analyses the electrons that have interacted with the sample, providing nanometer-resolution maps of specific elements in the sample. Given the high content of nitrogen in proteins, and that of phosphorus in nucleic acids, this tool has been explored for high-resolution studies of chromatin organization during interphase. In agreement with the early studies, chromatin seems to be organized mainly into 10 nm and 30 nm fibres, with varying local concentrations within the nucleus [53] (Figure 2c). Other higher-order structures, such as the 1 Mbp domain proposed in the CT-IC model, are not supported by ESI images. Instead, they are suggested to be randomly organized high concentrations of 10 nm and 30 nm fibres, which makes them irresolvable by lower-resolution methods [53]. However, the resolving power of ESI is challenged when it comes to heterochromatin, so that it can only be speculated whether heterochromatin reflects a high concentration of disorganized 10 nm and 30 nm fibres, or whether in this case chromatin folds into a particularly organized and compact conformation, forming a higher-order structure. The lattice model of chromosome organization was proposed based on these observations [53] (Figure 2d). It contradicts the CT-IC model because it argues against the presence of large chromatin-free channels within the nucleus, which are not seen by ESI; the ICD becomes simply the space within the lattice of 10 nm and 30 nm fibres. The latter conformation is in agreement with experiments that show that all of the nucleus is highly accessible to macromolecules moving by diffusion [55]. Because no large spaces, other than those occupied by bodies such as nucleoli and PML bodies, are observed, this model proposes that fibres from different chromosomes are able to intermingle to a certain extent at the edges of CTs, so that chromatin fibres form a nearly-continuous lattice across the whole nucleoplasm. A
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Figure 2. Three models of chromosome organization in the mammalian interphase nucleus. (a,b) The CT-IC model was initially proposed as the interchromosome domain (ICD) model (a) (modified, with kind permission of Springer Science and Business Media, from Ref. [35]), in which CTs were separated from each other by an interchromosomal space rich in the transcription machinery and processing factors; active genes were located at the periphery of CTs and inactive ones in the interior. In its current version, the CT-IC model (b) (modified, with kind permission of Springer Science and Business Media, from Ref. [49]) proposes that CTs are composed of ‘1 Mbp www.sciencedirect.com
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caveat in the ESI analysis is that phosphorus maps will have a contribution from the nuclear distribution of RNA, which might fill the gaps between chromatin domains. However, the secondary structure of RNAs and their association with proteins would have to make them indistinguishable from chromatin structures to enable their erroneous classification as chromatin in an interchromatin space. Combination of immunogold labelling with ESI showed that Pol II is found at the surface of chromatin fibres throughout the whole nucleus, with the exception of nucleoli and heterochromatin. In the lattice model, folding into a 30 nm fibre could be sufficient to regulate gene activity by limiting accessibility to its interior, without the need to invoke a more complex or compact structure such as the 100 kbp domain. The ICN model Several long-range interactions have recently been described between distal genomic loci within the same chromosome or in different chromosomes, and which are functionally relevant (see later) [13,21–27]. This suggests an additional layer of positional regulation involving chromosome organization. Intrachromosomal associations, established by means of chromatin looping, had already been suggested as a mechanism of action by which enhancer elements regulate genes located several kb away [56]. After the development of biochemical techniques enabling the detection of chromatin interactions [chromosome conformation capture (3C) and RNA tagging and recovery of associated proteins (RNA-TRAP) [57,58]], looping was first shown to occur in mammalian cells in the b-globin locus, where the locus control region (LCR) physically interacts with active genes lying 50 kb away [26]. Our notion of proximity based on the linear organization of the genome has since been challenged because a growing number of distant interactions are revealed. The mouse bglobin gene Hbb1 was shown to interact in fetal liver cells with other erythroid-specific genes lying several Mbp away, and the simultaneous colocalization with Pol II foci suggested a role of transcription factories as mediators for the interactions [13]. Intrachromosomal associations have also been shown to occur within the TH2 cytokine locus in mouse T cells, where a functional role is suggested by the dependence of these interactions on specific transcription factors [24] and on the presence of a hypersensitive site on the Rad50 gene [59]. Surprisingly, the same authors later reported an interchromosomal association between the TH2 locus on chromosome 11 and the Ifng gene on chromosome 10, which is also dependent on the same hypersensitive site, and is detected in naı¨ve T cells (where the genes
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are inactive) but not in differentiated TH1 and TH2 cells [25]. Several other examples of interactions between genes lying in different chromosomes have been characterized, such as those between a- and b-globin [13,60], Igf2/H19 and Wsb1/Nf1 [22], and both X inactivation centres during X inactivation onset [21,27]. An interesting example was recently reported, in which the enhancer element H interacts in each sensory neuron with only one of 1300 olfactory receptor genes distributed across several different chromosomes [23]. The interaction is in correlation with the gene that is active in each neuron, suggesting a mechanism for the choice of olfactory receptor gene expression that is dependent on positional information of chromatin. Furthermore, new technical developments have extended 3C studies to genome-wide approaches {open-ended 3C [28], chromosome conformation capture-on-chip (4C) [29], and circular chromosome conformation capture (also abbreviated 4C) [30]}, which have revealed extensive intraand interchromosomal interactions across the genome. The degree of genome flexibility and dynamics that is suggested by the detection of interchromosomal associations, points to a higher degree of intermingling between CTs than initially thought. This is in line with studies on chromatin motion, which show that chromatin moves mainly by constrained diffusion within 0.4 mm [61–63] but can exhibit movements up to 1.5 mm [62], and even directional movement [64]. Results from radiation biology have also been suggestive of chromosome intermingling because the frequency and complexity of translocations that are induced after irradiation of cells are not fully compatible with a separation of CTs, at least according to some models [65–67]. Using a novel high-resolution FISH method (cryo-FISH), we have recently shown that intermingling between CTs is significant, and estimated that 20% of the nuclear volume contains areas in which sequences from more than one chromosome are present [46]. Intermingling is confirmed at the ultrastructural level, after immunogold labelling of the painted chromosomes and imaging on the EM [46]. Therefore, a high potential for interchromosomal interactions exists, which will be reflected in the frequency of chromosome rearrangements. In fact, the amount of intermingling between pairs of CTs correlates with their translocation potential [46]. No difference in the average DNA concentration is detected between regions of intermingling and the interior of CTs [46], providing evidence that there is no clear distinction in terms of chromatin organization between the CT interior and its periphery, in agreement with the lattice model. In the absence of an interchromosomal space, genes that loop out from their own CTs [38,39] are likely to be intermingled
chromatin domains’, which are in turn made up by ‘100 kbp chromatin loop domains’; the ICD’’ or compartment extends into the interior of CTs, contacting the surface of the 100 kbp domains, where active genes are located. (c,d) The lattice model stems mainly from observations made using ESI (c), (modified, with kind permission of Springer Science and Business Media, from Ref. [53]), which provides high-resolution maps of particular elements such as phosphorus (enriched in nucleic acids) and nitrogen (enriched in proteins). At low magnification [(c) (i)], the nucleus seems to contain chromatin structures folded above the 30 nm fibre. At high resolution, chromatin is visualized as a network of 10 nm and 30 nm fibres in varying concentrations [(c) (ii) overlay of net phosphorus map in yellow and net nitrogen map in blue; (c) (iii) net phosphorus map]. The lattice model (d) (modified, with kind permission of Springer Science and Business Media, from Ref. [53]) proposes that interphase chromatin is made up solely by the latter fibres. There are no large channels or gaps devoid of chromatin, only the small spaces within the chromatin lattice that provide enough accessibility for macromolecules; chromatin fibres from different CTs are able to intermingle at CT boundaries. (e,f) The ICN model incorporates recent evidence for intraand interchromosomal associations into the context of chromosome organization. Some functional interactions between CTs might be mediated by RNA Pol II factories (e) because simultaneous labelling of chromosome 3 (Chr3, red), the whole genome except chromosome 3 (WG-3, blue) and RNA Pol II (Pol II, green), reveals that areas of intermingling between CTs can contain Pol II foci. The ICN model (f) proposes that functional interactions occurring within a CT and between CTs, and mediated by Pol II or other nuclear components, form a network that contributes to the organization of chromosomes within the nucleus. Together with other functional attachments and the physical properties of chromatin, this cell type-specific network will influence CT position and tissue-specific translocations. www.sciencedirect.com
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with neighbouring CTs. Indeed, the MHC II locus is seen more often within other chromosomes after looping out of its CT, following transcriptional activation [38,39], and interchromosomal interactions with HoxB1 (as detected by open-ended 3C) are more frequent after its differentiation-induced looping out [28]. Foci of active Pol II are detected within areas of intermingling (Figure 2e), and inhibition of transcription changes the pattern of intermingling across specific pairs of chromosomes [46]. If transcription factories stabilize some interchromatin associations, these results suggest that transcription helps to dictate chromosome conformation within the nucleus, and might help to explain why different cell types, with their specific transcriptional programmes, have distinct chromosome arrangements that are conserved in evolution [68,69]. The ICN model (Figure 2f) proposes that the balance between tissue-specific intra- and interchromosomal associations, together with functional tethering to other nuclear landmarks such as the lamina and nucleoli, will define chromosome position and conformation [46]. This will consequently establish which loci are close enough to have the potential to be misrepaired following DNA damage, thus determining tissue-specific translocations. Following exit from mitosis, as the transcription repertoire of the cell is re-established, a network of interactions forms across the genome, imposing spatial restrictions to the formation of other interactions, thereby functioning as a means of epigenetic regulation. Inevitably, the same spatial restrictions will also drive a passive proximity between chromatin segments not involved in functional interactions. Future efforts should aim to distinguish passive proximity from effective functional interactions. The ICN model brings the newly found interchromatin interactions into the context of general chromosome organization, suggesting that particular functional relationships might help to shape the genome. However, unlike the CTIC and lattice models, it incorporates few considerations on differences in structure and folding of active and inactive chromatin. Nevertheless, it suggests that the interaction of chromatin with transcription factories forms one kind of organizational unit, in which active sequences contact Pol II in the interior, and inactive chromatin loops out from the factories [70]. This structure would potentially resemble the 1 Mbp domains suggested in the CT-IC model but with the active genes located at the base of each 100 kbp loop and not at their periphery. Concluding remarks: converging views? Although the models presented here disagree in certain points, they are far from being mutually exclusive. Instead, they tend to describe different aspects of chromosome organization, pointing towards a unification of these views. It is accepted that chromosomes occupy territories and are not fully intertwined within the nucleus. The CT-IC model proposes that genes found within compact chromatin domains are silenced owing to inaccessibility of transcription factors, whereas active genes are in contact with an ICD, which contains the proteins required for transcription. The ICD is seen as a network of reasonably large www.sciencedirect.com
chromatin-free channels, branching off into progressively smaller channels that contact the surface of 100 kbp domains. The lattice model challenges this aspect and focuses on the organization of chromatin at the nanostructural level. By ESI, the majority of nuclear chromatin is found in the form of 10 nm and 30 nm fibres, with no evidence for large chromatin-free channels. However, different concentrations of these fibres are seen across the nucleus. This suggests that the ICD (as defined in the CT-IC model) might not be totally devoid of chromatin but simply contain a lower concentration of DNA fibres, thus appearing as gaps when seen by light microscopy or lower-sensitivity contrasting methods used for transmission EM [53]. Whether devoid of chromatin or not, there is now evidence that regions that are weakly stained by DNA dyes are distributed randomly in the nucleus and in relation to CTs, and do not show a preference for CT edges [46]. Finally, the ICN model mainly explores how functional interactions between chromatin segments across the genome, and with nuclear bodies or the lamina, might influence and be influenced by chromosome organization. It thus aims to add new functional views on the spatial regulation of gene expression to the context of nuclear organization. As predictions from each of these models are tested, new and refined views of chromosome organization will be proposed, helping to unravel the complex relationships between gene regulation and the spatial organization of the genome within the interphase nucleus. Acknowledgements We thank Drs Thomas Cremer and David Bazett-Jones for permission to reproduce published material and for providing us with high-resolution images. We thank the Medical Research Council (UK) and Fundac¸a˜o para a Cieˆncia e Tecnologia (Portugal) for funding.
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