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The yin and yang of chromatin spatial organization
Genome Biologyvolume 11, Article number: 204 (2010)
Spatial organization of the genome is non-random. Preferential chromatin interactions, both in cis and in trans and between transcriptionally active and silent regions, influence organization.
Long-range chromatin interactions can occur over many megabases, either between regions of the same chromosome (cis) or between different chromosomes (trans). Many chromatin clustering events involve preferential interactions between genomic loci and are cell type specific, indicating a functional role of genome organization in regulating gene expression. Many mechanisms are involved in establishing global organization, including transcription by specific sets of transcription factors or gene repression among similar epigenetically marked domains. Here, we discuss several examples of specific spatial organization patterns from transcriptionally active and silent chromatin and the potential mechanisms involved in their establishment.
Long-range chromatin interactions influence function
A growing number of specific long-range chromatin interactions have been identified, indicating that the three-dimensional organization of chromatin within the nucleus is not random. These interactions have been found using tools such as RNA and DNA fluorescence in situ hybridization (FISH) and the chromatin proximity-ligation assay chromosome conformation capture (3C) and its derivatives . In 3C, genomic regions in spatial proximity are cross-linked and digested with a restriction enzyme while in the nucleus. After nuclear lysis, the cross-linked chromatin complexes are diluted and ligated such that ends of restriction fragments in the same cross-linked complex form novel ligation junctions that can be detected by various methods. Numerous studies using these tools have shown that the three-dimensional organization of chromatin within the nucleus is not random. One of the best known and studied long-range interactions occurs between the erythroid-specific β-globin gene and its long-range enhancer, the distal locus control region (LCR). The mammalian β-globin LCR consists of five DNase I hypersensitive sites (HS1-HS5) distributed over 15 kb, located approximately 50 kb upstream of the β-globin gene. The LCR regulates β-globin gene transcription during erythroid development by physically interacting with the β-globin gene, leaving the intervening 50 kb of DNA looped out [2, 3] (Figure 1a). Deletion of the LCR, or ablation of specific transcription factors or cofactors required for the interaction, leads to dramatic decreases in β-globin gene transcription levels, highlighting the functional significance of the interaction [4–8].
Long-range interactions are also required for the processes of T cell receptor and V(D)J recombination in T cells and B cells. V(D)J recombination involves the selection of one of each gene from the V, D and J gene families of the immunoglobulin gene locus. A single V gene is selected from over 190 different V genes distributed over 2.5 Mb and is brought into close spatial proximity and physically linked to a previously recombined (D)J gene, creating a functional immunoglobulin gene . These findings show that chromatin or genes distally arranged on the same chromosome can interact in close physical proximity in three-dimensional space.
Interchromosomal or trans interactions have also been proposed to regulate gene activity. In murine naïve T cells the T helper cell 2 (TH2) LCR on chromosome 11 interacts with the interferon-γ (IFN-γ) promoter located on chromosome 10 [10, 11]. Following differentiation to effector TH1 or TH2 cells, these trans interactions are lost in favor of cis interactions: TH1 cells have interactions between the IFN-γ promoter and regulator elements located upstream to promote high levels of IFN-γ expression, whereas in TH2 cells the TH2 LCR interacts with three nearby interleukin (IL) genes, IL-4, IL-5 and IL-13, to enhance their expression (Figure 1b). In another example, the H19 imprinting control region, located on chromosome 7 in mice, drives the silencing of the maternally inherited insulin-like growth factor 2 receptor (Igf2r) allele and has been shown to interact in trans with up to four different chromosomes in embryonic tissue .
In the examples of the TH2 LCR and H19 imprinting control region mentioned above, deletion of genetic elements on one chromosome affected the expression of interacting genes on other chromosomes, indicating the functional significance of interchromosomal interactions. In contrast, conflicting reports surround the function of the mouse homology (H) enhancer, which engages in cis and trans interactions with odorant receptor genes. The H enhancer is located within the MOR28 odorant receptor gene cluster on mouse chromosome 14, while other odorant receptor gene clusters are scattered on multiple chromosomes. It has been proposed  that the choice of expression of a single mouse odorant receptor gene in a sensory neuron is determined by an interaction in cis or trans between the H enhancer and a single odorant receptor gene. However, two later reports [14, 15] showed that deletion of the H enhancer abolished expression of three flanking odorant receptor genes in the MOR28 cluster with no demonstrable effect on odorant receptor gene expression in trans.
Trans interactions may also be indirectly linked to diseases resulting from chromosomal translocations . The Myc and IgH loci (encoding a transcription factor and an immunoglobulin, respectively), which are located on different mouse chromosomes, are frequent breakpoints in chromosomal translocations, in which two different chromosomes are fused together through inappropriate DNA repair. In mouse B cells, Myc and IgH are found in close proximity in the nucleus only when transcribed, suggesting that transcriptional organization could affect their frequency of translocation . This finding is analogous to recent data indicating that, for androgen-receptor-regulated genes, a combination of irradiation-induced DNA breakage and transcription-induced proximity synergistically increases their chromosomal translocation frequency .
Architecture of association
Examination of nucleolar structure and function provides some of the first evidence for how clustering of specific genes in three-dimensional space could be achieved. Nucleoli are assembled through association of the nucleolar organizing regions (NORs) and various nucleolar proteins. Each of the five human NORs is composed of many tandemly repeated rRNA genes located on the acrocentric chromosomes 13, 14, 15, 21 and 22 (Figure 2). As cells exit mitosis, NORs are bound by the essential transcription protein upstream binding factor (UBF)  and coalesce into between one and four nucleolar structures. The NORs that are transcriptionally quiescent are not bound by UBF and are excluded from nucleoli, indicating that this transcription factor may be fundamental in the organization of these structures . Transcription is also fundamental to the organization of nucleoli. Inhibition of the nucleolar RNA polymerase (RNAPI) with actinomycin D (which intercalates into DNA that is being transcribed and immobilizes the polymerase) results in the formation of 'mini-nucleoli' when cells exit mitosis . Mini-nucleoli contain NORs, but other nucleolar components are distributed to discrete structures, or 'caps', on the mini-nucleolar surface. Removal of actinomycin D and the initiation of RNAPI transcription restores nucleolar morphology, showing that transcription itself has an important role in the organization of nuclear architecture. The nucleolus may represent the first observed specialized 'transcription factory' that can form a trans interaction network with a specific function.
RNA polymerase II (RNAPII)-transcribed genes, which represent the majority of protein coding genes, also engage in long-range transcription-dependent associations [22, 23]. Transcriptionally active genes, such as those genes involved with globin synthesis and regulation, have been shown to colocalize with shared RNAPII foci [22, 24] (Figure 3a). Co-regulated genes in cis and in trans share RNAPII foci with each other at higher frequencies than they do with other transcribed genes, suggesting the presence of large-scale transcription networks . These preferential interactions occur at nuclear subcompartments containing high local concentrations of hyperphosphorylated RNAPII, called transcription factories. Described as protein rich structures of about 10 MDa with an average diameter of about 87 nm, transcription factories contain multiple active RNAPII complexes at one time [25–27]. Gene interactions at transcription factories rely on active transcription: heat-shock treatment, which blocks initiation and elongation, resulted in release of genes from factories and disruption of their long-range associations . Treatment with 5,6-dichloro- β-D-ribofuranosylbenzimidazole (DRB), which interferes with phosphorylation of the carboxy-terminal domain of RNAPII and thus inhibits transcriptional elongation but not initiation, did not affect the frequency of gene co-associations . Transcription initiation is therefore critical for the long-range association of genes that are being transcribed. Transcription factories remained after heat shock, consistent with previous results suggesting that factories are meta-stable structures . These findings indicate that the structure and function of transcription factories are fundamental to long-range interactions between genes being transcribed.
Gene clustering through specialized transcription factories
The idea of transcription factories being specialized to transcribe a specific subset of genes in order to achieve high-level gene transcription seems logical and reasonable, because no two regions within the nucleus will contain the same genes or proteins. Early investigations in human cells into the spatial distribution of certain transcription factors (glucocorticoid receptor, Oct1 and E2f-1) revealed only a slight overlap with RNAPII and sites of transcription [29, 30], which the authors [29, 30] argued as evidence against transcription factory specialization. Contrary to this, the Oct1/PTF/transcription (OPT) domain was the first example of a nuclear compartment to be shown to contain high concentrations of interacting transcription factors (PTF1 and Oct1) at a transcription factory, which specifically recruited regions from human chromosomes 6 or 7 in early G1 phase . This suggests that specialization of transcription factories could provide a level of control over genome organization by encouraging specific genes to reside in the same factory. This, along with other studies, gives strong evidence in favor of transcription factory specialization. Examination of cotransfected plasmids in COS7 monkey cells showed that constructs with identical promoters colocalized to the same transcription factory to a higher degree than those with heterologous promoters . Furthermore, the finding that the erythroid transcription factor Klf1 mediates preferential co-associations of Klf1-regulated genes at Klf1-specialized transcription factories provided the first functional evidence that transcription factors could be responsible for the organization of a specific subset of genes at transcription factories  (Figure 3b,c).
Despite recent demonstrations of spatial clustering in three dimensions by 3C-based methods and RNA and DNA FISH [12, 24, 33, 34], it is still unclear whether association influences gene transcriptional output. Hu et al.  noted the appearance of larger RNA FISH signals in primary human breast epithelial cells from spatially associated genes induced by estrogen receptor (ER)α, suggesting increased transcriptional output from clustered alleles. In addition, long-range association of transcription factor binding sites or co-regulated genes correlated with an increased probability of transcriptional activity of the clustered alleles, suggesting that clustered alleles were more likely to show higher transcriptional activity [24, 36].
Spatial organization of silent chromatin
There are obvious potential incentives to cluster specific genes and chromatin regions. For example, clustering of co-regulated genes in specialized factories may be more efficient in terms of the machinery needed for their expression. The clustering of silent chromatin in the nucleus could also decrease the amount of machinery needed for maintenance. Indeed, heterochromatin has long been observed to form clusters that are distinct from euchromatin within the nucleoplasm. For example, centromeres cluster into chromocenters, visualized by staining with the DNA stain 4',6-diamidino-2-phenylindole (DAPI) or immuno-labeling of centromeric proteins. Clustering of centromeres is unusually pronounced in rodent rod cells, where these regions are gathered in the center of the nucleus surrounded by heterochromatin, which is suggested to reduce diffraction and permit more efficient passing of photons . This clustering demonstrates an extraordinary spatial organization of chromatin for a specific function. Silenced genes have also been observed clustering with pericentromeric heterochromatin . For example, the non-functional, rearranged IgH locus is recruited to centromeres concurrent with transcriptional silencing of its V genes in B cells [39, 40]. This relocalization correlates with dramatic deacetylation of the locus , but it is currently unclear whether this deacetylation occurs before or after localization to chromocenters. Telomeres are regions of transcriptionally silent chromatin and have been reported to cluster throughout the nucleoplasm . However, human telomeres with NORs located in their short acrocentric arms cluster separately at the perinucleolar compartment , again highlighting spatial localization.
Chromatin clustering may also be mediated through long non-coding RNAs (lncRNAs) such as Xist, Air and Kcnq1ot1, which range in size from 17 to 108 kb. The most studied of these lncRNAs is Xist. Transcription of Xist [43, 44] from one of the two X chromosomes results in the inactivation of that X chromosome in female mammals. The Xist RNA (about 17 kb in length) interacts with the future inactive X chromosome to create a nuclear domain devoid of RNAPII and basal transcription factors such as TFIIH and TFIIF. X-linked genes are recruited into this nuclear domain, correlating with their transcriptional silencing . This internal repositioning of previously active genes is the first structural change following Xist accumulation. Intriguingly, genes that escape X-inactivation are located on the periphery of, or outside the Xist domain , presumably interacting with RNAPII and various transcription factors.
lncRNAs have also been implicated in the regulation of imprinted gene clusters. Imprinted genes show effects specific to the parent of origin, in which a single allele (maternal or paternal) is epigenetically silenced during development. Imprinted repression of a selected allele may occur in a similar mechanism to that of Xist. For example, the murine Air (antisense to Igf2r) lncRNA is essential for imprinted allele-specific silencing of the cis-linked solute carrier genes Slc22a3 and Slc22a2 together with Igf2r from the paternal chromosome 17 . The Air RNA forms a cloud within nuclei and interacts, by an unknown mechanism, with the Slc22a3 promoter. Air is also required to target the histone H3 lysine 9 histone methyltransferase G9a to the Slc22a3 promoter . It seems plausible that the Air cloud recruits specific genes into the volume it occupies to induce silencing. Unlike Xist, which induces silencing over the entire X chromosome, Air's influence is restricted to a cluster of genes spanning a 300 kb region immediately adjacent to the Air gene. The structural aspects to how Air functions or what restricts the size of the Air compartment remains unclear. This effect is mirrored by the Kcnq1ot1 lncRNA, which also seems to create a repressive domain that is responsible for repression of a variable number of cis-linked genes in embryonic and placental tissues [48–51]. Kcnq1ot1 is an imprinted 50 kb lncRNA transcribed in the antisense direction from within the potassium voltage-gated channel gene, Kcnq1, on mouse chromosome 7. The Kcnq1ot1 repressive domain is larger in placental tissue than in embryonic tissue, and this may be correlated with a higher number of silenced genes in the placenta [49, 50].
lncRNA repression may also occur in trans. The 2.2 kb HOTAIR ncRNA, expressed from the HOXC locus on chromosome 12 in humans, has been shown to be necessary for repression of the HOXD locus, present on chromosome 2 . Although loss of the HOTAIR lncRNA results in the reactivation of the HOXD locus, indicating a potential trans mechanism of gene repression , no direct interaction between HOTAIR and the HOXD locus has been observed.
Establishing spatial organization
Spatial genome organization implies movement. The tissue-specific clustering of specific genomic elements requires that at some stage chromatin regions must move towards each other, in either a directed or a passive way. As cells exit mitosis and chromosomes decondense, large-scale movements of chromatin domains have been observed [53, 54]; these may result in the repositioning of chromosomal and sub-chromosomal regions to their generalized relative positions. Constrained diffusion  or chromatin movements mediated by nuclear actin and myosin [35, 56–58] may have a role in refining these positions throughout interphase (Figure 4).
The organization of the genome as it is transcribed is achieved to a large extent through interactions of genes with transcription factories. Although it is not known how factories form, the pulsatile nature of individual gene transcription during interphase [59, 60], which seems to involve dynamic gene associations with factories [17, 22], suggests two possible models to describe how specialized factories are established. In a deterministic factory model, specific key transcription factors (such as Klf1) are directed to or become concentrated at a subset of factories. Genes requiring that particular factor for transcription would then need to move to those factories to become active. In the second model, referred to as the self-organization model, genes and their bound regulatory factors stochastically engage factories in their local environment. Specialization may occur when several similarly regulated genes associate with the same factory simultaneously. This may stabilize their presence at the shared factory through factor sharing, in other words the increased local concentration of specific regulatory factors may increase occupancy at key regulatory sites on the clustered genes, thus promoting their reinitiation and stabilizing their co-association. There is little evidence in favor of either model at the moment. The deterministic model requires some mechanism to direct specific factors to a subset of factories, suggesting that differences in factories must precede their specialization. In the self-organization model, all factories may start out being equal but then may become specialized, perhaps transiently by character of the transcription units engaged there.
Evidence in favor of the self-organization model can be seen in a population of virally infected cells: the quickest cells to respond by producing IFN-β are those in which the IFN-β gene is in close physical proximity with other genetic loci that bind the NF-κB transcription factor . NF-κB induces the formation of the enhanceosome multiprotein complex, which binds upstream of the IFN-β promoter and interacts with the transcriptional machinery necessary for the induction of the IFN-β gene. The formation of the enhanceosome at the IFN-β promoter is more likely to occur if one NF-κB-dependent gene is in close physical proximity to another NF-κB-dependent gene, thereby enabling these loci to establish an environment that favors transcription . This supports a role for transcription factors mediating chromosomal interactions specific for the tissue and stimulus involved. Such transcriptional organization of genes may also be mediated by other proteins that are not part of the core transcriptional apparatus, such as the CCCTC-binding factor (CTCF) and Polycomb repressive complexes (PRCs).
Some proteins may have a structural role in maintenance of genome conformation. CTCF is a highly conserved vertebrate transcription regulator that has been reported to bind at many thousands of sites in multiple genomes [61–65]. This binding does not seem to correlate to specific networks of genes, but CTCF has been suggested to mediate chromatin interactomes . Indeed, CTCF binding has been suggested to silence the maternally inherited Igf2 allele , form active chromatin hubs , and establish cytokine-induced loops within the human MHC class II locus . Furthermore, CTCF interacts with a large number of nuclear proteins ranging from transcription factors to structural proteins . Cohesin, which is a key component for holding sister chromatids together and which is implicated in several diseases, has been shown to bind to about 70% of all CTCF sites in the human genome . Specifically, CTCF mediates cohesin binding , and this interaction has been suggested to impart cell-type-specific intrachromosomal interactions at the developmentally regulated human cytokine locus IFN-γ  and the apolipoprotein A1/C3/A4/A5 gene region on human chromosome 11 . These processes suggest a multifunctional role of CTCF in the organization of the genome, adding another organizational layer of complexity.
Repressive domains and complexes may also provide a structural component for establishing long-range interactions and organizing the genome. For example, genome-wide studies have revealed that PRCs associate with promoter regions of some developmentally regulated and silenced genes [74, 75]. Evidence to support long-range interactions through PRCs comes from studies investigating Polycomb response elements (PREs), which allow the recruitment of PRCs to target genes through DNA binding proteins . Fab-7 is a Drosophila regulatory element containing a PRE that contributes to regulated spatial transcription of the Abdominal-B gene of the Drosophila bithorax complex [77, 78]. The endogenous Fab-7 PRE has been shown to interact with transgenic Fab-7 elements inserted at heterologous sites , highlighting specific long-range PRE-mediated chromatin interactions. Similarly, Mcp, another PRE containing regulatory element from the Drosophila bithorax complex, can interact with other remote copies of Mcp elements in the genome . These results provided direct evidence that regulatory elements can promote sequence-specific long-range chromosomal interactions, suggesting that PRCs are likely to provide another mechanism for organizing the genome.
Recently, the roles of nuclear actin and myosin have generated considerable interest in the organization of the mammalian genome. Data strongly indicate that nuclear actin is involved in gene transcription by all three polymerases . Long-range directed interphase chromatin movement seems to require actin polymerization, as the expression of mutant actin that cannot polymerize prevents chromatin relocation [56, 57]. Nuclear actin and nuclear myosin I have also been implicated in mediating interchromosomal interactions between the ERα-dependent genes  and in repositioning of selected chromosomes during serum starvation .
Spatial organization and the future
Here, we have focused on the relationships between transcription, silencing and the three-dimensional organization of the genome (Figure 4). This is at the expense of other structures that also contribute to the genome's organization, such as the nuclear lamina [82, 83]. In summary, it is apparent that the genome is arranged in a non-random, cell- and tissue-specific manner that is suited for various nuclear functions. Highly expressed housekeeping genes are often organized in the linear genome in RIDGES (regions of increased gene expression) , but linear clustering of tissue-specific genes is not evident . Although clustering of housekeeping genes may be favored in a two-dimensional arrangement along the chromosome, clustering of tissue-specific genes is evident only in three dimensions across the nucleus [12, 24, 33], presumably reflecting transcriptional and other regulatory requirements. It is clear that the local folding of chromatin, for example between a gene and long-range enhancer or between PREs, is a critical determinant of gene expression. The way these regions interact with other regions of the same chromosome, some of which may be similarly regulated, also seems to be important for function. Similarly, the way these chromosomal regions interact with regions on other chromosomes will undoubtedly affect spatial genome organization, but it may also be important in contributing to tissue-specific gene expression programs. It is likely that three-dimensional organization is an important missing link in understanding how the genome is regulated; unraveling this organization is a major challenge for the future.
Simonis M, Kooren J, de Laat W: An evaluation of 3C-based methods to capture DNA interactions. Nat Methods. 2007, 4: 895-901. 10.1038/nmeth1114.
Carter D, Chakalova L, Osborne CS, Dai YF, Fraser P: Long-range chromatin regulatory interactions in vivo. Nat Genet. 2002, 32: 623-626. 10.1038/ng1051.
Tolhuis B, Palstra RJ, Splinter E, Grosveld F, de Laat W: Looping and interaction between hypersensitive sites in the active beta-globin locus. Mol Cell. 2002, 10: 1453-1465. 10.1016/S1097-2765(02)00781-5.
Pevny L, Simon MC, Robertson E, Klein WH, Tsai S-F, D'Agati V, Orkin SH, Costantini F: Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature. 1991, 349: 257-260. 10.1038/349257a0.
Starck J, Sarkar R, Romana M, Bhargava A, Scarpa AL, Tanaka M, Chamberlain JW, Weissman SM, Forget BG: Developmental regulation of human gamma-globin and beta-globin genes in the absence of the locus-control region. Blood. 1994, 84: 1656-1665.
Drissen R, Palstra RJ, Gillemans N, Splinter E, Grosveld F, Philipsen S, de Laat W: The active spatial organization of the beta-globin locus requires the transcription factor EKLF. Genes Dev. 2004, 18: 2485-2490. 10.1101/gad.317004.
Song SH, Hou CH, Dean A: A positive role for NLI/Ldb1 in long-range beta-globin locus control region function. Mol Cell. 2007, 28: 810-822. 10.1016/j.molcel.2007.09.025.
Kim SI, Bultman SJ, Kiefer CM, Dean A, Bresnick EH: BRG1 requirement for long-range interaction of a locus control region with a downstream promoter. Proc Natl Acad Sci USA. 2009, 106: 2259-2264. 10.1073/pnas.0806420106.
Bolland DJ, Wood AL, Corcoran AE: Large-scale chromatin remodeling at the immunoglobulin heavy chain locus: a paradigm for multigene regulation. Adv Exp Med Biol. 2009, 650: 59-72. full_text.
Spilianakis CG, Flavell RA: Long-range intrachromosomal interactions in the T helper type 2 cytokine locus. Nat Immunol. 2004, 5: 1017-1027. 10.1038/ni1115.
Spilianakis CG, Lalioti MD, Town T, Lee GR, Flavell RA: Interchromosomal associations between alternatively expressed loci. Nature. 2005, 435: 637-645. 10.1038/nature03574.
Zhao Z, Tavoosidana G, Sjolinder M, Gondor A, Mariano P, Wang S, Kanduri C, Lezcano M, Sandhu KS, Singh U, Pant V, Tiwari V, Kurukuti S, Ohlsson R: Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions. Nat Genet. 2006, 38: 1341-1347. 10.1038/ng1891.
Lomvardas S, Barnea G, Pisapia DJ, Mendelsohn M, Kirkland J, Axel R: Interchromosornal interactions and olfactory receptor choice. Cell. 2006, 126: 403-413. 10.1016/j.cell.2006.06.035.
Fuss SH, Omura M, Mombaerts P: Local and cis effects of the H element on expression of odorant receptor genes in mouse. Cell. 2007, 130: 373-384. 10.1016/j.cell.2007.06.023.
Nishizumi H, Kurnasaka K, Inoue N, Nakashima A, Sakano H: Deletion of the core-H region in mice abolishes the expression of three proximal odorant receptor genes in cis. Proc Natl Acad Sci USA. 2007, 104: 20067-20072. 10.1073/pnas.0706544105.
Rowley JD: Chromosomal translocations: revisited yet again. Blood. 2008, 112: 2183-2189. 10.1182/blood-2008-04-097931.
Osborne CS, Chakalova L, Mitchell JA, Horton A, Wood AL, Bolland DJ, Corcoran AE, Fraser P: Myc dynamically and preferentially relocates to a transcription factory occupied by IgH. PLoS Biol. 2007, 5: e192-10.1371/journal.pbio.0050192.
Lin C, Yang L, Tanasa B, Hutt K, Ju BG, Ohgi K, Zhang J, Rose DW, Fu XD, Glass CK, Rosenfeld MG: Nuclear receptor-induced chromosomal proximity and DNA breaks underlie specific translocations in cancer. Cell. 2009, 139: 1069-1083. 10.1016/j.cell.2009.11.030.
Prieto JL, McStay B: Nucleolar biogenesis: the first small steps. Biochem Soc Trans. 2005, 33: 1441-1443. 10.1042/BST20051441.
Kalmarova M, Smirnov E, Masata M, Koberna K, Ligasova A, Popov A, Raska I: Positioning of NORs and NOR-bearing chromosomes in relation to nucleoli. J Struct Biol. 2007, 160: 49-56. 10.1016/j.jsb.2007.06.012.
Dousset T, Wang C, Verheggen C, Chen DY, Hernandez-Verdun D, Huang S: Initiation of nucleolar assembly is independent of RNA polymerase I transcription. Mol Biol Cell. 2000, 11: 2705-2717.
Osborne CS, Chakalova L, Brown KE, Carter D, Horton A, Debrand E, Goyenechea B, Mitchell JA, Lopes S, Reik W, Fraser P: Active genes dynamically colocalize to shared sites of ongoing transcription. Nat Genet. 2004, 36: 1065-1071. 10.1038/ng1423.
Mitchell JA, Fraser P: Transcription factories are nuclear subcompartments that remain in the absence of transcription. Genes Dev. 2008, 22: 20-25. 10.1101/gad.454008.
Schoenfelder S, Sexton T, Chakalova L, Cope NF, Horton A, Andrews S, Kurukuti S, Mitchell JA, Umlauf D, Dimitrova DS, Eskiw CH, Luo Y, Wei CL, Ruan Y, Bieker JJ, Fraser P: Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells. Nat Genet. 2010, 42: 53-61. 10.1038/ng.496.
Eskiw CH, Rapp A, Carter DRF, Cook PR: RNA polymerase II activity is located on the surface of protein-rich transcription factories. J Cell Sci. 2008, 121: 1999-2007. 10.1242/jcs.027250.
Iborra FJ, Pombo A, Jackson DA, Cook PR: Active RNA polymerases are localized within discrete transcription 'factories' in human nuclei. J Cell Sci. 1996, 109: 1427-1436.
Pombo A, Hollinshead M, Cook PR: Bridging the resolution gap: imaging the same transcription factories in cryosections by light and electron microscopy. J Histochem Cytochem. 1999, 47: 471-480.
Kimura H, Sugaya K, Cook PR: The transcription cycle of RNA polymerase II in living cells. J Cell Biol. 2002, 159: 777-782. 10.1083/jcb.200206019.
Grande MA, vanderKraan I, deJong L, vanDriel R: Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II. J Cell Sci. 1997, 110: 1781-1791.
vanSteensel B, vanBinnendijk EP, Hornsby CD, vanderVoort HTM, Krozowski ZS, deKloet ER, vanDriel R: Partial colocalization of glucocorticoid and mineralocorticoid receptors in discrete compartments in nuclei of rat hippocampus neurons. J Cell Sci. 1996, 109: 787-792.
Pombo A, Cuello P, Schul W, Yoon JB, Roeder RG, Cook PR, Murphy S: Regional and temporal specialization in the nucleus: a transcriptionally-active nuclear domain rich in PTF, Oct1 and PIKA antigens associates with specific chromosomes early in the cell cycle. EMBO J. 1998, 17: 1768-1778. 10.1093/emboj/17.6.1768.
Xu M, Cook PR: Similar active genes cluster in specialized transcription factories. J Cell Biol. 2008, 181: 615-623. 10.1083/jcb.200710053.
Fullwood MJ, Liu MH, Pan YF, Liu J, Xu H, Mohamed YB, Orlov YL, Velkov S, Ho A, Mei PH, Chew EG, Huang PY, Welboren WJ, Han Y, Ooi HS, Ariyaratne PN, Vega VB, Luo Y, Tan PY, Choy PY, Wansa KD, Zhao B, Lim KS, Leow SC, Yow JS, Joseph R, Li H, Desai KV, Thomsen JS, Lee YK, et al: An oestrogen-receptor-alpha-bound human chromatin interactome. Nature. 2009, 462: 58-64. 10.1038/nature08497.
Simonis M, Klous P, Splinter E, Moshkin Y, Willemsen R, de Wit E, van Steensel B, de Laat W: Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C). Nat Genet. 2006, 38: 1348-1354. 10.1038/ng1896.
Hu Q, Kwon YS, Nunez E, Cardamone MD, Hutt KR, Ohgi KA, Garcia-Bassets I, Rose DW, Glass CK, Rosenfeld MG, Fu XD: Enhancing nuclear receptor-induced transcription requires nuclear motor and LSD1-dependent gene networking in interchromatin granules. Proc Natl Acad Sci USA. 2008, 105: 19199-19204. 10.1073/pnas.0810634105.
Apostolou E, Thanos D: Virus infection induces NF-kappa B-dependent interchromosomal associations mediating monoallelic IFN-beta gene expression. Cell. 2008, 134: 85-96. 10.1016/j.cell.2008.05.052.
Solovei I, Kreysing M, Lanctot C, Kosem S, Peichl L, Cremer T, Guck J, Joffe B: Nuclear architecture of rod photoreceptor cells adapts to vision in mammalian evolution. Cell. 2009, 137: 356-368. 10.1016/j.cell.2009.01.052.
Brown KE, Guest SS, Smale ST, Hahm K, Merkenschlager M, Fisher AG: Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin. Cell. 1997, 91: 845-854. 10.1016/S0092-8674(00)80472-9.
Skok JA, Brown KE, Azuara V, Caparros ML, Baxter J, Takacs K, Dillon N, Gray D, Perry RP, Merkenschlager M, Fisher AG: Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes. Nat Immunol. 2001, 2: 848-854. 10.1038/ni0901-848.
Roldan E, Fuxa M, Chong W, Martinez D, Novatchkova M, Busslinger M, Skok JA: Locus 'decontraction' and centromeric recruitment contribute to allelic exclusion of the immunoglobulin heavy-chain gene. Nat Immunol. 2005, 6: 31-41. 10.1038/ni1150.
Chowdhury D, Sen R: Transient IL-7/IL-7R signaling provides a mechanism for feedback inhibition of immunoglobulin heavy chain gene rearrangements. Immunity. 2003, 18: 229-241. 10.1016/S1074-7613(03)00030-X.
Ramirez MJ, Surralles J: Laser confocal microscopy analysis of human interphase nuclei by three-dimensional FISH reveals dynamic perinucleolar clustering of telomeres. Cytogenet Genome Res. 2008, 122: 237-242. 10.1159/000167809.
Avner P, Heard E: X-chromosome inactivation: counting, choice and initiation. Nat Rev Genet. 2001, 2: 59-67. 10.1038/35047580.
Cohen DE, Lee JT: X-chromosome inactivation and the search for chromosome-wide silencers. Curr Opin Genet Dev. 2002, 12: 219-224. 10.1016/S0959-437X(02)00289-7.
Chaumeil J, Le Baccon P, Wutz A, Heard E: A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced. Genes Dev. 2006, 20: 2223-2237. 10.1101/gad.380906.
Sleutels F, Zwart R, Barlow DP: The non-coding Air RNA is required for silencing autosomal imprinted genes. Nature. 2002, 415: 810-813.
Nagano T, Mitchell JA, Sanz LA, Pauler FM, Ferguson-Smith AC, Feil R, Fraser P: The air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science. 2008, 322: 1717-1720. 10.1126/science.1163802.
Fitzpatrick GV, Soloway PD, Higgins MJ: Regional loss of imprinting and growth deficiency in mice with a targeted deletion of KvDMR1. Nat Genet. 2002, 32: 426-431. 10.1038/ng988.
Pandey RR, Mondal T, Mohammad F, Enroth S, Redrup L, Komorowski J, Nagano T, Mancini-DiNardo D, Kanduri C: Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol Cell. 2008, 32: 232-246. 10.1016/j.molcel.2008.08.022.
Redrup L, Branco MR, Perdeaux ER, Krueger C, Lewis A, Santos F, Nagano T, Cobb BS, Fraser P, Reik W: The long noncoding RNA Kcnq1ot1 organises a lineage-specific nuclear domain for epigenetic gene silencing. Development. 2009, 136: 525-530. 10.1242/dev.031328.
Terranova R, Yokobayashi S, Stadler MB, Otte AP, van Lohuizen M, Orkin SH, Peters A: Polycomb group proteins Ezh2 and Rnf2 direct genomic contraction and imprinted repression in early mouse embryos. Dev Cell. 2008, 15: 668-679. 10.1016/j.devcel.2008.08.015.
Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E, Chang HY: Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell. 2007, 129: 1311-1323. 10.1016/j.cell.2007.05.022.
Walter J, Schermelleh L, Cremer M, Tashiro S, Cremer T: Chromosome order in HeLa cells changes during mitosis and early G1, but is stably maintained during subsequent interphase stages. J Cell Biol. 2003, 160: 685-697. 10.1083/jcb.200211103.
Thomson I, Gilchrist S, Bickmore WA, Chubb JR: The radial positioning of chromatin is not inherited through mitosis but is established de novo in early G1. Curr Biol. 2004, 14: 166-172. 10.1016/j.cub.2003.12.024.
Chubb JR, Boyle S, Perry P, Bickmore WA: Chromatin motion is constrained by association with nuclear compartments in human cells. Curr Biol. 2002, 12: 439-445. 10.1016/S0960-9822(02)00695-4.
Chuang CH, Carpenter AE, Fuchsova B, Johnson T, de Lanerolle P, Belmont AS: Long-range directional movement of an interphase chromosome site. Curr Biol. 2006, 16: 825-831. 10.1016/j.cub.2006.03.059.
Dundr M, Ospina JK, Sung MH, John S, Upender M, Ried T, Hager GL, Matera AG: Actin-dependent intranuclear repositioning of an active gene locus in vivo. J Cell Biol. 2007, 179: 1095-1103. 10.1083/jcb.200710058.
Mehta IS, Amira M, Harvey AJ, Bridger JM: Rapid chromosome territory relocation by nuclear motor activity in response to serum removal in primary human fibroblasts. Genome Biol. 2010, 11: R5-10.1186/gb-2010-11-1-r5.
Chubb JR, Trcek T, Shenoy SM, Singer RH: Transcriptional pulsing of a developmental gene. Curr Biol. 2006, 16: 1018-1025. 10.1016/j.cub.2006.03.092.
Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S: Stochastic mRNA synthesis in mammalian cells. PLoS Biol. 2006, 4: e309-10.1371/journal.pbio.0040309.
Xie XH, Mikkelsen TS, Gnirke A, Lindblad-Toh K, Kellis M, Lander ES: Systematic discovery of regulatory motifs in conserved regions of the human genome, including thousands of CTCF insulator sites. Proc Natl Acad Sci USA. 2007, 104: 7145-7150. 10.1073/pnas.0701811104.
Kim TH, Abdullaev ZK, Smith AD, Ching KA, Loukinov DI, Green RD, Zhang MQ, Lobanenkov VV, Ren B: Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome. Cell. 2007, 128: 1231-1245. 10.1016/j.cell.2006.12.048.
Jothi R, Cuddapah S, Barski A, Cui K, Zhao K: Genome-wide identification of in vivo protein-DNA binding sites from ChIP-Seq data. Nucleic Acids Res. 2008, 36: 5221-5231. 10.1093/nar/gkn488.
Cuddapah S, Jothi R, Schones DE, Roh TY, Cui KR, Zhao KJ: Global analysis of the insulator binding protein CTCF in chromatin barrier regions reveals demarcation of active and repressive domains. Genome Res. 2009, 19: 24-32. 10.1101/gr.082800.108.
Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, Wong E, Orlov YL, Zhang W, Jiang J, Loh YH, Yeo HC, Yeo ZX, Narang V, Govindarajan KR, Leong B, Shahab A, Ruan Y, Bourque G, Sung WK, Clarke ND, Wei CL, Ng HH: Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell. 2008, 133: 1106-1117. 10.1016/j.cell.2008.04.043.
Phillips JE, Corces VG: CTCF: Master weaver of the genome. Cell. 2009, 137: 1194-1211. 10.1016/j.cell.2009.06.001.
Kurukuti S, Tiwari VK, Tavoosidana G, Pugacheva E, Murrell A, Zhao ZH, Lobanenkov V, Reik W, Ohlsson R: CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2. Proc Natl Acad Sci USA. 2006, 103: 10684-10689. 10.1073/pnas.0600326103.
Splinter E, Heath H, Kooren J, Palstra RJ, Klous P, Grosveld F, Galjart N, de Laat W: CTCF mediates long-range chromatin looping and local histone modification in the beta-globin locus. Genes Dev. 2006, 20: 2349-2354. 10.1101/gad.399506.
Majumder P, Gomez JA, Chadwick BP, Boss JM: The insulator factor CTCF controls MHC class II gene expression and is required for the formation of long-distance chromatin interactions. J Exp Med. 2008, 205: 785-798. 10.1084/jem.20071843.
Zlatanova J, Caiafa P: CTCF and its protein partners: divide and rule?. J Cell Sci. 2009, 122: 1275-1284. 10.1242/jcs.039990.
Parelho V, Hadjur S, Spivakov M, Leleu M, Sauer S, Gregson HC, Jarmuz A, Canzonetta C, Webster Z, Nesterova T, Cobb BS, Yokomori K, Dillon N, Aragon L, Fisher AG, Merkenschlager M: Cohesins functionally associate with CTCF on mammalian chromosome arms. Cell. 2008, 132: 422-433. 10.1016/j.cell.2008.01.011.
Hadjur S, Williams LM, Ryan NK, Cobb BS, Sexton T, Fraser P, Fisher AG, Merkenschlager M: Cohesins form chromosomal cis-interactions at the developmentally regulated IFNG locus. Nature. 2009, 460: 410-413.
Mishiro T, Ishihara K, Hino S, Tsutsumi S, Aburatani H, Shirahige K, Kinoshita Y, Nakao M: Architectural roles of multiple chromatin insulators at the human apolipoprotein gene cluster. EMBO J. 2009, 28: 1234-1245. 10.1038/emboj.2009.81.
Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G: Genome regulation by polycomb and trithorax proteins. Cell. 2007, 128: 735-745. 10.1016/j.cell.2007.02.009.
Schwartz YB, Pirrotta V: Polycomb complexes and epigenetic states. Curr Opin Cell Biol. 2008, 20: 266-273. 10.1016/j.ceb.2008.03.002.
Muller J, Kassis JA: Polycomb response elements and targeting of Polycomb group proteins in Drosophila. Curr Opin Genet Dev. 2006, 16: 476-484. 10.1016/j.gde.2006.08.005.
Gyurkovics H, Gausz J, Kummer J, Karch F: A new homeotic mutation in the Drosophila bithorax complex removes a boundary separating 2 domains of regulation. EMBO J. 1990, 9: 2579-2585.
Busturia A, Bienz M: Silencers in abdominal-B, a homeotic Drosophila gene. EMBO J. 1993, 12: 1415-1425.
Bantignies F, Grimaud C, Lavrov S, Gabut M, Cavalli G: Inheritance of Polycomb-dependent chromosomal interactions in Drosophila. Genes Dev. 2003, 17: 2406-2420. 10.1101/gad.269503.
Vazquez J, Muller M, Pirrotta V, Sedat JW: The Mcp element mediates stable long-range chromosome-chromosome interactions in Drosophila. Mol Biol Cell. 2006, 17: 2158-2165. 10.1091/mbc.E06-01-0049.
Percipalle P: The long journey of actin and actin-associated proteins from genes to polysomes. Cell Mol Life Sci. 2009, 66: 2151-2165. 10.1007/s00018-009-0012-8.
Pickersgill H, Kalverda B, de Wit E, Talhout W, Fornerod M, van Steensel B: Characterization of the Drosophila melanogaster genome at the nuclear lamina. Nat Genet. 2006, 38: 1005-1014. 10.1038/ng1852.
Guelen L, Pagie L, Brasset E, Meuleman W, Faza MB, Talhout W, Eussen BH, de Klein A, Wessels L, de Laat W, van Steensel B: Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature. 2008, 453: 948-951. 10.1038/nature06947.
Caron H, van Schaik B, Mee van der M, Baas F, Riggins G, van Sluis P, Hermus MC, van Asperen R, Boon K, Voûte PA, Heisterkamp S, van Kampen A, Versteeg R: The human transcriptome map: clustering of highly expressed genes in chromosomal domains. Science. 2001, 291: 1289-1292. 10.1126/science.1056794.
Lercher MJ, Urrutia AO, Hurst LD: Clustering of housekeeping genes provides a unified model of gene order in the human genome. Nat Genet. 2002, 31: 180-183. 10.1038/ng887.
We thank all members of the Laboratory of Chromatin and Gene Expression for their help and advice, and also thank Lyubomira Chakalova, Claire Joyce and Nicole Shoaf for critical reading of the manuscript. This work was supported by the Medical Research Council and the Biotechnology and Biological Sciences Research Council, UK.