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Peter R. Cook - One of the best experts on this subject based on the ideXlab platform.
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Dissecting the nascent human transcriptome by analysing the RNA content of Transcription Factories.
Nucleic acids research, 2015Co-Authors: Maiwen Caudron-herger, Peter R. Cook, Karsten Rippe, Argyris PapantonisAbstract:While mapping total and poly-adenylated human transcriptomes has now become routine, characterizing nascent transcripts remains challenging, largely because nascent RNAs have such short half-lives. Here, we describe a simple, fast and cost-effective method to isolate RNA associated with Transcription Factories, the sites responsible for the majority of nuclear Transcription. Following stimulation of human endothelial cells with the pro-inflammatory cytokine TNFα, we isolate and analyse the RNA content of Factories by sequencing. Comparison with total, poly(A)+ and chromatin RNA fractions reveals that sequencing of purified factory RNA maps the complete nascent transcriptome; it is rich in intronic unprocessed transcript, as well as long intergenic non-coding (lincRNAs) and enhancer-associated RNAs (eRNAs), micro-RNA precursors and repeat-derived RNAs. Hence, we verify that Transcription Factories produce most nascent RNA and confer a regulatory role via their association with a set of specifically-retained non-coding transcripts.
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Promoter type influences Transcriptional topography by targeting genes to distinct nucleoplasmic sites
Journal of Cell Science, 2013Co-Authors: Joshua D. Larkin, Argyris Papantonis, Peter R. CookAbstract:Both the sequence of a promoter and the position of a gene in 3D nuclear space play crucial roles in gene regulation, but few studies address their inter-relationship. Using human and viral promoters on mini-chromosomes and RNA fluorescence in situ hybridization coupled to 'high-precision' localization, we show that promoters binding the same Transcription factors and responding to the same signaling pathways tend to be co-transcribed in the same Transcription Factories. We go on to suggest how such spatial co-association might drive co-regulation of genes under the control of similar cis-elements.
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Transcription Factories, chromatin loops, and the dysregulation of gene expression in malignancy
Seminars in cancer biology, 2012Co-Authors: Binwei Deng, Svitlana Melnik, Peter R. CookAbstract:Abstract Pathologists recognize and classify cancers according to nuclear morphology, but there remains little scientific explanation of why malignant nuclei possess their characteristic features, or how those features are related to dysregulated function. This essay will discuss a basic structure–function axis that connects one central architectural motif in the nucleus–the chromatin loop–to the vital nuclear function of Transcription. The loop is attached to a “Transcription factory” through components of the Transcription machinery (either polymerases or Transcriptional activators/repressors), and the position of a gene within a loop determines how often that gene is transcribed. Then, dysregulated Transcription is tightly coupled to alterations in structure, and vice versa. We also speculate on how the experimental approaches being used to analyze loops and Factories might be applied to study the problems of tumour initiation and progression.
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Transcriptional initiation frequency bursting and Transcription Factories
2011Co-Authors: Kieran Finan, Peter R. CookAbstract:We know a great deal about the relative population-averaged rates of Transcriptional initiation at many promoters, but little about absolute rates, and even less about the temporal distributions of initiations at single loci. Such data has now begun to accumulate; recently developed techniques such as RNA fluorescence in situ hybridization (FISH) and the MS2-GFP transcript-tagging system now allow single transcripts to be counted in single cells and in real time. Recent efforts have combined these methods with mathematical modeling to provide evidence that the Transcriptional activity of a given gene can vary widely from cell to cell and from minute to minute; many so-called “active” genes seem to spend much of their time inactive, before switching to produce a brief “burst” of transcripts. We briefly review the basic mechanisms of Transcription, before focusing on initiation rates. We discuss recent studies of initiation and relate findings to known mechanisms of regulation (concentrating on results obtained in mammalian systems).
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Genome Organization and Function in the Cell Nucleus - Transcriptional Initiation: Frequency, Bursting, and Transcription Factories
Genome Organization and Function in the Cell Nucleus, 2011Co-Authors: Kieran Finan, Peter R. CookAbstract:We know a great deal about the relative population-averaged rates of Transcriptional initiation at many promoters, but little about absolute rates, and even less about the temporal distributions of initiations at single loci. Such data has now begun to accumulate; recently developed techniques such as RNA fluorescence in situ hybridization (FISH) and the MS2-GFP transcript-tagging system now allow single transcripts to be counted in single cells and in real time. Recent efforts have combined these methods with mathematical modeling to provide evidence that the Transcriptional activity of a given gene can vary widely from cell to cell and from minute to minute; many so-called “active” genes seem to spend much of their time inactive, before switching to produce a brief “burst” of transcripts. We briefly review the basic mechanisms of Transcription, before focusing on initiation rates. We discuss recent studies of initiation and relate findings to known mechanisms of regulation (concentrating on results obtained in mammalian systems).
Ana Pombo - One of the best experts on this subject based on the ideXlab platform.
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Poised Transcription Factories prime silent uPA gene prior to activation.
PLoS biology, 2010Co-Authors: Carmelo Ferrai, Ana Pombo, Sheila Q. Xie, Paolo Luraghi, Davide Munari, Francisco Ramirez, Miguel R. Branco, Massimo P. CrippaAbstract:The position of genes in the interphase nucleus and their association with functional landmarks correlate with active and/or silent states of expression. Gene activation can induce chromatin looping from chromosome territories (CTs) and is thought to require de novo association with Transcription Factories. We identify two types of factory: "poised Transcription Factories," containing RNA polymerase II phosphorylated on Ser5, but not Ser2, residues, which differ from "active Factories" associated with phosphorylation on both residues. Using the urokinase-type plasminogen activator (uPA) gene as a model system, we find that this inducible gene is predominantly associated with poised (S5p(+)S2p(-)) Factories prior to activation and localized at the CT interior. Shortly after induction, the uPA locus is found associated with active (S5p(+)S2p(+)) Factories and loops out from its CT. However, the levels of gene association with poised or active Transcription Factories, before and after activation, are independent of locus positioning relative to its CT. RNA-FISH analyses show that, after activation, the uPA gene is transcribed with the same frequency at each CT position. Unexpectedly, prior to activation, the uPA loci internal to the CT are seldom Transcriptionally active, while the smaller number of uPA loci found outside their CT are transcribed as frequently as after induction. The association of inducible genes with poised Transcription Factories prior to activation is likely to contribute to the rapid and robust induction of gene expression in response to external stimuli, whereas gene positioning at the CT interior may be important to reinforce silencing mechanisms prior to induction.
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Transcription and chromatin organization of a housekeeping gene cluster containing an integrated β-globin locus control region
PLoS genetics, 2008Co-Authors: Daan Noordermeer, Ana Pombo, Miguel R. Branco, Wilfred F. J. Van Ijcken, Erik Splinter, Petra Klous, Sigrid M. A. Swagemakers, Manousos Koutsourakis, Peter J. Van Der Spek, Wouter De LaatAbstract:The activity of locus control regions (LCR) has been correlated with chromatin decondensation, spreading of active chromatin marks, locus repositioning away from its chromosome territory (CT), increased association with Transcription Factories, and long-range interactions via chromatin looping. To investigate the relative importance of these events in the regulation of gene expression, we targeted the human β-globin LCR in two opposite orientations to a gene-dense region in the mouse genome containing mostly housekeeping genes. We found that each oppositely oriented LCR influenced gene expression on both sides of the integration site and over a maximum distance of 150 kilobases. A subset of genes was Transcriptionally enhanced, some of which in an LCR orientation-dependent manner. The locus resides mostly at the edge of its CT and integration of the LCR in either orientation caused a more frequent positioning of the locus away from its CT. Locus association with Transcription Factories increased moderately, both for loci at the edge and outside of the CT. These results show that nuclear repositioning is not sufficient to increase Transcription of any given gene in this region. We identified long-range interactions between the LCR and two upregulated genes and propose that LCR-gene contacts via chromatin looping determine which genes are Transcriptionally enhanced.
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intermingling of chromosome territories in interphase suggests role in translocations and Transcription dependent associations
PLOS Biology, 2006Co-Authors: Miguel R. Branco, Ana PomboAbstract:After mitosis, mammalian chromosomes partially decondense to occupy distinct territories in the cell nucleus. Current models propose that territories are separated by an interchromatin domain, rich in soluble nuclear machinery, where only rare interchromosomal interactions can occur via extended chromatin loops. In contrast, recent evidence for chromatin mobility and high frequency of chromosome translocations are consistent with significant levels of chromosome intermingling, with important consequences for genome function and stability. Here we use a novel high-resolution in situ hybridization procedure that preserves chromatin nanostructure to show that chromosome territories intermingle significantly in the nucleus of human cells. The degree of intermingling between specific chromosome pairs in human lymphocytes correlates with the frequency of chromosome translocations in the same cell type, implying that double-strand breaks formed within areas of intermingling are more likely to participate in interchromosomal rearrangements. The presence of Transcription Factories in regions of intermingling and the effect of Transcription impairment on the interactions between chromosomes shows that Transcription-dependent interchromosomal associations shape chromosome organization in mammalian cells. These findings suggest that local chromatin conformation and gene Transcription influence the extent with which chromosomes interact and affect their overall properties, with direct consequences for cell-type specific genome stability.
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Transcription Factories: quantitative studies of nanostructures in the mammalian nucleus
Chromosome Research, 2003Co-Authors: Sonya Martin, Ana PomboAbstract:Transcription by the three nuclear RNA polymerases is carried out in Transcription Factories. This conclusion has been drawn from estimates of the total number of nascent transcripts or active polymerase molecules and the number of Transcription sites within a cell. Here we summarise the variety of methods used to determine these parameters, discuss their associated problems and outline future prospects.
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Specialized Transcription Factories within mammalian nuclei.
Critical reviews in eukaryotic gene expression, 2000Co-Authors: Ana Pombo, Francisco J. Iborra, Peter R. Cook, Emma Jones, Hiroshi Kimura, Kimihiko Sugaya, Dean A JacksonAbstract:Recent evidence suggests that active RNA polymerases are concentrated in discrete 'Factories' where they work together on many different templates. The evidence that such Factories specialize in the Transcription of particular groups of genes is reviewed.
Peter Fraser - One of the best experts on this subject based on the ideXlab platform.
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Transcription Factories genetic programming in three dimensions
Current Opinion in Genetics & Development, 2012Co-Authors: Lucas Brandon Edelman, Peter FraserAbstract:Among the most intensively studied systems in molecular biology is the eukaryotic Transcriptional apparatus, which expresses genes in a regulated manner across hundreds of different cell types. Several studies over the past few years have added weight to the concept that Transcription takes place within discrete ‘Transcription Factories’ assembled inside the cell nucleus. These studies apply innovative technical approaches to gain insights into the molecular constituents, dynamical behaviour and organizational regulators of Transcription Factories, providing exciting insights into the spatial dimension of Transcriptional control.
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Ultrastructural study of Transcription Factories in mouse erythroblasts.
Journal of Cell Science, 2011Co-Authors: Christopher Eskiw, Peter FraserAbstract:RNA polymerase II (RNAPII) Transcription has been proposed to occur at Transcription Factories; nuclear focal accumulations of the active, phosphorylated forms of RNAPII. The low ratio of Transcription Factories to active genes and Transcription units suggests that genes must share Factories. Our previous analyses using light microscopy have indicated that multiple genes could share the same factory. Furthermore, we found that a small number of specialized Transcription Factories containing high levels of the erythroid-specific Transcription factor KLF1 preferentially transcribed a network of KLF1-regulated genes. Here we used correlative light microscopy in combination with energy filtering transmission electron microscopy (EFTEM) and electron microscopy in situ hybridization (EMISH) to analyse Transcription Factories, transcribing genes, and their nuclear environments at the ultrastructural level in ex vivo mouse foetal liver erythroblasts. We show that Transcription Factories in this tissue can be recognized as large nitrogen-rich structures with a mean diameter of 130 nm, which is considerably larger than that previously seen in transformed cultured cell lines. We show that KLF1-specialized Factories are significantly larger, with the majority of measured Factories occupying the upper 25th percentile of this distribution with an average diameter of 174 nm. In addition, we show that very highly transcribed genes associated with erythroid differentiation tend to occupy and share the largest Factories with an average diameter of 198 nm. Our results suggest that individual Factories are dynamically organized and able to respond to the increased Transcriptional load imposed by multiple highly transcribed genes by significantly increasing in size.
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Transcription Factories and nuclear organization of the genome.
Cold Spring Harbor symposia on quantitative biology, 2010Co-Authors: Christopher Eskiw, Nathan F Cope, Ieuan Clay, Stefan Schoenfelder, Takashi Nagano, Peter FraserAbstract:The dynamic compartmental organization of the Transcriptional machinery in mammalian nuclei places particular constraints on the spatial organization of the genome. The clustering of active RNA polymerase I Transcription units from several chromo somes at nucleoli is probably the best-characterized and universally accepted example. RNA polymerase II localization in mam malian nuclei occurs in distinct concentrated foci that are several-fold fewer in number compared to the number of active genes and Transcription units. Individual transcribed genes cluster at these shared Transcription Factories in a nonrandom manner, pref erentially associating with heterologous, coregulated genes. We suggest that the three-dimensional (3D) conformation and relative arrangement of chromosomes in the nucleus has a major role in delivering tissue-specific gene-expression programs.
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Nuclear Transcription Factories.
Blood, 2009Co-Authors: Peter FraserAbstract:Abstract SCI-16 The discovery of long-range intrachromosomal and interchromosomal interactions in higher eukaryotes (1, 2) points to a functional interplay between genome architecture and gene expression, challenging the view of Transcription as a one-dimensional process. However, the extent of such interactions and the underlying mechanisms are unknown. Here we present the first genome-wide analysis of interactions between Transcriptionally active genes at Transcription Factories using the mouse globin genes in erythroid tissues. Our results show that the transcripitonally active globin genes associate with hundreds of other transcribed genes, revealing extensive and preferential intra- and interchromosomal Transcription interactomes. We show that the erythroid-specific Transcription factor Klf1 mediates preferential co-associations of Klf1-regulated genes at a limited number of specialized Transcription Factories. Our results establish a new gene expression paradigm, suggesting that active co-regulated genes and their regulatory factors cooperate to create specialized nuclear hotspots optimized for efficient and coordinated Transcriptional control. 1. Carter D, Chakalova L, Osborne CS, Dai YF, Fraser P. Long-range chromatin regulatory interactions in vivo. Nat. Genet. 2002;32:623-626. 2. Osborne CS, Chakalova L, Brown KE, et al. Active genes dynamically co-localize to shared sites of ongoing Transcription. Nat. Genet. 2004;36:1065-1071. Disclosures No relevant conflicts of interest to declare.
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Transcription Factories are nuclear subcompartments that remain in the absence of Transcription
Genes & development, 2008Co-Authors: Jennifer A Mitchell, Peter FraserAbstract:Nascent Transcription occurs at nuclear foci of concentrated, hyperphosphorylated RNA polymerase II (RNAPII). We investigate RNAPII localization, distal gene coassociation, and Hbb locus conformation during inhibition of Transcription. Our results show distal active genes remain associated with RNAPII foci and each other in the absence of elongation. When initiation is inhibited, active genes dissociate from RNAPII foci and each other, suggesting initiation is necessary to tether distal active genes to shared foci. In the absence of Transcription RNAPII foci remain, indicating they are not simple accumulations of RNAPII on transcribed genes but exist as independent nuclear subcompartments.
Vincenzo Pirrotta - One of the best experts on this subject based on the ideXlab platform.
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Insulators target active genes to Transcription Factories and polycomb-repressed genes to polycomb bodies.
PLoS genetics, 2013Co-Authors: Katsuhito Ohno, Hongxing Gui, Vincenzo PirrottaAbstract:Polycomb bodies are foci of Polycomb proteins in which different Polycomb target genes are thought to co-localize in the nucleus, looping out from their chromosomal context. We have shown previously that insulators, not Polycomb response elements (PREs), mediate associations among Polycomb Group (PcG) targets to form Polycomb bodies. Here we use live imaging and 3C interactions to show that transgenes containing PREs and endogenous PcG-regulated genes are targeted by insulator proteins to different nuclear structures depending on their state of activity. When two genes are repressed, they co-localize in Polycomb bodies. When both are active, they are targeted to Transcription Factories in a fashion dependent on Trithorax and enhancer specificity as well as the insulator protein CTCF. In the absence of CTCF, assembly of Polycomb bodies is essentially reduced to those representing genomic clusters of Polycomb target genes. The critical role of Trithorax suggests that stable association with a specialized Transcription factory underlies the cellular memory of the active state.
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A view of nuclear Polycomb bodies
Current opinion in genetics & development, 2011Co-Authors: Vincenzo PirrottaAbstract:Polycomb group (PcG) proteins are concentrated in nuclear foci called PcG bodies. Although some of these foci are due to the tendency of PcG binding sites in the genome to occur in linear clusters, distant PcG sites can contact one another and in some cases congregate in the same PcG body when they are repressed. Experiments using transgenes containing PcG binding sites reveal that co-localization depends on the presence of insulator elements rather than of Polycomb Response Elements (PREs) and that it can occur also when the transgenes are in the active state. A model is proposed according to which insulator proteins mediate shuttling of PcG target genes between PcG bodies when repressed to Transcription Factories when Transcriptionally active.
Christopher Eskiw - One of the best experts on this subject based on the ideXlab platform.
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Ultrastructural study of Transcription Factories in mouse erythroblasts.
Journal of Cell Science, 2011Co-Authors: Christopher Eskiw, Peter FraserAbstract:RNA polymerase II (RNAPII) Transcription has been proposed to occur at Transcription Factories; nuclear focal accumulations of the active, phosphorylated forms of RNAPII. The low ratio of Transcription Factories to active genes and Transcription units suggests that genes must share Factories. Our previous analyses using light microscopy have indicated that multiple genes could share the same factory. Furthermore, we found that a small number of specialized Transcription Factories containing high levels of the erythroid-specific Transcription factor KLF1 preferentially transcribed a network of KLF1-regulated genes. Here we used correlative light microscopy in combination with energy filtering transmission electron microscopy (EFTEM) and electron microscopy in situ hybridization (EMISH) to analyse Transcription Factories, transcribing genes, and their nuclear environments at the ultrastructural level in ex vivo mouse foetal liver erythroblasts. We show that Transcription Factories in this tissue can be recognized as large nitrogen-rich structures with a mean diameter of 130 nm, which is considerably larger than that previously seen in transformed cultured cell lines. We show that KLF1-specialized Factories are significantly larger, with the majority of measured Factories occupying the upper 25th percentile of this distribution with an average diameter of 174 nm. In addition, we show that very highly transcribed genes associated with erythroid differentiation tend to occupy and share the largest Factories with an average diameter of 198 nm. Our results suggest that individual Factories are dynamically organized and able to respond to the increased Transcriptional load imposed by multiple highly transcribed genes by significantly increasing in size.
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Transcription Factories and nuclear organization of the genome.
Cold Spring Harbor symposia on quantitative biology, 2010Co-Authors: Christopher Eskiw, Nathan F Cope, Ieuan Clay, Stefan Schoenfelder, Takashi Nagano, Peter FraserAbstract:The dynamic compartmental organization of the Transcriptional machinery in mammalian nuclei places particular constraints on the spatial organization of the genome. The clustering of active RNA polymerase I Transcription units from several chromo somes at nucleoli is probably the best-characterized and universally accepted example. RNA polymerase II localization in mam malian nuclei occurs in distinct concentrated foci that are several-fold fewer in number compared to the number of active genes and Transcription units. Individual transcribed genes cluster at these shared Transcription Factories in a nonrandom manner, pref erentially associating with heterologous, coregulated genes. We suggest that the three-dimensional (3D) conformation and relative arrangement of chromosomes in the nucleus has a major role in delivering tissue-specific gene-expression programs.
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Transcription Factories
Biochemical Society Transactions, 2008Co-Authors: David r.f. Carter, Christopher Eskiw, Peter r. CookAbstract:There is increasing evidence that different Transcription units are transcribed together in discrete nuclear structures known as Transcription Factories. Various new techniques enable us to detect and characterize these structures. We review the latest findings and discuss how they support a model for Transcription and chromosome organization.
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RNA polymerase II activity is located on the surface of protein-rich Transcription Factories.
Journal of cell science, 2008Co-Authors: Christopher Eskiw, David R. F. Carter, Alexander Rapp, Peter R. CookAbstract:We used electron spectroscopic imaging to map nucleoplasmic Transcription sites in human cells at unprecedented resolution. HeLa cells were permeabilised, nascent transcripts were extended in BrUTP by approximately 40 nucleotides and the resulting BrRNA immunolabelled with gold particles before structures were viewed. Nascent RNA is almost invariably associated with polymorphic and nitrogen-rich (but phosphorus-poor) structures with a diameter of approximately 87 nm and mass of 10 MDa (calculated by reference to nucleosomes with known numbers of phosphorus and nitrogen atoms). Structures with similar atomic signatures and diameters were observed using correlative microscopy and in unpermeabilised cells. Our results are consistent with RNA synthesis occurring on the surface of these huge protein-rich Transcription Factories.
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Specialized Transcription Factories.
Biochemical Society symposium, 2006Co-Authors: Jon Bartlett, Christopher Eskiw, Jelena Blagojevic, David R. F. Carter, Maud Fromaget, Christy Job, Monee Shamsher, Inês Faro Trindade, Peter R. CookAbstract:We have previously suggested a model for the eukaryotic genome based on the structure of the bacterial nucleoid where active RNA polymerases cluster to loop the intervening DNA. This organization of polymerases into clusters – which we call Transcription ‘Factories’ – has important consequences. For example, in the nucleus of a HeLa cell the concentration of soluble RNA polymerase II is ∼1 mM, but the local concentration in a factory is 1000-fold higher. Because a promoter can diffuse ∼100 nm in 15 s, one lying near a factory is likely to initiate; moreover, when released at termination, it will still lie near a factory, and the movement and modifications (e.g. acetylation) accompanying elongation will leave it in an ‘open’ conformation. Another promoter out in a long loop is less likely to initiate, because the promoter concentration falls off with the cube of the distance from the factory. Moreover, a long tether will buffer it from Transcription-induced movement, making it prone to deacetylation, deposition of HP1 (heterochromatin protein 1), and incorporation into heterochromatin. The context around a promoter will then be self-sustaining: productive collisions of an active promoter with the factory will attract factors increasing the frequency of initiation, and the longer an inactive promoter remains inactive, the more it becomes embedded in heterochromatin. We review here the evidence that different Factories may specialize in the Transcription of different groups of genes.