transcription factories: quantitative studies of nanostructures in the mammalian nucleus

Transcription factories: quantitative studies of nanostructuresin the mammalian nucleus

Sonya Martin & Ana Pombo*MRC ^ Clinical Sciences Centre, Faculty of Medicine, Imperial College School of Science, Technology andMedicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK; Tel: (þ 0/44) 2083838232; Fax: (þ 0/44) 20 83838306; E-mail: [email protected]*Correspondence

Key words: nascent transcripts, nucleolus, RNA polymerase, transcription sites

Abstract

Transcription by the three nuclear RNA polymerases is carried out in transcription factories. This con-clusion has been drawn from estimates of the total number of nascent transcripts or active polymerasemolecules and the number of transcription sites within a cell. Here we summarise the variety of methodsused to determine these parameters, discuss their associated problems and outline future prospects.

Introduction

Gene expression is a multifaceted and highlyorganized process, the mechanisms of which areintricately linked through a complex network ofregulatory factors (Orphanides & Reinberg 2002).In human cells, the*2 m genome is packaged intoa nucleus *10 mm across through a complex,hierarchical system of folding (Horn & Peterson2002), which involves formation of nucleosomes,higher-order chromatin structures and chromo-somes. During interphase, chromosomes areorganized into territories throughout the nucleus(Ferreira et al. 1997, Croft et al. 1999, Cremer &Cremer 2001, Sadoni et al. 2001) that becomehighly compacted duringmitosis. Gene positioningwithin chromatin is thought to be amajor regulatorof transcription (Dillon & Festenstein 2002). Itshigher-order structure is repressive to processesthat require access to the DNA such that, beforetranscription occurs, chromatin is remodelledthrough interactions with transcriptional activa-tors and chromatin remodelling complexes.

Transcriptional control is maintained by a multi-layered network of more than 10 000 positivelyand negatively acting factors (Lemon & Tjian2000). These include sequence speci¢c activators(e.g. SpI), accessory factors (e.g. TFIID) andnumerous co-activators and repressors. Manysequence-speci¢c activators and a number ofco-regulators show cell-type-speci¢c expressionpatterns (Lai & Darnell 1991, Dikstein et al. 1996).Various nuclear bodies, including Cajal bodies,splicing speckles and the OPT domain, have beenshown to contain speci¢c sets of transcriptionfactors leading to speculation that factors arecompartmentalized in an organized system whichfacilitates transcription (Dundr & Misteli 2001,Spector 2001). Transcription by the three nuclearRNA polymerases (pols) is carried out in discretesites that are speci¢c for each type of polymerase,with pol I active in the nucleolus and pols II and IIIin the nucleoplasm (Figure 1a, b). These sites havebeen termed transcription factories and, in thisreview, we focus on the study of pol II transcriptionfactories.

Chromosome Research 11: 461^470, 2003. 461# 2003 Kluwer Academic Publishers. Printed in the Netherlands

RNA polymerases

Transcription in eukaryotic nuclei is carried out bythree enzymatic activities, RNA polymerase I, IIand III, which can be distinguished by theirsubunit composition, drug sensitivity and nuclearlocation. Pol I is highly specialized with multipleenzymes simultaneously transcribing each of themany active 45S rRNA genes which are needed tomaintain ribosome numbers during the cell cycle(Miller & Bakken 1972). Its nuclear organization is

the best characterized of the polymerases astranscription of the rRNA genes initiates theformation of nucleoli. Nucleoli contain sub-structures visible on the electron microscope (EM)including a number of pale ¢brillar centres (FCs),each surrounded by a dense ¢brillar component(DFC), embedded in a granular component (GC).The current understanding is that pol I andassociated co-factors are contained within FCs(Reimer et al. 1987, Jordan et al. 1996, Mosgoelleret al. 1998), template rDNA is at the interface

Figure 1. Transcription occurs in discrete sites throughout the nucleus. Single confocal optical sections of whole cells (a^d) andcryosections (e, f ). (a, b) Nascent transcripts produced by all three nuclear RNA pols were labelled by incubating permeabilizedHeLa cells in BrUTP and the other nucleotides. After cell ¢xation, newly made BrRNA was indirectly immunolabelled with AlexaFluor 488 (b) and nucleic acids counterstained with TOTO-3 (a). BrRNA made by pol I is detected in nucleoli (no) whilst that madeby pols II and III is detected in the nucleoplasm (nu). (c, d) Pol II, phosphorylated at Ser2, was detected in ¢xed HeLa cells byindirect immunolabelling using antibody H5 and Alexa Fluor 488 (d) and cells counterstained with TOTO-3 (c). Pol II is foundin many discrete sites throughout the nucleoplasm, in a similar distribution to sites containing BrRNA (b). (e, f) Nascent transcriptswere labelled as for (b), cells were then ¢xed, frozen and cryosectioned (*100 nm) and BrRNA indirectly immunolabelled withCy3 (f) and sections counterstained with TOTO-3 (e). Note reduced complexity of labelling compared with whole cells (b). Bars 2 mm.

462 S. Martin & A. Pombo

between FCs and DFCs (Thirty & Goessens 1992),nascent rRNA extends into the DFCs, where it isprocessed, and maturing ribosomes are found inthe GC (Hozak et al. 1994b, Puvion-Dutilleul et al.1997). The human (subtetraploid) HeLa cellcontains *30 FCs and an estimated 120 activerRNA genes, with each FC containing *4 activegenes and *500 active pols plus associated nas-cent transcripts (Miller & Bakken 1972, Jacksonet al. 1998). The FCs and associated DFCs can bedescribed as ‘transcription factories’ where manycopies of the same rRNA are made and processed.

The activities of pols II and III are nucleo-plasmic: pol II is responsible for the transcriptionof protein-coding mRNA as well as snRNAs and agrowing number of other non-coding RNAs(Szymanski & Barciszewski 2002) whilst pol IIItranscribes genes encoding other small structuralRNAs, including tRNAs and 5S rRNA. Pol II isan evolutionarily conserved protein composed oftwo major, pol II-speci¢c subunits, RPB1 andRPB2, in conjunction with 10 smaller subunits(Dahmus 1996). RPB1 contains an unusual car-boxy-terminal domain (CTD) composed of aheptapeptide repeat (52 in mammals) withconsensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. The CTD plays a pivotal role in theproduction of mRNA; complex cycles of phos-phorylation and dephosphorylation mediate itsfunction as a nucleating centre that attracts factorsrequired for transcription as well as co-transcrip-tional events, such as RNA capping, splicing andpolyadenylation (Proudfoot 2000, Bentley 2002,Maniatis & Reed 2002).

Transcription by pol II and pol III also occurs indiscrete sites or transcription factories. In contrastwith pol I factories, which can measure up to*500 nm, pol II and pol III factories are verysmall,*50 nm, and so cannot be recognized at theultrastructural level in the absence of antibodylabeling (Wansink et al. 1993, Iborra et al. 1996a,Wansink et al. 1996, Pombo et al. 1999b). Theycontain a number of polymerase molecules actingon a number of di¡erent transcription units,similar to pol I transcription in the nucleolus. Thisconclusion has been drawn from estimates of thetotal number of nascent transcripts or activepolymerase molecules and the number of tran-scription sites within a cell. Although a variety ofmethods have been used, all with inherent

problems, very similar results have been reported.Here we summarise how the di¡erent parametershave been determined, discuss associated problemsand outline future prospects.

Numbers of polymerases and nascent transcripts

So far, estimates of the total numbers of poly-merase molecules have largely been limited tostudies of pol II. Initial experiments involving[3H]amanitin, which binds to the active centre ofpol II present in RPB1 (Bushnell et al. 2002),estimated a total of*200 000 pols in a diploid cell(Chambon 1974). A di¡erent approach by Kimuraet al. (1999), using quantitative immunoblotting,yielded a similar result: *320 000 pol II moleculesin the subtetraploid HeLa cell. Using sarkosyl todiscriminate active pol II indicated that *25%(*85 000) of total pol II is engaged (Jackson et al.1998, Kimura et al. 1999). This number is con-sistent with numbers of active polymerasesrequired to double hnRNA levels during the cellcycle, determined from in vivo synthetic rates of2 kb/min and knowledge of the average size oftranscripts made by pol II (Jackson et al. 1998).The number of active pol II molecules was initiallydetermined biochemically by comparing the rateof RNA synthesis with the rate given by a knownnumber of pure polymerase molecules (Sugden &Keller 1973) and incorporation of [3H]UTP intonascent RNA using puri¢ed nuclei (Cox 1976).These two methods gave results in the same orderof magnitude: 20 000^100 000. More recently, thenumber of active polymerases was determined insitu after incorporation of [32P]UTP in permea-bilized cells (Jackson et al. 1998). With thismethod, endogenous pools of nucleotides areremoved to simplify estimates of speci¢c activityand the rate of elongation is reduced to minimizetranscript completion and re-initiation duringlabelling. HeLa cells were found to contain*75 000 nascent pol II and pol III transcripts,*65 000 of which are due to pol II (Jackson et al.1998, Pombo et al. 1999b). Due to the short length(80^120 bp) of pol III transcripts, they are thoughtto be associated with only one polymerase;therefore, it is estimated that there are *10 000active pol III transcription units in a HeLa cell.Early studies using Miller spreads, where

Transcription factories 463

chromatin is spread and individual transcriptionunits can be imaged by EM, showed that, inmammalian cells, non-ribosomal transcriptionunits were associated with only a few widelydispersed active complexes (Miller & Bakken1972). More recently, Jackson et al. (1998) visu-alized nascent BrRNA on chromatin ¢bres fromHeLa cells and also concluded that each extra-nucleolar transcription unit is probably associatedwith only one pol. These results suggest that mostof the *65 000 active pols II are associated withone transcription unit and hence that, on average,*65 000 units are active; these will include bona¢de genes and also non-coding units.

Taken together, this wide variety of methodssuggests that a cell contains tens of thousands ofactive pol II molecules. This is not unexpected asthe haploid genome contains at least 30 000 pro-tein-coding genes (Lander et al. 2001), and pos-sibly the same number of non-genic transcriptionunits (Szymanski & Barciszewski 2002). In anunsynchronized population of HeLa cells (sub-tetraploid; 3n in G1 and 6n in G2), the average cellwill contain 140 000 protein-coding genes andmany more transcription units; it is therefore notsurprising that aHeLa cell should contain*65 000active pol II transcription units at any one time.

Labelling sites of transcription

The nuclear organization of active polymerasesbecame apparent after visualizing sites of tran-scription; the tens of thousands of active pols arefound distributed throughout the nucleoplasmconcentrated in discrete sites. Some e¡ort has beenput into counting the number of sites to determinewhether each site corresponds to one or a numberof polymerases, like pol I in FCs. Active sites havebeen localized to perichromatin ¢brils at the edgeof condensed chromatin (Fakan & Puvion 1980,Puvion & Puvion-Dutilleul 1996, Cmarko et al.1999); unfortunately these studies do not providequantitative information in relation to the numberof sites. High-resolution techniques to label nas-cent transcripts involving incorporation of labellednucleotides and immunolabelling with £uoro-chromes or gold particles have provided adetailed analysis of sites of transcription (Jacksonet al. 1993, Wansink et al. 1993, Hozak et al.

1994a, Iborra et al. 1996b, Wansink et al. 1996,Pombo et al. 1999b; see Figure 1). Improvementsin the methodology led to an increased number ofsites being seen; e.g. compare *300^500 sites(Jackson et al. 1993, Wansink et al. 1993) with*2000^6000 (Iborra et al. 1996a, Fay et al. 1997)and *10 000 sites (Wansink et al. 1996, Pomboet al. 1999b). With the latest methods, it is unlikelythat any more sites are missed as the same numberis seen even after increasing incubation time withthe labelled analogues (Iborra et al. 1996a, Pomboet al. 1999a). Additionally, active pol II can nowbe detected using antibodies to phosphorylatedepitopes in the absence of cell permeabilizationand in vitro bu¡ers and the same number andorganization of sites is obtained (A.P., D. Mason,S.M., P. R. Cook unpublished observations). Inorder to obtain accurate numbers, transcripts muststay at the synthetic site and not move down thetransport pathway. In a number of studies,microinjection of BrUTP was used to accelerateuptake of label into cells (Wansink et al. 1993,Wansink et al. 1994a, Fay et al. 1997, Cmarkoet al. 1999). However, this does not overcome theadditional problem of controlling the rate ofsynthesis and also only a small population oflabelled cells is obtained, making it inappropriatefor biochemical analysis. Permeabilization of cellsand washing away internal nucleotide pools allowsthe rate of synthesis to be controlled and thereforethe detection of transcripts at their synthetic site(Jackson et al. 1993). Also, complete substitutionof UTP by BrUTP inhibits splicing (Wansink et al.1994b) such that the resulting BrRNA is unable tomove away from its site of synthesis.

Initially, the sensitivity of these techniques onlyallowed detection of pol II transcription and notpol III, which is present to a much smaller extent(*10% of total polymerizing activity comparedwith *70% for pol II; Jackson et al. 1998, Pomboet al. 1999b). Modi¢cation of the method ofJackson et al. (1993), to improve preservation ofthe cellular ultrastructure, allowed increasedsensitivity manifested as the ability to label pol IIItranscripts as well as pol II; this modi¢ed bu¡eralso preserves pol II phosphorylation and dis-tribution (A.P., D. Mason, S.M., P. R. Cookunpublished observations). Here, there were*10 000 sites of transcription per cell, of which*20% (i.e.*2000) were speci¢c for pol III and the


remaining *8000 contained only pol II. There-fore, at the highest resolution, *65 000 active polII molecules and associated nascent transcripts aredistributed throughout the nucleoplasm in *8000discrete sites. These sites have thus been termed‘factories’ as they contain *8 pols along with *8transcripts.

But do these in vitro methods actually label allthe transcription sites that are present in vivo?Jackson et al. (1998) addressed this problem usingdouble labelling of transcription sites ¢rst in vivo,then in vitro. Cells were grown in BrU to mark sitesof transcription in vivo, then permeabilized andallowed to extend already growing nascent tran-scripts in BioCTP, thus labelling sites of tran-scription in vitro. Subsequent immunolabelling ofthe resulting BrRNA and BioRNA showed thatthe size and numbers of sites labelled in vivo (Br)and in vitro (Bio) were essentially the same.

An alternative approach to label active sitescontaining pol II may become possible with theemergence of antibodies speci¢c for the activeform of the enzyme itself. Elongating pol II isphosphorylated at the Ser2 residues of the CTD(Dahmus 1996, Komarnitsky et al. 2000) and soantibodies to this epitope may speci¢cally markactive sites of transcription by pol II. For example,the monoclonal antibody H5 (Warren et al. 1992,Patturajan et al. 1998) is a good candidate, as itlabels sites distributed throughout the nucleo-plasm that are found close to, and in some casesoverlapping, sites containing nascent BrRNA(Grande et al. 1997, Zeng et al. 1997, Pombo et al.1999b; see Figure 1c, d). However, a small pro-portion of this form is sensitive to sarkosyl andtherefore is not actively engaged in transcription(Jackson et al. 1998, Kimura et al. 1999).

Transcription by pol III is also carried out indedicated factories as*10 000 pols and transcriptsare distributed in only *2000 sites i.e. *5 polsand associated transcripts per site (Pombo et al.1999b). The identi¢cation of sites of pol IIItranscription requires inhibition of pol II activity,which can be achieved using low concentrations ofa-amanitin. Experiments in vivo have been com-plicated by the low permeability of a-amanitin inliving cells and also transcripts made by pol IIIare so short that they are completed in seconds andmay leave synthetic sites during the course of theexperiment. However, the improved in vitro

assays outlined above made possible the visuali-zation of sites containing nascent RNA made bypol III. Antibody-blocking assays showed that nodetectable pol II was found in pol III factories, andvice versa (Pombo et al. 1999b), suggesting that polII and III act in their own dedicated factories, likepol I in nucleoli. These experiments also highlightthe sensitivity of the immunolabelling proceduresused. The short BrRNAs (*100 bp) made by polIII can be detected after just one transcriptioncycle (Pombo et al. 1999b); detection of a singlepol II transcript, after a short incubation time toallow extension of *1000 bp, would therefore bestraightforward.

Visualizing sites of transcription

Estimation of the number of transcription sitesrequires methods of visualization that are sensitiveand have enough resolution to distinguish adjacentsites. Transcription by pols II and III occurs inthousands of very small sites, such that, on theconventional light microscope (LM), they canproduce a di¡use pattern with only a small numberof brighter foci (Jackson et al. 1993, Pombo et al.1999a). The process of deconvolution has beenused to subtract ‘out-of-focus’ £are from a seriesof optical sections. However, the algorithmsinvolved require subtraction of a ‘background’level that may lead to loss of low-level signals; themaximum number of sites obtained was <4000(Fay et al. 1997, Wei et al. 1999). Using theconfocal laser scanning microscope, more sites canbe seen as discrete foci but patterns are stillcomplicated by ‘out-of-focus’ £are from sitesabove and below the focal plane; optical sectioningreduces this problem but does not eliminate it. Sofar, the only way to resolve individual sites hasbeen to use ultrathin sections of resin- or sucrose-embedded material (Iborra et al. 1996a, Wansinket al. 1996, Pombo et al. 1999b). Physical sec-tioning eliminates ‘out-of-focus’ £are on the LM(compare Figure 1f with 1b) as all signal mustcome from sites within the section; it simpli¢espatterns as only a small volume of the cell isquanti¢ed, and it allows analysis on the EM(Pombo et al. 1999a).

For higher resolution studies, post- and pre-embedding labelling of resin-embedded sampleshas been used for EM (Iborra et al. 1996a,


Wansink et al. 1996, Jackson et al. 1998, Cmarkoet al. 1999). Post-embedding labelling has theobvious disadvantage of only labelling the surfaceof sections as antibodies cannot enter the resin.Pre-embedding labelling is an improvement(Iborra et al. 1996a) but it still requires dehy-dration of cells before embedding in resin and acomparatively weak ¢xation to allow access ofgold probes; both of which may a¡ect transcrip-tion sites. Tokuyasu’s cryosectioning method(1980) preserves ultrastructure and allows goodpenetration of antibodies (Gri⁄ths 1993, Pomboet al. 1999b). Visualization of sites of transcriptionin cryosections does not require dehydration ortreatment with detergents until after the cells havebeen ¢xed, cryosectioned and transferred to a solidsupport. Sites can be imaged ¢rst by LM then byEM; £uorescent foci (LM) and gold clusters (EM)colocalize, but EM is the most sensitive techniqueas it resolves sites that appear merged on the LM(Pombo et al. 1999a). It had been reported pre-viously that large gold probes do not peneratecryosections e⁄ciently (e.g. Stierhof & Schwarz1989) so the immunolabelling method was mod-i¢ed by treating sections with a mild detergent andincreasing labelling times (Pombo et al. 1999b).Gold conjugates of up to 10 nm penetrate cryo-sections as e⁄ciently as immunoglobulin^£uoro-chrome conjugates.

Size measurements of nanostructures

Sites of transcription have been measured insections using EM with diameters in the range of40^70 nm (Iborra et al. 1996a, Pombo et al.1999b), well beyond the resolution limit of the LM.Obtaining accurate measurements of smallstructures within cells is a major goal for biologistsand much work is currently underway to ¢ndsuitable methods. It is also crucial for obtainingaccurate estimates of the total number of sites in acell from the number measured in 2D sections asthe density per unit volume depends on the size.During sectioning, some sites will be cut across togive polar caps that can lead to an underestimationof size (Weibel 1979, 1980). The proportion ofpolar caps depends on the size of the objects andthe thickness of the section, and can be accountedfor mathematically using, for example, thesequential subtraction method (Weibel 1979, 1980,

Pombo et al. 1999a). Section thickness can bededuced from interference colour as they are beingcut. Alternative methods, such as atomic forcemicroscopy (Muller et al. 2002) may, in the future,provide more accurate values.

Another di⁄culty in the study of nano-structures, when they are not visible per se, is theuse of antibodies, which are large molecules (IgG*9 nm, IgM*20 nm). In post-embedding surfacelabelling, the antibody is free to move such that itmay collapse in or outward with respect to theepitope, leading to possible under- or over-estimation of size and possibly giving a distortedimpression of shape. Errors in size measurementrange from 20^50% when using probes with sizesin the same magnitude as the structures themselves(Iborra & Cook 1998). Such investigations havenot been carried out on cryosections whereimmunoprobes penetrate through the thickness ofthe section, possibly being more restrained. In fact,smaller sizes for transcription sites have beendetermined from cryosections (45 nm; Pomboet al. 1999b) than after surface labelling (71 nm;Iborra et al. 1996a), and larger gold particlesinevitably overestimate the size of the structure.

To enable more accurate methods of measuringnanostructures, a variety of novel light microscopytechniques are currently under development.These include spectral precision distance micro-scopy (SPDM), spatially modulated illumination(SMI) microscopy and other techniques involvingpoint spread function engineering as well as singlemolecule detection techniques. Virtual microscopystudies have shown that SMI can determine sizesas small as *20 nm (Albrecht et al. 2002, Faillaet al. 2002a). Experimentally, it has been used toaccurately measure the sizes of £uorescentlylabelled beads with diameters ranging from 40 to100 nm (Failla et al. 2002b). It has also provensuccessful in detecting and measuring the size ofpol II sites in cryosections from HeLa cells (S.M.,A. V. Failla, U. Spoeri, C. Cremer, A.P.unpublished) indicating that SMI microscopy is anexciting new prospect for size analysis of nano-structures.

Polymerase studies in vivo

Studies using GFP-tagged polymerase or trans-cription factors, in conjunction with £uorescence


recovery after photobleaching (FRAP) and kineticmodelling, have provided important insightsinto how the transcription machinery may beassembled/disassembled (Mistelli 2001b, Beckeret al. 2002, Dundr et al. 2002a, 2002b, Kimuraet al. 2002). Kimura et al. (2002) and Becker et al.(2002) show that pol II is present in the living cellin two fractions: a highly mobile fraction, which isprobably free pol, and a transiently immobilefraction, corresponding to engaged pol II. Theseresults, along with ¢ndings from similar experi-ments carried out using GFP-tagged pol I (Dundret al. 2002a), suggest that assembly of the tran-scription machinery is a stochastic process and isunlikely to involve the existence of pre-madeholoenzymes. The implication is that polymerase,associated factors and DNA template mightassociate randomly until the fortuitous associationof all the appropriate elements allows initiation oftranscription. Kimura et al. (2002) found that 20^25% of total pol II is engaged, which is in line withprevious biochemical estimates (Jackson et al.1998, Kimura et al. 1999), and that elongation ofthe transcript takes up *27% of each round oftranscription. Initiation and termination appear tobe extremely rapid events and the rest (>60%) ofthe transcription cycle is taken up by the exchangeof pol II and transcription factors from thenucleoplasm to promoters. Dundr et al. (2002a)showed that the pol I machinery is also highly

dynamic. They demonstrated that re-initiationoccurs every *1.4 s and that there is a rapidexchange of more than 95% of the polymerasesubunits and associated proteins from FCs to thenucleoplasm, suggesting that the pol I machineryis reassembled with each round of transcription.These and other studies of protein dynamics showthat many proteins in the cell nucleus are highlymobile (mostly inactive proteins) whilst others areless so (Stenoien et al. 2002) but that overall astable architectural framework is preserved(Misteli 2001a, 2001b).

Transcription factory models

Historically it has been thought that the poly-merase moves along the template as it transcribes;alternative models suggest that the active poly-merase remains immobilized whilst the template ismechanically moved through it (for review, seeCook 1999). The sheer size of the transcriptioncomplex makes it unlikely that it could track alongthe tangled DNA template but the polymerasewould need to be capable of acting as a molecularmotor in order to pull the template; this has in factbeen demonstrated experimentally (e.g. Gelles &Landick 1998). The idea of polymerase moleculestracking along the template is also inconsistentwith the data on transcription factories, where

Figure 2. Transcription factory models. Each factory contains a number of polymerase molecules (red circles) and associated nascenttranscripts (green wavy lines), which are produced as the DNA (blue line) moves across the surface of the factory. Pol I factories (a)contain many polymerase molecules and measure*200^500 nm. Their organization has, to some extent, been revealed by EM studieswhere pol I and associated factors are seen in ¢brillar centres (FC) and nascent transcripts in the dense ¢brillar component (DFC). PolII and pol III factories (b) contain *5^8 pols and measure *50 nm. Whether nascent transcripts are extruded into the centre of thefactory (b; top) or are formed around a cluster of pols (b; bottom) remains to be determined.


active polymerases and nascent transcripts arerestricted to an area of only *50 nm in diameter.In the case of pol I, it is known that *4 genes aresimultaneously transcribed at the same FC(Jackson et al. 1998, Dundr et al. 2002b). In orderfor these genes to remain associated, the activepolymerases must be attached to some underlyingfeature. Proposed models of how transcriptionfactories are organized are shown in Figure 2. Themodel of pol I factories (Figure 2a) comes fromultrastructural studies (see above) which suggestthat pol I and its associated factors are present inFCs and that the nascent transcripts are extrudedinto DFCs. Since FCs do not appear to containDNA, an inevitable conclusion is that the activepols lie at the surface of the FC, and the rDNAmoves across them. Current models of pol II andpol III transcription factories involve a number ofpols (*5^8) associated with a number of di¡erenttranscription units (*5^8). Based on the experi-mental evidence outlined above, Figure 2b showstwo models of how pol II (and pol III) factoriesmay function. In both models, the DNA is loopedto facilitate transcription by immobilized poly-merases. Active pols might be dispersed aroundthe growing nascent transcripts (Figure 2b, top)or, alternatively, DNA and nascent transcriptsmay surround clustered pols (Figure 2b, bottom).However, in EM micrographs of pol II andBrUTP, the nascent transcripts seem to be adja-cent to the polymerases (although not evenlydistributed around them), in line with the secondmodel (Iborra et al. 1996a, Pombo et al. 1999b).

What might secure the pols in a factory isunknown but protein : protein, DNA : protein andRNA : protein interactions may be enough to holdthe factory together whilst transcription isunderway. Whether factories retain their structurein the absence of transcription remains to be seen.Nuclear subcompartments are thought to be self-organizing as a result of a continuous tra⁄c ofproteins (Misteli 2001a); this could also hold truefor transcription factories.

In both models, DNA sequences placed closetogether in the genome are transcribed in thesame factory; this is in line with data suggest-ing that active ‘housekeeping’ genes are clusteredin the genome (Caron et al. 2001, Lercher et al.2002). Pol I, pol II and pol III genes are tran-scribed in separate factories. Pol I transcription

factories specialize in the transcription of rDNA;could this level of organization extend to thenature of genes transcribed by individual pol II orpol III factories? With the continuing developmentof methods to analyse polymerases, nascenttranscripts and active genes, it should be possibleto address this question. Further advances in livecell techniques and high-resolution microscopywill be invaluable in expanding our understandingof the intricate and highly complex process that isgene expression.

Acknowledgements

We thank the Medical Research Council (UK) forsupport.

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transcription factories: quantitative studies of nanostructures in the mammalian nucleus

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