A

vcdti©

K

C

1

olnamas

1d

Seminars in Cell & Developmental Biology 18 (2007) 691–697

Review

The role of transcription factories in large-scale structure and dynamics ofinterphase chromatin

Tom Sexton, David Umlauf, Sreenivasulu Kurukuti, Peter Fraser ∗Laboratory of Chromatin and Gene Expression, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK

Available online 25 August 2007

bstract

The genome is spatially organized inside nuclei, with chromosomes and genes occupying preferential positions relative to each other and toarious nuclear landmarks. What drives this organization is unclear, but recent findings suggest there are extensive intra- and inter-chromosomalommunications between various genomic regions that appear to play important roles in genome function. Here we review transcription factories,

istinct sub-nuclear foci where nascent transcription occurs. We argue that the spatially restricted, limited number of transcription sites compelsranscribed regions of the genome to dynamically self-organize into tissue-specific conformations, thus playing a major role in the three-dimensionalnterphase organization of the genome.

2007 Elsevier Ltd. All rights reserved.

eywords: Transcription; Nuclear organization; Transcription factories

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6912. Chromatin in motion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6923. Transcription dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6934. Why factories? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6935. Limited transcription factories: a bottleneck for regulation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6946. Transcription factories and genome organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6947. Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 695

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696

. Introduction

Transcription factories were first identified as nuclear focif nascent transcription by visualizing incorporation of pulse-abeled bromo-uridine [1,2]. Transcriptional output from theseon-nucleolar sites was shown to be sensitive to alpha-amanitin,

nuclear aggregations or compartments dedicated to transcriptionof multiple genes. Examination of individual active genes showsthat specific combinations of genes from different chromosomescan share the same factory with high frequency [5], suggestingthat active genes have preferred transcription partners. Thus,the transcriptional program of a cell may be reflected by, or may

nd found to contain high local concentrations of RNA poly-erase II (RNAPII) [3] (Fig. 1). It has been shown that genes that

re megabase pairs apart, or even on different chromosomes, canhare the same factory [4,5], suggesting that factories are sub-

∗ Corresponding author. Tel.: +44 1223 496644; fax: +44 1223 496022.E-mail address: peter.fraser@bbsrc.ac.uk (P. Fraser).

eTttnaao

084-9521/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcdb.2007.08.008

ven be dependent upon, the spatial organization of the genome.he appreciation that a very large proportion of the genome is

ranscribed [6–8] with relatively few transcription sites suggestshat transcription plays a major role in shaping the nuclear orga-

ization of the genome. In this review, we discuss recent findingsnd gaps in our knowledge regarding transcription factoriesnd argue that the spatial restraints imposed by aggregationf the transcriptional machinery affects the three-dimensional

692 T. Sexton et al. / Seminars in Cell & Develo

Fig. 1. Transcription factories are concentrated foci of active RNA polymerase.Immuno-detection of the hyper-phosphorylated form of RNAPII reveals theirfv5

oo

2

(tpngldvsamBntGctu

sttitdl

rrblpsfmantfvwstnfifipasfi

aal[gtgtatcmtocoalgei1t2bi[ii

ocal existence in a limiting number of transcription factories. Shown is a decon-oluted, single optical section of a mouse E12.5 fetal liver nucleus. Scale bar,�m. Image courtesy of L. Chakalova.

rganization of genomic regions that need to take advantagef it.

. Chromatin in motion

Interphase chromosomes occupy discrete territoriesreviewed in Ref. [9]) and are non-randomly arranged inhe nucleus. Individual chromosomes adopt tissue-specificreferential positions with respect to each other [10] and theuclear periphery [11]. Insertion of lac-operator repeats into theenome, and subsequent visualization with bound GFP-taggedac-repressor protein, reveals that individual sub-chromosomalomains are capable of significant movement within constrainedolumes—a few hundred nanometers over the time scale ofeconds, and micron movements over minutes [12–14] (seelso the paper of Chuang and Belmont in this issue). Thisobility was shown to be passive [13], occurring mainly viarownian motion, and is reduced if a locus is tethered to theuclear periphery or to nucleoli [12]. Large movements of upo 3 �m have been observed but appear to be restricted to early1 phases of the cell cycle [14]. The above measurements of

hromatin mobility were obtained without assessment of theranscriptional activity around the integrated locus, and it isnclear whether these bulk patterns apply to individual genes.

A more direct approach to link chromatin mobility with tran-cription involved targeting a transcriptional activation domaino the region bound by the lac-repressor protein, which increasedranscription of linked genes and caused the locus to adopt a more

nternal position in the nucleus [15,16]. In cells where the activa-ion domain was fused to the lac-repressor, the same constrainediffusion was observed as for previously described genomicoci [16]. However, directional movement was observed when

alst

pmental Biology 18 (2007) 691–697

ecruitment of a heterologous activation domain to the lac-epressor was induced, allowing visualization of the transitionetween the ‘silenced’ and ‘active’ forms of the linked genomicocus [15]. Mobility of the induced locus was bimodal, withreviously characterized patterns of constrained diffusion inter-persed with rapid, large-scale movements, predominantly awayrom the nuclear periphery [15]. Interestingly, these large-scaleovements of induced genes were found to be dependent on

ctin and nuclear myosin I [15]. Actin is implicated in variousuclear processes, such as chromatin remodeling, transcrip-ion and mRNA export (reviewed in Ref. [17]), and is requiredor transcription by all three RNA polymerases in vivo and initro [18,19]. However, for most of these functions it is unclearhether monomeric or polymeric actin is involved. It may be

peculated that one function of actin is in transcriptional induc-ion, whereby genes are actively transported to factories alonguclear actin filaments. It has been shown that nuclear actinlaments exist and have similar dynamics to cytoplasmic actinbers [20], and that transcription is reduced by inhibitors of actinolymerization [20,21]. However, the existence of a ‘nuclearctin network’ that can actively move chromatin has yet to behown, and will require the specific visualization of nuclear actinlaments and gene associations.

Fluorescent in situ hybridization (FISH) has also been used tossess indirectly chromatin movements in association with genectivation. In some cases, large gene-rich regions are found tooop considerable distances outside their chromosome territories22,23], while in other cases such as the tightly regulated HOXene locus, individual genes have been seen to loop out concomi-ant with activation [24]. A major question is where are theseenes going and why? A recent study by Osborne et al. [5] inves-igated the nuclear positioning of the immediate early genes Fosnd Myc and examined their spatial relationships with transcrip-ion factories and other genes in non-stimulated and stimulated Bells. Upon induction, previously silent immediate early allelesove rapidly to transcription factories and engage in transcrip-

ion. Remarkably, a quarter of the newly activated Myc allelesn chromosome 15 enter the same transcription factory as theonstitutively active Igh (immunoglobulin heavy chain) locusn chromosome 12 [5]. Based on estimates of factory numbersnd the numbers of genes transcribed at any given time, a co-ocalization frequency of only 1–2% would be predicted if theseenes can engage in any factory at random (unpublished). How-ver, this high co-localization frequency between genes in transs not just due to a preferential neighboring of chromosomes2 and 15 in lymphocytes (reported in Ref. [10]), as it is morehan double that between Igh and the gene Eif3s6, located only0 megabases away from Myc [5]. Chromosomal translocationsetween the Myc proto-oncogene and the Igh locus are foundn the majority of mouse plasmacytomas [25]. Osborne et al.5] suggest that the preferred inter-chromosomal juxtaposition-ng of these genes in a shared transcription factory provides anncreased opportunity for a chromosomal translocation event

nd thus may be one of the first steps in the generation ofeukemias. However, the reason why these genes preferentiallyhare transcription factories is unknown. The fact that activelyranscribed genes often share factories implies that factories may

evelo

nt

3

ipgci[cgeaifptsihp(tTtaft[catcpif

etor

ttf[bophsAstfmwIadg

4

tCmasfm

Fsst(I

T. Sexton et al. / Seminars in Cell & D

ot be assembled de novo on genes, but that genes may migrateo pre-assembled transcription sites [4,5].

. Transcription dynamics

RNA FISH visualization of transcription at the level ofndividual genes shows that ‘active’ genes are not continuallyroducing transcripts in expressing cells. Analysis of an activeene in a population of cells shows that none, one or two allelesan be active at any given time, suggesting that transcriptions dynamic and occurs in bursts rather than a steady output4,26,27]. Live-cell imaging of transcript production from a spe-ific gene in Dictyostelium supports this model, showing thatene transcription occurs in pulses of activity in vivo [28]. Rajt al. [29] visualized individual mRNA molecules arising fromreporter gene and showed that active genes are transcribed in

nfrequent bursts. Induced increases in gene expression resultrom increases in burst size (i.e. amount of transcript produceder burst) rather than the frequency of the bursts of RNA produc-ion. RNA FISH analyses of a limited number of specific geneshow that transcription of individual alleles is almost alwaysn association with a transcription factory [4,5,30]. Alleles thatave the potential to be active in a particular cell but are tem-orarily inactive are located away from transcription factoriesFig. 2) [4]. Thus, the reported transcriptional bursts are likelyo reflect periods of gene engagement in a transcription factory.he size of the burst will depend on the number of polymerases

ranscribing a gene at any given time, the rate of transcriptionlong the gene, and the amount of time the gene spends in theactory. By comparing RNA FISH frequencies with quantita-ive PCR-based detection of primary transcripts, Osborne et al.5] showed that increases in immediate early gene expressionould be accounted for completely by increased recruitment oflleles to transcription factories, rather than through ‘turning up’he transcription rate of all alleles, as is popularly believed. With

onventional RNA FISH, which takes a ‘snapshot’ of cells at theoint when they are fixed, it cannot be determined whether thisncreased gene recruitment to factories is a result of increasedrequency of engagement, an increased residence time once

altf

ig. 2. Actively transcribed genes co-localize at shared transcription factories. (a) Spleen cell, showing Hbb (�-globin; green), Eraf (erythroid associated factor; red) ahown in the side panels. On the left of the main panel, an Hbb signal alone associatehe same RNAPII focus. These three alleles are thus actively transcribing, and one Eb) A separate optical section of the same cell showing the second Eraf allele, whichmage taken from Ref. [4].

pmental Biology 18 (2007) 691–697 693

ngaged in a factory (i.e. a larger ‘burst’ phase of transcrip-ion), or a combination of both. Live imaging of the dynamicsf both genes and nascent transcripts in and out of factories isequired to resolve these models.

Earlier studies have correlated transcription of genes withheir presence in the periphery or outside of the main body ofheir chromosome territory [22–24,31], but both transcriptionactories and expressed genes can be found within territories32,33]. Until recently, it was believed that trans interactionsetween genes co-localizing at a common factory could onlyccur in the limited volume within the ‘interchromatin com-artment’ between chromosome territories [9]. However, recentigh-resolution visualization of chromosome territories hashown that territories intermingle extensively in the nucleus [34].pproximately 40% of the chromosome territory volume mea-

ured intermingles with other chromosome territories, providinghe nuclear space in which genes in trans can share transcriptionactories. The extent of intermingling between specific chro-osome pairs varies approximately 20-fold [34], in agreementith observations of tissue-specific chromosome neighbors [10].

nterestingly, inhibition of RNAPII transcription significantlyltered the intermingling volumes of some chromosome pairs,emonstrating that transcription can have a direct effect onenome organization [34].

. Why factories?

There are a number of biophysical arguments for the forma-ion of transcription factories that are not mutually exclusive.lustering of large complexes, such as elongating RNA poly-erases, may free up more nuclear volume in which chromatin

nd nuclear proteins outside of the factories can arrange them-elves [35]. The resultant entropic gain may be a contributingorce in the establishment of transcription factories [35]. Macro-olecular crowding in the nucleus, which is thought to play

role in the formation of nucleoli and PML (promyelocytic

eukemia) bodies [36], may also promote formation of transcrip-ion factories (see also the paper of Hancock in this issue). Apartrom polymerases and nascent transcripts, very little is known

ingle optical section of a triple-label DNA immuno-FISH on a mouse anemicnd RNAPII foci (blue). The merged and separate channels of the signals ares with an RNAPII focus. On the right, two co-localizing signals associate withraf allele shares a transcription factory with one Hbb allele. Scale bar, 5 �m.does not associate with an RNAPII focus and is thus not actively transcribing.

6 evelopmental Biology 18 (2007) 691–697

asad[mo

eeolwbwtnm1piIietraicmSggswdpocaitiemetit

5r

i2v

Fig. 3. Potentially active genes in cis and trans engage dynamically in transcrip-tion factories. For simplicity, transcription factories are represented as spheres inbetween chromosome territories (CTs), although factories can also exist withinCTs. Most ‘active’ genes spend the majority of their time outside factories andaCc

trswTut[ttswusfgassdictr

6

94 T. Sexton et al. / Seminars in Cell & D

bout the biochemical composition of transcription factories pere. Indirect early experiments suggest that nascent transcriptsre produced at a nucleoskeleton that is resistant to extensiveigestion and removal of the majority of chromatin fragments37]. Transcription factories themselves survive similar treat-ents [1,2], suggesting that they may be attached to some form

f nuclear ‘scaffold’.Much work has been done to characterize biochemically the

longating form of RNAPII (reviewed in Ref. [38]). Activelylongating RNA polymerase, which is hyper-phosphorylatedn the heptapeptide repeat of the C-terminal domain of theargest polymerase subunit (CTD), is resistant to extractionith saponin detergent [39]. Fluorescence recovery after photo-leaching and fluorescence loss in photobleaching experimentsith GFP-tagged RNAPII show that this active form is rela-

ively immobile with a half-life of 20 min [40], whereas theon-phosphorylated, inactive form of RNAPII is extremelyobile [41]. Inhibition of elongation with DRB (5,6-dichloro-

-�-d-ribofuranosylbenzimidazole), which specifically inhibitshosphorylation of serine-2 of the RNAPII CTD, results inncreased mobility of the immobile RNAPII fraction [40,41].nhibition of both initiation and elongation by heat shock resultsn even greater mobility, consistent with the concept that thengaged RNAPII is relatively immobile [41]. Taken together,hese results support a model whereby transcription facto-ies comprise elongating RNA polymerase complexes that arettached to a nucleoskeletal component. The apparent immobil-ty of elongating polymerases suggests that they may ‘ratchet’hromatin through the factory during transcription, rather thanoving along the chromatin template (proposed in Ref. [42]).tudies of transcriptomes show that a very large proportion of theenome is transcribed, much more than can be accounted for byenes alone [6–8]. Even if only a proportion of this genome tran-cription occurs at any given time, a mobile polymerase modelould predict tens if not hundreds of thousands of polymerasesistributed evenly throughout the nucleoplasm. This is not sup-orted by immunofluorescence analysis of RNAPII localizationr pulse-label detection of nascent transcription, which show aomparatively limited number of focal sites [1–3]. The avail-ble evidence points in favor of a relatively immobile factoryn which multiple genomic regions are transcribed at any givenime. Analysis of the tractor power of bacterial RNA polymerasendicates that it is a powerful motor protein, capable of gen-rating forces greater than those of more established ATPaseotors, such as myosin, and sufficient for the proposed ratch-

ting of chromatin [43,44]. However, it cannot be discountedhat elongating RNAPII moves along the transcribed gene, buts constrained predominantly to the small nuclear domain of theranscription factory.

. Limited transcription factories: a bottleneck foregulation?

Original transcription factory number estimates were made inmmortalized, fibroblast-like cell lines, counting approximately000 foci per nucleus [3]. Immunostaining for RNAPII in exivo primary cells from several mouse tissues reveals that fac-

tsm

re transcriptionally inactive, but may be present in chromatin loops out of theirT when poised for engagement in a factory. The limiting numbers of factoriesompel transcribed genes to share factories. Figure adapted from Ref. [56].

ory numbers are an order of magnitude lower [4]. This mayeflect the decreased nuclear volume in cells from tissues withpherical nuclei, as opposed to cells that flatten out in cultureith greatly expanded nuclear diameters and nuclear volumes.here appears to be a clear link between increased nuclear vol-me and increased number of transcription factories, whereashe density of factories per unit volume remains fairly constant45]. Thus, in spherical nuclei with a limited number of transcrip-ion factories and maximal packing of chromosome territories,housands of genes and transcription units may be serviced byharing a limited number of transcription sites. In flat cells,hich are in essence one chromosome territory thick, individ-al genes will have limited opportunities to share transcriptionites with other than their immediate neighbors. Thus, increasedactory numbers may be required to service the same number ofenes and transcription units in flat cells. We estimate that therere hundreds of transcription units for each factory in cells withpherical nuclei (unpublished). Even though they are not all tran-cribing at once the collective data suggests transcription unitsynamically co-associate within factories (Fig. 3). This concepts supported by a genome-wide application of 3C (chromosomeonformation capture [46]) which provides additional evidencehat active genes occupy a common ‘nuclear zone’ [47], possiblyeflecting their occupancy in shared transcription factories.

. Transcription factories and genome organization

Until recently, little work had assessed the role ofranscription factories in developmental switches in gene expres-ion. Analysis of �-globin expression during erythroid cellaturation [30] and X-chromosome inactivation in mouse

evelo

fIedmtMdsntiptfoitrsptbm

sTRTiXeoaIpt

rctsscuttaoaaoffen[

gaDepnpbfi

oTtSiittstrcssapcAcitniat

7

tsateikbpecct

T. Sexton et al. / Seminars in Cell & D

emale embryonic cells [48] has provided interesting insights.mmunostaining for RNAPII in mouse fetal liver cells at differ-nt stages of erythropoesis showed that transcription factoriesecrease in number and adopt more internal positions duringaturation [30]. This is likely to reflect progressive transcrip-

ional shutdown of mature erythrocytes prior to enucleation.ost cells keep their nuclei and do not undergo such drastic

evelopmental changes. For example, differentiating embryonictem cells halve their transcription factory numbers as theiruclear volume reduces, but the density of transcription fac-ories and their distribution in the nuclear periphery and interiors unchanged [45]. The �-globin gene adopts a more internalosition during erythroid maturation, but RNA FISH shows thatranscription in early erythroid cells also occurs in transcriptionactories close to the nuclear periphery [30]. Thus, ‘relocation’f the �-globin gene occurs after transcriptional activation, ands likely to reflect the predominance of transcription factories inhe nuclear interior in late-stage erythroid cells. Kosak et al. [49]eported the opposite finding for immunoglobulin gene expres-ion in developing lymphocytes, whereby nuclear repositioningreceded transcription. However, the organization of transcrip-ion factories was not studied, and transcription was not assessedy RNA FISH, so it is difficult to tell if this reflects a differentechanism to �-globin expression in erythrocytes.A change in nuclear organization also occurs during tran-

criptional silencing of genes on the inactivated X-chromosome.he first step in X-inactivation involves the non-coding XistNA, which coats the imminently silenced X-chromosome.his appears to set up an RNAPII exclusion zone, as RNAPII

s no longer detectable by immunostaining in the core of theist-coated chromosome territory [48]. Before inactivation,

xpressed X-linked genes are initially located at the periphery orutside this silenced core, however upon inactivation these genesre drawn into the RNAPII-free compartment and silenced.nterestingly, genes that escape X-inactivation remain on theeriphery of the Xist domain [48], and can presumably accessranscription factories in the adjacent regions of the nucleus.

Chromosomes adopt preferential, tissue-specific positionselative to each other [10] that are partly maintained acrossell divisions [50,51], but these may also be determined byhe transcriptional program of the cell, organizing the genomeuch that co-expressed genes are proximal and can share tran-cription factories. Conversely, the combinations of genes thatommonly share transcription factories could be dictated by thenderlying chromosome organization. Although not addressinghis ‘chicken and egg’ situation directly, an attractive modelo help explain the intimate link between genome structurend global transcription patterns is the concept of ‘self-rganization’ (reviewed in Ref. [52]). Meta-stable structuresrise as a consequence of the steady state between associationnd dissociation of highly dynamic components. In the contextf individual transcription factories, it may be predicted thatactories represent high local concentrations of factors required

or transcription elongation, and so genes are more likely tongage successfully on pre-existing factories than to form deovo [42]. As most elongating complexes are stable for minutes40], there is a large window of opportunity for other induced

tfir

pmental Biology 18 (2007) 691–697 695

enes to contact this transcriptionally competent environment,nd so maintain the factory for further transcriptional cycles.ue to their highly dynamic nature, it is difficult to show

xperimentally that a nuclear body is self-organizing, buterturbation of RNA polymerase I has been shown to disruptucleolar structure [53]. Analysis of factories after similarerturbations of RNAPII, a more complete understanding of theiochemical components required for a productive transcriptionactory, and live imaging to test the ‘stability’ of single factories,s required to test this model.

In the context of the entire genome, it can be speculated thatngoing transcription may self-organize chromosome structure.he ‘steady state’ conformation of the genome would position

he most highly expressed genes closer to transcription factories.uch an attractive model is even more difficult to assess exper-

mentally, not least because it is unlikely to be the sole factorn modulating genome structure. A few clues suggest that theranscriptional program of a cell is not just the consequence ofhe underlying nuclear structure, and thus may have a role inhaping the genome. First, induction of transcription was showno promote movement of the CFTR locus into the nuclear inte-ior, but altered nuclear localization of CFTR was insufficient toause transcription [54]. Second, inhibition of transcription washown to alter the intermingling volumes of specific chromo-ome pairs [34], showing a causative link between transcriptionnd nuclear structure. Third, RNA FISH studies show that tworoximal genes on one chromosome can have very differento-localization frequencies with the same gene in trans [5].lthough there are many possible causes for these different

o-localization frequencies, and only three genes have been stud-ed, this argues against the simplistic notion that genes shareranscription factories just because they reside in commonlyeighboring chromosome territories. These results are intrigu-ng, but a much greater understanding of transcription factoriesnd the extent to which transcription modulates genome struc-ure is required to probe the link between structure and function.

. Perspective

What evolutionary advantage could be gained from limitinghe numbers of transcription factories? Limiting the nuclearpace in which efficient transcription can take place may preventberrant expression of inappropriate genes. Additionally, mul-iple genes sharing a transcription factory may allow for morefficient sharing of regulatory factors by creating a localizedncreased concentration of the necessary factors. It is not yetnown if different transcription factories contain different com-inations of regulatory factors. If this were the case, it wouldrovide the mechanistic framework to link the observed prefer-nce of gene partners in transcription factories to their functionalo-ordination of gene expression. There are massive gaps in oururrent understanding of how transcription is organized withinhe nucleus, which will be filled when we understand more of

he fundamentals of transcription factories. Immunostainingor transcription factors and other regulatory proteins may givendirect clues as to the biochemical nature of transcription facto-ies. However, the limited subset of transcription factors looked

6 evelo

affettiiudtwdwOstml

A

GlritiB

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

96 T. Sexton et al. / Seminars in Cell & D

t so far has shown only limited co-staining with transcriptionactories [55]. It is unclear whether this represents specializedactories or a very transient binding of initiating factors tolongating complexes. Full biochemical characterization ofranscription factories will determine whether specialized fac-ories can exist, and may also determine whether transcriptionnitiation occurs before or on attachment to a factory. Live-cellmaging of gene recruitment to factories is also crucial innderstanding the dynamics of transcription, in particular toetermine how genes get to factories, and how far they are ableo move to get there. Finally, the extent of gene co-localizationsithin shared factories needs to be assessed fully in order toefine the transcriptional ‘network’ of the nucleus, and to seehat factors are responsible for establishing such a network.nce these begin to be addressed, we will move from under-

tanding the regulation of individual genes to the regulation ofhe entire transcriptional program of a cell. Similarly, we will

ove from understanding the chromatin structure of individualoci to gaining insight into how the entire genome is organized.

cknowledgements

We thank all members of the Laboratory of Chromatin andene Expression for helpful discussion and for sharing unpub-

ished data. In particular, we thank Cameron Osborne for criticaleading of the manuscript and Lyubomira Chakalova for provid-ng the image for Fig. 1. Work in the laboratory is supported byhe Medical Research Council, the Biotechnology and Biolog-cal Sciences Research Council and the European Moleculariology Organization.

eferences

[1] Jackson DA, Hassan AB, Errington RJ, Cook PR. Visualization of focalsites of transcription within human nuclei. EMBO J 1993;12:1059–65.

[2] Wansink DG, Schul W, van der Kraan I, van Steensel B, van Driel R, deJong L. Fluorescent labeling of nascent RNA reveals transcription by RNApolymerase II in domains scattered throughout the nucleus. J Cell Biol1993;122:283–93.

[3] Iborra FJ, Pombo A, Jackson DA, Cook PR. Active RNA polymerases arelocalized within discrete transcription “factories’ in human nuclei. J CellSci 1996;109:1427–36.

[4] Osborne CS, Chakalova L, Brown KE, Carter D, Horton A, Debrand E,et al. Active genes dynamically colocalize to shared sites of ongoing tran-scription. Nat Genet 2004;36:1065–71.

[5] Osborne CS, Chakalova L, Mitchell JA, Horton A, Wood AL, Bolland DJ,et al. Myc dynamically and preferentially relocates to a transcription factoryoccupied by Igh. PLoS Biol 2007;5:e192.

[6] Kapranov P, Cawley SE, Drenkow J, Bekiranov S, Strausberg RL, FodorSP, et al. Large-scale transcriptional activity in chromosomes 21 and 22.Science 2002;296:916–9.

[7] Bertone P, Stolc V, Royce TE, Rozowsky JS, Urban AE, Zhu X, et al. Globalidentification of human transcribed sequences with genome tiling arrays.Science 2004;306:2242–6.

[8] Cheng J, Kapranov P, Drenkow J, Dike S, Brubaker S, Patel S, et al. Tran-scriptional maps of 10 human chromosomes at 5-nucleotide resolution.

Science 2005;308:1149–54.

[9] Cremer T, Cremer C. Chromosome territories, nuclear architecture andgene regulation in mammalian cells. Nat Rev Genet 2001;2:292–301.

10] Parada LA, McQueen PG, Misteli T. Tissue-specific spatial organizationof genomes. Genome Biol 2004;5:R44.

[

pmental Biology 18 (2007) 691–697

11] Boyle S, Gilchrist S, Bridger JM, Mahy NL, Ellis JA, Bickmore WA. Thespatial organization of human chromosomes within the nuclei of normaland emerin-mutant cells. Hum Mol Genet 2001;10:211–9.

12] Chubb JR, Boyle S, Perry P, Bickmore WA. Chromatin motion is con-strained by association with nuclear compartments in human cells. CurrBiol 2002;12:439–45.

13] Marshall WF, Straight A, Marko JF, Swedlow J, Dernburg A, Belmont A,et al. Interphase chromosomes undergo constrained diffusional motion inliving cells. Curr Biol 1997;7:930–9.

14] Vazquez J, Belmont AS, Sedat JW. Multiple regimes of constrained chro-mosome motion are regulated in the interphase Drosophila nucleus. CurrBiol 2001;11:1227–39.

15] Chuang CH, Carpenter AE, Fuchsova B, Johnson T, de Lanerolle P, Bel-mont AS. Long-range directional movement of an interphase chromosomesite. Curr Biol 2006;16:825–31.

16] Tumbar T, Belmont AS. Interphase movements of a DNA chromo-some region modulated by VP16 transcriptional activator. Nat Cell Biol2001;3:134–9.

17] Bettinger BT, Gilbert DM, Amberg DC. Actin up in the nucleus. Nat RevMol Cell Biol 2004;5:410–5.

18] Hofmann WA, Stojiljkovic L, Fuchsova B, Vargas GM, Mavromma-tis E, Philimonenko V, et al. Actin is part of pre-initiation complexesand is necessary for transcription by RNA polymerase II. Nat Cell Biol2004;6:1094–101.

19] Philimonenko VV, Zhao J, Iben S, Dingova H, Kysela K, Kahle M, et al.Nuclear actin and myosin I are required for RNA polymerase I transcription.Nat Cell Biol 2004;6:1165–72.

20] McDonald D, Carrero G, Andrin C, de Vries G, Hendzel MJ. Nucleo-plasmic beta-actin exists in a dynamic equilibrium between low-mobilitypolymeric species and rapidly diffusing populations. J Cell Biol2006;172:541–52.

21] Wu X, Yoo Y, Okuhama NN, Tucker PW, Liu G, Guan JL. Regulation ofRNA-polymerase-II-dependent transcription by N-WASP and its nuclear-binding partners. Nat Cell Biol 2006;8:756–63.

22] Mahy NL, Perry PE, Bickmore WA. Gene density and transcription influ-ence the localization of chromatin outside of chromosome territoriesdetectable by FISH. J Cell Biol 2002;159:753–63.

23] Volpi EV, Chevret E, Jones T, Vatcheva R, Williamson J, Beck S, etal. Large-scale chromatin organization of the major histocompatibilitycomplex and other regions of human chromosome 6 and its response tointerferon in interphase nuclei. J Cell Sci 2000;113:1565–76.

24] Chambeyron S, Bickmore WA. Chromatin decondensation and nuclearreorganization of the HoxB locus upon induction of transcription. GenesDev 2004;18:1119–30.

25] Potter M. Neoplastic development in plasma cells. Immunol Rev2003;194:177–95.

26] Levsky JM, Shenoy SM, Pezo RC, Singer RH. Single-cell gene expressionprofiling. Science 2002;297:836–40.

27] Wijgerde M, Grosveld F, Fraser P. Transcription complex stability andchromatin dynamics in vivo. Nature 1995;377:209–13.

28] Chubb JR, Trcek T, Shenoy SM, Singer RH. Transcriptional pulsing of adevelopmental gene. Curr Biol 2006;16:1018–25.

29] Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S. Stochastic mRNAsynthesis in mammalian cells. PLoS Biol 2006;4:e309.

30] Ragoczy T, Bender MA, Telling A, Byron R, Groudine M. The locus controlregion is required for association of the murine beta-globin locus withengaged transcription factories during erythroid maturation. Genes Dev2006;20:1447–57.

31] Dietzel S, Schiebel K, Little G, Edelmann P, Rappold GA, Eils R, et al.The 3D positioning of ANT2 and ANT3 genes within female X chromo-some territories correlates with gene activity. Exp Cell Res 1999;252:363–75.

32] Verschure PJ, van Der Kraan I, Manders EM, van Driel R. Spatial relation-

ship between transcription sites and chromosome territories. J Cell Biol1999;147:13–24.

33] Mahy NL, Perry PE, Gilchrist S, Baldock RA, Bickmore WA. Spatialorganization of active and inactive genes and noncoding DNA withinchromosome territories. J Cell Biol 2002;157:579–89.

evelo

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

T. Sexton et al. / Seminars in Cell & D

34] Branco MR, Pombo A. Intermingling of chromosome territories ininterphase suggests role in translocations and transcription-dependent asso-ciations. PLoS Biol 2006;4:e138.

35] Marenduzzo D, Micheletti C, Cook PR. Entropy-driven genome organiza-tion. Biophys J 2006;90:3712–21.

36] Hancock R. A role for macromolecular crowding effects in the assem-bly and function of compartments in the nucleus. J Struct Biol2004;146:281–90.

37] Jackson DA, Cook PR. Transcription occurs at a nucleoskeleton. EMBO J1985;4:919–25.

38] Sims 3rd RJ, Belotserkovskaya R, Reinberg D. Elongation by RNApolymerase II: the short and long of it. Genes Dev 2004;18:2437–68.

39] Kimura H, Tao Y, Roeder RG, Cook PR. Quantitation of RNA polymeraseII and its transcription factors in an HeLa cell: little soluble holoenzymebut significant amounts of polymerases attached to the nuclear substructure.Mol Cell Biol 1999;19:5383–92.

40] Kimura H, Sugaya K, Cook PR. The transcription cycle of RNA polymeraseII in living cells. J Cell Biol 2002;159:777–82.

41] Hieda M, Winstanley H, Maini P, Iborra FJ, Cook PR. Different popula-tions of RNA polymerase II in living mammalian cells. Chromosome Res2005;13:135–44.

42] Cook PR. Predicting three-dimensional genome structure from transcrip-tional activity. Nat Genet 2002;32:347–52.

43] Wang HY, Elston T, Mogilner A, Oster G. Force generation in RNA poly-merase. Biophys J 1998;74:1186–202.

44] Yin H, Wang MD, Svoboda K, Landick R, Block SM, Gelles J. Transcrip-tion against an applied force. Science 1995;270:1653–7.

45] Faro-Trindade I, Cook PR. A conserved organization of transcription duringembryonic stem cell differentiation and in cells with high C value. Mol BiolCell 2006;17:2910–20.

[

[

pmental Biology 18 (2007) 691–697 697

46] Dekker J, Rippe K, Dekker M, Kleckner N. Capturing chromosome con-formation. Science 2002;295:1306–11.

47] Simonis M, Klous P, Splinter E, Moshkin Y, Willemsen R, de Wit E,et al. Nuclear organization of active and inactive chromatin domainsuncovered by chromosome conformation capture-on-chip (4C). Nat Genet2006;38:1348–54.

48] Chaumeil J, Le Baccon P, Wutz A, Heard E. A novel role for Xist RNAin the formation of a repressive nuclear compartment into which genes arerecruited when silenced. Genes Dev 2006;20:2223–37.

49] Kosak ST, Skok JA, Medina KL, Riblet R, Le Beau MM, Fisher AG, et al.Subnuclear compartmentalization of immunoglobulin loci during lympho-cyte development. Science 2002;296:158–62.

50] Gerlich D, Beaudouin J, Kalbfuss B, Daigle N, Eils R, Ellenberg J. Globalchromosome positions are transmitted through mitosis in mammalian cells.Cell 2003;112:751–64.

51] Walter J, Schermelleh L, Cremer M, Tashiro S, Cremer T. Chromosomeorder in HeLa cells changes during mitosis and early G1, but is stably main-tained during subsequent interphase stages. J Cell Biol 2003;160:685–97.

52] Misteli T. The concept of self-organization in cellular architecture. J CellBiol 2001;155:181–5.

53] Oakes M, Nogi Y, Clark MW, Nomura M. Structural alterations of the nucle-olus in mutants of Saccharomyces cerevisiae defective in RNA polymeraseI. Mol Cell Biol 1993;13:2441–55.

54] Zink D, Amaral MD, Englmann A, Lang S, Clarke LA, Rudolph C, et al.Transcription-dependent spatial arrangements of CFTR and adjacent genesin human cell nuclei. J Cell Biol 2004;166:815–25.

55] Grande MA, van der Kraan I, de Jong L, van Driel R. Nuclear distribu-tion of transcription factors in relation to sites of transcription and RNApolymerase II. J Cell Sci 1997;110(Pt 15):1781–91.

56] Fraser P. Transcriptional control thrown for a loop. Curr Opin Genet Dev2006;16:490–5.