organization of transcriptioncshperspectives.cshlp.org/content/2/9/a000729.full.pdf · organization...

Organization of Transcription

Lyubomira Chakalova1,2 and Peter Fraser1

1Laboratory of Chromatin and Gene Expression, The Babraham Institute, Babraham Research Campus,Cambridge, CB22 3AT, United Kingdom

2Research Centre for Genetic Engineering and Biotechnology, Macedonian Academy of Sciences and Arts,Skopje 1000, Republic of Macedonia

Correspondence: [email protected]

Investigations into the organization of transcription have their origins in cell biology. Earlystudies characterized nascent transcription in relation to discernable nuclear structuresand components. Advances in light microscopy, immunofluorescence, and in situ hybridi-zation helped to begin the difficult task of naming the countless individual players and com-ponents of transcription and placing them in context. With the completion of mammaliangenome sequences, the seemingly boundless task of understanding transcription of thegenome became finite and began a new period of rapid advance. Here we focus on theorganization of transcription in mammals drawing upon information from lower organismswhere necessary. The emerging picture is one of a highly organized nucleus with specificconformations of the genome adapted for tissue-specific programs of transcription andgene expression.

Much of what is known about eukaryotictranscription is dominated by decades of

advances in in vitro biochemistry with whole-cell extracts, or subfractionated and recom-binant proteins on purified DNA templates.These reductionist approaches have lead toseminal findings describing the basic DNA se-quence regulatory elements and enzymatic ma-chinery of transcription. More recent researchincorporating genetic approaches has added tothe complexity of transcription, involving liter-ally hundreds of factors, cofactors, remodelingcomplexes, histone modifiers, and elongation-,splicing- and termination-factors required foror associated with a single transcriptionalevent. Furthermore the discovery of, sometimes

distant, sequence elements required for regu-lated transcription of some genes has added tothe intricacy of the transcriptional process thatoccurs in vivo. Though there is still much tolearn, the difficult task of integrating this infor-mation and placing it in the context of the nu-cleus is gathering momentum.

The widely held view of transcriptionalmechanics, of the RNA polymerase complexsliding along a template to generate a transcriptis also dominated by biochemistry. Textbooksare full of descriptions of promoter bound fac-tors recruiting RNA polymerase, which initiatestranscription before sliding along the transcrip-tion unit. Indeed, single molecules of prokary-otic RNA polymerase have been visualized in

Editors: Tom Misteli and David L. Spector

Additional Perspectives on The Nucleus available at www.cshperspectives.org

Copyright # 2010 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a000729

Cite this article as Cold Spring Harb Perspect Biol 2010;2:a000729

1

on August 18, 2020 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from

http://cshperspectives.cshlp.org/

vitro, sliding along a fixed DNA template duringa one-dimensional diffusional search for a pro-moter (Kabata et al. 1993; Guthold et al. 1999;Harada et al. 1999) or during transcription(Schafer et al. 1991; Wang et al. 1998; Gutholdet al. 1999; Davenport et al. 2000). However,which molecule actually moves, the polymeraseor the DNA depends on which is fixed (Iborraet al. 1996b). Several studies have shown thatan anchored polymerase generates considerablepulling force on a DNA template, rotating thedouble helix in a clockwise manner as it threadsthe strand through the protein during transcrip-tion (Kabata et al. 1993; Wang et al. 1998; Gut-hold et al. 1999). It is impossible to know fromthese in vitro studies what actually happensin vivo. Although the answer to this questionmay seem trivial, it has profound implicationsfor our understanding of transcription andgenome function, and can only be answeredby examining evidence of transcription in thenucleus.

Fakan and colleagues have studied nascenttranscripts at high resolution for decades(Fakan and Bernhard 1971; Fakan et al. 1976;Fakan 2004). They found that nascent RNA islocated in perichromatin fibrils (PF). PF arestructures observed using electron microscopyby specific contrasting methods, and most oftenlocated in the perichromatin region, the boun-daries between condensed and decondensedchromatin (Fakan and Bernhard 1971). Earlyautoradiographic studies combined with laterfindings indicated that PF are the in situ formof nascent RNA complexed with processing fac-tors (Nash et al. 1975; Fakan et al. 1976; Cremeret al. 2004).

Seminal studies on transcription in mam-malian nuclei were carried out by Jackson andCook (Jackson et al. 1981; Jackson and Cook1985). They uniformly labeled DNA of HeLacells with 14C in vivo, and encapsulated themin agarose beads before a short incubationwith [3H]uridine to label nascent RNA. Thecells were then lysed in an isotonic solutionand chromatin was digested with a restrictionenzyme or DNase followed by electrophoresisto remove the digested chromatin. They foundthat over 90% of nascent RNA is retained in

the beads after as much as 98% of the DNA/chromatin had been removed. The chromatinreleased from the beads by electrophoresis wasassayed further and found to be considerablylarger than an RNAPII holocomplex, suggest-ing that nonattached transcribing complexesshould have been released with the chromatin.However, they found that 60% of the originalRNA polymerase activity was retained in thebeads after loss of 75% of the chromatin. Theseresults suggested a model whereby newly syn-thesized RNA and the transcriptional machi-nery are retained in the nucleus by a structurethat is resistant to nuclease digestion. Indeedanalysis of the DNA retained in the beads indi-cated that it was enriched in active genes. Jack-son and Cook proposed the involvement of a“nucleoskeleton” in the process of transcrip-tion, suggesting attachment of the polymerasecomplex, and arguing against the concept offree RNA polymerase complexes tracking DNAtemplates. These were controversial studies intheir time, and the merits and conclusions arestill debated today. Though Jackson and Cookwere careful to maintain physiological con-ditions during their experiments (Jackson andCook 1985), critics argued that the systememployed may have created an artificial networkof proteins that retained active gene sequences.

TRANSCRIPTION OCCURS IN FACTORIES

These studies were expanded and advanced byJackson et al. (1993), and particularly Wansinket al. (1993) in which transcription sites werevisualized by indirect immunofluorescenceafter pulse-labeling or microinjecting cells withhalogenated ribonucleoside precursors. The useof in vivo labeling methods excluded potentialartifacts, which may have been introduced bythe earlier run-on procedures. These studies re-vealed that nascent transcripts were not equallydistributed throughout the nucleoplasm, butthat transcription occurred in a limited numberof discrete sites or foci that were sensitiveto transcriptional inhibitors. An importantobservation was that increased labeling timesdid not result in detection of more foci, onlyincreased intensity suggesting that all sites of

L. Chakalova and P. Fraser

2 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a000729



transcription were being detected. Estimates ofthe number of transcription foci vary betweenseveral hundred and several thousand per nu-cleus depending on the cell type (Jacksonet al. 1993; Iborra et al. 1996a; Pombo et al.1999; Osborne et al. 2004). The total numberof sites appeared to be several times lower thanthe number of active transcription units sug-gesting that each site contained multiple genes(Jackson et al. 1993; Jackson et al. 1998).The term “transcription factories” was coined(Iborra et al. 1996a) to reflect the potential gath-ering of several transcription units to each fac-tory in similarity to replication factories,which form during the S phase of the cell cycle,each accommodating several replicons at once.Fakan and colleagues have often observed thatimmunogold detection of nascent RNAs aftershort labeling is most often represented byindividual gold particles associated with PF(Cmarko et al. 1999). This was taken as evidencethat transcription takes place in individuallocalized sites rather than in factories. Howeverquantitative estimates based on immunogoldlabeling on the surface of EM sections is ten-uous. Though PF and transcription factorieshave not been linked directly, probably becausewith the various imaging techniques used onecan only see what one attempts to detect, itseems likely that PFs do represent nascent tran-scripts (Fakan 1994) produced at focal RNAPIIfactories (Cmarko et al. 1999).

Both groups (Jackson et al. 1993; Wansinket al. 1993), investigated the localization ofsplicing components relative to nascent tran-scription sites. Large, intensely labeled SC-35domains localized close to some nascent tran-scription sites but did not overlap with them,whereas some colocalization was observed be-tween transcription sites and weak SC-35 foci(Wansink et al. 1993). These observations areconsistent with suggestions that large SC-35domains are storage sites containing no tran-scriptional activity (Iborra et al. 1996a; Pomboand Cook 1996; Fay et al. 1997), whereas smallSC-35 foci may result from recruitment of splic-ing components to transcription sites forcotran-scriptional splicing (Huang and Spector 1996;Misteli et al. 1997; Lamond and Spector 2003).

Building on previous work, Cook and col-leagues employed electron microscopy (EM)to study nascent transcripts in pulse labeled cells(Iborra et al. 1996a) using indirect labeling withimmuno-gold particles. They found that goldparticles marking nascent transcripts appearedas clusters, which increased in particle numberwith increased labeling times suggesting thatthe clusters marked synthetic sites. Once again,the total number of clusters did not change withincreasing labeling times, which they inter-preted as evidence that the technique was sensi-tive enough to detect all sites. Importantly, theyfound that the diameter of nascent transcriptclusters remained fairly constant (at �75 nm)regardless of the labeling time and increasedelongation, even during extended chase peri-ods. Iborra et al. (1996a) suggest that this resultis contrary to models in which the polymeraseslides along the template during transcription.They argue that such a polymerase trackingmechanism would result in nascent transcriptsoccupying an increased volume with increasingelongation. Instead they proposed their data tobe consistent with a model in which the DNAtemplate slides through a polymerase that isimmobilized in a factory. This has been one ofthe most difficult concepts for many in the tran-scription field to accept, and perhaps rightly sobecause it contradicts the textbook view thatholds the gene as the central scaffold to whichthe transcription complex is recruited. Interest-ingly, in the case of DNA replication, it is widelyaccepted that DNA is drawn through and ex-truded from discrete replication factories con-taining many active polymerases (Berezneyet al. 2000). Though this is not proof that a sim-ilar mechanism occurs during transcription, itappears to be the most plausible given the avail-able evidence in favor of this concept and rela-tive lack of evidence to the contrary.

Iborra et al. (1996a) also examined the rela-tionship between nascent transcript sites andRNAPII by combining detection of nascentRNA and RNAPII with immno-gold particlesof different sizes. Gold particles markingRNAPII were also found in discrete clustersthroughout the nucleus and averaged 56 nm indiameter. They showed that nascent transcript


Cite this article as Cold Spring Harb Perspect Biol 2010;2:a000729 3



gold clusters were intimately associated withRNAPII clusters but did not overlap exactly.The centers of nascent transcript clusters andRNAPII clusters were shifted relative to eachother by an average of 24 nm, consistent withtwo overlapping zones, one rich in transcripts,and the other rich in RNA polymerase II. Si-multaneous immunofluorescent detection ofRNAPII and nascent transcripts combinedwith confocal microscopy confirmed this asso-ciation between focal RNAPII sites and tran-scriptional activity (Grande et al. 1997).RNAPII-containing foci colocalized with sitesof nascent transcript labeling and vice versa,however, there were also many sites stronglylabeled for RNAPII that contained little or noBrUTP label, and vice versa. With hindsight,this might be taken as one of the first hints ofspecialized polymerase factories and the possi-bility that not all factories are equally active(see below).

What actually constitutes the factory is aquestion open to interpretation. Nascent tran-scripts are undoubtedly the product but theenzymatic machinery is in essence the factory.The most recent and probably most accuratemeasurements of transcription factories sizeemployed electron spectroscopic imaging (ESI)(Eskiw et al. 2008). Rather than measure thesize of a cluster of gold particles, ESI recordsatomic signatures (Fig. 1). Transcription facto-ries appear as large proteinacious (nitrogenrich) structures with an average diameter of 87nm. Chromatin and nascent transcripts withassociated RNPs appear as fibrous, relativelyphosphorus-rich structures located at the pe-riphery of factories.

A GFP-tagged form of the human catalyticsubunit of RNAPII (Sugaya et al. 2000) wasused to monitor RNAPII kinetics in living cellsby fluorescence recovery after photobleaching(FRAP) and fluorescence loss in photobleach-ing (FLIP) (Kimura et al. 2002; Hieda et al.2005). GFP-RNAPII dynamics were consistentwith the existence of at least two populationsof RNAPII in nuclei. Most of the tagged RNAPII(75–80% of the pool) had a very short recoverytime on the order of seconds characteristic ofrapid free diffusion as has been seen for other

nuclear factors. The transcriptionally engagedfraction (20–25%) had a considerably longerrecovery time (t1/2 approx. 20 min) consistentwith relative immobilization. The latter fractioncould be further subdivided into initiating andelongating subfractions of decreasing turnoverrates and differential sensitivity to transcrip-tional inhibitors of initiation and elongation.These results suggested that RNAPII and most

ch

* ***

***

* *** *

* * *

**

***

*

IG

100 nmch

B

A

Figure 1. (A) Schematic diagram of transcription ofmultiple genes at a nuclear RNAPII transcriptionfactory. RNAPII factory shown as central blue circlewith three transcribing genes and their associatedtranscription factors (small colored circles). Nascenttranscripts are shown in red, chromatin is dark blue,and splicing components are depicted as small blackcircles with orange halo. (B) Electron spectroscopicimaging of HeLa cell nucleus. Phosphorous-richstructures are colored red and nitrogen green.Arrows point to nitrogen-rich transcription factory.White dots are immunogold detection of BrdUpulse-labeled nascent transcripts. Asterisks outline asmall region of interchromatin granules (IG) and chdenotes regions of relatively compact chromatin.Image courtesy of Dr. Christopher Eskiw.





likely other factory components undergo con-tinual but relatively slow dynamic exchangewith freely diffusing components, but that theposition of the factories themselves may bemore stable. Under conditions in which newinitiation is specifically and globally inhibited(Allen et al. 2004; Espinoza et al. 2004; Marineret al. 2008), it was found that expressed genesdisengage from transcription factories but focalsites of Ser5-RNAPII were still present after 30min (Mitchell and Fraser 2008). These findingssuggest that factories are not simply aggregatesof RNAPII on active genes but appear to be gen-uine subnuclear compartments. Consistentwith this model, inhibition with drugs whichfreeze or slow the elongating polymerase (Pal-stra et al. 2008), did not lead to genomereorganization.

HOW MANY FACTORIES ARE THERE?

The number of transcription sites per cellnucleus has been determined in a number ofways in many different cell and tissue types.The earliest methods used nascent transcriptlabeling in commonly used cultured cell linessuch as HeLa and other fibroblastic cells anddetermined that there were 100–500 sites ofnascent RNA per nucleus (Jackson et al. 1993;Wansink et al. 1993). In those days, the mostreliable estimates put the number of eukaryo-tic genes at over 100,000 with the number ofexpressed or active genes in a given cell typearound 10,000–30,000, far greater than thenumber of RNA synthesis sites. Wansink et al.(1993) suggested two possible reasons to explainthis discrepancy. Because few eukaryotic genesare transcribed at relatively high rates andmost are transcribed at low rates, the smallnumber of nascent RNA sites could be causedby detection of only the most highly transcribedgenes, and a failure to detect the bulk of activegenes making few nascent transcripts. In thisscenario there would be no evidence in favorof a factory model because each gene could betranscribing in isolation. Another possibility,particularly favored by Jackson and Cook wasthat each transcription site was occupied by anumber of actively transcribed genes. Over the

subsequent decade, refinements in nascenttranscript labeling and detection have led toimproved estimates of the number of factoriesper nucleus (Martin and Pombo 2003). Approx-imately 2100 (Iborra et al. 1996a) and 2400(Jackson et al. 1998) nascent transcript siteswere detected respectively in HeLa nuclei andan equivalent number of RNAPII sites wereobserved (Iborra et al. 1996a). Fay et al. (1997)had similar results but noted considerable var-iations in the number of nascent transcript sites(849–3888) in individual human fibroblasts.Pombo et al. (1999) using cryosectioning foundevidence for 10,000 nucleoplasmic factories inHeLa cells with separate factories for RNAPIIand RNAPIII; approximately 8000 RNAPIIand 2000 RNAPIII factories. As the number oftranscription sites increased, estimates of thenumber of eukaryotic genes in the genomeand the number of active genes in a particularcell type decreased. However, the new apprecia-tion of the extent of nongenic noncoding tran-scription has again increased the total numberof transcription units and may now in fact ex-ceed even the early estimates of gene numbers.So we are left with the realization that the num-ber of active transcription units greatly out-weighs the number of transcription sites percell nucleus even with the highest estimates offactory numbers from HeLa cells.

Some of the reports detailing the number offactories per nucleus make claims of highly sen-sitive techniques enabling the detection of allnascent transcription sites. Part of the variationin factory numbers observed may be because ofdifferences in the ability to detect weak nascentRNA sites, but it also seems that there maybe substantial variation within a cell type andbetween different cell types. In many casesHeLa or fibroblastic cells, which flatten out inculture were used, resulting in a substantialincrease in nuclear diameter and nuclear vol-ume when compared to cells with sphericalnuclei. Osborne et al. (2004) used immuno-fluorescence to detect nuclear sites with highconcentrations of the active form of RNAPIIphosphorylated on Serine 5 (Ser5-RNAPII).They found approximately 2000 Ser5-RNAPIIsites in the extended and flattened nuclei of





mouse embryo fibroblast, consistent with pre-vious nascent transcript labeling studies in fibro-blastic cells (Fig. 2). In contrast, they found thaterythroblasts, B-cells, T-cells, and fetal braincells, which have spherical nuclei with signifi-cantly smaller radii and nuclear volumes, havedramatically fewer Ser5-RNAPII sites (100–300 per nucleus). Though the claim of detectingall RNAPII transcription sites was not made, theRNAPII detection threshold used by two differ-ent groups was sufficient to show that 90–99%of nascent RNA FISH signals for a variety of dif-ferent genes overlapped with RNAPII factories(Osborne et al. 2004; Ragoczy et al. 2006;Osborne et al. 2007; Schoenfelder et al. 2010).Thus, although some RNAPII sites may havebeen missed because of the RNAPII detectionthreshold, the fact that nearly all nascent genetranscript signals colocalize with the RNAPIIsites detected suggest that the majority of siteswere detected. These results support the con-cept that all gene transcription occurs in RNA-PII factories, consistent with conclusions fromnascent transcript labeling studies. The largedifferences in factory numbers seen in nucleifrom tissues versus cells grown on a surfaceappear genuine and may be a consequence ofa reduced potential for intrachromosomal andespecially interchromosomal sharing of fac-tories in flattened cell nuclei. More factoriesmay be required as an adaptation to servicethe same number of genes in flattened nuclei.

Interestingly, changes in cell and nuclear mor-phology have been demonstrated to result inwidespread changes in gene expression (Dalbyet al. 2007; Chang and Hughes-Fulford 2009).

HIGHER-ORDER CHROMATIN FOLDING,TRANSCRIPTION AND SHAREDRNAPII SITES

Since the discovery of transcriptional enhanc-ers, which function to increase gene expressionfrom remote genomic positions and independ-ent of orientation to gene promoters, debate hascentered on their mechanism. Most now agreethat long-range gene control by remote enhan-cers and locus control regions involves directinteraction between chromatin at the enhancersites and gene local regulatory elements (Carteret al. 2002; Tolhuis et al. 2002). It appears thatDNA binding factors, transcription factors,and associated factors are required to eithercreate an appropriate chromatin structure fordistal elements to engage in interactions or me-diate interactions by acting as bridging mole-cules (Drissen et al. 2004; Vakoc et al. 2005;Kurukuti et al. 2006; Splinter et al. 2006; Songet al. 2007; Majumder et al. 2008; Hadjur et al.2009). Though we are still in the dark as towhat specific transcriptional advantage suchfolding endows to a particular gene locus, thereis little doubt that higher-order chromatin fold-ing plays a critical role in transcriptional regula-tion over genomic distances of up to a megabaseor more (Chakalova et al. 2005a; Kleinjan andvan Heyningen 2005). One line of reasoningsuggests that the increased local concentrationof transcription factors brought into proximityof the promoter by the distal enhancer results inincreased recruitment of (or to) the transcrip-tional machinery. A logical extension then leadsto the question of what effect associations orclustering of distal actively transcribed genesand their complexed enhancers at transcriptionfactories would have?

A critical tenet of the transcription factorymodel is that each site has the potential to beoccupied by a number of actively transcribedgenes or transcription units. Cook (2002) pro-posed that transcription factories might form

10 um

Figure 2. Maximum intensity projections of Ser5-RNAPII factories in splenic B cell (left) and prim-ary mouse embryo fibroblast (right) nuclei. FromOsborne et al., 2004.





by the aggregation of a local cluster of transcrip-tionally active genes and their associated poly-merases. Logic suggested that active genes inproximity in the primary DNA sequence wouldbe more likely to engage in the same factory thanactive genes separated by long stretches of inac-tive genomic sequence. This may be true, butthe original demonstration that actively tran-scribed genes could indeed share the same fac-tory greatly exceeded this expectation. Osborneet al. (2004) showed using primary transcriptRNA FISH and immunofluorescence (RNAFISH) for Ser5-RNAPII that genes separated by25–40 megabases of chromosomal DNA couldshare factories at remarkably high frequenciesin mouse erythroid progenitors. Chromosomeconformation capture (3C) (Dekker et al.2002) was used to verify the spatial associationbetween distal transcribed sequences. It wasalso apparent that active genes on separate chro-mosomes could share factories but appeared todo so at reduced frequency compared to linkedtranscribed genes. These results, coupled witha realization of the transcriptional behavior of“active” genes in vivo lead to some importantconclusions.

Several lines of evidence suggest that tran-scription of “active” genes in a particular cellor tissue type is not continuous. Genes appearto be transcribed in bursts or pulses of transcrip-tional activity separated by variable periods ofinactivity (Chubb et al. 2006; Raj et al. 2006).Similarly, RNA FISH for most “active” genesresults in signals for only a portion of allelesacross a population of expressing cell, with indi-vidual cells displaying none, one, or two activelytranscribed alleles (Wijgerde et al. 1995; Levskyet al. 2002; Osborne et al. 2004; Osborne et al.2007), There are a handful of so called “supergenes,” which appear to defy this general rule(Fraser 2006). For example, the a- and b-globingenes (Hba and Hbb) in erythroid cells (Wij-gerde et al. 1995) and the immunoglobulingenes (Igh, Igk, and Igl) in B-cells (Bollandet al. 2004; Osborne et al. 2007) tend to be con-tinually or constitutively active with nearly allalleles in an interphase population associatedwith transcription factories (Osborne et al.2004; Osborne et al. 2007). For other “expressed

genes,” which appear to be less frequently tran-scribed, inactive alleles were located away fromRNAPII factories. Thus the high degree of co-localization between transcribed genes sug-gested that upon activation a significantly highproportion of newly transcribed genes join pre-established transcription sites containing otheractive genes rather than assemble their owntranscription site de novo. This was furtherinvestigated by studying the dynamics of theimmediate early genes Fos and Myc in restingsplenic mouse B-cells (Osborne et al. 2007).Most alleles for these genes are transcriptionallyinactive and located away from factories in rest-ing cells. However, in cells induced for 5 min,the opposite is found, most Fos and Myc allelesare associated with factories and transcription-ally active. A high percentage of the newly activeFos alleles associated with the same factory asthe constitutively transcribed Igh locus locatedapproximately 30 Mb away on chromosome12. Most interestingly, 25% of the newly activeMyc alleles from chromosome 15 were alsofound to be associated with the same factoryas the Igh locus. This appeared to be a highlypreferential interchromosomal association be-cause RNA FISH analyses of many other genesin trans had significantly lower interchromoso-mal association frequencies with Igh. Previousstudies had shown that chromosomes 12 and15 are preferred neighbors in mouse B-cells(Roix et al. 2003; Parada et al. 2004) and thata significant degree of intermingling occurs be-tween chromosome territories (Branco andPombo 2006). That MYC and IGH come intoproximity in B-cells is without doubt, becausethe two genes are the most common transloca-tion partners in Burkitt’s Lymphoma in hu-mans and plasmacytomas in mouse (Hechtand Aster 2000; Potter 2003). Osborne’s workshowed that induction of MYC correlated witha measurable shift in position of MYC allelestowards IGH alleles, indicating that activationof transcription involves short-range chromatinmovements over distances of 0.5–1.5 micronsto access a factory, and suggesting that prefer-ential interchromosomal transcription factorycoassociations could be involved in the mech-anism of chromosomal translocations. Others





have also documented long-range intra- andinterchromosomal interactions between loci orcoassociations with specific nuclear subcom-partments (Spilianakis et al. 2005; Bacheret al. 2006; Brown et al. 2006; Ling et al. 2006;Lomvardas et al. 2006; Simonis et al. 2006; Wur-tele and Chartrand 2006; Xu et al. 2006; Zhaoet al. 2006; Apostolou and Thanos 2008; Brownet al. 2008; Hu et al. 2008) and in many casescorrelated clustering or proximity with effectson gene expression. This remains a very contro-versial area, and even those who promote suchconcepts, in many cases cannot agree on how,where and to what effect proximity in nuclearspace has a functional role.

CHROMATIN DYNAMICS ANDTRANSCRIPTION

Constrained Brownian motion of chromatincould in theory account for the rapid, short-range chromatin movement (Chakalova et al.2005a) of induced Myc alleles toward the Ighfactory mentioned earlier. The Myc gene in rest-ing B-cells is not transcriptionally naıve. Mycexpression is involved in stimulating prolifera-tion of immature B-cells and would have beeninduced at various points in B-cell differentia-tion concurrent with Igh transcription. How-ever, that is not to say that active or directedprocesses are not involved in reorganization ofchromatin and transcription in vivo. More dra-matic chromatin movements may be involved,and have been suggested in the initial activationof a locus from a completely silent state. Gro-und-breaking live cell studies (Chuang et al.2006) reported dramatic vectorial movementsof chromatin in response to targeting a tran-scriptional activator to a silent transgene array.Migration occurred 1–2 h after targeting atspeeds of up to 0.9 microns/min over distancesof 1–5 microns. Rapid vectorial movementsappeared to be punctuated by brief periods ofrandomdiffusionalmotion.Chuangetal.(2006)showed that repositioning did not requiretranscription and did not appear to involveextensive decondensation of the transgenearray structure. Actin and nuclear myosin were

required for directed movements and the analy-sis of an actin point mutant defective in actinpolymerization suggested that filamentous, F-actin may be involved. Dundr et al. (2007)also showed actin-dependent, long-range repo-sitioning using a tagged array of inducible U2snRNA genes. Again, hours rather than minutesafter induction, they observed vectorial move-ment of the transgene array toward relativelystably positioned Cajal bodies that are normallyfound in association with snRNAs and histonegene loci. Expression of a dominant negativemutant of b-actin markedly inhibited reposi-tioning, supporting a role for nuclear actin inlong-range chromatin movements.

Large-scale chromatin movement has notbeen seen in every instance of transcriptionalactivation in live cells. Kumaran and Spector(Kumaran and Spector 2008) targeted a trans-gene array to the peripheral nuclear lamina.Transcriptional induction resulted in activationat the periphery with a clear increase in localRNAPII concentration. It was not clear whetherthe transgenes used local factories, which in-creased in size to accommodate the 200-copyarray, or whether RNAPII was recruited toeach promoter in the array de novo. Howeverlong-range movements were not observed. Sim-ilarly, Yao et al. (Yao et al. 2007) observed re-cruitment of polymerase components to heatshock loci in live cells on Drosophila polytenechromosomes creating characteristic puffs.Apart from local swelling, movement of theheat shock loci on the giant polytene chromo-somes was not observed upon activation oftranscription. Clearly live cell imaging holdsthe most potential for understanding the dy-namic relationships between genes, transcrip-tional components and transcription factories.The studies of Kumaran and Spector (2008)and Yao et al. (2007) are probably the best avail-able evidence in favor of the textbook model oftranscription, in which RNAPII is recruited togene promoters. However, aside from theirobvious use, it remains to be seen whether thehundreds of gene copies in transgene arrays orgiant polytene chromosomes are good modelsthat accurately reflect the behavior of an iso-lated single copy gene in a diploid nucleus. It





is possible that these localized multi-gene as-semblies are in themselves super factories thatalter the dynamic exchange of polymerase com-ponents (Kimura et al. 2002) to create a steadystate structure that is several times larger thanthe average transcription factory.

TRANSCRIPTIONAL INTERACTOME ANDTRANSCRIPTION HOTSPOTS

The number of transcription units sharing a fac-tory at any one time has been estimated to bebetween two and 30 in HeLa cell nuclei (Jacksonet al. 1998; Pombo et al. 1999). Mouse erythroidprecursors, which have a few hundred factories,approximately 13,000 expressed alleles (6500active genes assuming that most are bi-allelicallyexpressed) with approximately half activelytranscribing at any given moment are estimatedto hold about 10–30 genes per factory (Schoen-felder et al. 2010). Obviously the large amountof nongenic transcription must be factoredinto this calculation, but most studies suggestthat a substantial fraction of these transcriptsoccur in the vicinity of active genes (Kapranovet al. 2007) and in the case of the globin genesare found in a relatively small fraction of theerythroid cells concomitant with gene tran-scription (Ashe et al. 1997; Gribnau et al. 2000;Chakalova et al. 2005b; Miles et al. 2007). Theobservation that preferential interchromosomalgene associations at factories can occur raises thepossibility that the combination of genes at aparticular factory could be meaningful in termsof transcriptional output of the assembledgenes. Recent studies examining the intranu-clear localization of transcriptionally activemini-chromosomes suggested that similarlyregulated genes cluster at a limited number offactories. Xu and Cook (2008) used expressionconstructs carrying transcription units drivenby different promoters. The multicopy con-structs were assembled into nucleosomes in cellsand the resulting mini-chromosomes under-went rounds of transcription and replication.RNA immuno-FISH experiments showed thattranscriptionally active copies of mini-chro-mosomes clustered at a subset of the available

transcription factories. Moreover, the mini-chromosome appeared to share transcriptionsites with endogenous genes suggesting the in-troduced constructs were nonrandomly accom-modated by host sites. The authors went on tocotransfect pairs of different constructs to testwhether plasmids containing functionally sim-ilar genes and regulatory elements would sharenuclear factories more often than functionallyunrelated transcription units. Firstly, mini-chro-mosomes carrying RNAPI, II, or III transcrip-tion units separated into different factories,specialized in RNAPI, II, or III transcription,respectively. RNAPI genes acquired nucleolarlocalization, whereas RNAPII and III plasmidswere spread in nonoverlapping foci throughoutthe nucleoplasm. These results were in agree-ment with earlier findings showing separateRNAPII and RNAPIII nucleoplasmic transcrip-tion sites (Pombo et al. 1999), and supportedthe view that the mini-chromosomes mimickedthe behavior of endogenous chromosomal loci.Secondly, evidence for specialization was foundwithin the RNAPII factory subclass. Mini-chromosomes carrying different genes drivenby identical RNAPII promoters co-occupiedthe same foci at high frequencies. Conversely,plasmids carrying different promoters weremostly transcribed in separate factories. Thesepreferences extended to endogenous genestoo, because an edogenous gene was more likelyto be in close proximity to its plasmid-bornecounterpart than an unrelated gene. Taken to-gether, these results suggested that factoriesare not functionally equal, and may be special-ized to transcribe similarly regulated genes.

However, important questions remain: Doesthe tendency for factory preference or special-ization result in a non-random organizationof native or chromosomally integrated transcri-ption units? Schoenfelder et al. (2010) answeredthese questions in two ways. First they observedthe nuclear localization of ectopically inte-grated, transcriptionally active human HBBtransgenes in erythroid cells from several differ-ent lines of transgenic mice. They found that theHBB transgene locus had a three- to 15-foldpreference to be transcribed in the same factoryas the endogenous mouse Hbb genes compared





to the endogenous Hba locus. The fact that theectopically integrated transgene in all six linestested showed preferential interchromosomalassociations, in one case occurring in up to35% of cells, suggested that preferential factoryco-associations had the potential to reorganizethe genome in the nucleus. They next developedan enhanced 4C assay to screen the entire ge-nome for genes sharing transcription factorieswith the Hbb or Hba genes. Supported by exten-sive FISH and 3C analyses they revealed twooverlapping transcription networks, each in-volving hundreds of preferred transcriptionpartners in cis and trans. Genes regulated bythe erythroid-specific transcription factor,Klf1 (erythroid Kruppel-like factor) were over-represented in the globin transcription net-works suggesting that coregulated genespreferentially transcribed in shared factories.Klf1 was previously shown to be required forhigh-level transcription of the Hbb gene indefinitive erythroid cells (Nuez et al. 1995; Per-kins et al. 1995) and also appears to contributeto Hba regulation (Shyu et al. 2006; Vernimmenet al. 2007). Schoenfelder et al. (2010) usedimmunofluorescence to show that, Klf1 local-ized to 30-40 small foci in erythroid nucleiand that nearly all overlapped with Ser5-RNA-PII foci. These results suggested the possibilitythat a subset of factories were specializedbecause of an increased concentration of a par-ticular transcription factor; a phenomena seenbefore for other factors (Grande et al. 1997).Actively transcribed alleles of Klf1-regulatedgenes were more often found in associationwith factories containing high levels of Klf1 sug-gesting that they had a higher probability oftranscription when associated with a specializedfactory. Furthermore, they found that manyKlf1-regulated genes were preferentially clusteredat these sites. This data provided strong evidencefor functional specialization of transcriptionfactories, and suggested that individual factoriescould become hotspots for optimal transcriptionof a subset of coregulated genes. There seemed tobe no specific requirement for the globin genes totranscribe in the same factory with any other par-ticular gene, but that in most, if not all, erythroidcells the globin genes associated with a varied

subset of other Klf1 regulated genes (Schoen-felder et al. 2010).

PULLING SOME STRINGS

The emerging evidence provides strong supportfor the transcription factory model where dis-tant genes in cis and trans nonrandomly associ-ate during transcription. A key feature of thismodel as originally proposed by Cook is thattranscribed genes are reeled through factoriesby the relatively immobilized polymerase whileextruding nascent RNA, rather than the poly-merase tracking along the template. Cook andcolleagues recently tested this prediction byassessing nuclear proximity of two distal, rap-idly induced genes, one long and one short.SAMD4A is 221 kb long and located approxi-mately 50 Mb from TNFAIP2—a relatively shortgene of 11 kb. Both genes are activated simulta-neously by tumor necrosis factor a(TNFa). In-duction of SAMD4A has been shown to resultin a fairly synchronous wave of transcriptionand RNAPII traversing the transcription unitover a 70-min period (Wada et al. 2009). The3C assay was used to investigate long-range asso-ciations over this time period between the short,repeatedly transcribed TNFAIP2 gene and sev-eral regions along the length of SAMD4A. Beforeinduction there was no evidence for long-rangeassociation between these genes, but upon acti-vation they found that proximity betweenTNFAIP2 and the SAMD4A promoter was rap-idly induced. As the wave of RNAPII movedalong SAMD4A, contacts between the promoterand TNFAIP2 were lost in favor of new contactsbetween TNFAIP2 and successive downstreamregions of SAMD4A. In other words, it appearedthat the long genewas sliding past the distal shortgene with a temporal pattern that matched theprogress of the transcribing polymerase on thelong gene. These results showing that differentparts of transcription units are brought intoproximity are consistent with the idea thattranscribed genes are reeled through factoriesand very difficult to reconcile with the textbookmodel of RNAPII sliding along a relativelyimmobile template.





CONCLUDING REMARKS

The realization that the active form of RNAPII iscompartmentalized in the nucleus into a finitenumber of discrete transcription factories andthat actively transcribed genes are non-ran-domly organized around these sites has wide-spread implications in understanding nuclearorganization of the genome and genome fun-ction. This turns the classical view of genetranscription on its head, and with this newunderstanding come new insights and newquestions. Rather than the requirement fornear simultaneous recruitment and assemblyof hundreds of factors required for regulatedactivation of transcription at the site of individ-ual genes, this model suggests that many tran-scriptional components are preassembled intoactive sites which genes migrate to for transcrip-tion. There is still of course ample scope for reg-ulatory factors and transcription componentsbeing recruited to genes while outside factories.For example, transcription factors, cofactors,chromatin remodeling complexes, and varioushistone modifying activities may act as licensingfactors that affect genes before factory entryto prepare them for productive engagementswith an RNAPII complex. The hypophosphory-lated or initial binding form of RNAPII mayalso engage genes outside factories. The chanceor directed coassociation of coregulated genes ata factory may favor re-initiation of those genesthrough sharing of dynamically binding orinteracting factors, which will in turn stabilizethe presence of those genes at the factory foras long as it takes to transcribe them. Thismay then become a favorable place for othercoregulated genes to alight leading to a dynam-ically self-organizing hotspot of transcriptionfor a specific subgroup or network of genes.Within the factory, epigenetic modificationsmay also be laid down or re-affirmed in tran-scribed regions, which may further promotere-initiation and/or serve as a memory for theactivation state. Obviously chromatin mobility isa potentially important component of this newunderstanding. Factors or activities that com-pact chromatin or tether them to “repressive”nuclear domains may serve to restrict mobility

and access to transcription factories. Genes oralleles not able to reach one of their hotspotsmay disengage after relative few rounds oftranscription at a generic factory because of arelatively low local concentration of specific fac-tors required for re-initiation and stabilizationat a factory.

There is still much to do and many of theimportant questions will best be answered bylive cell experiments on single copy or endoge-nous genes. For example, the transcriptionalperiod of individual genes needs to be assessed.Are factory encounters transient and highlydynamic, potentially reshuffling at every tran-scriptional cycle, or once established, do activealleles/genes remain associated with factoriesthroughout a cell cycle. It is likely the answerswill vary widely for different genes and mayencompass the entire range, potentially influ-enced by the character of long-range regulatoryelements that individual active genes are com-plexed with as well as other transcription unitsthey may encounter in factories. Also, howdoes the genome end up in such highly-organ-ized, tissue-specific conformations that allowspecific subgroups of genes the opportunity tocluster at specialized factories? There is contro-versy surrounding evidence suggesting chromo-some positions may be partially inheritedthrough mitosis (Gerlich et al. 2003; Walteret al. 2003; Cvackova et al. 2009), and that invery early G1 phase genome reorganization ismore dramatic than in the rest of interphase(Dimitrova and Gilbert 1999). It appears likelythat mitosis offers an essential opportunity forlarge-scale, though imperfect, genome reorgan-ization which may be further refined in earlyG1. If the organized associations of co-regulatedgenes are beneficial then we may expect consid-erable pressure to be exerted on the primaryorganization of the genome, such that individ-ual genes are optimally placed to take advantageof their eventual folding in 3 dimensions in thevarious tissues in which they will be expressed.These are exciting times and the advent of newtechnologies coupled with deep sequencingalong with advanced microscopy hold greatpromise for further exciting advances in un-derstanding the relationships between genome





organization and transcriptional control as wellas other genome functions. And hopefully,future textbooks will reflect these importantaspects of the transcriptional mechanism sothat the next generation of genome investigatorswill be more adequately prepared to advancethe understanding of transcription inside thecomplex world of the nucleus.

ACKNOWLEDGMENTS

We would like to thank in particular C. Eskiwand other members of the Laboratory of Chro-matin and Gene Expression for many helpfuldiscussions. We are also grateful to A. Papanto-nis and P. Cook for generously sharing unpub-lished data.

REFERENCES

Allen TA, Von Kaenel S, Goodrich JA, Kugel JF. 2004.The SINE-encoded mouse B2 RNA represses mRNAtranscription in response to heat shock. Nat Struct MolBiol 11: 816–821.

Apostolou E, Thanos D. 2008. Virus Infection InducesNF-kB-dependent interchromosomal associationsmediating monoallelic IFN-b gene expression. Cell 134:85–96.

Ashe HL, Monks J, Wijgerde M, Fraser P, Proudfoot NJ.1997. Intergenic transcription and transinduction ofthe human b-globin locus. Genes Dev 11: 2494–2509.

Bacher CP, Guggiari M, Brors B, Augui S, Clerc P, Avner P,Eils R, Heard E. 2006. Transient colocalization ofX-inactivation centres accompanies the initiation of Xinactivation. Nat Cell Biol 8: 293–299.

Berezney R, Dubey DD, Huberman JA. 2000. Heterogeneityof eukaryotic replicons, replicon clusters, and replicationfoci. Chromosoma 108: 471–484.

Bolland DJ, Wood AL, Johnston CM, Bunting SF, Morgan G,Chakalova L, Fraser PJ, Corcoran AE. 2004. Antisenseintergenic transcription in V(D)J recombination. NatImmunol 5: 630–637.

Branco MR, Pombo A. 2006. Intermingling of chromosometerritories in interphase suggests role in translocationsand transcription-dependent associations. PLoS Biology4: e138.

Brown JM, Green J, das Neves RP, Wallace HA, Smith AJ,Hughes J, Gray N, Taylor S, Wood WG, Higgs DR, et al.2008. Association between active genes occurs at nuclearspeckles and is modulated by chromatin environment.J Cell Biol 182: 1083–1097.

Brown JM, Leach J, Reittie JE, Atzberger A, Lee-Prudhoe J,Wood WG, Higgs DR, Iborra FJ, Buckle VJ. 2006. Core-gulated human globin genes are frequently in spatialproximity when active. J Cell Biol 172: 177–187.

Carter D, Chakalova L, Osborne CS, Dai YF, Fraser P. 2002.Long-range chromatin regulatory interactions in vivo.Nat Genet 32: 623–626.

Chakalova L, Debrand E, Mitchell JA, Osborne CS, Fraser P.2005a. Replication and transcription: Shaping the land-scape of the genome. Nat Rev Genet 6: 669–677.

Chakalova L, Osborne CS, Dai YF, Goyenechea B,Metaxotou-Mavromati A, Kattamis A, Kattamis C, FraserP. 2005b. The Corfu db thalassemia deletion disruptsg-globin gene silencing and reveals post-transcriptionalregulation of HbF expression. Blood 105: 2154–2160.

Chang TT, Hughes-Fulford M. 2009. Monolayer and sphe-roid culture of human liver hepatocellular carcinomacell line cells demonstrate distinct global gene expressionpatterns and functional phenotypes. Tissue Eng Part A 15:559–567.

Chuang CH, Carpenter AE, Fuchsova B, Johnson T, deLanerolle P, Belmont AS. 2006. Long-range directionalmovement of an interphase chromosome site. Curr Biol16: 825–831.

Chubb JR, Trcek T, Shenoy SM, Singer RH. 2006. Transcrip-tional pulsing of a developmental gene. Curr Biol 16:1018–1025.

Cmarko D, Verschure PJ, Martin TE, Dahmus ME, Krause S,Fu XD, van Driel R, Fakan S. 1999. Ultrastructural anal-ysis of transcription and splicing in the cell nucleus afterbromo-UTP microinjection. Mol Biol Cell 10: 211–223.

Cook PR. 2002. Predicting three-dimensional genomestructure from transcriptional activity. Nat Genet 32:347–352.

Cremer T, Kupper K, Dietzel S, Fakan S. 2004. Higher orderchromatin architecture in the cell nucleus: On the wayfrom structure to function. Biol Cell 96: 555–567.

Cvackova Z, Masata M, Stanek D, Fidlerova H, Raska I.2009. Chromatin position in human HepG2 cells:Although being non-random, significantly changed indaughter cells. J Struct Biol. 165:107–117.

Dalby MJ, Gadegaard N, Herzyk P, Sutherland D, Agheli H,Wilkinson CD, Curtis AS. 2007. Nanomechanotrans-duction and interphase nuclear organization influenceon genomic control. J Cell Biochem 102: 1234–1244.

Davenport RJ, Wuite GJ, Landick R, Bustamante C. 2000.Single-molecule study of transcriptional pausing andarrest by E. coli RNA polymerase. Science 287: 2497–2500.

Dekker J, Rippe K, Dekker M, Kleckner N. 2002. Capturingchromosome conformation. Science 295: 1306–1311.

Dimitrova DS, Gilbert DM. 1999. The spatial position andreplication timing of chromosomal domains are bothestablished in early G1 phase. Mol Cell 4: 983–993.

Drissen R, Palstra RJ, Gillemans N, Splinter E, Grosveld F,Philipsen S, de Laat W. 2004. The active spatial organiza-tion of the b-globin locus requires the transcription fac-tor EKLF. Genes Dev 18: 2485–2490.

Dundr M, Ospina JK, Sung MH, John S, Upender M, Ried T,Hager GL, Matera AG. 2007. Actin-dependent intranu-clear repositioning of an active gene locus in vivo. J CellBiol 179: 1095–1103.

Eskiw CH, Rapp A, Carter DR, Cook PR. 2008. RNA poly-merase II activity is located on the surface of protein-richtranscription factories. J Cell Sci 121: 1999–2007.





Espinoza CA, Allen TA, Hieb AR, Kugel JF, Goodrich JA.2004. B2 RNA binds directly to RNA polymerase II torepress transcript synthesis. Nat Struct Mol Biol 11:822–829.

Fakan S. 1994. Perichromatin fibrils are in situ forms ofnascent transcripts. Trends Cell Biol 4: 86–90.

Fakan S. 2004. The functional architecture of the nucleus asanalysed by ultrastructural cytochemistry. Histochem CellBiol 122: 83–93.

Fakan S, Bernhard W. 1971. Localisation of rapidly andslowly labelled nuclear RNA as visualized by high resolu-tion autoradiography. Exp Cell Res 67: 129–141.

Fakan S, Puvion E, Sphor G. 1976. Localization and charac-terization of newly synthesized nuclear RNA in isolate rathepatocytes. Exp Cell Res 99: 155–164.

Fay FS, Taneja KL, Shenoy S, Lifshitz L, Singer RH. 1997.Quantitative digital analysis of diffuse and concentratednuclear distributions of nascent transcripts, SC35 andpoly(A). Exp Cell Res 231: 27–37.

Fraser P. 2006. Transcriptional control thrown for a loop.Curr Opin Genet Dev 16: 490–495.

Gerlich D, Beaudouin J, Kalbfuss B, Daigle N, Eils R, Ellen-berg J. 2003. Global chromosome positions are trans-mitted through mitosis in mammalian cells. Cell 112:751–764.

Grande MA, van der Kraan I, de Jong L, van Driel R. 1997.Nuclear distribution of transcription factors in relationto sites of transcription and RNA polymerase II. J CellSci 110: 1781–1791.

Gribnau J, Diderich K, Pruzina S, Calzolari R, Fraser P. 2000.Intergenic transcription and developmental remodelingof chromatin subdomains in the human b-globin locus.Mol Cell 5: 377–386.

Guthold M, Zhu X, Rivetti C, Yang G, Thomson NH, KasasS, Hansma HG, Smith B, Hansma PK, Bustamante C.1999. Direct observation of one-dimensional diffusionand transcription by Escherichia coli RNA polymerase.Biophys J 77: 2284–2294.

Hadjur S, Williams LM, Ryan NK, Cobb BS, Sexton T, FraserP, Fisher AG, Merkenschlager M. 2009. Cohesins formchromosomal cis-interactions at the developmentallyregulated IFNG locus. Nature 460: 410–413.

Harada Y, Funatsu T, Murakami K, Nonoyama Y, IshihamaA, Yanagida T. 1999. Single-molecule imaging of RNApolymerase-DNA interactions in real time. Biophys J 76:709–715.

Hecht JL, Aster JC. 2000. Molecular biology of Burkitt’slymphoma. J Clin Oncol 18: 3707–3721.

Hieda M, Winstanley H, Maini P, Iborra FJ, Cook PR. 2005.Different populations of RNA polymerase II in livingmammalian cells. Chromosome Res 13: 135–144.

Hu Q, Kwon YS, Nunez E, Cardamone MD, Hutt KR, OhgiKA, Garcia-Bassets I, Rose DW, Glass CK, Rosenfeld MG,et al. 2008. Enhancing nuclear receptor-induced tran-scription requires nuclear motor and LSD1-dependentgene networking in interchromatin granules. Proc NatlAcad Sci 105: 19199–19204.

Huang S, Spector DL. 1996. Dynamic organization of pre-mRNA splicing factors. J Cell Biochem 62: 191–197.

Iborra FJ, Pombo A, Jackson DA, Cook PR. 1996a. Ac-tive RNA polymerases are localized within discrete

transcription “factories” in human nuclei. J Cell Sci109: 1427–1436.

Iborra FJ, Pombo A, McManus J, Jackson DA, Cook PR.1996b. The topology of transcription by immobilizedpolymerases. Exp Cell Res 229: 167–173.

Jackson DA, Cook PR. 1985. Transcription occurs at a nucle-oskeleton. EMBO J 4: 919–925.

Jackson DA, McCready SJ, Cook PR. 1981. RNA is synthe-sized at the nuclear cage. Nature 292: 552–555.

Jackson DA, Hassan AB, Errington RJ, Cook PR. 1993.Visualization of focal sites of transcription within humannuclei. Embo J 12: 1059–1065.

Jackson DA, Iborra FJ, Manders EM, Cook PR. 1998.Numbers and organization of RNA polymerases, nascenttranscripts, and transcription units in HeLa nuclei. MolBiol Cell 9: 1523–1536.

Kabata H, Kurosawa O, Arai I, Washizu M, Margarson SA,Glass RE, Shimamoto N. 1993. Visualization of singlemolecules of RNA polymerase sliding along DNA.Science 262: 1561–1563.

Kapranov P, Willingham AT, Gingeras TR. 2007. Genome-wide transcription and the implications for genomicorganization. Nat Rev Genet 8: 413–423.

Kimura H, Sugaya K, Cook PR. 2002. The transcriptioncycle of RNA polymerase II in living cells. J Cell Biol159: 777–782.

Kleinjan DA, van Heyningen V. 2005. Long-range control ofgene expression: Emerging mechanisms and disruptionin disease. Am J Hum Genet 76: 8–32.

Kumaran RI, Spector DL. 2008. A genetic locus targeted tothe nuclear periphery in living cells maintains its tran-scriptional competence. J Cell Biol 180: 51–65.

Kurukuti S, Tiwari VK, Tavoosidana G, Pugacheva E,Murrell A, Zhao Z, Lobanenkov V, Reik W, Ohlsson R.2006. CTCF binding at the H19 imprinting controlregion mediates maternally inherited higher-orderchromatin conformation to restrict enhancer access toIgf2. Proc Natl Acad Sci 103: 10684–10689.

Lamond AI, Spector DL. 2003. Nuclear speckles: A modelfor nuclear organelles. Nat Rev Mol Cell Biol 4: 605–612.

Levsky JM, Shenoy SM, Pezo RC, Singer RH. 2002. Single-cell gene expression profiling. Science 297: 836–840.

Ling JQ, Li T, Hu JF, Vu TH, Chen HL, Qiu XW, Cherry AM,Hoffman AR. 2006. CTCF mediates interchromosomalcolocalization between Igf2/H19 and Wsb1/Nf1. Science312: 269–272.

Lomvardas S, Barnea G, Pisapia DJ, Mendelsohn M, Kirk-land J, Axel R. 2006. Interchromosomal interactionsand olfactory receptor choice. Cell 126: 403–413.

Majumder P, Gomez JA, Chadwick BP, Boss JM. 2008. Theinsulator factor CTCF controls MHC class II gene expres-sion and is required for the formation of long-distancechromatin interactions. J Exp Med 205: 785–798.

Mariner PD, Walters RD, Espinoza CA, Drullinger LF,Wagner SD, Kugel JF, Goodrich JA. 2008. Human AluRNA is a modular transacting repressor of mRNA tran-scription during heat shock. Mol Cell 29: 499–509.

Martin S, Pombo A. 2003. Transcription factories: Quantita-tive studies of nanostructures in the mammalian nucleus.Chromosome Res 11: 461–470.





Miles J, Mitchell JA, Chakalova L, Goyenechea B, OsborneCS, O’Neill L, Tanimoto K, Engel JD, Fraser P. 2007.Intergenic transcription, cell-cycle and the developmen-tally regulated epigenetic profile of the human b-globinlocus. PLoS One 2: e630.

Misteli T, Caceres JF, Spector DL. 1997. The dynamics of apre-mRNA splicing factor in living cells. Nature 387:523–527.

Mitchell JA, Fraser P. 2008. Transcription factories arenuclear subcompartments that remain in the absence oftranscription. Genes Dev 22: 20–25.

Nash RE, Puvion E, Bernhard W. 1975. Perichromatin fibrilsas components of rapidly labeled extranucleolar RNA.J Ultrastruct Res 53: 395–405.

Nuez B, Michalovich D, Bygrave A, Ploemacher R, GrosveldF. 1995. Defective haematopoiesis in fetal liver resultingfrom inactivation of the EKLF gene. Nature 375: 316–318.

Osborne CS, Chakalova L, Brown KE, Carter D, Horton A,Debrand E, Goyenechea B, Mitchell JA, Lopes S, Reik W,et al. 2004. Active genes dynamically colocalize to sharedsites of ongoing transcription. Nat Genet 36: 1065–1071.

Osborne CS, Chakalova L, Mitchell JA, Horton A, Wood AL,Bolland DJ, Corcoran AE, Fraser P. 2007. Myc dynami-cally and preferentially relocates to a transcription factoryoccupied by igh. PLoS Biol 5: e192.

Palstra RJ, Simonis M, Klous P, Brasset E, Eijkelkamp B, deLaat W. 2008. Maintenance of long-range DNA interac-tions after inhibition of ongoing RNA polymerase II tran-scription. PLoS One3: e1661.

Parada LA, McQueen PG, Misteli T. 2004. Tissue-specificspatial organization of genomes. Genome Biol 5: R44.

Perkins AC, Sharpe AH, Orkin SH. 1995. Lethalb-thalassae-mia in mice lacking the erythroid CACCC-transcriptionfactor EKLF. Nature 375: 318–322.

Pombo A, Cook PR. 1996. The localization of sites contain-ing nascent RNA and splicing factors. Exp Cell Res 229:201–203.

Pombo A, Jackson DA, Hollinshead M, Wang Z, Roeder RG,Cook PR. 1999. Regional specialization in human nuclei:Visualization of discrete sites of transcription by RNApolymerase III. EMBO J 18: 2241–2253.

Potter M. 2003. Neoplastic development in plasma cells.Immunol Rev 194: 177–195.

Ragoczy T, Bender MA, Telling A, Byron R, Groudine M.2006. The locus control region is required for associationof the murine b-globin locus with engaged transcriptionfactories during erythroid maturation. Genes Dev 20:1447–1457.

Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S. 2006. Sto-chastic mRNA synthesis in mammalian cells. PLoS Biol 4:e309.

Roix JJ, McQueen PG, Munson PJ, Parada LA, Misteli T.2003. Spatial proximity of translocation-prone gene lociin human lymphomas. Nat Genet 34: 287–291.

Schafer DA, Gelles J, Sheetz MP, Landick R. 1991. Transcrip-tion by single molecules of RNA polymerase observed bylight microscopy. Nature 352: 444–448.

Schoenfelder S, Sexton T, Chakalova L, Cope NF, Horton A,Andrews S, Kurukuti S, Mitchell JA, Umlauf D, Dimi-trova DS, et al. 2010. Preferential associations between

co-regulated genes reveal a transcriptional interactomein erythroid cells. Nat Genet 42:53–61.

Shyu YC, Wen SC, Lee TL, Chen X, Hsu CT, Chen H, ChenRL, Hwang JL, Shen CK. 2006. Chromatin-binding invivo of the erythroid kruppel-like factor, EKLF, in themurine globin loci. Cell Res 16: 347–355.

Simonis M, Klous P, Splinter E, Moshkin Y, Willemsen R, deWit E, van Steensel B, de Laat W. 2006. Nuclear organiza-tion of active and inactive chromatin domains uncoveredby chromosome conformation capture-on-chip (4C).Nat Genet 38: 1348–1354.

Song SH, Hou C, Dean A. 2007. A positive role for NLI/Ldb1 in long-range b-globin locus control region func-tion. Mol Cell 28: 810–822.

Spilianakis CG, Lalioti MD, Town T, Lee GR, Flavell RA.2005. Interchromosomal associations between alterna-tively expressed loci. Nature 435: 637–645.

Splinter E, Heath H, Kooren J, Palstra RJ, Klous P, Grosveld F,Galjart N, de Laat W. 2006. CTCF mediates long-rangechromatin looping and local histone modification inthe b-globin locus. Genes Dev 20: 2349–2354.

Sugaya K, Vigneron M, Cook PR. 2000. Mammalian celllines expressing functional RNA polymerase II taggedwith the green fluorescent protein. J Cell Sci 113: 2679–2683.

Tolhuis B, Palstra RJ, Splinter E, Grosveld F, de Laat W. 2002.Looping and interaction between hypersensitive sites inthe active b-globin locus. Mol Cell 10: 1453–1465.

Vakoc CR, Letting DL, Gheldof N, Sawado T, Bender MA,Groudine M, Weiss MJ, Dekker J, Blobel GA. 2005. Prox-imity among distant regulatory elements at the b-globinlocus requires GATA-1 and FOG-1. Mol Cell 17: 453–462.

Vernimmen D, De Gobbi M, Sloane-Stanley JA, Wood WG,Higgs DR. 2007. Long-range chromosomal interactionsregulate the timing of the transition between poisedand active gene expression. Embo J 26: 2041–2051.

Wada Y, Ohta Y, Xu M, Tsutsumi S, Minami T, Inoue K,Komura D, Kitakami J, Oshida N, Papantonis A, et al.2009. Awave of nascent transcription on activated humangenes. Proc Natl Acad Sci 106:18357–18361.

Walter J, Schermelleh L, Cremer M, Tashiro S, Cremer T.2003. Chromosome order in HeLa cells changes duringmitosis and early G1, but is stably maintained duringsubsequent interphase stages. J Cell Biol 160:685–697.

Wang MD, Schnitzer MJ, Yin H, Landick R, Gelles J, BlockSM. 1998. Force and velocity measured for single mole-cules of RNA polymerase. Science 282: 902–907.

Wansink DG, Schul W, van der Kraan I, van Steensel B, vanDriel R, de Jong L. 1993. Fluorescent labeling of nascentRNA reveals transcription by RNA polymerase II indomains scattered throughout the nucleus. J Cell Biol122: 283–293.

Wijgerde M, Grosveld F, Fraser P. 1995. Transcription com-plex stability and chromatin dynamics in vivo. Nature377: 209–213.

Wurtele H, Chartrand P. 2006. Genome-wide scanning ofHoxB1-associated loci in mouse ES cells using an open-ended Chromosome Conformation Capture methodol-ogy. Chromosome Res 14: 477–495.





Xu M, Cook PR. 2008. Similar active genes cluster inspecialized transcription factories. J Cell Biol 181:615–623.

Xu N, Tsai CL, Lee JT. 2006. Transient homologous chromo-some pairing marks the onset of X inactivation. Science311: 1149–1152.

Yao J, Ardehali MB, Fecko CJ, Webb WW, Lis JT. 2007. Intra-nuclear distribution and local dynamics of RNA

polymerase II during transcription activation. Mol Cell28: 978–990.

Zhao Z, Tavoosidana G, Sjolinder M, Gondor A, Mariano P,Wang S, Kanduri C, Lezcano M, Sandhu KS, Singh U,et al. 2006. Circular chromosome conformation capture(4C) uncovers extensive networks of epigenetically regu-lated intra- and interchromosomal interactions. NatGenet 38: 1341–1347.





28, 20102010; doi: 10.1101/cshperspect.a000729 originally published online JulyCold Spring Harb Perspect Biol

Lyubomira Chakalova and Peter Fraser Organization of Transcription

Subject Collection The Nucleus

Developments in RNA Splicing and Disease

Charizanis, et al.Michael G. Poulos, Ranjan Batra, Konstantinos

DNA Damage Response

VermeulenGiuseppina Giglia-Mari, Angelika Zotter and Wim

Nuclear Stress BodiesGiuseppe Biamonti and Claire Vourc'h

Gene Positioning

Lavitas, et al.Carmelo Ferrai, Inês Jesus de Castro, Liron

Nuclear Functions of ActinNeus Visa and Piergiorgio Percipalle

Lamin-binding ProteinsKatherine L. Wilson and Roland Foisner

Organization of DNA Replication

CardosoVadim O. Chagin, Jeffrey H. Stear and M. Cristina

The Nucleus IntroducedThoru Pederson

The NucleolusThoru Pederson

Nuclear SpecklesDavid L. Spector and Angus I. Lamond

RNA Processing and Export

GrünwaldSami Hocine, Robert H. Singer and David

Biogenesis of Nuclear BodiesMiroslav Dundr and Tom Misteli

DiseaseHigher-order Genome Organization in Human

Tom Misteli

The Budding Yeast Nucleus

GasserAngela Taddei, Heiko Schober and Susan M.

Organization of TranscriptionLyubomira Chakalova and Peter Fraser

Orphan Nuclear Bodies

Miguel LafargaMaria Carmo-Fonseca, Maria T. Berciano and

http://cshperspectives.cshlp.org/cgi/collection/ For additional articles in this collection, see

Copyright © 2010 Cold Spring Harbor Laboratory Press; all rights reserved


http://cshperspectives.cshlp.org/cgi/collection/

http://cshperspectives.cshlp.org/cgi/collection/


organization of transcriptioncshperspectives.cshlp.org/content/2/9/a000729.full.pdf · organization...

Documents