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Dynamics of chromatin, proteins, and bodies within the cell nucleus

Andrew Belmont
A new and still evolving paradigm of a highly dynamic nucleus has emerged in recent years. This paradigm includes an inherently high turnover rate of histone and non-histone protein modications, targeted turnover and/or displacement of stable core histone proteins, constant ux of macromolecules through chromosomes and nuclear bodies, including transcription factors and co-activators, and an energy-dependent facilitation of nuclear-protein complex formation and disassembly. Also included are fast, local movements of chromosomes, together with slower but long-range movements of chromosomes and nuclear bodies.
Addresses Department of Cell and Structural Biology, University of Illinois, Urbana-Champaign B107, Chemical and Life Science Laboratory, 601 South Goodwin Avenue, Urbana, IL 61801, USA e-mail: asbel@uiuc.edu

with chromatin proteins such as histones showing little turnover. Moreover, biochemical in vitro reconstitution experiments led to sequential models of assembly for transcription pre-initiation complexes, over time periods of tens of minutes, with the idea that these complexes, once assembled, would be stable for tens of minutes to hours, as observed for transcription factorDNA interactions in vitro. Here, I review recent experiments that are beginning to reveal instead a highly dynamic cell nucleus whose components are in constant ux and movement.

Dynamics of chromosomal proteins

DNA folding on the nucleosome surface inhibits binding of most chromosomal proteins. Slow, intrinsic nucleosome dynamics occurs through a breathing mechanism, most likely involving progressive uncoiling from the DNA entry or exit points on the core particle, leading to transient release of DNA from the histone surface [1]. Although this breathing allows DNA binding of a wide range of proteins, the binding rate is low. Pioneer transcription factors capable of binding to compacted nucleosome arrays and directly remodelling these arrays have been identied [2]. Other transcription factors that bind early in the activation process lead to chromatin remodelling through recruitment of chromatin-remodelling and histone-modication complexes. In both cases, chromatin remodelling and histone modications may, through unmasking binding sites, facilitate binding of other regulatory proteins. Histone modications can recruit other specic proteins to the site, according, to a combinatorial or histone code [3], while in some cases changing structural properties of individual nucleosomes or their folding into higher-order chromatin structures. What is new over the past year is a growing appreciation for the existence of global histone acetylation and deacetylation activity, acting over large regions of the genome in addition to locally targeted histone-modication activities [4]. This allows rapid reversal of targeted histone acetylation or deacetylation at regulatory regions after removal of the targeting signal. In yeast, using a TetR-targeted VP16 activation domain, recovery to baseline histone acetylation levels occurred within 2 min after removal of targeted histone acetyltransferases (HATs) and 6 min for targeted Rpd3 histone deacetylase [5]. This implies an inherently high turnover cycle of core-histone modications, in turn implying a constitutive high capacity for dynamic chromatin remodelling throughout the genome. In higher eukaryotic cells, there are indications that particular histone modications might be targeted not
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Current Opinion in Cell Biology 2003, 15:304310 This review comes from a themed issue on Nucleus and gene expression Edited by Jeanne Lawrence and Gordon Hager 0955-0674/03/$ see front matter 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0955-0674(03)00045-0

Abbreviations ChIP chromatin immunoprecipitation ER oestrogen receptor FRAP uorescence recovery after photobleaching GFP green uorescent protein GR glucocorticoid receptor HAT histone acetyltransferase PML promyelocytic leukaemia pol RNA polymerase SMN survival motor neuron

Introduction
Over the past two decades, the dynamics of the cytoskeleton and cytoplasmic organelles have emerged as central themes in cell biology. By contrast, the interphase nucleus appeared to be the last refuge for the concept of stable cell structures and compartmentalisation. Transmission light microscopy of living cells revealed nuclei whose shape and internal substructures showed little apparent movement. At the molecular level, epigenetic programmes of gene activity, such as X chromosome inactivation, were demonstrated as being stable, once established, for many cell generations, even in the absence of factors required for initiation of silencing,
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Dynamics of chromatin, proteins and bodies within the cell nucleus Belmont 305

only to local DNA regions surrounding regulatory regions but to large genome regions as well, hundreds to thousands of kilobases in size. This includes not only large heterochromatin domains, enriched in repetitive sequences, but also euchromatic regions; for example, domains of increased histone H3 acetylation are observed within interphase nuclei [6]. Recently, functional genomics analysis has revealed clustering of coordinately expressed genes in organisms as diverse as Drosophila, Caenorhabditis elegans and humans [79]. Therefore, it is not clear to what degree these locally enriched chromosomal patches reect targeting to large genome regions per se, versus the integrated signal from several different gene loci sharing similar activity states. However, one can distinguish between these two possibilities in the case of DNA repair. UV irradiation leads to nuclear foci enriched in DNA-repair proteins; each individual focus represents the site of a DNA break. One of the earliest markers for these foci is Ser139 phosphorylation within 110 min in the H2AX histone variant. Phosphorylation of H2AX occurs in microscopically large foci corresponding to megabasepair DNA domains [10]. Changes in chromatin structure resulting from DNA breaks, rather than the break itself, have been proposed as the initial signalling event leading to this highly delocalised response [11]. Given the emergence of the central role of core-histone modications in regulating DNA function over the past few years, it is natural that the dynamics of these modications have been the focus of much work. However, the recent discovery of core-histone methylation as a key biological regulatory mechanism for transcriptional activation and silencing [3], together with the failure to identify any histone demethylases, has turned attention back to what had been thought was minimal turnover of the core histones themselves. In vitro experiments, however, have demonstrated displacement of H2AH2B dimers during transcription [12], and in vivo uorescence recovery after photobleaching (FRAP) experiments with green uorescent protein (GFP)-labelled histones demonstrates increased turnover of H2A and H2B relative to H3 and H4, which decreases with transcriptional inhibition [13]. The discovery of a histone H3 variant, H3.3, which is assembled into chromatin in a pathway independent of replication, has suggested a possible mechanism for turnover of methylated histone H3 [14]. Recent studies reveal a replication-independent deposition of histone H3.3 over transcriptionally active ribosomal genes [15]. Thus, there exists a fast turnover of histone modications such as acetylation and phosphorylation, on a timescale of minutes, and a slow turnover of core histones and variant core histones, on a timescale of hours, which may contribute to a longer-term epigenetic memory. In some species, DNA methylation might further extend this epigenetic memory. In between these two timescales is
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the ux of linker histones H1 and high-mobility group protein (HMG) through both open and condensed chromatin on a timescale of tens of seconds [16], which, at least in the case of histone H1, can be modied by phosphorylation [17]. Interestingly, application of FRAP to topoisomerase II, a component of the nuclear matrix and chromosome scaffold, revealed rapid in vivo exchange rates on a timescale of just 1020 s within interphase nuclei and mitotic chromosomes [18,19]. These results challenge the proposed role of topoisomerase II as a structural component of chromosomes.

Dynamics of the transcriptional machinery

An obvious research direction has been to temporally order chromatin modication events during gene activation at specic loci. Using chromatin immunoprecipitation (ChIP), several groups have successfully demonstrated a specic sequence of histone modications, together with recruitment of different chromatin regulatory complexes and co-activators, over a timescale of minutes to tens of minutes, as reviewed recently [20]. Although several genes investigated show recruitment of the same chromatin-modication complexes, their order of recruitment may differ; for instance, SWISNF recruitment follows GCN5 recruitment and histone H3 acetylation for the interferon-b promoter, but precedes recruitment of the GCN5-containing SAGA (Spt-Ada-Gcn5-acetyltransferase) complex for the yeast HO promoter. These results might suggest that multiple paths can lead to the same activated state. Alternatively, the temporal order may matter very much, with different orders of recruitment resulting in physiologically relevant differences in activation temporal proles or susceptibility to epigenetic inuences. For example, many yeast genes expressed near mitosis require SWISNF activity followed by GCN5 action; in the absence of SWISNF remodelling, mitotic chromosome condensation might block GCN5 activity [21]. Biochemical purication of chromatin regulatory complexes as distinct structural entities in the megadalton size range has raised the natural question of how such large complexes access condensed chromatin. An immunouorescence approach was used to monitor co-activator recruitment and histone modications following targeting of the VP16 acidic activation domain to an engineered heterochromatic chromosome region. Unexpectedly, different subcomponents of both SWISNF and HAT complexes showed quite different temporal recruitment patterns, with some subcomponents accumulating within several minutes after VP16 targeting and others after tens of minutes [22]. These results suggest that rather than diffusing into condensed chromatin as intact entities, large chromatin-modifying complexes might assemble on the gene target, possibly with individual subunits or
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subcomplexes acting as pioneer factors, opening their target for subsequent binding of the intact complex. Similarly, despite purication of a large RNA polymerase II (pol II) holoenzyme containing the mediator complex, CBP, and other proteins, ChIP has shown separate timing of pol II and mediator recruitment [20] Sequential recruitment also has been suggested for components of DNA-repair holocomplexes [23]. Bigger surprises have come from in vivo FRAP measurements, which have demonstrated off rates for transcription factors binding to their target sequences, and co-activators binding to their target proteins, orders of magnitude faster than in vitro measured rates [24]. Intranuclear recovery half lives of seconds to tens of seconds have been measured for glucocorticoid receptor (GR) [25] and the GR-interacting protein 1 (GRIP-1) co-activator [26] on a chromosome array containing a GR-responsive MMTV promoter, leading to a hitand-run model of transcriptional activation. Similar dynamics have been observed for intranuclear oestrogen receptor (ER) and the steroid receptor co-activator 1 (SRC-1) binding to ER in the presence of ligand [27,28], with ER becoming immobilised in the nucleoplasm after exposure to proteosome inhibitors or hormone antagonist [28]. The general transcription factor TFIIB shows rapid turnover in vivo over seconds, but TATA-binding protein (TBP) turnover is relatively slow with a FRAP half life of minutes [29]; however, ChIP experiments reveal TBP dissociation coincident with removal of targeted VP16, within the 12 min experimental time resolution [5]. FRAP of pol II subunits shows a predominant component which slowly exchanges over 1020 min; this component corresponds to elongating polymerases [26,30]. A faster component, which recovers over seconds to tens of seconds, might correspond to turnover at the promoter [26]. Recently, an elegant FRAP analysis of pol I subunits and transcription factors revealed similarly rapid dynamics over ribosomal genes [31]. On the basis of a combination of experiments and data modelling, it was concluded that the pol I holoenzyme did not diffuse to its target promoters as a pre-assembled structural entity in vivo, but rather assembled at the promoter. These data highlight the current paradox implicit in trying to reconcile in vitro and in vivo results. Specically, a GFP-tagged pol I holoenzyme could be isolated as a distinct, stable structural entity from cells expressing an epitope-tagged subunit, but in vivo subunits showed dynamics inconsistent with the idea of a pre-assembled holoenzyme. One explanation for this paradox would be if the dynamic behaviour observed in vivo depended on specic, energydependent mechanisms facilitating exchange and turnover. Using an in vitro chromatin reconstitution system, Hager and co-workers [32] demonstrated that
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the rapid turnover of GR on the MMTV promoter is ATP-dependent and can be reproduced at least partially by puried SWISNF, suggesting a turnover mechanism involving chromatin-remodelling complexes. An alternative mechanism has emerged from studies showing repression of ER transcriptional activation by the molecular chaperone p23, suggesting a role for disassembly complexes fostering active turnover of large transcription complexes [33].

Dynamics of chromosomes
One might expect passive chromosome movements simply as the result of chromatin decondensation/condensation; however, recent results indicate rapid and active chromosome movements. Using chromosomes tagged with GFPlac-repressor bound to lac-operator repeats, a rapid, but locally constrained, motion has been observed in yeast, Drosophila and mammalian cells [34,35], This motion is rapid so rapid that current sampling rates on the order of tens to hundreds of milliseconds are still too slow adequately to follow the motion but localised to excursions of a few hundred nanometres. Sites associated with the nuclear periphery or nucleolus show lower amplitude movements [36]. The motion varies according to the physiological state of the cell, and ATP-depletion experiments suggest that the motion is energy-dependent [37]. In yeast, motion is reduced in S-phase cells, which might reect associations of chromosomes with the DNA replication machinery and/or elements of nuclear structure [38]. In Drosophila and yeast, long-range motions over greater distances are observed over a longer timescale. In Drosophila, this long-range motion is developmentally regulated, disappearing during later stages of spermatocyte differentiation [39]. Absence of long-range chromosome movements appears to be the general rule in mammalian tissue-culture cells. However, at specic times during S phase, long-range motions of specic, engineered chromosome regions have been observed [40,41]. In mammalian cells, targeting of the acidic activator VP1 changed the position of a peripheral chromosome site to the interior in stable transformants [41]. More recent data show this induced motion occurs at a dened time after VP16 targeting independent of cell cycle position (C Chuang and A Belmont, unpublished data). In addition, uncoiling of large-scale chromatin structure is observed with VP16 targeting [42], similar to that induced by GR in a chromosome array containing a GR-responsive promoter [43]. These changes in position and conformation may recapitulate physiological changes in chromosome position and conformation observed for wild-type chromosomes. Peripheral versus interior localisation of gene-poor versus gene-rich chromosomes have been described, as have changes in chromosome positioning as a function of cell cycle progression [44].
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Dynamics of chromatin, proteins and bodies within the cell nucleus Belmont 307

Dynamics of nuclear bodies

Long-range chromosome movements naturally raise questions concerning whether these movements are targeted to specic nuclear bodies. The list of distinct nuclear suborganelles or compartments is growing, with the addition over the past year of clastosomes [45] and paraspeckles [46] to the established list of interchromatin granule clusters (speckles), PML (promyelocytic leukaemia), SMN (survival motor neuron) and Cajal bodies, gems and nucleoli, as well as centromeric heterochromatin and the nuclear periphery. Apparent localisation of a new multiple bridging protein to connections between gems and SMN bodies [47] could suggest functional connections between several of these bodies. There is a long-standing observation of the association of many active genes with interchromatin granule clusters (splicing speckles, Sc-35 bodies); recent data indicate association of mRNA from these genes with these bodies [48]. A growing literature points to concentrations of specic and general transcription factors within PML and Cajal bodies, as well as physiological regulation of this targeting (reviewed by Isogai and Tijan, this issue), and both the histone and U2 gene loci specically associate with Cajal bodies [49,50]. A clear demonstration of a role of Cajal bodies in the maturation of certain proteins and ribonucleoprotein complexes [51,52,53,54] has recently emerged, as well as a protein maturation pathway from speckles to nucleoli [55]. Rapid trafcking of proteins between nuclear bodies and the nucleoplasm has also been demonstrated recently [46,56]. In addition to protein trafcking into and out of nuclear bodies, the bodies themselves move within the nucleus. PML bodies move discontinuously with stationary periods interspersed with long-range movements at maximum velocities of 18 mm/min [57]. Their movements are decreased by ATP depletion and reduced by the myosin inhibitor 2,3-butanedione monoxime. By contrast, Cajal bodies show an increase in long-range diffusive movements and a decrease in immobile periods correlated with chromatin associations within live cells after ATP depletion or transcriptional inhibition, suggesting that transient associations with chromatin might be active processes dependent on transcription [58].

activation initially localises to these same centromeric regions, with delocalisation to the nucleoplasm coinciding with movements of the locus to the nuclear interior and gene activation [61]. In this single example of regulated gene activation, we connect dynamics of chromatin modication, both local and long-range chromatin movements, and transcription-factor trafcking. Similar connections have emerged between compartmentalisation of transcription repressors, gene silencing, and intranuclear localisation of gene loci [62]. Our future challenge will be to understand how these different dynamic processes together accomplish regulation of nuclear processes. This will require continued development of new technological approaches suitable for in vivo observations of nuclear dynamics.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest Anderson JD, Thastrom A, Widom J: Spontaneous access of proteins to buried nucleosomal DNA target sites occurs via a mechanism that is distinct from nucleosome translocation. Mol Cell Biol 2002, 22:7147-7157. This study demonstrates that spontaneous access of DNA bound to nucleosome core proteins is produced by DNA breathing, produced by changes in nucleosome conformation, as opposed to nucleosome sliding. Cirillo LA, Lin FR, Cuesta I, Friedman D, Jarnik M, Zaret KS: Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol Cell 2002, 9:279-289. Using in vitro reconstituted chromatin including linker histone H1, this paper demonstrates the capability of certain transcription factors to bind and remodel nucleosome arrays without the help of ATP-dependent chromatin-modifying complexes. 3. 4.  Turner BM: Cellular memory and the histone code. Cell 2002, 111:285-291. 2.  1. 

Robyr D, Suka Y, Xenarios I, Kurdistani SK, Wang A, Suka N, Grunstein M: Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases. Cell 2002, 109:437-446. Chromatin immunoprecipitation is used with DNA chip analysis to compare histone acetylation patterns between wild-type yeast cells and knockouts of specic histone deacetylases (HDACs). This approach identies large subsets of genes whose histone acetylation patterns, and expression, are regulated by specic HDACs. Interestingly, Hda1 appears to act preferentially over large 1025 kb genomic domains anking telomeres. Katan-Khaykovich Y, Struhl K: Dynamics of global histone acetylation and deacetylation in vivo: rapid restoration of normal histone acetylation status upon removal of activators and repressors. Genes Dev 2002, 16:743-752. In this work in yeast, very rapid global acetylation or deacetylation back to steady-state levels is observed within several minutes after removal of targeted repressor (Ume6) or activator (VP16 acidic activator), respectively. The authors also show that TATA-binding protein (TBP) binding correlates closely with activator binding, rather than with histone acetylation. 6. Hendzel MJ, Kruhlak MJ, Bazett-Jones DP: Organization of highly acetylated chromatin around sites of heterogeneous nuclear RNA accumulation. Mol Biol Cell 1998, 9:2491-2507. Lercher MJ, Urrutia AO, Hurst LD: Clustering of housekeeping genes provides a unied model of gene order in the human genome. Nat Genet 2002, 31:180-183. Cohen BA, Mitra RD, Hughes JD, Church GM: A computational analysis of whole-genome expression data reveals chromosomal domains of gene expression. Nat Genet 2000, 26:183-186. Current Opinion in Cell Biology 2003, 15:304310 5. 

Conclusions and future directions

In this review, I have discussed dynamics of nuclear proteins, chromosome loci, and nuclear bodies as if they were distinct physiological processes. There already exist strong hints that these processes are connected functionally. For instance, recent data point to longrange associations of elements within the globin locus control region with distant promoter regions [59]. However, these same regulatory elements also regulate chromatin modications, as well positioning of the globin locus relative to centromeric heterochromatin [60]. A specic transcription factor essential for globin gene
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7.

8.

308 Nucleus and gene expression

9.

Spellman PT, Rubin GM: Evidence for large domains of similarly expressed genes in the Drosophila genome. J Biol 2002, 1:5.

10. Rogakou EP, Boon C, Redon C, Bonner WM: Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 1999, 146:905-916. 11. Bakkenist CJ, Kastan MB: DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 2003, 421:499-506. 12. Kireeva ML, Walter W, Tchernajenko V, Bondarenko V, Kashlev M,  Studitsky VM: Nucleosome remodeling induced by RNA polymerase II: loss of the H2A/H2B dimer during transcription. Mol Cell 2002, 9:541-552. In vitro transcription through nucleosome core particles by RNA polymerase II leads to a loss of one H2AH2B dimer, providing a possible mechanism for in vivo turnover of core histones H2A and H2B that is independent of replication but linked to transcription. 13. Kimura H, Cook PR: Kinetics of core histones in living human cells: little exchange of H3 and H4 and some rapid exchange of H2B. J Cell Biol 2001, 153:1341-1353. 14. Ahmad K, Henikoff S: Histone H3 variants specify modes  of chromatin assembly. Proc Natl Acad Sci USA 2002, 99(Suppl 4):16477-16484. The authors demonstrate replication-independent deposition for the two Drosophila histone H3 variants H3.3 and Cid using a Drosophila cell line system. H3.3 marks active chromatin and Cid marks centromeric regions. Both are incorporated into chromatin throughout the cell cycle, implying mechanisms for core-histone displacement and/or turnover. 15. Ahmad K, Henikoff S: The histone variant H3.3 marks active  chromatin by replication-independent nucleosome assembly. Mol Cell 2002, 9:1191-1200. Replication-independent incorporation of H3.3 into the active ribosomal gene array was demonstrated. Under favourable growth conditions, additional ribosomal arrays became active and these were found to incorporate H3.3 and to show reductions of methylated histone H3. Replication-independent incorporation was shown to be conferred upon normal histone H3 by amino acid changes towards H3.3. 16. Catez F, Brown DT, Misteli T, Bustin M: Competition between  histone H1 and HMGN proteins for chromatin binding sites. EMBO Rep 2002, 3:760-766. Fluorescence recovery after photobleaching analysis indicates competition between HMGN (high-mobility group protein N) and histone H1 for binding sites. 17. Dou Y, Bowen J, Liu Y, Gorovsky MA: Phosphorylation and an  ATP-dependent process increase the dynamic exchange of H1 in chromatin. J Cell Biol 2002, 158:1161-1170. Linker-histone phosphorylation leads to a more rapid uorescence recovery after photobleaching, suggesting decreased binding of the phosphorylated protein. Interestingly, ATP depletion in vivo results in decreased linker-histone mobility. 18. Christensen MO, Larsen MK, Barthelmes HU, Hock R, Andersen  CL, Kjeldsen E, Knudsen BR, Westergaard O, Boege F, Mielke C: Dynamics of human DNA topoisomerases IIalpha and IIbeta in living cells. J Cell Biol 2002, 157:31-44. Topoisomerase II has been proposed to be a major component of the chromosome scaffold and also the nuclear matrix. However, in vivo uorescence recovery after photobleaching experiments indicate rapid turnover of topoisomerase II on a timescale of seconds in both nuclei and mitotic chromosomes. If a more stably bound topoisomerase II fraction is present as part of a stable structural scaffold, it must correspond to a small fraction of the total enzyme. 19. Tavormina PA, Come MG, Hudson JR, Mo YY, Beck WT,  Gorbsky GJ: Rapid exchange of mammalian topoisomerase II alpha at kinetochores and chromosome arms in mitosis. J Cell Biol 2002, 158:23-29. This is an independent uorescence recovery after photobleaching study of topoisomerase II in vivo. The authors come to a similar conclusion as in Christensen et al. [18], that there is rapid turnover in nuclei and mitotic chromosomes. 20. Featherstone M: Coactivators in transcription initiation: here are  your orders. Curr Opin Genet Dev 2002, 12:149-155. This is a nice review of ordered recruitment of different components of the transcriptional machinery after gene activation of specic loci. Data from several sources argue against one-step recruitment of entire holoenyzmes, as dened by biochemical isolation. Current Opinion in Cell Biology 2003, 15:304310

21. Krebs JE, Fry CJ, Samuels ML, Peterson CL: Global role for chromatin remodeling enzymes in mitotic gene expression. Cell 2000, 102:587-598. 22. Memedula S, Belmont AS: Sequential recruitment of HAT and  SWI/SNF components to condensed chromatin by VP16. Curr Biol 2003, 13:241-246. Using a microscopy-based immunouorescence approach, recruitment of various SWISNF and histone acetyltransferase (HAT) components are monitored, together with histone acetylation, after targeting of VP16 acidic activator to a condensed chromosome region. Different subunits of SWISNF and HAT complexes accumulate at the chromosome site with very different kinetics, arguing against a simple one-step recruitment of large chromatin-modifying complexes to condensed chromatin. 23. Essers J, Houtsmuller AB, van Veelen L, Paulusma C, Nigg AL,  Pastink A, Vermeulen W, Hoeijmakers JH, Kanaar R: Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage. EMBO J 2002, 21:2030-2037. Using uorescence recovery after photobleaching analysis, this study demonstrates stable association of Rad51 with radiation-induced foci, but rapid turnover of Rad52 and Rad54. In cells not exposed to radiation, different turnover rates for these proteins argues against stable preexisting holoenzymes. 24. Hager GL, Elbi C, Becker M: Protein dynamics in the nuclear compartment. Curr Opin Genet Dev 2002, 12:137-141. 25. McNally JG, Muller WG, Walker D, Wolford R, Hager GL: The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. Science 2000, 287:1262-1265. 26. Becker M, Baumann C, John S, Walker DA, Vigneron M, McNally  JG, Hager GL: Dynamic behavior of transcription factors on a natural promoter in living cells. EMBO Rep 2002, 3:1188-1194. This is a continuation of groundbreaking work [25], providing additional support for the hit-and-run mechanism underlying transcription factor binding and action in vivo. Using the same chromosome array system as that previously described by McNally et al. [25], the Hager group demonstrated rapid turnover of the co-activator GRIP-1 at similar rates to glucocorticoid receptor. By contrast, the RPB1 RNA polymerase subunit recovers over a much longer timescale (13 min), consistent with the processive property of elongating polymerase. A faster recovering component suggests the possibility of more rapid turnover of promoter-bound RNA polymerase II. Accumulation of RNA polymerase II peaks at 2030 min after ligand addition, dropping substantially afterwards. 27. Stenoien DL, Nye AC, Mancini MG, Patel K, Dutertre M, OMalley BW, Smith CL, Belmont AS, Mancini MA: Ligand-mediated assembly and real-time cellular dynamics of estrogen receptor alpha-coactivator complexes in living cells. Mol Cell Biol 2001, 21:4404-4412. 28. Stenoien DL, Patel K, Mancini MG, Dutertre M, Smith CL, OMalley BW, Mancini MA: FRAP reveals that mobility of oestrogen receptor-alpha is ligand- and proteasome-dependent. Nat Cell Biol 2001, 3:15-23. 29. Chen D, Hinkley CS, Henry RW, Huang S: TBP dynamics in living  human cells: constitutive association of TBP with mitotic chromosomes. Mol Biol Cell 2002, 13:276-284. The authors describe uorescence recovery after photobleaching (FRAP) analysis of TATA-binding protein (TBP) in mitotic and interphase cells. No recovery of photobleaching is seen in mitotic chromosomes, demonstrating stable association of TBP. Within interphase nuclei, TBP shows slow recovery over a timescale of minutes (i.e. 120). This recovery was independent of transcriptional inhibition, suggesting that TBP is stably bound over multiple initiation rounds. 30. Kimura H, Sugaya K, Cook PR: The transcription cycle of RNA  polymerase II in living cells. J Cell Biol 2002, 159:777-782. Fluorescence recovery after photobleaching analysis of the Large RNA polymerase II (pol II) reveals a transiently immobile fraction correlated with transcriptional elongation. Little pol II is immobile in the presence of transcriptional inhibition, arguing against stable binding of a signicant fraction of enzyme in transcription factories. 31. Dundr M, Hoffmann-Rohrer U, Hu Q, Grummt I, Rothblum LI,  Phair RD, Misteli T: A kinetic framework for a mammalian RNA polymerase in vivo. Science 2002, 298:1623-1626. Fluorescence recovery after photobleaching analysis of multiple components of the RNA polymerase I (pol I) transcriptional apparatus are described. Recovery over tens of seconds was observed for several pre-initiation factors. Four pol I subunits showed biphasic recovery www.current-opinion.com

Dynamics of chromatin, proteins and bodies within the cell nucleus Belmont 309

curves, with a slow recovery phase associated with transcription elongation. Differences in the early rapid recovery rates for the four pol I subunits was used to argue against recruitment of a pre-assembled holoenzyme. 32. Fletcher TM, Xiao N, Mautino G, Baumann CT, Wolford R,  Warren BS, Hager GL: ATP-dependent mobilization of the glucocorticoid receptor during chromatin remodeling. Mol Cell Biol 2002, 22:3255-3263. Using an in vitro assembled template, glucocorticoid receptor (GR)mediated, ATP-dependent chromatin remodelling was produced by exposure to nuclear extract or puried human SWISNF. This remodelling was accompanied by ATP-dependent GR displacement. 33. Freeman BC, Yamamoto KR: Disassembly of transcriptional  regulatory complexes by molecular chaperones. Science 2002, 296:2232-2235. The p23 molecular chaperone was demonstrated to localise in vivo to nuclear receptor response elements. Moreover, tethering p23 disrupted receptor-mediated transcriptional activation. Hsp90 displayed similar, but weaker, effects. These results suggest a role for disassembly complexes in transcription complex turnover. 34. Carmo-Fonseca M, Platani M, Swedlow JR: Macromolecular mobility inside the cell nucleus. Trends Cell Biol 2002, 12:491-495. 35. Gasser SM: Visualizing chromatin dynamics in interphase nuclei. Science 2002, 296:1412-1416. 36. Chubb JR, Boyle S, Perry P, Bickmore WA: Chromatin motion is  constrained by association with nuclear compartments in human cells. Curr Biol 2002, 12:439-445. Fast but constrained motion of chromatin is observed in mammalian tissue culture cells, as previously observed in yeast and Drosophila. This local motion is reduced at chromosome sites associated with nucleoli or with the nuclear periphery. 37. Heun P, Laroche T, Shimada K, Furrer P, Gasser SM: Chromosome dynamics in the yeast interphase nucleus. Science 2001, 294:2181-2186. 38. Heun P, Laroche T, Raghuraman MK, Gasser SM: The positioning and dynamics of origins of replication in the budding yeast nucleus. J Cell Biol 2001, 152:385-400. 39. Vazquez J, Belmont AS, Sedat JW: Multiple regimes of constrained chromosome motion are regulated in the interphase Drosophila nucleus. Curr Biol 2001, 11:1227-1239. 40. Belmont AS: Visualizing chromosome dynamics with GFP. Trends Cell Biol 2001, 11:250-257. 41. Tumbar T, Belmont AS: Interphase movements of a DNA chromosome region modulated by VP16 transcriptional activator. Nat Cell Biol 2001, 3:134-139. 42. Tumbar T, Sudlow G, Belmont AS: Large-scale chromatin unfolding and remodeling induced by VP16 acidic activation domain. J Cell Biol 1999, 145:1341-1354. 43. Muller WG, Walker D, Hager GL, McNally JG: Large-scale chromatin decondensation and recondensation regulated by transcription from a natural promoter. J Cell Biol 2001, 154:33-48. 44. Mahy NL, Bickmore WA, Tumbar T, Belmont AS: Linking largescale chromatin structure with nuclear function. In Chromatin Structure and Gene Expression, 2nd edn. Edited by Elgin SCR, Workman JL. Oxford: Oxford University Press; 2000:300-321. [Hames BD, Glover DM (Series Editor): Frontiers in Molecular Biology]. 45. Lafarga M, Berciano MT, Pena E, Mayo I, Castano JG, Bohmann D,  Rodrigues JP, Tavanez JP, Carmo-Fonseca M: Clastosome: a subtype of nuclear body enriched in 19S and 20S proteasomes, ubiquitin, and protein substrates of proteasome. Mol Biol Cell 2002, 13:2771-2782. This is a report of a new class of nuclear body enriched in components of the ubiquitinproteosome pathway. Normally rare, these bodies increase in number under conditions in which proteolysis is stimulated. Protein substrates of the proteosome found in this body include c-Fos, c-Jun and PML. 46. Fox AH, Lam YW, Leung AK, Lyon CE, Andersen J, Mann M,  Lamond AI: Paraspeckles: a novel nuclear domain. Curr Biol 2002, 12:13-25. www.current-opinion.com

Proteomic analysis of nucleolar proteins revealed a novel protein, PSP1, whose concentration into 1020 spots in a large number of primary and transformed human cell types reveals a new class of nuclear bodies known as paraspeckles. Paraspeckles distribute near splicing speckles but move to the nucleolar periphery within several hours of transcriptional inhibition. PSP1 dynamically exchanges between paraspeckles and nucleoplasm. 47. Wang IF, Reddy NM, Shen CKJ: Higher order arrangement of the  eukaryotic nuclear bodies. Proc Natl Acad Sci USA 2002, 99:13583-13588. TDP is a protein associated with transcriptional repression and alternative splicing. TDP staining reveals TDP bodies, a new class of nuclear bodies. Staining shows overlap of TDP bodies with splicing speckles, gems, PML and Cajal bodies. Data are intriguing, but proper statistical analysis is needed to access signicance of overlap. 48. Shopland LS, Johnson CV, Lawrence JB: Evidence that all SC-35  domains contain mRNAs and that transcripts can be structurally constrained within these domains. J Struct Biol 2002, 140:131-139. The authors demonstrate that SC35 domains contain specic mRNAs. Transcriptional inhibition leads to their long-term association with SC35 domains, suggesting that a transcription-coupled maturation step might be required for their release. 49. Shopland LS, Byron M, Stein JL, Lian JB, Stein GS, Lawrence JB: Replication-dependent histone gene expression is related to Cajal body (CB) association but does not require sustained CB contact. Mol Biol Cell 2001, 12:565-576. 50. Smith KP, Lawrence JB: Interactions of U2 gene loci and their nuclear transcripts with Cajal (coiled) bodies: evidence for PreU2 within Cajal bodies. Mol Biol Cell 2000, 11:2987-2998. 51. Sleeman JE, Lamond AI: Newly assembled snRNPs associate with coiled bodies before speckles, suggesting a nuclear snRNP maturation pathway. Curr Biol 1999, 9:1065-1074. 52. Sleeman JE, Ajuh P, Lamond AI: snRNP protein expression  enhances the formation of Cajal bodies containing p80-coilin and SMN. J Cell Sci 2002, 114:4407-4419. Continuation of studies indicating that Cajal bodies are sites of small nuclear ribonucleoprotein particle (snRNP) maturation or assembly. Higher levels of snRNP protein expression and snRNP assembly might lead to Cajal body formation. 53. Verheggen C, Lafontaine DLJ, Samarsky D, Mouaikel J, Blanchard JM, Bordonne R, Bertrand E: Mammalian and yeast U3 snoRNPs are matured in specic and related nuclear compartments. EMBO J 2002, 21:2736-2745. 54. Darzacq X, Jady BE, Verheggen C, Kiss AM, Bertrand E, Kiss T:  Cajal body-specic small nuclear RNAs: a novel class of 20 -Omethylation and pseudouridylation guide RNAs. EMBO J 2002, 21:2746-2756. A novel class of small nuclear RNAs (snRNAs) that localise to Cajal bodies have been shown or are predicted to function as guide RNAs for sitespecic modication of snRNAs. 55. Leung AK, Lamond AI: In vivo analysis of NHPX reveals a  novel nucleolar localization pathway involving a transient accumulation in splicing speckles. J Cell Biol 2002, 157:615-629. NHPX, a nucleolar factor that binds to an RNA sequence found in nucleolar box C/D small nucleolar RNAs (snoRNAs) and in U4 small nuclear RNA (snRNA), accumulates in nucleoli and Cajal bodies in vivo. NHPX nucleolar accumulation is preceded by transient accumulation in splicing speckles. 56. Wiesmeijer K, Molenaar C, Bekeer IMLA, Tanke HJ, Dirks RW:  Mobile foci of Sp100 do not contain PML: PML bodies are immobile but PML and Sp100 proteins are not. J Struct Biol 2002, 140:180-188. Sp100 identies nuclear bodies, of which a subset contain promyelocytic leukaemia protein (PML). The subset containing PML are relatively immobile; those lacking PML show mobility that may correspond to the mobile PML bodies described by Darzacq et al. (2002) [54]. SP100 and PML show turnover with nucleoplasmic pools on a timescale of several minutes. 57. Muratani M, Gerlich D, Janicki SM, Gebhard M, Eils R, Spector DL: Metabolic-energy-dependent movement of PML bodies within the mammalian cell nucleus. Nat Cell Biol 2001, 4:106-110. Current Opinion in Cell Biology 2003, 15:304310

310 Nucleus and gene expression

58. Platani M, Goldberg I, Lamond AI, Swedlow JR: Cajal body  dynamics and association with chromatin are ATP-dependent. Nat Cell Biol 2002, 4:502-508. Cajal bodies are demonstrated to show intranuclear movements, with most showing mobility consistent with constrained diffusion. ATP depletion and transcriptional inhibition increases movements and decreases chromatin associations. 59. Carter D, Chakalova L, Osborne CS, Dai YF, Fraser P: Long-range  chromatin regulatory interactions in vivo. Nat Genet 2002, 32:623-626. A novel method, dependent on generation of free radicals by labelled antibodies to generate biotinylated DNA, allows probing of the spatial relationship between the b-globin locus control region (LCR) and globin

open reading frames. The data support movement of certain regulatory regions of LCR near the promoter during globin gene activation. 60. Francastel C, Schubeler D, Martin DI, Groudine M: Nuclear compartmentalization and gene activity. Nat Rev Mol Cell Biol 2000, 1:137-143. 61. Francastel C, Magis W, Groudine M: Nuclear relocation of a transactivator subunit precedes target gene activation. Proc Natl Acad Sci USA 2001, 98:12120-12125. 62. Fisher AG, Merkenschlager M: Gene silencing, cell fate and nuclear organisation. Curr Opin Genet Dev 2002, 12:193-197.

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