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Dynamics of chromatin, proteins, and bodies within thecell nucleusAndrew 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

modifications, targeted turnover and/or displacement of stable

core histone proteins, constant flux 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, USAe-mail: [email protected]

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 immunoprecipitationER oestrogen receptorFRAP fluorescence recovery after photobleachingGFP green fluorescent protein

GR glucocorticoid receptor

HAT histone acetyltransferasePML promyelocytic leukaemiapol RNA polymerase

SMN survival motor neuron

Introduction

Over the past two decades, the dynamics of the cyto-skeleton 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. Trans-

mission 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,

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 interac-

tions in vitro. Here, I review recent experiments that are

beginning to reveal instead a highly dynamic cell nucleus

whose components are in constant flux and movement.

Dynamics of chromosomal proteinsDNA folding on the nucleosome surface inhibits binding

of most chromosomal proteins. Slow, intrinsic nucleo-

some 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 identified [2]. Other transcription factors that

bind early in the activation process lead to chromatinremodelling through recruitment of chromatin-remodel-

ling and histone-modification complexes. In both cases,

chromatin remodelling and histone modifications may,through unmasking binding sites, facilitate binding of

other regulatory proteins. Histone modifications can

recruit other specific 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 deacetyla-

tion activity, acting over large regions of the genome in

addition to locally targeted histone-modification activities

[4]. This allows rapid reversal of targeted histone acet-

ylation or deacetylation at regulatory regions after removalof 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 modifi-

cations, in turn implying a constitutive high capacity for

dynamic chromatin remodelling throughout the genome.

In higher eukaryotic cells, there are indications that

particular histone modifications might be targeted not

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only to local DNA regions surrounding regulatory regions

but to large genome regions as well, hundreds to thou-

sands of kilobases in size. This includes not only large

heterochromatin domains, enriched in repetitive seq-

uences, but also euchromatic regions; for example,

domains of increased histone H3 acetylation are observedwithin interphase nuclei [6]. Recently, functional geno-

mics 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 chromo-

somal patches reflect 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 enrichedin 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

modifications in regulating DNA function over the pastfew years, it is natural that the dynamics of these mod-

ifications have been the focus of much work. However,

the recent discovery of core-histone methylation as a key

biological regulatory mechanism for transcriptional acti-

vation and silencing [3], together with the failure toidentify any histone demethylases, has turned attention

back to what had been thought was minimal turnover of

the core histones themselves. In vitro experiments, how-

ever, have demonstrated displacement of H2AH2B

dimers during transcription [12], and in vivo fluores-

cence recovery after photobleaching (FRAP) experi-

ments with green fluorescent protein (GFP)-labelledhistones demonstrates increased turnover of H2A and

H2B relative to H3 and H4, which decreases with tran-

scriptional 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 histoneH3 [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 modifications

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 con-

tribute to a longer-term epigenetic memory. In some

species, DNA methylation might further extend this

epigenetic memory. In between these two timescales is

the flux of linker histones H1 and high-mobility group

protein (HMG) through both open and condensed chro-

matin on a timescale of tens of seconds [16], which, at

least in the case of histone H1, can be modified 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 time-

scale of just 1020 s within interphase nuclei and mitotic

chromosomes [18,19]. These results challenge the

proposed role of topoisomerase II as a structural compo-

nent of chromosomes.

Dynamics of the transcriptional machineryAn obvious researchdirection has been to temporally orderchromatin modification events during gene activation

at specific loci. Using chromatin immunoprecipitation

(ChIP), several groups have successfully demonstrated

a specific sequence of histone modifications, together

with recruitment of different chromatin regulatory com-

plexes 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-modification complexes, their order of recruit-

ment may differ; for instance, SWISNF recruitment

follows GCN5 recruitment and histone H3 acetylation

for the interferon-b promoter, but precedes recruitmentof the GCN5-containing SAGA (Spt-Ada-Gcn5-acetyl-

transferase) 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 recruit-ment resulting in physiologically relevant differences in

activation temporal profiles or susceptibility to epigenetic

influences. 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 purification of chromatin regulatory com-

plexes as distinct structural entities in the megadalton

size range has raised the natural question of how such

large complexes access condensed chromatin. An immu-

nofluorescence approach was used to monitor co-activatorrecruitment and histone modifications following targeting

of the VP16 acidic activation domain to an engineered

heterochromatic chromosome region. Unexpectedly, dif-

ferent subcomponents of both SWISNF and HAT com-

plexes 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 purification 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

] Sequentialrecruitment also has been suggested for components of

DNA-repair holocomplexes [23].

Bigger surprises have come from in vivo FRAP mea-

surements, which have demonstrated off rates for trans-

cription factors binding to their target sequences, and

co-activators binding to their target proteins, orders of

magnitude faster than in vitro measured rates [24]. Intra-

nuclear 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-acti-

vator [26] on a chromosome array containing a

GR-responsive MMTV promoter, leading to a hit-

and-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 nucleo-

plasm after exposure to proteosome inhibitors or hor-

mone antagonist [28]. The general transcription factor

TFIIB shows rapid turnover in vivo over seconds, but

TATA-binding protein (TBP) turnover is relatively slowwith a FRAP half life of minutes [29]; however, ChIP

experiments reveal TBP dissociation coincident with

removal of targeted VP16, within the 12 min experi-

mental time resolution [5]. FRAP of pol II subunits

shows a predominant component which slowly ex-changes 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 dynamicsover 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 pro-

moters 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 vitroand in vivo results. Specifically, a GFP-tagged pol I

holoenzyme could be isolated as a distinct, stable struc-

tural entity from cells expressing an epitope-tagged sub-

unit, but in vivo subunits showed dynamics inconsistent

with the idea of a pre-assembled holoenzyme.

Oneexplanationfor this paradox would be if thedynamic

behaviour observed in vivo depended on specific, energy-

dependent mechanisms facilitating exchange and

turnover. Using an in vitro chromatin reconstitution

system, Hager and co-workers [32] demonstrated that

the rapid turnover of GR on the MMTV promoter is

ATP-dependent and can be reproduced at least partially

by purified SWISNF, suggesting a turnover mechanism

involving chromatin-remodelling complexes. An alter-

native mechanism has emerged from studies showing

repression of ER transcriptional activation by the mole-cular chaperone p23, suggesting a role for disassembly

complexes fostering active turnover of large transcrip-

tion complexes [33].

Dynamics of chromosomesOne might expect passive chromosome movements sim-

ply as the result of chromatin decondensation/condensa-

tion; however, recent results indicate rapid and active

chromosome movements. Using chromosomes tagged

with GFPlac-repressor bound to lac-operator repeats, arapid, 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-depen-

dent [37]. In yeast, motion is reduced in S-phase cells,

which might reflect associations of chromosomes withthe 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 Dro-sophila, this long-range motion is developmentally regu-

lated, 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 specific times during S

phase, long-range motions of specific, engineered chro-

mosome 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 defined time after

VP16 targeting independent of cell cycle position(C Chuang and A Belmont, unpublished data). In addi-

tion, 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 conforma-

tion may recapitulate physiological changes in chromo-

some 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 nuclear bodiesLong-range chromosome movements naturally raise

questions concerning whether these movements are tar-

geted to specific nuclear bodies. The list of distinct

nuclear suborganelles or compartments is growing, with

the addition over the past year of clastosomes [45

] andparaspeckles [46] to the established list of interchroma-

tin granule clusters (speckles), PML (promyelocytic

leukaemia), SMN (survival motor neuron) and Cajal

bodies, gems and nucleoli, as well as centromeric hetero-

chromatin and the nuclear periphery. Apparent localisa-

tion 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

specific and general transcriptionfactors 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 specifically 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 path-

way from speckles to nucleoli [55]. Rapid trafficking ofproteins between nuclear bodies and the nucleoplasm

has also been demonstrated recently [46,56].

In addition to protein trafficking into and out of nuclear

bodies, the bodies themselves move within the nucleus.PML bodies move discontinuously with stationary peri-

ods interspersed with long-range movements at maxi-

mum 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 corre-lated 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].

Conclusions and future directionsIn 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 long-

range associations of elements within the globin locus

control region with distant promoter regions [59].

However, these same regulatory elements also regulate

chromatin modifications, as well positioning of the globin

locus relative to centromeric heterochromatin [60].

A specific transcription factor essential for globin gene

activation initially localises to these same centromeric

regions, with delocalisation to the nucleoplasm coincid-

ing with movements of the locus to the nuclear interior

and gene activation [61]. In this single example of regu-

lated gene activation, we connect dynamics of chromatin

modification, both local and long-range chromatin move-ments, and transcription-factor trafficking. Similar con-

nections have emerged between compartmentalisation

of transcription repressors, gene silencing, and intranuc-

lear localisation of gene loci [62]. Our future challenge

will be to understand how these different dynamic pro-

cesses together accomplish regulation of nuclear pro-

cesses. This will require continued development of

new technological approaches suitable for in vivo obser-

vations of nuclear dynamics.

References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:

of special interestof outstanding interest

1.

Anderson JD, Thastrom A, Widom J: Spontaneous access ofproteins to buried nucleosomal DNA target sites occurs viaa mechanism that is distinct from nucleosome translocation.Mol Cell Biol 2002, 22:7147-7157.

This study demonstrates that spontaneous access of DNA bound tonucleosome core proteins is produced by DNA breathing, produced bychanges in nucleosome conformation, as opposed to nucleosome sliding.

2.

Cirillo LA, Lin FR, Cuesta I, Friedman D, Jarnik M, Zaret KS:Opening of compacted chromatin by early developmentaltranscription factors HNF3 (FoxA) and GATA-4. Mol Cell 2002,9:279-289.

Using in vitro reconstituted chromatin including linker histone H1, thispaper demonstrates the capability of certain transcription factors to bindand remodel nucleosome arrays without the help of ATP-dependentchromatin-modifying complexes.

3. Turner BM: Cellular memory and the histone code. Cell 2002,111:285-291.

4.

Robyr D, Suka Y, Xenarios I, Kurdistani SK, Wang A, Suka N,Grunstein M: Microarray deacetylation maps determinegenome-wide functions for yeast histone deacetylases.Cell 2002, 109:437-446.

Chromatin immunoprecipitation is used with DNA chip analysis to com-pare histone acetylation patterns between wild-type yeast cells andknockouts of specific histone deacetylases (HDACs). This approachidentifies large subsets of genes whose histone acetylation patterns,and expression, are regulated by specific HDACs. Interestingly, Hda1appears to act preferentially over large 1025 kb genomic domainsflanking telomeres.

5.

Katan-Khaykovich Y, Struhl K: Dynamics of global histoneacetylation and deacetylation in vivo: rapid restoration ofnormal histone acetylation status upon removal of activatorsand repressors. Genes Dev 2002, 16:743-752.

In this work in yeast, very rapid global acetylation or deacetylation back tosteady-state levels is observed within several minutes after removal oftargetedrepressor (Ume6) or activator (VP16 acidic activator), respectively.The authors also show that TATA-binding protein (TBP) binding correlatesclosely with activator binding, rather than with histone acetylation.

6. Hendzel MJ,Kruhlak MJ,Bazett-JonesDP: Organization of highlyacetylated chromatin around sites of heterogeneous nuclearRNA accumulation. Mol Biol Cell 1998, 9:2491-2507.

7. Lercher MJ, Urrutia AO, Hurst LD: Clustering of housekeepinggenes provides a unified model of gene order in the humangenome. Nat Genet 2002, 31:180-183.

8. Cohen BA, Mitra RD, Hughes JD, Church GM: A computationalanalysis of whole-genome expression data revealschromosomal domains of gene expression. Nat Genet 2000,26:183-186.



8/6/2019 15-304

5/7

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

10. Rogakou EP, Boon C, Redon C, Bonner WM: Megabasechromatin 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 throughintermolecular 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 RNApolymerase II: loss of the H2A/H2B dimer during transcription.Mol Cell 2002, 9:541-552.

In vitro transcription through nucleosome core particles by RNA poly-merase II leads to a loss of one H2AH2B dimer, providing a possiblemechanism for in vivo turnover of core histones H2A and H2B that isindependent of replication but linked to transcription.

13. Kimura H, Cook PR: Kinetics of core histones in living humancells: little exchange of H3 and H4 and some rapid exchange ofH2B. J Cell Biol 2001, 153:1341-1353.

14.

Ahmad K, Henikoff S: Histone H3 variants specify modesof 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 cellline system. H3.3 marks active chromatin and Cid marks centromericregions. 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 activechromatin by replication-independent nucleosome assembly.Mol Cell 2002, 9:1191-1200.

Replication-independent incorporation of H3.3 into the active ribosomalgene array was demonstrated. Under favourable growth conditions,additional ribosomal arrays became active and these were found toincorporate H3.3 and to show reductions of methylated histone H3.Replication-independent incorporation was shown to be conferred uponnormal histone H3 by amino acid changes towards H3.3.

16.

Catez F, Brown DT, Misteli T, Bustin M: Competition betweenhistone H1 and HMGN proteins for chromatin binding sites.EMBO Rep 2002, 3:760-766.

Fluorescence recovery after photobleaching analysis indicates competi-tion between HMGN (high-mobility group protein N) and histone H1 for

binding sites.

17.

Dou Y, Bowen J, Liu Y, Gorovsky MA: Phosphorylation and anATP-dependent process increase the dynamic exchange of H1in chromatin. J Cell Biol 2002, 158:1161-1170.

Linker-histone phosphorylation leads to a more rapid fluorescencerecovery after photobleaching, suggesting decreased binding of thephosphorylated protein. Interestingly, ATP depletion in vivo results indecreased linker-histone mobility.

18.

Christensen MO, Larsen MK, Barthelmes HU, Hock R, AndersenCL, Kjeldsen E, Knudsen BR, Westergaard O, Boege F, Mielke C:Dynamics of human DNA topoisomerases IIalpha and IIbeta inliving cells. J Cell Biol 2002, 157:31-44.

Topoisomerase II has been proposed to be a major component of thechromosome scaffold and also the nuclear matrix. However, in vivofluorescence recovery after photobleaching experiments indicate rapidturnover of topoisomerase II on a timescale of seconds in both nuclei andmitotic chromosomes. If a more stably bound topoisomerase II fraction ispresent as part of a stable structural scaffold, it must correspond to asmall fraction of the total enzyme.

19.

Tavormina PA, Come MG, Hudson JR, Mo YY, Beck WT,Gorbsky GJ: Rapid exchange of mammalian topoisomerase IIalpha at kinetochores and chromosome arms in mitosis.J Cell Biol 2002, 158:23-29.

This is an independent fluorescence recovery after photobleaching studyof topoisomerase IIin vivo. Theauthors come to a similar conclusion as inChristensen et al. [18], that there is rapid turnover in nuclei and mitoticchromosomes.

20.

Featherstone M: Coactivators in transcription initiation: here are your orders. Curr Opin Genet Dev 2002, 12:149-155.

This is a nice reviewof ordered recruitment of differentcomponents of thetranscriptional machinery after gene activation of specific loci. Data fromseveral sources argue against one-step recruitment of entire holo-enyzmes, as defined by biochemical isolation.

21. Krebs JE, Fry CJ, Samuels ML, Peterson CL: Global role forchromatin remodeling enzymes in mitotic gene expression.Cell 2000, 102:587-598.

22.

Memedula S, Belmont AS: Sequential recruitment of HAT andSWI/SNF components to condensed chromatin by VP16.Curr Biol 2003, 13:241-246.

Using a microscopy-based immunofluorescence approach, recruitment

of various SWISNF and histone acetyltransferase (HAT) components aremonitored, together with histone acetylation, after targeting of VP16acidic activator to a condensed chromosome region. Different subunitsof SWISNF and HAT complexes accumulate at the chromosome sitewith very different kinetics, arguing against a simple one-step recruitmentof 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: Nucleardynamics of RAD52 group homologous recombinationproteins in response to DNA damage. EMBO J 2002,21:2030-2037.

Using fluorescence recovery after photobleaching analysis, this studydemonstrates 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 pre-existing holoenzymes.

24. Hager GL, Elbi C, Becker M: Protein dynamics in the nuclearcompartment. Curr Opin Genet Dev 2002, 12:137-141.

25. McNally JG, Muller WG, Walker D, Wolford R, Hager GL: Theglucocorticoid receptor: rapid exchange with regulatory sitesin living cells. Science 2000, 287:1262-1265.

26.

Becker M, Baumann C, John S, Walker DA, Vigneron M, McNallyJG, Hager GL: Dynamic behavior of transcription factors on anatural promoter in living cells. EMBO Rep 2002, 3:1188-1194.

This is a continuation of groundbreaking work [25], providing additionalsupport for the hit-and-run mechanism underlying transcription factorbinding and action in vivo. Using the same chromosome array system asthat previously described by McNally et al. [25], the Hager group demon-strated rapid turnover of the co-activator GRIP-1 at similar rates toglucocorticoid receptor. By contrast, the RPB1 RNA polymerase subunitrecovers over a much longer timescale (13 min), consistent with theprocessive property of elongating polymerase. A faster recovering com-ponent suggests the possibility of more rapidturnover of promoter-boundRNA polymerase II. Accumulation of RNA polymerase II peaks at2030 min after ligand addition, dropping substantially afterwards.

27. Stenoien DL, Nye AC, Mancini MG, Patel K, Dutertre M, OMalleyBW, Smith CL, Belmont AS, Mancini MA: Ligand-mediatedassembly and real-time cellular dynamics of estrogen receptoralpha-coactivator complexes in living cells. Mol Cell Biol 2001,21:4404-4412.

28. Stenoien DL, Patel K, Mancini MG, Dutertre M, Smith CL, OMalleyBW, Mancini MA: FRAP reveals that mobility of oestrogenreceptor-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 livinghuman cells: constitutive association of TBP with mitoticchromosomes. Mol Biol Cell 2002, 13:276-284.

The authors describe fluorescence recovery after photobleaching (FRAP)analysis of TATA-binding protein (TBP) in mitotic and interphase cells. Norecovery of photobleaching is seen in mitotic chromosomes, demonstrat-ing stable association of TBP. Within interphase nuclei, TBP shows slowrecovery over a timescale of minutes (i.e. 120). This recovery wasindependent of transcriptional inhibition, suggesting that TBP is stablybound over multiple initiation rounds.

30.

Kimura H, Sugaya K, Cook PR: The transcription cycle of RNApolymerase II in living cells. J Cell Biol 2002, 159:777-782.

Fluorescence recovery after photobleaching analysis of the Large RNApolymeraseII (pol II) reveals a transientlyimmobile fraction correlated withtranscriptional elongation. Little pol II is immobile in the presence oftranscriptional inhibition, arguing against stable binding of a signi ficantfraction 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 RNApolymerase in vivo. Science 2002, 298:1623-1626.

Fluorescence recovery after photobleaching analysis of multiple compo-nents of the RNA polymerase I (pol I) transcriptional apparatus aredescribed. Recovery over tens of seconds was observed for severalpre-initiation factors. Four pol I subunits showed biphasic recovery



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curves, with a slow recovery phase associated with transcription elonga-tion. Differences in theearlyrapid recovery rates forthe four polI subunitswas 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 theglucocorticoid 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 byexposure to nuclear extract or purified human SWISNF. Thisremodellingwas accompanied by ATP-dependent GR displacement.

33.

Freeman BC, Yamamoto KR: Disassembly of transcriptionalregulatory complexes by molecular chaperones. Science 2002,296:2232-2235.

The p23 molecular chaperone was demonstrated to localise in vivo tonuclear receptor response elements. Moreover, tethering p23 disruptedreceptor-mediated transcriptional activation. Hsp90 displayed similar,but weaker, effects. These results suggest a role for disassembly com-plexes in transcription complex turnover.

34. Carmo-Fonseca M, Platani M, Swedlow JR: Macromolecularmobility inside the cell nucleus. Trends Cell Biol 2002,12:491-495.

35. Gasser SM: Visualizing chromatin dynamics in interphasenuclei. Science 2002, 296:1412-1416.

36.

Chubb JR, Boyle S, Perry P, Bickmore WA: Chromatin motion isconstrained by association with nuclear compartments inhuman cells. Curr Biol 2002, 12:439-445.

Fast but constrained motion of chromatin is observed in mammaliantissue culture cells, as previously observed in yeast and Drosophila. Thislocal motion is reduced at chromosome sites associated with nucleoli orwith 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 positioningand dynamics of origins of replication in the budding yeastnucleus. J Cell Biol 2001, 152:385-400.

39. Vazquez J, Belmont AS, Sedat JW: Multiple regimes ofconstrained chromosome motion are regulated in theinterphase 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 DNAchromosome region modulated by VP16 transcriptionalactivator. Nat Cell Biol 2001, 3:134-139.

42. Tumbar T, Sudlow G, Belmont AS: Large-scale chromatinunfolding and remodeling induced by VP16 acidic activationdomain. J Cell Biol 1999, 145:1341-1354.

43. Muller WG, Walker D, Hager GL, McNally JG: Large-scalechromatin decondensation and recondensation regulated bytranscription from a natural promoter. J Cell Biol 2001,154:33-48.

44. Mahy NL, Bickmore WA, Tumbar T, Belmont AS: Linking large-scale chromatin structure with nuclear function. In ChromatinStructure 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 MolecularBiology].

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 20Sproteasomes, ubiquitin, and proteinsubstrates of proteasome.Mol Biol Cell 2002, 13:2771-2782.

This is a report of a new class of nuclear body enriched in components ofthe ubiquitinproteosome pathway. Normally rare, these bodies increasein number under conditions in which proteolysis is stimulated. Proteinsubstrates of the proteosome found in this body include c-Fos, c-Junand 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.

Proteomic analysis of nucleolar proteins revealed a novel protein, PSP1,whose concentration into 1020 spots in a large number of primary andtransformed human cell types reveals a new class of nuclear bodiesknown as paraspeckles. Paraspeckles distribute near splicing specklesbutmove to thenucleolar periphery withinseveral hours of transcriptionalinhibition. PSP1 dynamically exchanges between paraspeckles andnucleoplasm.

47. Wang IF, Reddy NM, Shen CKJ: Higher order arrangementof theeukaryotic nuclear bodies. Proc Natl Acad Sci USA 2002,99:13583-13588.

TDP is a protein associated with transcriptional repression and alternativesplicing. TDP staining reveals TDP bodies, a new class of nuclearbodies. Staining shows overlap of TDP bodies with splicing speckles,gems, PML and Cajal bodies. Data are intriguing, but proper statisticalanalysis is needed to access significance of overlap.

48.

Shopland LS, Johnson CV, Lawrence JB: Evidence that all SC-35domains contain mRNAs and that transcripts can bestructurally constrained within these domains.J Struct Biol 2002, 140:131-139.

The authors demonstrate that SC35 domains contain specific mRNAs.Transcriptional inhibition leads to their long-term association with SC35domains, suggesting that a transcription-coupled maturation step mightbe 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 CBcontact. Mol Biol Cell 2001, 12:565-576.

50. Smith KP, Lawrence JB: Interactions of U2 gene loci andtheir nuclear transcripts with Cajal (coiled) bodies: evidencefor PreU2 within Cajal bodies. Mol Biol Cell 2000,11:2987-2998.

51. Sleeman JE, Lamond AI: Newly assembled snRNPs associatewith coiled bodies before speckles, suggesting a nuclearsnRNP maturation pathway. Curr Biol 1999, 9:1065-1074.

52.

Sleeman JE, Ajuh P, Lamond AI: snRNP protein expressionenhances the formation of Cajal bodies containing p80-coilinand SMN. J Cell Sci 2002, 114:4407-4419.

Continuation of studies indicating that Cajal bodies are sites of smallnuclear ribonucleoprotein particle (snRNP) maturation or assembly.Higher levels of snRNP protein expression and snRNP assembly mightlead to Cajal body formation.

53. Verheggen C, Lafontaine DLJ, Samarsky D, Mouaikel J, BlanchardJM, Bordonne R, Bertrand E: Mammalian and yeast U3 snoRNPsare matured in specific 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-specific small nuclear RNAs: a novel class of 20-O-methylation and pseudouridylation guide RNAs. EMBO J2002,21:2746-2756.

A novel class of small nuclear RNAs (snRNAs) that localise to Cajal bodieshave been shown or are predicted to function as guide RNAs for site-specific modification of snRNAs.

55.

Leung AK, Lamond AI: In vivo analysis of NHPX reveals anovel nucleolar localization pathway involving a transientaccumulation in splicing speckles. J Cell Biol 2002,157:615-629.

NHPX, a nucleolar factor that binds to an RNA sequence found innucleolar box C/D small nucleolar RNAs (snoRNAs) and in U4 smallnuclear RNA (snRNA), accumulates in nucleoli and Cajal bodies in vivo.NHPX nucleolar accumulation is preceded by transient accumulation insplicing speckles.

56.

Wiesmeijer K, Molenaar C, Bekeer IMLA, Tanke HJ, Dirks RW:Mobile foci of Sp100 do not contain PML: PML bodies areimmobile but PML and Sp100 proteins are not . J Struct Biol2002, 140:180-188.

Sp100 identifies nuclear bodies, of which a subset contain promyelocyticleukaemia protein (PML). The subset containing PML are relativelyimmobile; those lacking PML show mobility that may correspond tothe mobile PML bodies described by Darzacq et al. (2002) [54].SP100 and PML show turnover with nucleoplasmic pools on a timescaleof several minutes.

57. Muratani M, Gerlich D, Janicki SM, Gebhard M, Eils R, Spector DL:Metabolic-energy-dependent movement of PML bodies withinthe mammalian cell nucleus. Nat Cell Biol 2001, 4:106-110.



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58.

Platani M, Goldberg I, Lamond AI, Swedlow JR: Cajal bodydynamics and association with chromatin are ATP-dependent.Nat Cell Biol 2002, 4:502-508.

Cajal bodies are demonstrated to show intranuclear movements, withmost showing mobility consistent with constrained diffusion. ATP deple-tion and transcriptional inhibition increases movements and decreaseschromatin associations.

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

A novel method, dependent on generation of free radicals by labelledantibodies to generate biotinylated DNA, allows probing of the spatialrelationship between the b-globin locus control region (LCR) and globin

open reading frames. The data support movement of certain regulatoryregions of LCR near the promoter during globin gene activation.

60. Francastel C, Schubeler D, Martin DI, Groudine M: Nuclearcompartmentalization and gene activity. Nat Rev Mol Cell Biol2000, 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 fateand nuclear organisation. Curr Opin Genet Dev 2002,12:193-197.



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