Genome organization in and aroundthe nucleolusAttila Nemeth and Gernot Langst

Department of Biochemistry III, University of Regensburg, Universitatsstr. 31, D-93053 Regensburg, Germany

Review

Glossary

Chromatin domains: dynamic nucleoprotein structures assembled

on interphase chromosomes, which specifically interact with each

other depending on the physiological state of the cell.

Chromosome conformation capturing (3C): a method developed for

the detection of high-resolution genomic interaction maps [69–73].

The technique includes in situ fixation of the nuclear architecture by

formaldehyde and subsequent intramolecular ligation and PCR-

based detection of captured DNA fragments.

Chromosome territories: subspaces of the interphase nucleus,

which are occupied by single chromosomes [4]. Chromosome terri-

tories interact with each other at their borders, but the structural

organization of such interactions is not fully understood.

DNA adenine methyltransferase identification (DamID): a method

used to map the binding sites of chromatin-interacting proteins in

eukaryotes [74,75]. The assay is based on the low-level expression of

target proteins tagged with a bacterial DNA adenine methyltransfer-

ase enzyme and the subsequent methylation-sensitive analysis of

the modified DNA.

Histone modifications: histones, the protein components of nucleo-

somes, can undergo post-translational, enzymatic modifications by

covalent attachment of acetyl, methyl, phosphate, ubiquitin, small

ubiquitin-like modifier (SUMO) or ADP-ribose groups to the side

chains of specific amino acids, which alters their interactions with

other proteins and nucleic acids.

Nucleolus- and lamina-associated domains (NAD and LAD): defined

chromatin domains that dynamically interact with nucleoli and the

nuclear lamina, respectively. Given that many of them have been

mapped to overlapping chromosomal regions, they might change

their position between these compartments.

Nucleolus organizer regions (NORs): contain multiple copies of

ribosomal RNA genes (rDNA) around which nucleoli form. They

are located on the short arms of the acrocentric chromosomes

(chromosomes 13, 14, 15, 21 and 22 in humans) and appear as

secondary constrictions on metaphase chromosome stains.

Nuclear bodies: nucleoprotein assemblies within the interphase

nucleus (Figure 1, main text).

Nuclear compartments: morphologically and functionally distinct

parts of the interphase nucleus (e.g. nucleoli and nuclear lamina).

They are assembled from chromatin domains and nuclear bodies.

Nucleosome: the basic building unit of chromatin. Nucleosomes

contain approximately 147 bp DNA wrapped around a histone pro-

tein octamer consisting of a pair each of the core histones H2A, H2B,

H3 and H4.

Perinucleolar heterochromatin: the nucleolus is generally sur-

rounded by a shell of heterochromatin that frequently contains

centromeric and pericentromeric chromosomal regions. This peri-

nucleolar heterochromatin constitutes the major part of the identi-

fied NADs. The perinucleolar heterochromatin is not identical to the

perinucleolar compartment, which is rather typical for cancer-de-

The nucleolus is the largest compartment of the cellnucleus and is where ribosomal RNAs (rRNAs) are syn-thesized, processed and assembled with ribosomal pro-teins. In addition to rRNA gene clusters that build the coreof this subnuclear structure, nucleoli are associated withcondensed chromatin. Although the higher order struc-tures of rRNA genes and nucleolus-associated chromatinhave been studied for decades, detailed molecularinsights into the constituents and organization of thenucleolar genome are only beginning to emerge. Here,we summarize current views on the structural organiza-tion of nucleolar DNA and on the targeting and anchoringof chromatin domains to this subnuclear compartment.

Nuclear architecture and genome organizationprinciplesThe interphase nucleus consists of morphologically andfunctionally distinct substructures, called nuclear bodies(seeGlossary) and chromatin domains [1,2]. The chromatindomains are parts of chromosome territories [3,4], whereasmost of the nuclear bodies [5] are mainly protein assem-blies (Figure 1). These structures are built at the mitoticexit of the cell cycle and are maintained until the onset ofthe next mitosis. They are in steady state during theinterphase, when their individual components can be ex-changed [6]. The largest subnuclear structure is the nucle-olus, which is built around rRNA genes. The primaryactivity of the interphase nucleus is the synthesis of nucleicacids. In eukaryotic cells, up to 80% of RNA synthesis isdevoted to rRNA transcription, which is indispensable tothe preservation of ribosome production and thus proteinsynthesis capacity. This powerful biosynthetic processoccurs in the nucleolus, which consequently has a centralregulatory role in cell growth. Recent studies revealed theinvolvement of the nucleolus inmultiple biological process-es, such as senescence, cell cycle regulation and stresssensing (Box 1) [7] and explored the nucleolar proteome[8,9]. During the past few years, significant advances havebeen made in understanding of the higher order organiza-tion of the nucleolar chromatin. Here, we address theimpact of these recent results and discuss novel methodsdeveloped to study the nuclear and nucleolar architecture.

One of the working hypotheses of the ENCODE (Ency-clopedia of DNA elements) project [10] postulates that thefunctional domains of chromosomes are the principal com-ponents that define the nuclear genome architecture.

Corresponding authors: Nemeth, A. (attila.nemeth@vkl.uni-regensburg.de);Langst, G. (gernot.laengst@vkl.uni-regensburg.de)

0168-9525/$ – see front matter � 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2011.0

Genome-wide, population average-based studies stronglysupport this hypothesis. The high-resolution profiling of

rived cell lines and contains RNA polymerase III transcripts and the

polypyrimidine tract binding protein [76–78].

1.002 Trends in Genetics, April 2011, Vol. 27, No. 4 149

[()TD$FIG]

Nuclear bodiesKey:

Paraspeckle

Polycomb bodyPML body

Nuclear speckleNucleolus

NucleolusFibrillar center

Dense fibrillar component

Granular component

Perinucleolar heterochromatin

(a) (b)

Cajal body

Transcription factory

TRENDS in Genetics

Figure 1. Compartmentalization of the mammalian nucleus. The mammalian

nucleus contains a variety of nucleoprotein assemblies called nuclear bodies. (a)

Schematic overview of the nuclear bodies of the mammalian nucleus. Cajal bodies

are implicated in the biogenesis of small nuclear and nucleolar ribonucleoprotein

complexes, nuclear speckles are involved in pre-mRNA processing, nucleoli in

ribosome biogenesis and the transcription factories in RNA synthesis by the RNA

polymerase II enzyme. The other subnuclear bodies are functionally less well

characterized: paraspeckles are formed around long non-coding RNA, PML bodies

might sequester and post-translationally modify associated proteins and

Polycomb bodies contain Polycomb group complex proteins associated with

pericentric heterochromatin. (b) High-resolution structure of the nucleolus. The

mammalian nucleolus has a tripartite composition. Primary rRNA transcripts are

localized at the fibrillar center–dense fibrillar component border and are post-

transcriptionally modified in the dense fibrillar component. The early steps of

ribosome assembly take place in the granular component of the nucleolus. The

nucleoli are surrounded mainly by condensed heterochromatin.

Review Trends in Genetics April 2011, Vol. 27, No. 4

histone methylationmarks throughout the human genomeclearly supports the existence of large chromosomaldomains, in which the nucleosomes are tailored with spe-cific combinations of post-translational modifications [11].The integration of several high-throughput, genome-wideanalyses of the localization of histone modifications,histone variants, chromatin regulator proteins and nucle-osome positions have provided a detailed picture of

Box 1. The multifunctional nucleolus and human diseases

The core function of the nucleolus is the synthesis of ribosomal RNA

and the first steps of ribosome assembly; however, it is also

involved in numerous other cellular functions, including senes-

cence, RNA modification, cell cycle regulation and stress sensing

[7,30]. Alterations of the nucleolus might be linked to different

human diseases, including viral infections. The interaction between

viruses and the nucleolus is a pan-virus phenomenon that has been

shown for DNA viruses, retroviruses and RNA viruses [79,80].

Furthermore, multiple genetic disorders have been mapped to

genes that encode proteins located in nucleoli under specific

conditions. These include Werner [81], fragile X [82,83], Treacher

Collins [84], Bloom [85], Rothmund–Thomson [86], dyskeratosis

congenita syndromes [87] and Diamond-Blackfan anemia [88]. High

proliferation activity of tumor cells coincides with high ribosome

biogenesis activity, thus highlighting the nucleolus as a promising

target in cancer therapy [89]. In addition, cell-type and function-

dependent nucleolar localization of tumor suppressor proteins, such

as p53 [90,91], MDM2 [92,93] or p14ARF [94] and the proto-

oncogene protein c-Myc [95] indicates a role for the nucleolus in

carcinogenesis. Indeed, on the basis of the high ribosome biogen-

esis activity of tumor cells, nucleolus-targeting agents that selec-

tively kill cancer cells have been developed, and quarfloxin is the

first to begin clinical trials [96].

150

functional chromatin domains along the linear sequenceof the chromosomes [12–14]. In the most comprehensivestudy, the integrative analysis of the genome-wide distri-bution of 53 chromatin-associated proteins revealed fiveprincipal classes of chromatin types in the nucleus ofDrosophila melanogaster cells [12].

Recent investigations have revealed how chromatindomains are arranged to form chromosome territories,leading to an interphase genome organization that canbe described as a fractal globule [15,16]. The fractal glob-ule model describes the genome as a knot-free polymer, inwhich the rapid unfolding and folding of specific genomicloci is facilitated and compatible with the dense packagingof the genome. Thismodel of genome architecture explainshow the genome can achieve its dual function of storageand expression, which is mirrored by the active and inac-tive chromatin domains of the interphase nucleus [15].Furthermore, it supports and extends the fractal chromo-some organization hypothesis, which was proposed morethan two decades ago [17]. This hypothesis was developedto clarify topological problems of genome replication andwas based on the assumption of self-similarity betweenthe shapes of lower and higher order structures of thechromosome.

Current studies have also addressed the dynamics andassembly principles of the Cajal body [18], which is impli-cated in the biogenesis of small nuclear and nucleolarribonucleoprotein complexes; the promyelocytic leukemia(PML) nuclear body [19], whose function is not yet clari-fied; and the nucleolus [20–22]. In these studies, severalconserved nucleic acid and protein components were iden-tified as constituents of self-assemblymechanismsand theartificial assembly of these compartments was achieved invivo by ectopically introducing their molecular compo-nents into living cells. Despite these findings, there islittle known about the dynamics of the chromatin domainsand nuclear bodies, and the interplay between them. Thecombination of high-throughput genomics and spatiotem-poral analysis of genome architecture at the level of nu-clear compartments provides a novel tool to study thisinterplay. Pioneering work has been performed with thelargest nuclear compartments, the nuclear lamina [23,24]and the nucleolus [25,26]. The nuclear lamina, whosemain components are the Lamin A/C and Lamin B pro-teins, builds a proteinaceous network under the innernuclearmembrane. This nuclear compartment is a specialfeature of metazoan cells [27] and has an important role ingenome organization [23].

Here, we provide an overview of the current understand-ing of the interactions between chromatin domains andnuclear compartments, focusing on the nucleolus. We dis-cuss the intranucleolar chromatin organization and reviewthe association of chromatin domains with nucleoli and theregulatory factors that can mediate this association.

Organization of rRNA genes in the mammaliannucleolusNucleoli are formed around nucleolus organizer regions(NORs), which contain multiple copies of rRNA genes.NORs are located on the short arms of the acrocentricchromosomes (chromosomes 13, 14, 15, 21 and 22 in

[()TD$FIG]

Initiation

DNA

Termination

Histone H1

meCpG

Canonicalnucleosome

UBF

RNA polymerase I

‘Dynamic’nucleosome

Inactive

ActiveN

ucleo

lar interio

rc-Myc, TTF-I

TRENDS in Genetics

Key:

Figure 2. Activity-dependent chromatin conformations of rRNA genes. Transcriptionally active and inactive rRNA genes have different conformations and are localized in

different subnucleolar regions. The inactive rRNA genes are tightly packaged into chromatin and the DNA in their coding region is methylated. These genes are depleted in

RNA polymerase I and the architectural transcription factor UBF. The promoter and terminator elements of the inactive gene do not interact. The active genes are occupied

by RNA polymerase I and UBF; furthermore, the TTF-I and c-Myc transcription factors mediate the interaction between the promoter and terminator sequences. These genes

are localized in the internal regions of the nucleoli.

Review Trends in Genetics April 2011, Vol. 27, No. 4

humans) and appear as secondary constrictions on meta-phase chromosome stains. The rRNA genes are arrangedmainly, but not exclusively, in tandem repeated arrays,ordered in a head-to-tail fashion [28]. Owing to their highcopy number and repetitive organization, these rDNAunits are not fully sequenced and annotated. There areonly two archetype sequences ofmammalian rDNA repeatsin GenBank (Acc. No. U13369 and BK000964 for humanand mouse rDNA, respectively), although many sequencevariations occur in these rDNA units [29]. Each rDNA unitconsists of a gene and an intergenic spacer, and each geneis flanked by promoter and terminator sequence elements.The rRNA genes are transcribed by RNA polymerase I andencode a precursor transcript (47S pre-rRNA) that is pro-cessed into the three RNA species present in the ribosome:5.8S, 18S and 28S rRNA. The epigenetic marks of activeand inactive rRNA genes reflect their transcriptional sta-tus. The actively transcribed genes lack DNA methylationand are associated with the architectural RNA polymeraseI transcription factor, UBF (upstream binding factor; high-affinity binding sites for this protein are located immedi-ately upstream of the rRNA transcriptional start site). TheDNA of inactive genes is methylated and the associatedhistones carry repressive marks [30] (Figure 2).

The three-dimensional topology of rRNA genes withinhuman and mouse cells was recently studied using thechromosome conformation capturing (3C) method [31,32].These experiments revealed that the conformation of anrRNA gene is dependent on its activity. Active rRNA genesexhibit specific interactions between the promoter and theterminator, suggesting DNA looping between the sites oftranscription initiation and termination. DNA looping be-tween the beginning and the end of an actively transcribed

gene enables coregulation of transcription initiation andtermination, and also provides the structural basis forefficient re-initiation of the RNA polymerase.

It has been shown that the global transcriptional regu-lator c-Myc and the multifunctional RNA polymerase Itranscription factor TTF-I have essential roles in the es-tablishment of the looped rRNA gene conformation. c-Mycis a global regulator of protein synthesis and cell growththat has multiple binding sites close to the rRNA genepromoter and terminator [33–36]. The protein binds as adimer and its interaction with other proteins enables thebridging of distant binding sites [32]. TTF-I has DNAbinding sites within the rDNA intergenic spacer, at thepromoter and termination region. It was also shown tooligomerize and is thus able to link physically its bindingsites and mediate contacts between the beginning and theend of transcribed rRNA gene units [37]. It is unclearwhether c-Myc and TTF-I cooperate functionally andhow the DNA looping activity of TTF-I is regulated. Giventhat TTF-I is also required for rRNA gene silencing andremains bound to inactive genes [38], where promoter–

terminator interactions do not exist [31], a mechanism isrequired to explain the modulation of its oligomerizationactivity. Moreover, it remains to be determined whetherDNA loops are formed within individual genes, betweenneighboring genes of an array, or even between rRNAgenes on different acrocentric chromosomes. A well-designed 3C assay demonstrated that promoters of at leasttwo different repeats can be juxtaposed, suggesting thelatter scenario [32].

The mammalian nucleolus consists of three functionaland structural regions (Figure 1b). Transcription of the47S pre-rRNA takes place in the fibrillar centers or at the

151

Review Trends in Genetics April 2011, Vol. 27, No. 4

fibrillar center–dense fibrillar component border. TherRNA processing mainly occurs in the dense fibrillarcomponent and the granular component of the nucleolusis the place where the early steps of ribosome assemblyoccur. Hence, gene activity determines the subnuclearlocalization of rDNA, with active rRNA genes placed inthefibrillar centers or at the fibrillar center–dense fibrillarcomponent border and inactive rRNA genes located out-side of this area [30] (Figure 2). However, the exact locali-zation of the rRNA transcription is still amatter of debate.Moreover, rRNA genes exist not only as active and silentgenes, but also as poised genes. Poised rRNA genes pres-ent a transitional state between the active and silentchromatin conformations. The existence of poised genesis postulated based on the findings that: (i) not all loopedrRNA genes are free of DNA methylation (i.e. active) [31];(ii) depletion of UBF leads to a pseudo-silenced statewithout affecting the epigenetic state of the gene [39];and (iii) specific variants of rRNA genes are selectivelyactive in different tissues [40]. The non-transcribed,poised genes and inactive RNA polymerase I complexesare supposed to be localized in the fibrillar center and,upon activation, to move towards the fibrillar center–

dense fibrillar component border [41]. Themolecular stepsand the order of events during transcriptional activationstill remain to be determined. However, binding of UBF tothe transcription unit seems to be the crucial event inswitching the gene to the active status [39].

Chromosomal constitution of the human nucleolus-associated DNArRNA genes form higher order structures depending ongene activity and localize to different regions of the nucle-olus. In addition, the nucleolus also contains DNA otherthan rRNA genes. What are the genomic contributions tothe nucleolus formation and organization? On humanacrocentric chromosomes, satellite and low-copy numberD4Z4 repeats flank the rDNA repeats and belong to thecore of the nucleolar genome because of their physicalproximity [30,42]. Moreover, three decades ago, the fre-quent association of the centromeres of chromosomes 1 and9 and the heterochromatin of chromosome Y with nucleoliwas observed [43]. The cell cycle-dependent dynamics ofthe pericentromeric region of chromosome 1 associationwith the nucleolus was also described [44]. These studieshad already therefore suggested that a set of conservedgenomic regions interact with the mammalian nucleolus.

The recent high-resolution, genome-wide map of nucle-olus-associated chromatin was characterized in humanHeLa cervix carcinoma cells [25] andHT1080 fibrosarcomacells [26]. Nucleoli were isolated that consisted of thenucleolus itself and the nucleolar shell, a dense chromatinnetwork surrounding the nucleolus. The nucleoli wereprepared with [25] or without [26] formaldehyde treatmentof the cells and the co-purified DNA was subjected tomicroarray analysis and/or high-throughput sequencingto obtain a high confidence list of nucleolus-associatedDNA sequences. Importantly, the findings of both studiesare largely comparable, suggesting that the main resultsare robust and not sensitive to small differences in thesample preparation or bioinformatic analyses.

152

The detailed analysis of nucleolus-associated chromatindomains (NADs) [25] revealed that: (i) only a specific subsetof the genome is associated with nucleoli (4% without therDNA-containing short arms of the acrocentric chromo-somes); (ii) NADs are enriched in different sequence fea-tures [e.g. satellite repeats (mainly alpha-, beta-,(GAATG)n/(CATTC)n-types), members of the zinc-finger,olfactory receptor defensin and immunoglobulin protein-coding gene families, transcriptionally active 5S rRNAgenes and tRNA genes]; and (iii) the transcriptional statusand chromatin feature analysis of NADs shows that theycontain mainly inactive chromosomal regions.

The sequence composition of NADs suggests that spe-cific satellite repeats have a central role in the assembly ofheterochromatin at the nucleolar periphery. In turn, UBFbinding to the rRNA genes mediates the assembly of thenucleolus [20]. The essential role of UBF was shown by thestable integration of large arrays of UBF binding sites,isolated from the rDNA of Xenopus laevis, into the genomeof human cells. Around these arrays, the formation ofnucleolus-like structures was observed, suggesting thatUBF acts as a scaffold, initiating nucleolus formation evenin the absence of RNA polymerase I transcription. Thelatter observation reinforced earlier findings, whichshowed that nucleoli are formed after the mitosis beforethe onset of rRNA transcription [45]. In contrast to thenucleolus, the nucleolar periphery consists mainly of sat-ellite repeats, which might nucleate the assembly of theperinucleolar heterochromatin.

How do the different repetitive DNA sequences buildthe robustly linked structure that can be isolated from thecell and how are these sequences linked to the rDNA? Apreliminary answer comes from studies on the genomeorganization in Saccharomyces cerevisiae. A 3C-based ap-proach was performed to map genomic regions interactingwith the rRNA gene. Here, specific, repeated elements (i.e.Ty and Y0 elements) were identified. A model was proposedin which interactions between these DNA sequences orga-nize genome architecture by restricting the movement ofthese loci relative to the nucleolar interaction point [46],resulting in a high residence time of these loci close to thenucleolus. In addition, live cell imaging demonstratedfrequent contacts between the nucleolus and specific chro-matin domains [47]. These results are in good agreementwith earlier discoveries, showing that the movement ofchromatin domains is constrained at the nucleoli of humancells [48].

Comparison of nucleolus- and lamina-associatedchromatin domainsThe genomics of nuclear compartments is a new field innuclear architecture research that has been initiated bythe study of nuclear lamina-associated chromatin domains(LADs) [24]. It is becoming increasingly clear that thenuclear lamina has a role in genome organization andconsequently influences gene activity [49,50]. The nuclearlamina and the nucleolar shell are the inner and outersurfaces of sphere-like structures (i.e. the nucleus and thenucleolus). Therefore, a comparison of NADs and LADs ofhuman cells provides an opportunity to explore somefeatures of architectural genome organization. Human

[()TD$FIG]

1 2 3 4 5 6 7 8 9 10 11 12

13 14 15 16 17 18 19 20 21 22 X Y

NADsKey:LADs

Centromeres

NORs

TRENDS in Genetics

Figure 3. The distribution of NADs and LADs in the human genome. Specific chromosomal regions of the human genome are associated with nucleoli and the nuclear

lamina. NADs are indicated in red on the left sides of the chromosome schemes, whereas LADs are on the right in green. NORs are labeled in blue and centromeres in

orange. NADs occupy approximately 4% of the genome, whereas more than 40% of the genome was found to be associated with the nuclear lamina in cell population-

based analyses. There is remarkable overlap between NADs, LADs and the centromeric and pericentromeric regions of most chromosomes.

Review Trends in Genetics April 2011, Vol. 27, No. 4

LADswere identified using the DNA adeninemethyltrans-ferase identification (DamID) method in Tig3 fibroblastcells by analyzing the DNA that is in contact with Lamin Band emerin, two protein components of the nuclear lamina[23]. The genomic maps of NADs and LADs highlight theircommon and distinctive features (Figure 3). First, thenumber of LADs (1344) is larger than the number of NADs(97). LADs cover almost half of the genome, whereas NADsoccupy less than 5%. This discrepancy could be due to thedifferent techniques that were used for their identification.DamID cannot distinguish between transient and stableinteractions, whereas the DNA analysis of isolated nucleolireveals the most stably interacting chromatin domains.Furthermore, differences in the actual surface size of thenucleoli and the nuclear lamina can also influence the totalsize of the interacting part of the genome. Indeed, bothNADs and LADs contain the majority of the heterochro-matic, centromeric and pericentromeric chromosomalregions (Figure 3). Moreover, these chromatin domainsare approximately 0.1–1 Mb in size, enriched in repressivechromatin marks and contain a specific subset of protein-coding genes, RNA genes and repetitive DNA sequenceelements. The domain borders of NADs are less welldefined than are the borders of LADs, probably owing tothe different experimental approaches that were used tomap them. Taken together, these results have revealedwhich chromatin domains are associated with the laminaand the nucleolus, unraveling their main sequence and

chromatin features. Questions concerning their tetheringto dynamic chromatin networks can now be addressed.

Targeting and anchoring of chromatin domains to thenucleolusThe observation that only a specific subset of the genomeassociates with nucleoli raises the following question:which cis- and trans-acting factors are involved in theestablishment and maintenance of nucleolus–chromatindomain interactions within the interphase nucleus? Thefactors that are implicated in the recruitment of chromatindomains to nucleoli have been investigated in mammaliancells in two particular cases so far. First, the centromericalpha-satellite RNA, which is associated with the centro-mere-specific CENPC1 and INCENP proteins, has beenshown to be essential for the targeting of centromeres tothe nucleolus [51]. This observation fits well with thegenomic data on NADs, where specific satellite repeatshave been found to be highly enriched. Thus, satelliteRNAs might be essential for directing the majority ofthe identified DNA sequences towards the nucleolus. In-deed, the transcriptional activity of RNA polymerase II,which is required to synthesize satellite RNAs, was impli-cated in the maintenance of nucleolar structure [25,52].However, inhibition of RNA polymerase II transcriptionhas multiple effects and the disruption of nucleoli mightstill be an indirect effect. The second factor that has beenshown to mediate nucleolar tethering of DNA elements is

153

Review Trends in Genetics April 2011, Vol. 27, No. 4

the CCCTC-binding factor (CTCF), a genome organizerand DNA binding protein that interacts with the nucleolarnucleophosmin/B23 protein and is thus able to recruit itsDNA binding sites to the nucleolar periphery [53].

The list of putative mediators of chromatin domain–

nucleolus interactions can be extended to specific histonemodifications and the corresponding readers of thesemarks, as well as to the nuclear proteins TTF-I interactingprotein 5 (Tip5) and DNA methyltransferase 1 (Dnmt1).The following observations motivate these speculations.First, NADs have been found to be enriched in certainhistone modifications, most significantly H4K20me3,H3K9me3 and H3K27me3, providing a binding platformfor a specific subset of chromatin-interacting proteins.These include the heterochromatin protein 1 (HP1) andPolycomb group (PcG) proteins, which could act as a scaf-fold in mediating chromatin domain interactions. Second,Tip5, the large subunit of the nucleolar remodeling com-plex (NoRC) might also have a role in the targeting andanchoring of chromatin domains to the nucleolus. NoRC isa multifunctional chromatin-dependent regulator of rRNAgenes, which regulates nucleosome positioning, transcrip-tional repression, epigenetic silencing and replication tim-ing [54–59]. Tip5 has multiple AT-hooks that bind to theminor groove of DNA [57], possibly enabling Tip5 to bind todifferent DNA elements simultaneously. It was shown thatTip5, as well as binding to rDNA, also binds centromericrepeats and could therefore have a role in tethering them tothe nucleolus [60]. Third, Dnmt1 dysfunction alters theshape of nucleoli [61]. However, this could be an indirecteffect caused by changes in the DNA methylation status ofcertain genomic regions. It is well known that the methyl-ation of cytosine nucleotides at the gene promoter canrepress the transcriptional activity of the gene [62]. Thus,the change of the nucleolar structure could be because ofthe altered expression of chromatin regulators and notmediated directly by the dysfunction of Dnmt1.

So far, only a few factors have been suggested to partic-ipate in tethering the chromatin domains to the nucleolusand only the role of satellite RNA in mediating suchinteractions has been clarified in detail. As research inthis field continues, other regulators of chromatin domain–

nucleolus interactions will be identified and the molecularsteps of nucleolar targeting and anchoring of NADs will bedetermined.

Concluding remarks and future perspectivesIt is becoming increasingly clear that the genome organi-zation and location of genes in the nucleus is not random.Functionally related genes are often found next to eachother on the same chromosome and distant DNA elementsor DNA regions located on different chromosomes mightreside in specific nuclear compartments of the interphasenucleus. Major challenges now include deciphering themechanisms by which chromatin domains come togetherand understanding the dynamics and functional conse-quences of these associations. Given that numerous cellu-lar activities are linked to the nucleolus, the analysis of itschromatin environment provides a well-suited experimen-tal system to understand structure–function relationshipsof the genome organization.

154

Time variant analysis of nucleolus–chromatin domaininteractions concomitant with genome-wide gene expres-sion analyses will be one of the next steps in the under-standing of nucleolar functions at the system level. Forthis, research should move from the snapshot analysis ofasynchronous cell populations [25,26] to more sophisticat-ed cellular systems, where the dynamics of the nucleolarchromatin network could be contrasted with changes inthe physiological state of the cell involving the globalremodeling of the nuclear architecture. These states in-clude differentiation, senescence, carcinogenesis, nuclearreprogramming and cellular stress, all of which should becorrelated with genome function (i.e. transcription). More-over, the comparative analysis of NADs from differentorganisms should reveal the evolution of nucleolar target-ing signals. The technical challenges of such studies residein the optimization of nucleolus isolation protocols from avariety of cell types, the reduction of input material possi-bly to the single cell level, the further development of realquantitative high-throughput sequence analysis and theintroduction of live cell imaging to the investigation ofnucleolus–chromatin domain interactions. In summary,these techniques will enable researchers to discover themolecular mechanisms of nucleolar targeting and anchor-ing of NADs. The candidate proteins that might be in-volved in such processes could be selected from the list ofchromatin-related proteins of the Nucleolar Proteome Da-tabase (http://www.lamondlab.com/NOPdb3.0/). Other ex-perimental setups should address the interaction maps ofactive and inactive rRNA genes with other parts of thegenome, for example by using novel genomics tools [63]mainly based on 3C-related approaches.

In addition to further genomic analyses, an integrativeapproach monitoring nucleolar proteome–genome–tran-scriptome dynamics is envisioned. This would extend theaforementioned list of technical challenges with high-throughput mass spectrometry analyses. Recent studiesdelivered the first comprehensive analyses of the nucleolarproteome [64–66], demonstrating that such investigationsare technically possible.

The importance of transcription for nucleolar integritysuggests a significant role for RNA molecules as architec-tural factors. Two pioneering studies were carried out toisolate non-ribosomal nucleolar RNA from Arabidopsisthaliana cells [67,68], leading the way to identify RNAsthatmight be involved in nucleolar chromatin organization.These analyses identified a new class of small nucleolarRNAs with unknown function, which might be candidatesfor this function. Recently, the first data sets on thesubcellular compartmentalization of the transcriptomes(GSE24565 GEO data set series) of human cells furtherextended the available nucleolar RNA data. The foreseengenome–transcriptome–proteome meta-analysis of the nu-cleolar data sets could lead to the understanding of nucleo-lus and perinucleolar chromatin formation at themolecularlevel and give detailed insights into their functions in ge-nome organization.

References1 Lanctot, C. et al. (2007) Dynamic genome architecture in the nuclear

space: regulation of gene expression in three dimensions. Nat. Rev.Genet. 8, 104–115

Review Trends in Genetics April 2011, Vol. 27, No. 4

2 Misteli, T. (2005) Concepts in nuclear architecture. Bioessays 27, 477–

4873 Cremer, T. and Cremer, C. (2001) Chromosome territories, nuclear

architecture and gene regulation in mammalian cells. Nat. Rev. Genet.2, 292–301

4 Cremer, T. and Cremer, M. (2010) Chromosome territories. ColdSpring Harb. Perspect. Biol. 2, a003889

5 Spector, D.L. (2006) SnapShot: cellular bodies. Cell 127, 10716 Matera, A.G. et al. (2009) Nuclear bodies: random aggregates of sticky

proteins or crucibles of macromolecular assembly? Dev. Cell 17, 639–

6477 Boisvert, F.M. et al. (2007) The multifunctional nucleolus. Nat. Rev.

Mol. Cell Biol. 8, 574–5858 Mohamad, N. and Boden, M. (2010) The proteins of intra-nuclear

bodies: a data-driven analysis of sequence, interaction andexpression. BMC Syst. Biol. 4, 44

9 Boisvert, F.M. et al. (2010) A quantitative proteomics analysis ofsubcellular proteome localization and changes induced by DNAdamage. Mol. Cell. Proteomics 9, 457–470

10 Birney, E. et al. (2007) Identification and analysis of functionalelements in 1% of the human genome by the ENCODE pilot project.Nature 447, 799–816

11 Barski, A. et al. (2007) High-resolution profiling of histonemethylations in the human genome. Cell 129, 823–837

12 Filion, G.J. et al. (2010) Systematic protein location mapping revealsfive principal chromatin types in Drosophila cells. Cell 143, 212–224

13 Rando, O.J. and Chang, H.Y. (2009) Genome-wide views of chromatinstructure. Annu. Rev. Biochem. 78, 245–271

14 Zhou, V.W. et al. (2010) Charting histone modifications and thefunctional organization of mammalian genomes. Nat. Rev. Genet. 12,7–18

15 Lieberman-Aiden, E. et al. (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome.Science 326, 289–293

16 Bancaud, A. et al. (2009) Molecular crowding affects diffusion andbinding of nuclear proteins in heterochromatin and reveals thefractal organization of chromatin. EMBO J. 28, 3785–3798

17 Takahashi, M. (1989) A fractal model of chromosomes andchromosomal DNA replication. J. Theor. Biol. 141, 117–136

18 Kaiser, T.E. et al. (2008) De novo formation of a subnuclear body.Science 322, 1713–1717

19 Brand, P. et al. (2010) Assembly dynamics of PML nuclear bodies inliving cells. PMC Biophys. 3, 3

20 Mais, C. et al. (2005) UBF-binding site arrays form pseudo-NORs andsequester the RNA polymerase I transcription machinery. Genes Dev.19, 50–64

21 Prieto, J.L. and McStay, B. (2007) Recruitment of factors linkingtranscription and processing of pre-rRNA to NOR chromatin isUBF-dependent and occurs independent of transcription in humancells. Genes Dev. 21, 2041–2054

22 Prieto, J.L. and McStay, B. (2008) Pseudo-NORs: a novel model forstudying nucleoli. Biochim. Biophys. Acta 1783, 2116–2123

23 Guelen, L. et al. (2008) Domain organization of human chromosomesrevealed by mapping of nuclear lamina interactions. Nature 453, 948–

95124 Pickersgill, H. et al. (2006) Characterization of the Drosophila

melanogaster genome at the nuclear lamina.Nat. Genet. 38, 1005–101425 Nemeth, A. et al. (2010) Initial genomics of the human nucleolus. PLoS

Genet. 6, e100088926 van Koningsbruggen, S. et al. (2010) High-resolution whole-genome

sequencing reveals specific chromatin domains from most humanchromosomes associate with nucleoli. Mol. Biol. Cell 21, 3735–3748

27 Dechat, T. et al. (2008) Nuclear lamins: major factors in the structuralorganization and function of the nucleus and chromatin.Genes Dev. 22,832–853

28 Caburet, S. et al. (2005) Human ribosomal RNA gene arrays display abroad range of palindromic structures. Genome Res. 15, 1079–1085

29 Kuo, B.A. et al. (1996) Human ribosomal RNA variants from a singleindividual and their expression in different tissues. Nucleic Acids Res.24, 4817–4824

30 McStay, B. and Grummt, I. (2008) The epigenetics of rRNA genes: frommolecular to chromosome biology. Annu. Rev. Cell Dev. Biol. 24,131–157

31 Nemeth, A. et al. (2008) Epigenetic regulation of TTF-I-mediatedpromoter-terminator interactions of rRNA genes. EMBO J. 27,1255–1265

32 Shiue, C.N. et al. (2009) c-Myc induces changes in higher orderrDNA structure on stimulation of quiescent cells. Oncogene 28,1833–1842

33 Arabi, A. et al. (2005) c-Myc associates with ribosomal DNAand activates RNA polymerase I transcription. Nat. Cell Biol. 7,303–310

34 Gomez-Roman, N. et al. (2006) Activation by c-Myc of transcription byRNA polymerases I, II and III. Biochem. Soc. Symp. 141–154

35 Grandori, C. et al. (2005) c-Myc binds to human ribosomal DNA andstimulates transcription of rRNA genes by RNA polymerase I.Nat. CellBiol. 7, 311–318

36 Grewal, S.S. et al. (2005) Myc-dependent regulation of ribosomal RNAsynthesis during Drosophila development. Nat. Cell Biol. 7, 295–302

37 Sander, E.E. and Grummt, I. (1997) Oligomerization of thetranscription termination factor TTF-I: implications for thestructural organization of ribosomal transcription units. NucleicAcids Res. 25, 1142–1147

38 Nemeth, A. et al. (2004) The chromatin remodeling complex NoRC andTTF-I cooperate in the regulation of the mammalian rRNA genes invivo. Nucleic Acids Res. 32, 4091–4099

39 Sanij, E. et al. (2008) UBF levels determine the number of activeribosomal RNA genes in mammals. J. Cell Biol. 183, 1259–1274

40 Tseng, H. et al. (2008) Mouse ribosomal RNA genes contain multipledifferentially regulated variants. PLoS ONE 3, e1843

41 Raska, I. et al. (2006) Structure and function of the nucleolus in thespotlight. Curr. Opin. Cell Biol. 18, 325–334

42 Lyle, R. et al. (1995) The FSHD-associated repeat, D4Z4, is amember ofa dispersed family of homeobox-containing repeats, subsets of whichare clustered on the short arms of the acrocentric chromosomes.Genomics 28, 389–397

43 Stahl, A. et al. (1976) Chromosomal constitution of nucleolus-associated chromatin in man. Hum. Genet. 35, 27–34

44 Leger, I. et al. (1994) Interactive computer-assisted analysis ofchromosome 1 colocalization with nucleoli. Cytometry 16, 313–323

45 Dousset, T. et al. (2000) Initiation of nucleolar assembly is independentof RNA polymerase I transcription. Mol. Biol. Cell 11, 2705–2717

46 O’Sullivan, J.M. et al. (2009) Repeated elements coordinate the spatialorganization of the yeast genome. Yeast 26, 125–138

47 Berger, A.B. et al. (2008) High-resolution statistical mapping revealsgene territories in live yeast. Nat. Methods 5, 1031–1037

48 Chubb, J.R. et al. (2002) Chromatin motion is constrained byassociation with nuclear compartments in human cells. Curr. Biol.12, 439–445

49 Shimi, T. et al. (2008) The A- and B-type nuclear lamin networks:microdomains involved in chromatin organization and transcription.Genes Dev. 22, 3409–3421

50 Zhao, R. et al. (2009) Nuclear neighborhoods and gene expression.Curr. Opin. Genet. Dev. 19, 172–179

51 Wong, L.H. et al. (2007) Centromere RNA is a key component for theassembly of nucleoproteins at the nucleolus and centromere. GenomeRes. 17, 1146–1160

52 Haaf, T. and Ward, D.C. (1996) Inhibition of RNA polymerase IItranscription causes chromatin decondensation, loss of nucleolarstructure and dispersion of chromosomal domains. Exp. Cell Res.224, 163–173

53 Yusufzai, T.M. et al. (2004) CTCF tethers an insulator to subnuclearsites, suggesting shared insulatormechanisms across species.Mol. Cell13, 291–298

54 Li, J. et al. (2006) NoRC-dependent nucleosome positioning silencesrRNA genes. EMBO J. 25, 5735–5741

55 Li, J. et al. (2005) The chromatin remodeling complex NoRC controlsreplication timing of rRNA genes. EMBO J. 24, 120–127

56 Santoro, R. et al. (2002) The nucleolar remodeling complex NoRCmediates heterochromatin formation and silencing of ribosomal genetranscription. Nat. Genet. 32, 393–396

57 Strohner, R. et al. (2001) NoRC – a novel member of mammalian ISWI-containing chromatin remodeling machines. EMBO J. 20, 4892–4900

58 Strohner, R. et al. (2004) Recruitment of the nucleolar remodelingcomplexNoRC establishes ribosomal DNA silencing in chromatin.Mol.Cell. Biol. 24, 1791–1798

155

Review Trends in Genetics April 2011, Vol. 27, No. 4

59 Zhou, Y. et al. (2002) The chromatin remodeling complex NoRC targetsHDAC1 to the ribosomal gene promoter and represses RNApolymerase I transcription. EMBO J. 21, 4632–4640

60 Guetg, C. et al. (2010) The NoRC complex mediates theheterochromatin formation and stability of silent rRNA genes andcentromeric repeats. EMBO J. 29, 2135–2146

61 Espada, J. et al. (2007) Epigenetic disruption of ribosomal RNA genesand nucleolar architecture in DNA methyltransferase 1 (Dnmt1)deficient cells. Nucleic Acids Res. 35, 2191–2198

62 Klose, R.J. and Bird, A.P. (2006) Genomic DNA methylation: the markand its mediators. Trends Biochem. Sci. 31, 89–97

63 van Steensel, B. and Dekker, J. (2010) Genomics tools for unravelingchromosome architecture. Nat. Biotechnol. 28, 1089–1095

64 Andersen, J.S. et al. (2002) Directed proteomic analysis of the humannucleolus. Curr. Biol. 12, 1–11

65 Pendle, A.F. et al. (2005) Proteomic analysis of the Arabidopsisnucleolus suggests novel nucleolar functions. Mol. Biol. Cell 16,260–269

66 Scherl, A. et al. (2002) Functional proteomic analysis of humannucleolus. Mol. Biol. Cell 13, 4100–4109

67 Kim, S.H. et al. (2009) Aberrant mRNA transcripts and the nonsense-mediated decay proteins UPF2 and UPF3 are enriched in theArabidopsis nucleolus. Plant Cell 21, 2045–2057

68 Kim, S.H. et al. (2010) Plant U13 orthologues and orphan snoRNAsidentified by RNomics of RNA from Arabidopsis nucleoli.Nucleic AcidsRes. 38, 3054–3067

69 Dekker, J. et al. (2002) Capturing chromosome conformation. Science295, 1306–1311

70 Dostie, J. et al. (2006) Chromosome conformation capture carbon copy(5C): a massively parallel solution for mapping interactions betweengenomic elements. Genome Res. 16, 1299–1309

71 Gondor, A. et al. (2008) High-resolution circular chromosomeconformation capture assay. Nat. Protoc. 3, 303–313

72 Simonis, M. et al. (2006) Nuclear organization of active and inactivechromatin domains uncovered by chromosome conformation capture-on-chip (4C). Nat. Genet. 38, 1348–1354

73 Vassetzky, Y. et al. (2009) Chromosome conformation capture (from 3Cto 5C) and its ChIP-based modification. Methods Mol. Biol. 567, 171–

18874 van Steensel, B. and Henikoff, S. (2000) Identification of in vivo DNA

targets of chromatin proteins using tethered dam methyltransferase.Nat. Biotechnol. 18, 424–428

75 Vogel, M.J. et al. (2007) Detection of in vivo protein–DNA interactionsusing DamID in mammalian cells. Nat. Protoc. 2, 1467–1478

76 Ghetti, A. et al. (1992) hnRNP I, the polypyrimidine tract-bindingprotein: distinct nuclear localization and association with hnRNAs.Nucleic Acids Res. 20, 3671–3678

77 Huang, S. et al. (1997) The dynamic organization of the perinucleolarcompartment in the cell nucleus. J. Cell Biol. 137, 965–974

156

78 Matera, A.G. et al. (1995) A perinucleolar compartment contains severalRNA polymerase III transcripts as well as the polypyrimidine tract-binding protein, hnRNP I. J. Cell Biol. 129, 1181–1193

79 Hiscox, J.A. (2002) The nucleolus – a gateway to viral infection? Arch.Virol. 147, 1077–1089

80 Hiscox, J.A. (2007) RNA viruses: hijacking the dynamic nucleolus.Nat.Rev. Microbiol. 5, 119–127

81 Marciniak, R.A. et al. (1998) Nucleolar localization of the Wernersyndrome protein in human cells. Proc. Natl. Acad. Sci. U.S.A. 95,6887–6892

82 Tamanini, F. et al. (2000) The fragile X-related proteins FXR1P andFXR2P contain a functional nucleolar-targeting signal equivalent tothe HIV-1 regulatory proteins. Hum. Mol. Genet. 9, 1487–1493

83 Willemsen, R. et al. (1996) Association of FMRP with ribosomalprecursor particles in the nucleolus. Biochem. Biophys. Res.Commun. 225, 27–33

84 Isaac, C. et al. (2000) Characterization of the nucleolar gene product,treacle, in Treacher Collins syndrome. Mol. Biol. Cell 11, 3061–3071

85 Yankiwski, V. et al. (2000) Nuclear structure in normal and Bloomsyndrome cells. Proc. Natl. Acad. Sci. U.S.A. 97, 5214–5219

86 Woo, L.L. et al. (2006) The Rothmund-Thomson gene product RECQL4localizes to the nucleolus in response to oxidative stress. Exp. Cell Res.312, 3443–3457

87 Heiss, N.S. et al. (1999) Dyskerin localizes to the nucleolus and itsmislocalization is unlikely to play a role in the pathogenesis ofdyskeratosis congenita. Hum. Mol. Genet. 8, 2515–2524

88 Lipton, J.M. and Ellis, S.R. (2009) Diamond Blackfan anemia 2008-2009: broadening the scope of ribosome biogenesis disorders. Curr.Opin. Pediatr. 22, 12–19

89 Drygin, D. et al. (2010) TheRNA polymerase I transcriptionmachinery:an emerging target for the treatment of cancer. Annu. Rev. Pharmacol.Toxicol. 50, 131–156

90 Kruger, T. and Scheer, U. (2010) p53 localizes to intranucleolar regionsdistinct from the ribosome production compartments. J. Cell Sci. 123,1203–1208

91 Rubbi, C.P. and Milner, J. (2000) Non-activated p53 co-localizes withsites of transcription within both the nucleoplasm and the nucleolus.Oncogene 19, 85–96

92 Lohrum, M.A. et al. (2000) Identification of a cryptic nucleolar-localization signal in MDM2. Nat. Cell Biol. 2, 179–181

93 Tao, W. and Levine, A.J. (1999) P19(ARF) stabilizes p53 by blockingnucleo-cytoplasmic shuttling ofMdm2. Proc. Natl. Acad. Sci. U.S.A. 96,6937–6941

94 Rizos, H. et al. (2000) Two arginine rich domains in the p14ARF tumoursuppressor mediate nucleolar localization. Oncogene 19, 2978–2985

95 Schlosser, I. et al. (2003) A role for c-Myc in the regulation of ribosomalRNA processing. Nucleic Acids Res. 31, 6148–6156

96 Drygin, D. et al. (2009) Anticancer activity of CX-3543: a directinhibitor of rRNA biogenesis. Cancer Res. 69, 7653–7661