Nuclear Organization and Genome Function

CB28CH07-Corces ARI 11 September 2012 12:17

Nuclear Organization andGenome FunctionKevin Van Bortle and Victor G. CorcesDepartment of Biology, Emory University, Atlanta, Georgia 30322;email: [email protected]

Annu. Rev. Cell Dev. Biol. 2012. 28:163–87

First published online as a Review in Advance onAugust 17, 2012

The Annual Review of Cell and DevelopmentalBiology is online at cellbio.annualreviews.org

This article’s doi:10.1146/annurev-cellbio-101011-155824

Copyright c© 2012 by Annual Reviews.All rights reserved

1081-0706/12/1110-0163$20.00

Keywords

CTCF, polycomb, TFIIIC, cohesin, epigenetics

Abstract

Long-range interactions between transcription regulatory elementsplay an important role in gene activation, epigenetic silencing, andchromatin organization. Transcriptional activation or repression of de-velopmentally regulated genes is often accomplished through tissue-specific chromatin architecture and dynamic localization between ac-tive transcription factories and repressive Polycomb bodies. However,the mechanisms underlying the structural organization of chromatinand the coordination of physical interactions are not fully understood.Insulators and Polycomb group proteins form highly conserved multi-protein complexes that mediate functional long-range interactions andhave proposed roles in nuclear organization. In this review, we explorerecent findings that have broadened our understanding of the func-tion of these proteins and provide an integrative model for the roles ofinsulators in nuclear organization.

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Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 164NUCLEAR ORGANIZATION AND

GENOME FUNCTION . . . . . . . . . . 164Differentiation, Replication,

and Genome Stability . . . . . . . . . . . 165Genomic Strategies . . . . . . . . . . . . . . . . 165Mediators of Nuclear

Organization . . . . . . . . . . . . . . . . . . . 166INSULATORS. . . . . . . . . . . . . . . . . . . . . . . 166

Composition and Evolution . . . . . . . . 167Distribution and Chromatin

Structure . . . . . . . . . . . . . . . . . . . . . . . 169Long-Range Interactions . . . . . . . . . . . 170Role in Nuclear Organization . . . . . . 172

POLYCOMB . . . . . . . . . . . . . . . . . . . . . . . . 172Composition and Evolution . . . . . . . . 173Distribution and Chromatin

Structure . . . . . . . . . . . . . . . . . . . . . . . 173Long-Range Interactions . . . . . . . . . . . 174Role in Nuclear Organization . . . . . . 175

TRANSCRIPTION FACTORIES . . . . 176Composition and Evolution . . . . . . . . 176Enhancer-Promoter Interactions . . . 177

PERSPECTIVES . . . . . . . . . . . . . . . . . . . . 178

INTRODUCTION

Eukaryotic genomes are packaged into higher-order chromatin structures and ultimatelyorganized in a manner that functionally re-lates to gene expression. Understanding themechanisms and molecular players involved ingenome organization is therefore essential tofully comprehend the fundamental relationshipbetween nuclear organization and genomefunction. Insulators are multiprotein DNAcomplexes proposed to underlie nuclear archi-tecture on the basis of their ability to facilitatelong-range physical interactions, to interactwith nuclear substructures, and to cluster intonuclear foci termed insulator bodies. However,spatiotemporal expression and repression ofgenes pertinent to development and cell-typespecification involve the function of additionalregulatory elements, such as enhancers and

Polycomb response elements (PREs), whichsuggests additional factors may also play a rolein genome organization. It is then possible thatthe 3D arrangement of chromatin in the nuclearspace is in part a consequence of function, i.e.,the interaction of regulatory sequences withgene promoters, and in part due to structuralelements whose role is to establish interactionsbetween specific sequences to effect partic-ular patterns of gene expression. The recentdevelopment of unbiased, high-throughputmethods for mapping protein binding sitesand genome-wide interactions has allowed anunprecedented look into the inner workings ofgenome biology, and new studies have providedvaluable insights into the roles of insulators andchromatin structure in nuclear organization.In this review we highlight the relationshipbetween nuclear organization and genomefunction and emphasize the dynamic interplayamong chromatin insulators, transcriptionactivation, and Polycomb (Pc)-mediated re-pression in creating and/or maintaining a 3Darrangement of the chromatin that is con-ducive to the establishment of patterns of geneexpression required for cell-type specification.

NUCLEAR ORGANIZATION ANDGENOME FUNCTION

Eukaryotic cells are tasked with packaging thegenome several thousandfold into the confinesof the cell nucleus while maintaining geneaccessibility and chromatin structure that ac-commodates highly dynamic processes, includ-ing gene transcription, replication, and DNArepair. Interphase chromosomes are organizedinto discrete territories that are distributednonrandomly with respect to the nucleus andwith respect to other chromosomes, and whoseplacement can influence the potential for transinteractions and dictate whether a genomiclocus is in an active or repressive nuclear en-vironment (Cremer & Cremer 2010, Fraser &Bickmore 2007). The nucleus also harborsseveral discrete subnuclear foci, termed nu-clear bodies, which are dynamically regulatedstructures that facilitate greater efficiency of

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many nuclear processes (Mao et al. 2011). Forexample, active genes can relocate from chro-mosome territories (Branco & Pombo 2006,Chambeyron & Bickmore 2004) and clusterinto subnuclear foci termed transcription fac-tories for gene expression (Chakalova & Fraser2010). Gene silencing is also accomplishedthrough recruitment to repressive nuclearstructures, and most biological processes aresimilarly compartmentalized into analogousnuclear bodies, which indicates an importantrelationship between nuclear organization andgenome function.

Differentiation, Replication,and Genome Stability

The nonrandom order and significance ofgenome organization are perhaps best high-lighted by its relationship to cellular differen-tiation, replication, and genome stability. Thepathway from pluripotency to differentiated tis-sues is accompanied by changes in epigenomiclandscapes, genome compaction, and some de-gree of chromosomal reorganization (Ahmedet al. 2010, Mikkelsen et al. 2007, Vastenhouwet al. 2010, Wiblin et al. 2005). Developmentalgenes are differentially targeted to transcrip-tionally active or transcriptionally repressivenuclear substructures, and differentiation isassociated with restructuring of interactionsbetween chromatin and the nuclear lamina(Peric-Hupkes et al. 2010). An increase ingenome compaction may accommodate the or-ganization of nuclear foci associated with tran-scription, DNA repair, replication, and splicingwhile restricting the complexity of genomefunction by concealing irrelevant transcriptionfactor binding sites (Meister et al. 2011). Nu-clear organization also plays a critical role in or-ganizing DNA replication into discrete subnu-clear compartments (Berezney et al. 2000) andin maintaining genome integrity. DNA damagegives rise to the accumulation of repair andDNA damage checkpoint proteins concomitantto increased chromatin accessibility (Kruhlaket al. 2006, Lisby et al. 2003), and studiesin both yeast and human cells demonstratean important relationship between nuclear

organization, DNA repeat stability, andtelomere protection (for reviews see Mekhail& Moazed 2010, Nagai et al. 2011). Char-acterizing the mechanisms involved in 3Dgenome organization is therefore essentialfor understanding the apparent fundamentalrelationship between nuclear organization andcellular function.

Genomic Strategies

Microscopy studies have been invaluable inrevealing insights into the distribution and or-ganization of chromosomes in individual cellsand their relationship with gene regulation.However, to break down the 3D organizationof chromosomes and the relationship betweennuclear organization and underlying chro-matin proteins, new techniques were requiredthat exceeded the resolution and throughputlimits imposed by traditional light microscopy(Table 1). The advent of the chromosome con-formation capture (3C) technique described byDekker et al. (2002) marked the first approachto effectively map physical chromosomalinteractions across the genome. Although 3Chas been useful in identifying locus-specificinteractions between regulatory elements andtarget genes (Dekker et al. 2002, Tolhuis et al.2002), derivations of the 3C technique havebeen introduced to extend the approach inan unbiased, high-throughput, genome-widefashion. For example, the Hi-C methodintegrates an extended 3C protocol withmassively parallel DNA sequencing, therebycapturing all genome-wide interactions at aresolution limited by the depth of sequencing(Lieberman-Aiden et al. 2009). Initial Hi-Canalyses in a human lymphoblastoid cell lineprovided valuable insight into chromatin orga-nization, supporting the fractal globule modelin which chromosomes self-organize into ahierarchy of crumples, or series of globulesgoverned by topological constraints (Grosberget al. 1988, Lieberman-Aiden et al. 2009,Mirny 2011). Subsequent computational mod-eling supports the existence of chromosometerritories and transcriptional foci and revealed

www.annualreviews.org • Nuclear Organization and Genome Function 165

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Table 1 Genomic tools for assaying chromatin occupancy, structure, and organization

Method DescriptionChIP-PCR Identify binding status of chromatin-associated protein at selected genomic loci; PCR

against genomic region of interest in DNA fragments obtained by chromatinimmunoprecipitation (ChIP) against protein of interest

ChIP-chip Identify the genome-wide binding profile of a chromatin-associated protein; microarrayhybridization of protein-associated DNA fragments enriched for by ChIP

ChIP-seq Identify the genome-wide binding profile for chromatin-associated protein;high-throughput sequencing of protein-associated DNA regions enriched for by ChIP

3C Measure the interaction frequency between two selected genomic loci; quantitative PCRagainst ligated restriction fragments of interest

4C Map physical interactions between a selected locus and the entire genome; microarrayhybridization or high-throughput sequencing of interacting sequences captured byinverse PCR

5C Map physical interactions between genomic loci within a targeted locus;ligation-mediated amplification and quantitation of interacting DNA fragments

Hi-C Measure genome-wide interaction frequencies between all genomic loci;high-throughput sequencing of interacting sequences obtained by purification ofbiotin-marked ligation junctions

ChIA-PET Map genome-wide interactions bound by chromatin-associated protein; high-throughputsequencing of ligated fragments enriched for by ChIP against protein of interest

new insights into the relationship betweenchromatin organization and CCCTC-bindingfactor (CTCF) as well as the nuclear lamina(Yaffe & Tanay 2011). However, further con-clusions about the chromosome topology andnuclear organization of chromatin in humancells will require higher resolution, which islikely to be obtained in the near future withgreater sequencing depth.

Mediators of Nuclear Organization

Determination of how interphase chromo-somes are anchored within the nuclear spaceand which proteins mediate structural arrange-ments conducive to gene regulation and locusplasticity remains a critical hurdle to under-standing the mechanisms that regulate genomefunction. Fortunately, genome-wide mappingof chromatin-associated proteins has increasedat an extraordinary pace during the past fewyears thanks to the ENCyclopedia of DNAElements (ENCODE) Projects (Birney et al.2007, Celniker et al. 2009) and the increas-ingly affordable option of high-throughput

sequencing. Analyses combining the 3D or-ganization of interphase chromosomes—withgenome-wide binding profiles of chromatin-associated factors—may ultimately establishwhich proteins functionally mediate nuclearorganization. Information from microscopyand biochemical studies, combined with recentgenome-wide mapping, has implicated multi-ple factors, including chromatin insulators, Pccomplexes, and noncoding RNAs, as havingroles in domain formation and chromatinorganization that we consider in this review.

INSULATORS

Chromatin insulators originally were defined asregulatory elements that recruit proteins to es-tablish boundaries between adjacent chromatindomains. Insulators were also shown to blockthe communication between enhancers andnearby promoters in an orientation-dependentmanner, which led to intuitive models in whichinsulators limit the promiscuity of enhancers.However, further characterization of thesesequences from yeast to humans has revealed


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that insulators mediate long-range intra- andinterchromosomal interactions, colocalize tosubnuclear foci called insulator bodies as wellas transcription factories, and preferentiallycluster in trans, as revealed by Hi-C compu-tational modeling in both yeast and humans.These findings suggest that insulators serve agreater purpose in chromatin organization.

Composition and Evolution

Studies of chromatin insulators in mammalshave long been restricted to the highly con-served insulator protein CTCF, which untilrecently was the only characterized proteincapable of insulator activity in humans. How-ever, insulator activity in yeast involves tRNAgenes and the transcription factor TFIIIC,and this role now appears to be conserved inmammals (Ebersole et al. 2011, Raab et al.2012). Concurrent research on insulators inyeast, Drosophila, and mammalian systemstherefore suggests that these elements servean evolutionarily conserved role in generegulation and nuclear organization.

Insulator protein CTCF. CTCF contains acentral domain composed of 11 zinc fingersand is ubiquitously expressed (Klenova et al.1993). Early biochemical studies demonstrated>93% amino acid identity between human andchicken CTCF proteins (Filippova et al. 1996),and studies in Drosophila later identified an or-thologous CTCF factor with a similar domainstructure and conserved insulator function(Moon et al. 2005), which suggests this zinc-finger protein plays a vital, highly conservedrole in nuclear biology. Remarkably, CTCFprimarily targets a highly similar core consensussequence from Drosophila to humans, despiteits ability to bind a variety of DNA sequences(Holohan et al. 2007). The proteins with whichCTCF associates and the variant sequences it isable to bind have been suggested to underlie thenumerous roles in which CTCF has been im-plicated (Weth & Renkawitz 2011, Zlatanova &Caiafa 2009), such as X-chromosome inacti-vation, V(D)J rearrangement, and chromatin

insulation. Many CTCF binding sites recruitthe cohesin complex (Figure 1), which isrequired for functional CTCF insulator activ-ity (Nativio et al. 2009, Wendt et al. 2008). Thecohesin complex forms a ring-shaped struc-ture and mediates cohesion between sister chro-matids from S-phase until mitosis, which sug-gests that cohesin may specifically stabilizechromatin loops arranged by CTCF through asimilar mechanism. CTCF also interacts withYin and yang 1 (YY1), a transcription fac-tor involved in X-chromosome inactivation(Donohoe et al. 2007) capable of recruiting Pccomplexes (Wilkinson et al. 2006), and CTCF-mediated insulator activity at the H19/Igf2 re-quires the SNF2-like chromodomain helicaseprotein CHD8 (Figure 1) (Ishihara et al. 2006),the DEAD-box RNA helicase p68, and its as-sociated RNA (SRA) (Yao et al. 2010).

Insulators in Drosophila melanogaster.Drosophila insulator elements and their asso-ciated proteins have been particularly wellcharacterized thanks to in vivo insulatoractivity reporter assays made easy by the ge-netic manipulations available in the fly modelsystem. In addition to the Drosophila homologof CTCF (dCTCF), several insulator proteinshave been identified, including Suppressor ofHairy wing [Su(Hw)], Boundary Element As-sociated Factor (BEAF-32), and GAGA factor(GAF) (Figure 1) (Gurudatta & Corces 2009).Contrary to what happens in vertebrates,the Drosophila cohesin complex localizes totranscriptionally active genes independentlyof dCTCF (Misulovin et al. 2008). Insteadof cohesin, Drosophila insulator activity relieson fly-specific insulator proteins CentrosomalProtein 190 kDa (CP190) and Modifier ofModg4 [Mod(mdg4)], both of which con-tain BTB/POZ domains and are capable offorming stable multimers in vitro (Bonchuket al. 2011, Gerasimova et al. 2007, Ghoshet al. 2001). Genome-wide localization studiessuggest that dCTCF tandemly aligns withSu(Hw), BEAF-32, and CP190 at many sitesthroughout the genome, perhaps representinga unifying and synergistic role in facilitating


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GAGA insulator

GAGA

BEAF insulator

BEAF

dCTCF insulator

dCTCF

TFIIIC insulator

Su(Hw) insulator

Su(Hw)

b

a

CTCF insulator

CTCF TFIIIC

CHD8

CP190

Mod(mdg4)2.2

CP190

Mod(mdg4)??

CP190

Mod(mdg4)??

CP190

Mod(mdg4)2.2

Cohesin

YY1

Condensin

Figure 1Diagram showing the structure of different vertebrate and Drosophila insulators. (a) Structure of the vertebrate CCCTC-binding factor(CTCF) and TFIIIC insulators. Indicated are factors associated with CTCF, such as cohesin, CHD8, and YY1, and with TFIIIC.(b) Each Drosophila insulator subclass contains a different binding protein that may define the specific function of the correspondingsubclass. All insulators share the common protein Centrosomal Protein 190 kDa (CP190), although the role of this protein in thefunction of the GAGA insulator has not been demonstrated experimentally. In addition, all subclasses may also have one Modifier ofmdg4 [Mod(mdg4)] isoform. The gypsy/Suppressor of Hairy wing [Su(Hw)] insulator contains Mod(mdg4)2.2. The dCTCF andBoundary Element Associated Factor (BEAF) insulators lack this isoform but contain a different variant of Mod(mdg4). GAGA hasbeen shown to interact with Mod(mdg4)2.2.

chromosomal interactions and genome or-ganization (Negre et al. 2010, Van Bortleet al. 2012). Interestingly, insulator activityin Drosophila also relies on components of theRNA interference (RNAi) machinery (Lei &Corces 2006, Moshkovich et al. 2011), thoughthe mechanistic relationship remains poorlyunderstood.

tDNA and TFIIIC. tRNA genes were firstdemonstrated to function as boundary ele-ments flanking the repressed HMR locus inSaccharomyces cerevisiae and subsequently at thepericentromeric regions of Schizosaccharomycespombe (Donze et al. 1999, Scott et al. 2006).The conservation of tDNA as an insulatorelement has been further extended by thedemonstration that tRNA genes functionas barrier and enhancer-blocking insulatorsin transgenic reporter assays in human cells

(Raab et al. 2012). Analysis of the mat locusin S. pombe revealed that a repeat of B-boxelements, which are highly conserved in-tragenic promoter elements in tRNA genesthat recruit RNA polymerase III (RNAPIII)transcription initiation factor TFIIIC, were re-sponsible for barrier activity (Figure 1) (Nomaet al. 2006). Mutations in TFIIIC and TFIIIB,but not RNAPIII, affected insulator activityin S. cerevisiae, which suggests that insulatoractivity occurs independent of RNAPIII tran-scription (Donze & Kamakaka 2001). AlthoughTFIIIC is sufficient for gene insulation alone,insulator activity is strengthened by TFIIIBand utilizes chromatin remodelers, possibly toevict histones at tDNA insulators (Valenzuelaet al. 2009). Interestingly, in yeast and humans,TFIIIC binds many regions devoid ofRNAPIII, called Extra TFIIIC (ETC) loci(Moqtaderi & Struhl 2004), and these sitesare associated with the cohesin complex and


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localize near CTCF-binding sites in mouseembryonic stem cells and humans (Carriereet al. 2012, Moqtaderi et al. 2010). TFIIIC sitesand tRNA genes also function as loading sitesfor the highly conserved condensin complex inyeast, which suggests insulators serve an im-portant role in chromatin architecture duringboth interphase and mitosis (D’Ambrosio et al.2008).

Distribution and Chromatin Structure

Genome-wide localization studies by ChIP-chip and ChIP-seq have revealed that insulatorsare dispersed throughout eukaryotic genomes.CTCF is bound to thousands of independentsites in Drosophila (Bushey et al. 2009, Holohanet al. 2007, Negre et al. 2010) and tens ofthousands of sites in human cell lines (Barskiet al. 2007, Cuddapah et al. 2009, Kim et al.2007), consistent with a global role in genomefunction. ChIP-chip analysis further showedthat CTCF colocalizes with cohesin at morethan half of its sites (Parelho et al. 2008,Rubio et al. 2008, Wendt et al. 2008), whichsuggests many CTCF sites are likely capableof functional insulator activity. Meanwhile,recent genome-wide localization of TFIIICoccupancy in mouse embryonic stem cells re-vealed that although all three TFIIIC subunitsco-occupy <300 tRNA genes, as many as 2,200independent TFIIIC-bound ETC loci aredispersed throughout the mouse genome (Car-riere et al. 2012). Remarkably, as many as 85%of ETC loci lie within 20 kb of CTCF-bindingsites, and cohesin subunits Smc1A and Smc3were enriched at the ETC loci specifically,which suggests CTCF and TFIIIC distributionand insulator activity may be intimately asso-ciated. Evidence that suggests insulators mayindeed collaborate comes from recent map-ping of insulator proteins in D. melanogaster(Schwartz et al. 2012, Van Bortle et al. 2012).CTCF tandemly aligns with other classesof Drosophila insulators, including Su(Hw)and BEAF-32, and these multi-insulatorcomplexes then become enriched for CP190,Mod(mdg4), and additional co-factors (Van

Bortle et al. 2012). Alignment of insulatorssuggests CTCF may cluster with other distinctinsulator proteins to efficiently recruit essentialcofactors important for establishing a robustinsulator complex and perhaps capable of main-taining stable, long-range interactions. Futurestudies may uncover a similar relationshipbetween CTCF and TFIIIC in humans.

The distribution of CTCF, tDNA, andaligned insulators correlates with recentmapping of physical domain borders in bothDrosophila and mammals. For example, Cavalliand colleagues recently utilized an indepen-dent, high-throughput 3C derivative (3C-seq)to explore the 3D folding and functional or-ganization principles of the Drosophila genome(Sexton et al. 2012). Their data suggest eu-karyotic genomes are partitioned into physicaldomains that can be clustered on the basis ofstrong statistical association with linear epige-nomic profiles. Physical domains were cate-gorized as active, which correlates with activehistone marks; null, which comprises large tran-scriptionally repressive regions lacking silentchromatin marks; Pc domains associated withhistone H3 K27 trimethylation (H3K27me3)repression; or HP1/centromeric domains asso-ciated with classical heterochromatin. Physicaldomains are demarcated sharply, and contactswithin domains abruptly decay at positionscorresponding to physical domain edges.Remarkably, insulators are highly enriched atdomain borders, and hierarchical clusteringrevealed recurrent combinations of insulatorsand active histone marks that are present at allcombinations of physical boundaries (e.g., evenbetween two similarly annotated physical do-mains, such as null–null). Analogous mappingof physical domains reveals similar partitioningof the human genome as well as enrichment forCTCF and tRNA genes at chromatin domainborders (Dixon et al. 2012, Nora et al. 2012).

The enrichment of insulator proteins atall combinations of physical domain bordersbegs the question of what role insulators playin delimiting discrete chromatin domains.Earlier correlations for CTCF and tDNAs atthe borders of repressed chromatin domains,


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typically in the form of H3K27me3 (Cuddapahet al. 2009, Negre et al. 2010), have led manyto believe insulators function as chromatinboundaries, where they simply serve to preventthe spread of heterochromatin into flankingchromatin domains (Bartkuhn et al. 2009). Re-cent compounding evidence strongly suggeststhat despite this correlation, endogenous insu-lator proteins are not important for restrictingthe spread of silencing chromatin. For example,RNAi depletion of CTCF in D. melanogasterdoes not lead to significant alterations in chro-matin structure or gene activity (Schwartz et al.2012, Van Bortle et al. 2012). Similar findingsare reported for depletion of other insulatorproteins (Van Bortle et al. 2012), and mutationsin Drosophila insulator protein Su(Hw) havelittle consequence on several regions tested forgene activity (Soshnev et al. 2012). Meanwhile,CTCF is not required for barrier activity atthe well-characterized β-globin locus (Barkess& West 2012, Recillas-Targa et al. 2002, Yaoet al. 2003), together suggesting endogenousinsulator proteins are not involved in hetero-chromatin barrier formation. The discoverythat insulators are present at all combinationsof physical domain borders rather than justrepressive H3K27me3 domains perhaps rein-forces the notion that chromatin insulators areplaying a paramount role, beyond the scope ofbarrier activities observed in transgenic assaysthat remove insulators from their natural ge-nomic context. Instead, the ability to facilitatelong-range interactions appears to be the defin-ing feature of insulators, underlying their rolein nuclear organization and genome function.

Long-Range Interactions

Insulators have also been defined by theirability to impede the interaction betweenpromoters and distal enhancers in a direction-dependent manner. Meanwhile, observations atnumerous genomic loci across species suggestthat insulators influence chromatin structureby establishing chromatin loops throughphysical interactions. Concurrent modelstherefore proposed that insulators evolved to

ensure the fidelity of enhancers and their targetpromoters in vivo by establishing chromatinloops and thereby dictating the potential forenhancer-promoter interactions. However,accumulating data suggest that insulators facil-itate long-range inter- and intrachromosomalinteractions across the genome, includingbridging connections between distant en-hancers and target promoters, which suggeststhat local determination of enhancer-promoterinteraction represents only part of a moresignificant role in chromosome organization.

The enhancer-blocking activities of CTCFhave been best characterized at the chicken β-globin locus and the murine H19/Igf2 imprintedlocus, both of which have been extensivelyreviewed (Phillips & Corces 2009). These lociprovide ideal scenarios for studying the role ofinsulators in allele-specific and developmentalcell-type-specific gene regulation and chro-matin architecture, and 3C experiments suggestCTCF underlies chromatin contacts at both ge-nomic loci (Kurukuti et al. 2006, Splinter et al.2006). Recent application of 3C has revealedthat CTCF also underlies developmentalhigher-order architecture at the conservedhomeobox gene A (HOXA) locus in mouse andhumans (Kim et al. 2011). Specifically, CTCFand cohesin contribute to reorganization andselective gene activation at HOXA by parti-tioning silenced genes through chromatin loopformation upon differentiation. Furthermore,pluripotency factor OCT4 antagonizes cohesinloading at the CTCF binding site, therebydemonstrating developmental regulation ofinsulator activity and gene expression by in-hibiting chromosome loop formation. Studiesin D. melanogaster suggest insulators have a con-served role in developmental coordination ofgene expression and chromatin structure. Forexample, genome-wide mapping revealed thatdCTCF and Drosophila-specific insulator pro-teins are regulated through DNA binding andrecruitment of CP190 during the ecdysone hor-mone response in Kc cells (Wood et al. 2011). Inaddition, 3C analysis at the ecdysone-inducedEip75B gene further revealed a developmentallyregulated dCTCF site, wherein recruitment of


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CP190 and enhanced chromatin looping uponecdysone stimulation suggested alterations inEip57B locus chromatin organization.

Numerous studies have focused on the roleof insulators in locus-specific gene regulationand chromatin architecture, but the emergingpicture of insulators in genome-wide nuclearorganization requires global analyses of chro-matin interactions. The first genome-wide mapof CTCF-mediated functional interactionshas been obtained by combining ChIP withhigh-throughput sequencing of enriched chro-matin interactions (Handoko et al. 2011). Theauthors identified ∼1,500 cis and ∼330 transinteractions facilitated by CTCF in mouse em-bryonic stem cells and classified them into fivecategories on the basis of distinct epigeneticpatterns. CTCF interactions harbor chromatinloops enriched for active or repressive chro-matin signatures, which suggests that CTCFharnesses clusters of genes with coordinatedexpression (Figure 2). CTCF interactions also

create chromatin hubs conducive to enhancerand promoter activities, in support of recentevidence suggesting that CTCF and cohesinunderlie enhancer-promoter interactions atthe INFG and MHC-II loci (Hadjur et al.2009, Majumder & Boss 2011). The authorstherefore speculate that insulators may insteadfacilitate cell-type specific enhancer-promoterinteractions. Ultimately, Handoko et al. (2011)provide a genome-wide interrogation ofCTCF interactions and reveal several modesthrough which CTCF functionally organizesthe genome.

Genome-wide interrogations of intra-and interchromosomal interactions in yeastand humans independently demonstrate thattDNA insulators also underlie long-rangegenomic interactions. Noble and colleaguesrecently employed a high-throughput 3C-based technique to query the 3D organizationof the S. cerevisiae genome (Duan et al. 2010).tRNA genes were significantly enriched for

CTCF CTCFCTCF

a b c

Figure 2Structure of some of the domains created by interactions between CCCTC-binding factor (CTCF) insulators in mouse embryonicstem cells. Actively transcribed genes are represented by a blue arrow and silenced genes by a red inhibition line. Nucleosomes and thehistone tails are represented in gray; active histone modifications are indicated as blue spheres and repressive modifications as redspheres. DNA is represented in black and CTCF as brown ovals. (a) CTCF forms a loop to separate a domain containing active histonemodifications and transcribed genes from repressive marks and silenced genes. (b) CTCF forms a loop to separate a domain containingrepressive histone modifications and silenced genes from active marks and transcribed genes. (c) CTCF forms a loop containingnucleosomes enriched in mono- and dimethylated H3K4, and trimethylated H3K4 at the boundaries of the loops, whereas the activetranscription modification H3K36me3 and repressive H3K27me3 marks are observed outside the loops on opposite sides.


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interactions with other tRNA genes, whichsuggests that insulator-to-insulator interac-tions are a conserved feature of eukaryoticgenome organization. Hierarchical clusteringrevealed two clusters of colocalizing tRNAgenes, one consistent with previously de-scribed nucleolar localization (Thompson et al.2003) and another with centromeres. Similarmapping of interactions at a tDNA insulatorin humans revealed analogous long-rangeinteractions between tDNAs as well as ETCloci, which suggests tRNA genes and TFIIICplay a conserved role in genome organization.Recent findings indicate that TFIIIC bindingsites facilitate condensin binding in S. cerevisiaeand S. pombe (D’Ambrosio et al. 2008) andcolocalize with cohesin in mammals (Carriereet al. 2012), which suggests TFIIIC recruit-ment of condensin and cohesin complexes mayunderlie chromosomal interactions in yeastand humans analogous to those with CTCF.

Role in Nuclear Organization

Microscopy-based analyses of the physicaland functional organization of eukaryoticnuclei have led to the identification of severaldiscrete subnuclear organelles called nuclearbodies, which play host to a variety of nuclearprocesses, including transcription, splicing,processing, and epigenetic regulation (Maoet al. 2011). Nuclear staining of insulatorproteins has shown a clear propensity for insu-lators to concentrate into distinct nuclear foci,a feature that is conserved in yeast (Noma et al.2006), Drosophila (Gerasimova et al. 2000), andmammals (MacPherson et al. 2009). Insulatorsalso interact with and localize to nuclear sub-structures, including the nuclear and nucleolarperipheries, which suggests insulators tetherassociated chromatin to defined nuclear com-partments (Gerasimova et al. 2000, Yusufzaiet al. 2004). These findings, combined withknowledge about insulator-mediated functionalinteractions, have led to models proposing thatinsulators ultimately interact to partition chro-matin into structural and functional domainsthat are physically organized through insulatorbodies. Although tantalizing, the functional

importance and molecular underpinnings ofinsulator bodies remain poorly characterized.

Nuclear organization clearly involvesspatial arrangement of chromosomes whoseposition with respect to the nuclear peripherycorrelates with chromatin structure and geneexpression. Chromatin interactions at thenuclear periphery recently have been mappedin both Drosophila (Pickersgill et al. 2006) andhumans (Guelen et al. 2008), revealing large,sharply defined lamina-associated domains(LADs) that correlate with low gene densityand transcriptional repression. The bordersof LADs in humans are enriched for CTCF(Guelen et al. 2008, Zullo et al. 2012), whichsuggests this protein may separate chromatinenvironments at the nuclear periphery. Map-ping of insulators in D. melanogaster withrespect to the nuclear lamina has also revealeda significant enrichment for the Drosophila-specific Su(Hw) insulator at the borders ofLADs (van Bemmel et al. 2010). Handoko et al.(2011) independently identified the apparentrelationship between lamina and the CTCFinteraction network in mouse embryonic stemcells. Specifically, CTCF loops were depletedwithin LADs but enriched at LAD borders,which supports a model in which CTCForchestrates genome organization with respectto the nuclear lamina. An analogous role forTFIIIC in nuclear organization in yeast hasbeen proposed on the basis of perinuclearstaining of insulator bodies in S. pombe (Nomaet al. 2006). In support of this model, peri-nuclear localization and silencing of the HMRlocus in S. cerevisiae was recently shown torely on nuclear pore proteins that localize to atDNA barrier insulator (Ruben et al. 2011).

POLYCOMB

Genome plasticity and selective expression areessential features of multicellular development,and several early regulatory factors involved inbody patterning and segmentation need to bestrictly regulated to facilitate appropriate de-velopmental decisions. Pc group (PcG) pro-teins are evolutionarily conserved epigenetic


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transcriptional repressors that play an impor-tant role in establishing and maintaining cellfate by influencing the expression status of per-tinent genes. PcG proteins specifically medi-ate the repression of numerous developmentalgenes through posttranslational modification ofhistone proteins, and recent studies demon-strate that Pc activity is involved in a broadscope of cellular processes, including differ-entiation, cell cycle regulation, X-inactivation,and cell signaling (Sawarkar & Paro 2010). Mi-croscopy studies have demonstrated that PcGproteins concentrate into nuclear foci, called Pcbodies, which suggests PcG proteins may alsomediate the nuclear organization of their targetgenes. Here we review recent progress in deter-mining the relationship between Pc and nuclearorganization.


The PcG genes were first discovered aschromatin repressors that maintain silencingof the homeotic regulatory genes throughoutDrosophila development (Paro 1990). Furtherstudies in the fruit fly demonstrated that PcGproteins are recruited through cis-regulatoryelements called PREs (Simon et al. 1993),and a recently identified element at the HoxDlocus in humans facilitating Pc-dependenttranscriptional repression throughout celldifferentiation suggests the mechanism of Pcrecruitment may be conserved (Woo et al.2010). However, PREs are not easily brokeninto obvious DNA consensus sequences asdescribed for insulator proteins, and thefunctional mechanism of Pc targeting remainsunclear, though it appears to involve numerousplayers including DNA-binding proteins,histone posttranslational modification bindingproteins, RNAi machinery proteins, andnoncoding RNAs (Beisel & Paro 2011).

PcG proteins are present in two major com-plexes, Polycomb repressive complex 1 (PRC1)and 2 (PRC2), whose core components arelargely conserved from flies to humans. ThePcG family also includes several additionalproteins that allow for the formation of diverse

Pc chromatin-binding complexes with variableenzymatic activities (Simon & Kingston 2009).PRC2 catalyzes H3K27 di- and trimethyla-tion (H3K27me3, which is associated with tran-scriptional repression) through SET domain–containing subunit EZH1, as well as EZH2depending on cellular context (Beck et al. 2010,Margueron & Reinberg 2011). PRC2 recruit-ment and activity is regulated by core com-ponents and ancillary subunits, such as PHF1,JARID2, and AEBP2, which stimulate PRC2enzymatic activity (Beck et al. 2010, Kim et al.2009, Li et al. 2010, Sarma et al. 2008). PRC1subunits RING1B and BMI1 form a stable het-erodimer capable of catalyzing H2AK119 ubiq-uitylation (H2AK199Ub1) (Cao et al. 2005),which likely underlies PRC1-mediated Pc si-lencing (Wang et al. 2004). There is alsorecent evidence that histone modification–binding proteins containing malignant braintumor (MBT) modules contribute to Pc func-tion. For example, L3MBTL2 interacts withand is required for Pc-mediated repression by aPRC1-like complex in human cells (Trojer et al.2011). Interestingly, the Drosophila MBT pro-tein L(3)mbt localizes specifically to chromatininsulators (Richter et al. 2011), and as we discussbelow, recent evidence suggests insulator activ-ity may play an important role in Pc repression.

Distribution and Chromatin Structure

The genome-wide localization of PcG pro-teins has been studied in several independentChIP-chip and ChIP-seq experiments in bothDrosophila and mammals. Pc complexes localizeto putative PREs in Drosophila and correlatewith broad repressive H3K27me3 domains thatencompass genes involved in major develop-mental pathways (Schwartz et al. 2006, Tolhuiset al. 2006). Most PREs are co-occupied by themajor Pc complexes PRC1 and PRC2, and theoccupancy landscape of PcG proteins changesduring development, consistent with its rolein mediating cell fate restriction by differentialgene silencing (Negre et al. 2006, Oktaba et al.2008). Mapping of PRC1 and PRC2 complexesin mammals shows similar co-occupation of


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developmental pathway genes and displace-ment of PcG proteins during gene activation(Boyer et al. 2006, Bracken et al. 2006, Leeet al. 2006), which suggests Pc repression isa highly conserved feature of multicellulardevelopment. However, PcG proteins alsolocalize to bivalent domains characterizedby overlapping H3K27me3 and H3K4me3encompassing genes poised for activation orrepression upon cellular differentiation inmammals, a feature that is largely absent in flyembryos (Schuettengruber et al. 2009).

Genome-wide mapping of trithorax group(trxG) proteins, which catalyze H3K4 methyl-ation and antagonize Pc repression throughtranscriptional activation, suggests a dynamicinterplay between Pc repression and Trx acti-vation dependent on overlapping recruitmentproteins and the relative levels of Pc- and Trx-associated factors (Kwong et al. 2008, Schuet-tengruber et al. 2009, Schwartz et al. 2010).Interestingly, PRC2 also functionally associateswith numerous noncoding RNAs (ncRNAs)(Zhao et al. 2010). For example, the 2.2-kblong ncRNA HOTAIR serves as a scaffold forboth PRC2 and H3K4 demethylase LSD1complexes, and thereby coordinates targetingof Pc to chromatin while coupling H3K27trimethylation and H3K4 demethylation activ-ities for epigenetic repression (Tsai et al. 2010).Mapping of the genome-wide occupancy ofHOTAIR revealed >800 focal, transcriptionfactor–like binding sites that co-occupiedthe genomic binding profiles for the PRC2subunits EZH2, SUZ12, and H3K27me3.HOTAIR occupancy was maintained uponEZH2 depletion, which suggests HOTAIRactively binds chromatin and may underlie thenucleation of PRC2 domains (Chu et al. 2011).

Long-Range Interactions

PcG proteins have long been shown to concen-trate into nuclear foci called Pc bodies, whosenumber and size change upon cellular differen-tiation (Ficz et al. 2005, Grimaud et al. 2006),which suggests that Pc facilitates genome-wideinteractions that are further compartmentalized

within the nuclear space. Accumulating datasuggest that PcG proteins are indeed involvedin long-range interactions essential for Pcrepression. Combinatorial use of 3C and fluo-rescence in situ hybridization (FISH) showedthat PRE-PRE interaction occurs between thebxd and Fab-7 elements separated by ∼130kb in the Drosophila bithorax complex (BX-C)and that colocalization occurs specificallyin embryonic tissues and cell lines in whichboth the AbdA and Ubx genes are corepressed(Lanzuolo et al. 2007). Long-range associationsdependent on PRC2 core component EZH2at the mammalian GATA-4 locus, which issilenced in undifferentiated human TERA-2cells, have also been observed (Tiwari et al.2008b). Tiwari et al. (2008a) subsequentlydevised a 3C-based approach analogous tochromatin interaction analysis with paired-endtag sequencing (ChIA-PET) and demonstrateda limited number of intra- and interchromo-somal interactions in human TERA-2 cellsdependent on EZH2, which suggests thatPRC2 is involved in mediating long-rangeinteractions in mammals.

The formation of Pc bodies and iden-tification of long-range PRE interactionssupport a global role in genome function, butunderstanding the role of Pc complexes inmultigene regulation and nuclear organizationrequires genome-wide exploration of genomicinteractions. To this end, van Steensel andcolleagues recently adapted chromosome con-formation capture on chip (4C), a 3C derivativethat determines genome-wide interactions ofa given locus (Simonis et al. 2006), to explorewhere Pc domains are able to interact withinthe Drosophila genome (Tolhuis et al. 2011).The authors demonstrate long-range interac-tions between Pc target genes and independentPc and/or H3K27me3 domains in larval braintissue, and further show that Pc interactions aretopologically constrained to a single chromo-some arm. Accordingly, chromosome inversiondramatically altered the interaction profilesrevealed by 4C, but global gene expressionpatterns were relatively unchanged. Althoughspecific interaction partners were altered, Pc


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target genes nevertheless continue to interactwith independent Pc domains, which suggeststhat Pc interactions are flexibly amenable tonew partners for gene repression. Genome-wide mapping of chromosomal interactions bySexton et al. (2012) independently identified30 significant pairs of long-range interactionsbetween Pc domains, thus supporting the roleof PcG proteins in mediating specific long-range associations. However, the mechanismsby which these interactions are establishedand maintained and whether PcG proteinsare directly responsible for mediating physicalinteractions are not clear.

Role in Nuclear Organization

The correlation of long-range chromatin inter-actions with Pc domains provides compellingevidence in support of the possibility that Pccomplexes orchestrate the nuclear organizationof repressed genes, but whether PcG proteinsare required for long-range interactions hasbeen called into question recently. Co-stainingfor insulator protein CTCF and PcG proteinPc2 shows clear colocalization of Pc and insu-lator bodies in HeLa cells (MacPherson et al.2009), and accumulating evidence suggests thatinsulators underlie the colocalization and nu-clear organization of Pc domains (Pirrotta &Li 2011).

The best-studied examples of physical in-teractions involving Pc targets occur betweenelements within Drosophila Hox gene clustersthat have been shown to harbor insulatoractivity, and genome-wide localization studieshave characterized numerous dCTCF insu-lator sites within the Antennapedia (Antp)complex and BX-C (Holohan et al. 2007). Onlyrecently have Pirrotta and colleagues shownthat interactions between copies of the BX-CFab-7 or Mcp elements are not dependenton PREs (Li et al. 2011). Importantly, bothFab-7 and Mcp elements have been shown toharbor enhancer-blocking insulator activities(Gruzdeva et al. 2005, Zhou et al. 1996), and Liet al. (2011) now demonstrate that interactionsdepend on insulators flanking the Mcp and

Fab-7 PREs. A concurrent study independentlyrevealed that the Su(Hw) insulator is capableof dictating PRE-target interactions throughchromatin looping and topological constraintin D. melanogaster (Comet et al. 2011), con-sistent with earlier findings showing that theSu(Hw) insulator facilitates PRE-PRE contactsin trans (Sigrist & Pirrotta 1997). Interestingly,the maintenance of long-range interactionsbetween copies of the Fab-7 element alsorequires components of the RNAi machinery(Grimaud et al. 2006), including those requiredfor insulator activity (Lei & Corces 2006),and recent mapping of RNAi component Arg-onaute2 (AGO2) shows specific colocalizationwith insulator proteins dCTCF and CP190throughout the BX-C (Moshkovich et al. 2011).

Although insulators likely play an impor-tant role in previously described Pc interac-tions, Li et al. (2011) speculate that Pc com-plexes may contribute to the stability of physicalinteraction. In support of this theory, mutationsin PcG proteins significantly reduce the levelof Antp and Abd-B colocalization at Pc bod-ies (Bantignies et al. 2011). Meanwhile, currentmodels suggest Pc mechanistically facilitates re-pression through PRC1-mediated chromatincompaction (Grau et al. 2011), and mapping ofgenome accessibility in D. melanogaster revealsthat H3K27me3 domains are indeed the mostinaccessible (Bell et al. 2010). Together, thesefindings suggest that insulators mediate the in-teractions between Pc targets and that PRCslikely strengthen the association and repressionof Pc domains through histone modificationsand chromatin compaction. In support of thismodel, RNAi depletion of CTCF and otherDrosophila insulator proteins results in loss ofH3K27me3 within associated Pc domains (VanBortle et al. 2012), which suggests that insula-tors are important for maintaining appropriatechromatin architecture within these domains.

The interrogation of physical interactionsbetween distant Hox loci has provided new andimportant insight into the regulation of long-range interactions during development. InD. melanogaster, the homeotic genes Antp andAbd-B, which are corepressed and colocalize


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to Pc bodies in Drosophila embryo heads, arerecruited to separate nuclear compartmentswhen one gene becomes activated (Bantignieset al. 2011). In mammals, the HoxD clusterforms a single interaction domain in murineembryonic tissues in which all genes areinactive and transitions to a bimodal state inembryonic tissues in which HoxD genes aredifferentially expressed, with active genes seg-regated into an active domain (Noordermeeret al. 2011b). This suggests that developmen-tally regulated genes are dynamically targetedto specific nuclear subcompartments, suchas Pc bodies and transcription factories, fortranscriptional repression or activation. Theapparent role of insulators in Pc contacts sug-gests that insulators are likely involved in genelocalization to both transcriptionally repressiveand transcriptionally permissive environments.

TRANSCRIPTION FACTORIES

The organization of transcription withineukaryotic nuclei is far more complex thantraditional textbook models of polymeraserecruitment and gene tracking. Instead, tran-scription is spatially organized into discernablenuclear structures in which multiple RNApolymerases and active genes dynamically lo-calize into nuclear bodies termed transcriptionfactories. The formation of transcriptionallyactive subcompartments presumably allows formore efficient transcription by concentratingthe molecular players, reactants, and DNAsubstrates within a confined nuclear volume.Recent evidence suggests that transcriptionfactories are highly conserved features ofnuclear organization, that long-range chro-mosomal interactions are a hallmark of geneexpression, and that insulators likely play animportant role at transcription factories.


Transcription is a fundamental cellular processcarried out by highly conserved multisubunitRNA polymerases that share a high degree ofhomology in bacteria, archaea, and eukaryotes(Werner & Grohmann 2011). RNA polymerase

I transcription of ribosomal genes is organizedinto a strongly conserved and highly orga-nized nuclear substructure called the nucleolus(Thiry & Lafontaine 2005), which representsthe classical example of transcriptional cluster-ing into a factory structure. Meanwhile, mostprotein-coding genes are transcribed by RNApolymerase II (RNAPII), and several findingsnow support models proposing that RNAPIItranscription occurs at analogous factories. Thelocalization of transcription into discrete siteswas initially identified by detection of nascenttranscripts and by RNAPII staining, which re-vealed a limited number of foci unable to ac-count for the number of active genes in humannuclei (Iborra et al. 1996). Subsequent stud-ies further revealed that transcription factoriesare large and relatively immobile proteinaceousstructures whose numbers vary by cell typeand nuclear morphology (Chakalova & Fraser2010) and that active genes dynamically local-ize to factories for expression in a transcription-dependent manner (Osborne et al. 2004). Re-cent genome-wide interaction assays describedin this review also provide supporting evidencefor the existence of transcription factories andfurther demonstrate that clustering of activegenes is a highly conserved phenomenon. Hi-C modeling of chromosomal contacts revealspreferential clustering and interactions amongactively transcribed genes and active chromatindomains in S. pombe, Drosophila, and humans(Sexton et al. 2012, Tanizawa et al. 2010, Yaffe& Tanay 2011).

How transcription factories are physicallyorganized and how genes are dynamically tar-geted to them remains poorly understood. Tothis end, Cook and colleagues recently isolatedtranscription complexes from human nucleiand identified the proteome of RNAPI, II,and III factories by mass spectrometry (Melniket al. 2011). Each complex was shown to harbora characteristic set of unique proteins, thoughseveral proteins involved in DNA or RNAmetabolism were shared. Whereas most pro-teins isolated from RNAPI complexes overlapthose characteristic of nucleoli, RNAPII fac-tories contain general transcription factors and


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CTCF, consistent with the finding that CTCFunderlies organization of coregulated genes inmammals. Interestingly, RNAPII factories alsocontain repressive histone methyltransferases,including PRC2-core component EZH2,which suggests that the transition from epi-genetic repression to gene activation may notrequire the ejection of PcG proteins. In supportof this possibility, Pc2 was recently shown torelocate from Pc bodies to transcription facto-ries dependent on demethylase KDM4C (Yanget al. 2011). Targeting of Pc2 relies on ncRNAs,including TUG1, which interacts with PRC2and acts as a scaffold at PcG bodies, and NEAT2,which associates with epigenetic regulatorsinvolved in gene activation. Taken together,genes likely are directed to repressive Pcbodies or active transcription factories throughdynamic interplay of PcG and trxG proteins,whose long-range interactions require thefunction of ncRNAs and chromatin insulators.This model, recently highlighted by Pirrotta& Li (2011), is consistent with early findingsin D. melanogaster, wherein mutations in bothPcG and trxG genes were shown to modulatethe activity and nuclear organization mediatedby insulators (Gerasimova & Corces 1998).

Enhancer-Promoter Interactions

As the name suggests, enhancers are regulatoryelements functionally defined by their abilityto activate transcription and to do so regardlessof their location, distance, or orientation withrespect to gene promoters (Banerji et al. 1981).Enhancers underlie complex spatiotemporalregulation of tissue-specific gene expressionand have been characterized by chromatin andtranscription factor signatures in mammaliancells (Barski et al. 2007, Heintzman et al. 2007,Wang et al. 2008). Enhancers are commonlyseparated by large genomic distances from theirassociated promoters, which makes accurate as-signments of enhancer-promoter relationshipsdifficult and suggests that long-range interac-tions are a defining feature of gene regulation.Numerous 3C-based interaction studies havesupported enhancer looping models wherein

enhancers directly contact gene promotersfor activation, and recent models suggestchromatin signatures at enhancers may act asepigenetic signals for transcriptional activation(Ong & Corces 2011). Interestingly, a distinctclass of enhancer elements is also capable ofrecruiting RNAPII and is transcribed intoenhancer-derived RNAs (Kim et al. 2010,Wang et al. 2011). The transcriptional outputof neuronal activity-regulated enhancerspositively correlates with the expression levelsof associated genes (Kim et al. 2010), andalternative models of enhancer function havebeen proposed (Bulger & Groudine 2011),including ones in which enhancers and theirassociated promoters colocalize by virtue ofrecruitment to transcription factories.

Enhancer-promoter interactions and tran-scriptional clustering of active genes consistentwith transcription factory models are furthersupported by ChIA-PET analyses enrichingfor RNAPII-based chromosomal interactions(Li et al. 2012). As many as 65% of RNAPIIbinding sites have been shown to be involvedin a complex network of physical interactionsin human cell lines. RNAPII interactions wereintergenic (e.g., promoter-promoter), and ex-tragenic (e.g., promoter-enhancer), with mostcontacts aggregated into ∼1,500 interactioncomplexes. Multigene complexes typically con-sisted of related and coregulated genes, whichsuggests gene families functionally associatefor cotranscription, whereas single-gene com-plexes tended to associate with tissue-specificor developmentally regulated genes and cell-type-specific enhancer-promoter interactions.The authors ultimately reveal a complexorganization of transcription reflecting theimportance of long-range chromatin inter-actions between coregulated promoters andbetween enhancers and promoters, possibly attranscription factories.

Nevertheless, the role of long-rangeenhancer-promoter interactions in eukaryoticgene activation and how these interactions areorganized is not fully understood. Traditionalmodels propose that enhancers underlie re-cruitment and assembly of the transcription


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machinery at core promoters (Maston et al.2006). However, in the case of the humanFOSL1 gene, histone cross talk between anenhancer and promoter triggers transcrip-tion elongation (Zippo et al. 2009), whichsuggests some enhancers may function byreleasing RNAPII from promoter-proximalpausing. Meanwhile, recent characterization ofchromatin-associated proteins at the human α-globin genes and upstream MCS-R2 enhancersuggest distal enhancers stimulate gene expres-sion by reversing PcG activities (Vernimmenet al. 2011). The MCS-R2 enhancer is requiredfor recruitment of H3K27 demethylase JMJD3,although active chromatin marks indicativeof Trx activity (H3K4me3) were present inthe absence of the enhancer. This supportsa model wherein PcG and Trx complexesdynamically associate with target genes andenhancers promote gene induction by favoringTrx activity. This is consistent with ChIA-PETmapping of RNAPII contacts, which foundhigh enrichment of active chromatin marksH3K4me1 and H3K4me3 coupled with a lackof repressive marks at RNAPII interaction sites(Li et al. 2012).

Recent studies have also provided new in-sight into how enhancers interact with distanttarget promoters to induce gene transcriptionand have suggested roles for transcriptionfactors, chromatin insulators, and a uniquecohesin complex in enhancer-promoter orga-nization. In the case of the well characterizedβ-globin locus, whose expression levels aredevelopmentally regulated by a distal up-stream locus control region (LCR), long-rangeenhancer-promoter interactions require tran-scription factors EKLF and GATA-1 (Drissenet al. 2004, Vakoc et al. 2005), and genes coreg-ulated by EKLF preferentially cluster intoshared transcription factories (Schoenfelderet al. 2010). Ectopically integrated human LCRon a discrete chromosome in transgenic micepreferentially interacts in trans with EKLF- andGATA-1-regulated genes (Noordermeer et al.2011a), which suggests transcription factorscoordinate the specificity and organization ofenhancer-promoter interactions. Through the

powerful combination of 4C and FISH, Noor-dermeer et al. (2011a) also demonstrate that in-terchromosomal interactions between the LCRand β-globin genes are limited to specific “jack-pot” cells actively transcribing β-globin genes,which suggests interactions are cell specific andreflect genome conformations that are con-ducive to enhancer-promoter association. Inother words, enhancers preferentially interactwith genes through shared transcription fac-tors, but they do so stochastically in a restrictednuclear space that varies from cell to cell.

Insulators also play an important rolein facilitating cell-type-specific chromatinorganization conducive to enhancer-promoterinteractions, and recent mapping of CTCF in-teractions in pluripotent cells supports this pos-sibility (Handoko et al. 2011). CTCF-mediatedtissue-specific chromatin architecture has beencharacterized at the apolipoprotein gene clus-ter; the imprinted IGF2-H19 locus; and the de-velopmentally regulated IFNG, β-globin, MHC-II, and CFTR loci (Gillen & Harris 2011, Ong &Corces 2011). However, recent findings suggestthat cohesin complexes, which are recruited byCTCF, are also capable of stabilizing enhancer-promoter chromatin looping in the absenceof CTCF. For example, cell-type-specificenhancer-promoter interactions in murine em-bryonic stem cells require a unique cohesincomplex, including the transcriptional coac-tivator Mediator and cohesin loading factornipped B-like protein (NIPBL) (Kagey et al.2010). Cohesin and NIPBL were also recentlyshown to mediate interactions between the β-globin genes and upstream LCR, and whereasCTCF-coordinated organization at the locusdoes not directly influence gene expression(Splinter et al. 2006), the cohesin-mediatedLCR interaction regulates globin gene expres-sion in vivo and in vitro (Chien et al. 2011).

PERSPECTIVES

The power of chromatin profiling and3C-based genomic strategies for exploringgenome-wide interactions has led to substan-tial progress in our understanding of nuclear


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organization over the past few years. Mean-while, the recent identification of tDNAinsulator activity in humans (Raab et al. 2012),mapping of CTCF-mediated chromosomalinteractions in mammals (Handoko et al.2011), and identification of genome-foldingprinciples in human cells (Dixon et al. 2012,Lieberman-Aiden et al. 2009, Nora et al. 2012,Yaffe & Tanay 2011) and Drosophila embryos(Sexton et al. 2012) have significantly expandedour knowledge of insulator function and thehighly conserved role of insulators in genomeorganization. The requirement for insulatorsin mediating long-range interactions essentialfor Pc repression (Li et al. 2011) and their local-ization to transcription factories (Melnik et al.2011) suggest insulators underlie the dynamicinterplay between epigenetic gene repressionand gene induction associated with develop-mental gene regulation. These observations can

be used to derive a comprehensive model of therole of insulators and chromatin structure in nu-clear organization (Figure 3), with emphasis onthe evolutionarily conserved role of insulatorsin yeast, Drosophila, and mammals; the molec-ular players involved in each; and their role ingene localization to PcG bodies and transcrip-tion factories. Despite substantial progress,several important questions remain, includinghow chromosomal associations and underlyinginsulator activities are regulated. The discoveryof tDNA insulator activity in mammals raisesthe question of whether the highly conservedtDNA and CTCF insulators functionallycooperate. Finally, studies have only begun tocharacterize the new roles for ncRNAs in generegulation and nuclear organization.

Interactions mediated by insulators, PcGproteins, and enhancer/promoter-bound fac-tors result in the creation of a 3D arrangement

NucleolusNucleolus

Transcriptionfactory

Transcriptionfactory

Insulatorbody

Insulatorbody

LAD

LAD

TF

Cohesin/condensin

CTCF

TFIIIC

Pc

CP190/Mod(mdg4)

SuHw/dCTCF/BEAF

Nuclear lamina

Pc bodyPc body

Figure 3Comprehensive model for the highly conserved role of insulators in nuclear organization. Insulators in yeast (TFIIIC, orange),Drosophila [Su(Hw), dCTCF, BEAF, green; CP190, Mod(mdg4), yellow], and mammals (CTCF, brown; TFIIIC, orange) mediatelong-range inter- and intrachromosomal interactions important for gene regulation and cluster into subnuclear foci called insulatorbodies. Insulators underlie interactions necessary for Polycomb (Pc) body repression (blue) and localize with general transcriptionfactors (TF, pink) to transcription factories. Insulators localize to subnuclear structures, including the nuclear lamina (red ), where theyare enriched at the borders of lamina-associated domains (LADs) and the nucleolus ( gray). CTCF insulator activity in mammalsrequires cohesin ( purple), and TFIIIC insulator sites are associated with both cohesin and condensin ( purple). Insulator activity inDrosophila relies on recruitment of fly-specific proteins CP190 and Mod(mdg4).


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of the DNA that must represent a finger-print of the functional status of the nucleus.Therefore, a detailed understanding of allinter- and intrachromosomal interactions inthe nucleus together with information on thenature and function of the interacting loci canlead to the establishment of structure-basedfunctional maps of nuclear output that are arepresentation of cell identity. Some of theseinteractions may be a consequence of genomefunction, whereas others may be established

during cell differentiation to elicit specificpatterns of gene expression. As a consequence,the 3D architecture of the genetic materialmay carry epigenetic information in additionto that written into the 10-nm chromatinfiber. Understanding how this informationis maintained during the cell cycle and howthe 3D arrangement of chromosomes duringinterphase relates to their structure duringmitosis remains a major challenge for the nearfuture.

SUMMARY POINTS

1. Long-range interactions between regulatory elements often govern transcriptional regu-lation, in the form of both gene activation and repression, and are therefore a fundamentalprocess underlying nuclear organization and genome function.

2. Chromatin insulators, including tRNA genes and transcription factor TFIIIC, appearto be highly conserved features involved in the nuclear organization of all eukaryoticgenomes, from yeast to humans.

3. Insulators have outgrown the enhancer-blocking and barrier activities for which theywere defined. Endogenous insulators facilitate long-range interactions that both mediatefunctional contacts between regulatory elements, including enhancer-promoter interac-tions, and correlate with topological domains enriched for coregulated genes. Insulatorsdo not appear to be necessary to prevent the spread of heterochromatin, as proposedpreviously.

4. Colocalization of genes targeted by Pc group proteins for repression and maintenanceof H3K27me3 within Pc domains relies on insulators. Insulator proteins also localize totranscription factories, which suggests a role in directing the localization of target genesto nuclear substructures for regulation.

5. Transcription factors and insulator-independent complexes also contribute to the orga-nization of coregulated genes for coordinated expression, which suggests that nuclearorganization and appropriate genome function depend on numerous additional factors.

6. Preliminary findings suggest that insulators can collaborate, perhaps to establish robustcomplexes capable of facilitating stable long-range interactions. Insulators also appear tobe developmentally regulated by recruitment of both DNA-binding insulator proteinsand additional cofactors.

7. Understanding how regulatory elements contribute to cell type–specific nuclear archi-tecture, and how this information is maintained throughout the cell cycle, remains amajor challenge for the near future.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.


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ACKNOWLEDGMENTS

Work in the author’s lab is supported by US Public Health Service Award GM35463 from theNational Institutes of Health.

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CB28-FrontMatter ARI 11 September 2012 11:50

Annual Reviewof Cell andDevelopmentalBiology

Volume 28, 2012 Contents

A Man for All Seasons: Reflections on the Life and Legacyof George PaladeMarilyn G. Farquhar � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Cytokinesis in Animal CellsRebecca A. Green, Ewa Paluch, and Karen Oegema � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �29

Driving the Cell Cycle Through MetabolismLing Cai and Benjamin P. Tu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �59

Dynamic Reorganization of Metabolic Enzymesinto Intracellular BodiesJeremy D. O’Connell, Alice Zhao, Andrew D. Ellington,

and Edward M. Marcotte � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �89

Mechanisms of Intracellular ScalingDaniel L. Levy and Rebecca Heald � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 113

Inflammasomes and Their Roles in Health and DiseaseMohamed Lamkanfi and Vishva M. Dixit � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 137

Nuclear Organization and Genome FunctionKevin Van Bortle and Victor G. Corces � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 163

New Insights into the Troubles of AneuploidyJake J. Siegel and Angelika Amon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 189

Dynamic Organizing Principles of the Plasma Membrane thatRegulate Signal Transduction: Commemorating the FortiethAnniversary of Singer and Nicolson’s Fluid-Mosaic ModelAkihiro Kusumi, Takahiro K. Fujiwara, Rahul Chadda, Min Xie,

Taka A. Tsunoyama, Ziya Kalay, Rinshi S. Kasai, and Kenichi G.N. Suzuki � � � � � � � � 215

Structural Basis of the Unfolded Protein ResponseAlexei Korennykh and Peter Walter � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 251

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The Membrane Fusion Enigma: SNAREs, Sec1/Munc18 Proteins,and Their Accomplices—Guilty as Charged?Josep Rizo and Thomas C. Sudhof � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 279

Diversity of Clathrin Function: New Tricks for an Old ProteinFrances M. Brodsky � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 309

Multivesicular Body MorphogenesisPhyllis I. Hanson and Anil Cashikar � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 337

Beyond Homeostasis: A Predictive-Dynamic Frameworkfor Understanding Cellular BehaviorPeter L. Freddolino and Saeed Tavazoie � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 363

Bioengineering Methods for Analysis of Cells In VitroGregory H. Underhill, Peter Galie, Christopher S. Chen,

and Sangeeta N. Bhatia � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 385

Emerging Roles for Lipid Droplets in Immunityand Host-Pathogen InteractionsHector Alex Saka and Raphael Valdivia � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 411

Second Messenger Regulation of Biofilm Formation:Breakthroughs in Understanding c-di-GMP Effector SystemsChelsea D. Boyd and George A. O’Toole � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 439

Hormonal Interactions in the Regulation of Plant DevelopmentMarleen Vanstraelen and Eva Benkova � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 463

Hormonal Modulation of Plant ImmunityCorne M.J. Pieterse, Dieuwertje Van der Does, Christos Zamioudis,

Antonio Leon-Reyes, and Saskia C.M. Van Wees � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 489

Functional Diversity of LamininsAnna Domogatskaya, Sergey Rodin, and Karl Tryggvason � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 523

LINE-1 Retrotransposition in the Nervous SystemCharles A. Thomas, Apua C.M. Paquola, and Alysson R. Muotri � � � � � � � � � � � � � � � � � � � � � � � 555

Axon Degeneration and Regeneration: Insights from Drosophila Modelsof Nerve InjuryYanshan Fang and Nancy M. Bonini � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 575

Cell Polarity as a Regulator of Cancer Cell Behavior PlasticitySenthil K. Muthuswamy and Bin Xue � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 599

Planar Cell Polarity and the Developmental Control of Cell Behaviorin Vertebrate EmbryosJohn B. Wallingford � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 627

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The Apical Polarity Protein Network in Drosophila Epithelial Cells:Regulation of Polarity, Junctions, Morphogenesis, Cell Growth,and SurvivalUlrich Tepass � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 655

Gastrulation: Making and Shaping Germ LayersLila Solnica-Krezel and Diane S. Sepich � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 687

Cardiac Regenerative Capacity and MechanismsKazu Kikuchi and Kenneth D. Poss � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 719

Paths Less Traveled: Evo-Devo Approaches to Investigating AnimalMorphological EvolutionRicardo Mallarino and Arhat Abzhanov � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 743

Indexes

Cumulative Index of Contributing Authors, Volumes 24–28 � � � � � � � � � � � � � � � � � � � � � � � � � � � 765

Cumulative Index of Chapter Titles, Volumes 24–28 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 768

Errata

An online log of corrections to Annual Review of Cell and Developmental Biology articlesmay be found at http://cellbio.annualreviews.org/errata.shtml

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Documents

Nuclear Organization and Genome Function