Biochimica et Biophysica Acta 1839 (2014) 178–190

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Biochimica et Biophysica Acta

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Review A Crowdsourced nucleus: Understanding nuclear organization in terms of dynamically networked function☆☆

Ashley M. Wood, Arturo G. Garza-Gongora, Steven T. Kosak ⁎

Department of and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA article info abstract

Article history: The spatial organization of the nucleus results in a compartmentalized structure that affects all aspects of nuclear Received 2 August 2013 function. This compartmentalization involves genome organization as well as the formation of Received in revised form 30 December 2013 and plays a role in many functions, including gene regulation, genome stability, replication, and RNA processing. Accepted 2 January 2014 Here we review the recent findings associated with the spatial organization of the nucleus and reveal that a com- Available online 9 January 2014 mon theme for nuclear is their ability to participate in a variety of functions and pathways. We consider this multiplicity of function in terms of Crowdsourcing, a recent phenomenon in the world of information tech- Keywords: Nuclear organization nology, and suggest that this model provides a novel way to synthesize the many intersections between nuclear Genome organization organization and function. This article is part of a Special Issue entitled: and epigenetic regulation of Nuclear body animal development. © 2014 The Authors. Published by Elsevier B.V. Open access under CC BY-NC-SA license.

1. Introduction integrated into the genome [2]. Similarly, NBs have been created de novo using the same strategy [3]. We have shown that the principles The nucleus is functionally compartmentalized. For example, there is of self-organization describe the cell-specific chromosomal topologies ample evidence for the deterministic positioning of gene loci, chroma- that arise through coordinate gene regulation during cellular differenti- tin, and even chromosomes according to physical and functional charac- ation [4]. Thus, the nucleus is an open system not at equilibrium, and teristics. In particular, the radial distribution of genes and chromosomes rather than the dynamic association of nuclear proteins and modified has been shown to have a defined pattern, with active and chromatin devolving into ever greater entropy, they form functional gene rich chromosomes occupying the nuclear interior, and gene poor centers and characteristic organizational patterns. chromosomes and silenced, developmentally regulated gene loci posi- There is an inherent promiscuity of nuclear proteins with many tioned at the nuclear periphery. Moreover, the nucleus is unique as an being involved in a wide range of networks and functions. A prime in that many of its functions are compartmentalized in non- example of this is the nuclear intermediate filament protein lamin A/C membranous assemblies, referred to as nuclear bodies (NBs). These (encoded by LMNA), a central component of the . Muta- NBs are themselves spatially localized non-randomly within the nucleus. tions in LMNA have been implicated in a wide variety of human disease The mechanistic basis for the arrangement of the genome and its func- phenotypes, collectively referred to as laminopathies [5]. The basis for a tional outcome remain to be fully understood. single protein being involved in myriad functions, from replication, to The idea of self-organization has been implemented as a means to transcription, to cell signaling, remains a perplexing problem. Of course, conceive the dynamic organization of the genome. Self-organization in lamin A/C's multi-functionality can at least partially be attributed to its the context of nuclear cell biology has been understood as a non- role in the nucleoskeleton, a network of laminar and other proteins hierarchical association of factors that result in functionally competent that is thought to provide a substrate for nuclear activities. As reviewed stable-state structures [1]. For example, double stranded DNA repair below, however, there are many such examples of a foci have been generated in the absence of DNA breaks simply by teth- intersecting numerous and varied functional pathways. While self- ering individual components of the pathway to a lac operator array organization provides a useful model to describe the functional dynamics of the genome, it may be inadequate to address the multi-functionality of regulatory and structural nuclear proteins. In particular, self-organizing systems are comprised of defined components, albeit non-hierarchical in their association. As we review extensively below, the dynamic organi- zation of genome function more closely resembles a multi-agent system, ☆☆ This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of with the factors involved in a particular function originating from diverse animal development. and often unexpected sources. ⁎ Corresponding author at: 303 E. Chicago Ave., Ward 8-132, Chicago, IL 60611 USA. Tel.: +1 312 503 9582. For many decades the genetic code and its central dogma have pro- E-mail address: [email protected] (S.T. Kosak). vided a colorful metaphor for understanding computer technology. In a

1874-9399 © 2014 The Authors. Published by Elsevier B.V. Open access under CC BY-NC-SA license. http://dx.doi.org/10.1016/j.bbagrm.2014.01.003 A.M. Wood et al. / Biochimica et Biophysica Acta 1839 (2014) 178–190 179 structural sense, the hard disk drive has been considered something of a of nuclear proteins and genome function. A particular nuclear activity, genome (storage of information); the central processing unit the such as coordinate gene regulation, is both seeker and problem—a machinery that replicates and transcribes (processes information); task that requires (solicits) factors for its action. In this vein, proximity and random access memory the proteins that carry out cellular function (which does not exist for the W3, as we are all connected) is the avail- (running programs). Beyond the clear parallels of information storage, ability of any given agent. On the other hand, ability is the potential that processing, and function, modeling computer technology from the an agent can contribute to the initiated function. Thus, the striking standpoint of molecular biology provides a comparison to a deeply com- multi-functionality of nuclear proteins can be considered a multi- plex system, in a sense predicting the potential of information technol- agent system, in which problems/functions are addressed by a ‘crowd’ ogy (IT). Intriguingly, the relatively recent emergence of the Web 2.0, of proteins based upon their individual availability and ability (Fig. 1B). which comprises the myriad social uses of the internet that harness As physicists and mathematicians begin to analyze the nature of the possibilities of the billions of World Wide Web (W3) users, may Crowdsourcing, the principles behind it may yield useful models for turn the tables and provide models for biological insight. The behaviors understanding the dynamic networks involved in protein multi- that emerge from the interactions of ‘agents’ in the W3 provide a labo- functionality. In any event, we offer the idea of Crowdsourcing as a ratory to explore dynamic biological systems in ways that we are not guide for our review of the dynamic complexity and interconnected currently capable. In particular, what can the Web 2.0 inform us about nature of genome organization. the multiplicity of functional associations of nuclear proteins and the genome? 2. Genome organization We offer that the nascent Web 2.0 phenomenon of Crowdsourcing may provide a useful analytical model to address the multi-functionality A central focus of the study of nuclear spatial organization involves of nuclear proteins. Crowdsourcing, in the context of the W3, is com- the physical positioning of the genome within the nuclear space. The prised of three central elements: seeker, problem, and solvers. The seek- features of nuclear topology include the localization patterns of chro- er is a company or agency that is involved in a given purpose (from mosomes, genes, and other DNA elements, as well as the interactions fi commercial to non-pro t), which is in need of a solution to a problem that occur between these components (Fig. 2). An important aspect of ‘ ’ (Fig. 1A). Traditionally, solutions were sought in-house ; however, the spatial organization of the genome is that the observed patterns through Crowdsourcing the seeker promulgates the problem through change during cell differentiation and regulation of . the W3. Thus, the problem is introduced to myriad potential solvers, Here we discuss the recent findings associated with dynamic genome with varying availability and ability (Fig. 1A). By harnessing the power organization and the proteins involved in this process. of this diverse community, a robust resolution is often achieved [6,7]. We suggest that this template is observable in the dynamic interplay 2.1. Genome organizers

When describing the spatial organization of the genome, there are a few key factors that repeatedly appear. These proteins can influence many different functions and are involved in numerous pathways, with the common thread being their ability to influence genome archi- tecture. Here we describe three of the most prominent genome orga- nizers: CTCF, , and nuclear lamins.

2.1.1. CTCF CTCF is a ubiquitously expressed zinc finger DNA-binding protein that was first described as a transcription factor based on its role in reg- ulation of c-myc gene expression ][8–10 . CTCF was later shown to block the communication between enhancers and promoters (enhancer blocking activity) and also to act as a boundary to the spread of chroma- tin domains (barrier function), and was therefore additionally defined as an insulator protein [11–13]. Genome-wide analysis revealed CTCF binding throughout the genome, and found that the majority of sites are invariant across various cell-types [14]. Many of these sites are in- volved in CTCF-mediated chromatin loops that correlate with chromatin domains, lamin-associated domains (LADs), and enhancer–promoter interactions [15]. In order to incorporate this array of functions, a more general view describes CTCF as a facilitator of chromatin looping and a global genome organizer [16]. In this role, CTCF participates in a variety of different pathways including regulation of transcription, im- printing, alternative splicing, X inactivation, end protection, Fig. 1. Depiction of nuclear Crowdsourcing. (A) In traditional Crowdsourcing, company or and somatic recombination (Fig. 3 and Table S1) [17–22].Thismaybe agency (the seeker), advertises a given problem to the W3. The immense ‘crowd’ of individ- possible due to its vast number of interacting partners including tran- uals on the W3 then participate in creating a solution to the problem based upon their avail- scription factors and chromatin regulatory proteins (YB1, Kaiso, and ability (they are aware of the challenge) and ability (they are capable of contributing to its YY1), chromatin remodeling factors (CHD8), nucleolar components solution). For example, red arrows represent solvers who are aware but unable to help, whereas green arrows indicate solvers who are both available and able. (B) Crowdsourcing (nucleophosmin, UBF), and proteins involved in other nuclear functions of nuclear function would be similar, with the notable exception of the seeker and the prob- (cohesin, PARP1, Suz12, RNAPII) [23,24]. How interacting partners are lem being inextricable, as the problem is the seeking entity. In parallel with (A) the solvers determined at specific genomic locations is not understood, but it is of the function/‘problem’ would also have varying availability and ability. In this case avail- likely influenced by the protein composition of the local nuclear envi- ability would be whether a given agent (protein) is proximal (or dynamic enough) to ronment. Based on its functional multiplicity, it is not surprising that engage the problem, and ability would be its capacity to function in the given process or fi pathway. Circles, squares, and triangles represent different classes of protein agents, with CTCF is an essential protein and that CTCF de cient mice result in red and green shapes indicating proteins either available and/or able to participate. early embryonic lethality [25,26]. 180 A.M. Wood et al. / Biochimica et Biophysica Acta 1839 (2014) 178–190

Fig. 2. Various forms of nuclear organization. (A) Immunofluorescent staining of a human retinal pigmented epithelial cell (RPE1) nucleus with anti-lamin B1 (red) shows the localization of the nuclear lamina. (B) FISH staining of a human fetal lung fibroblast (IMR90) nucleus with a chromosome 1 paint (red) and the region around the MYOG gene on chromosome 1 (green) shows the radial positioning of CTs and individual loci as well as the relationship between these elements. Image courtesy of D. Neems. (C) Immunofluorescent staining of an IMR90 nucleus with anti-PML (green) and anti-nucleolin (red) shows the organization of PML nuclear bodies and nucleoli respectively. (D) FISH staining of dividing murine hematopoietic pro- genitor cells with 4n DNA content. Chromosome 11 (red) and chromosome 2 (green) are depicted. This image shows maintained organization of chromosome positioning in daughter cells during mitosis. In B–D, DAPI is used to visualize DNA.

2.1.2. Cohesin promoter interactions through co-localization with transcription factors, Traditionally, the cohesin complex has been considered for its role in mediator, and the cohesin loading factor Nipbl [30–32]. maintaining sister chromatid cohesion after DNA replication [27].Itis composed of four core subunits (Smc1, Smc3, Scc1/Rad21, Scc3/SA1/ 2.1.3. Lamins SA2), three of which (Smc1, Smc3, Scc1/Rad21) are thought to form a Lamins are nucleus-specific class V intermediate filament proteins tripartite ring around sister chromatids after replication. Additionally, that form the major component of the nuclear lamina, the proteina- there are many accessory proteins that associate with the core cohesin ceous layer found on the nucleoplasmic side of the , subunits, mainly involved in loading and dissociation of the cohesin as well as throughout the (Fig. 2A). This distribution sug- ring [28,29]. Beyond its role in chromosome segregation, cohesin pro- gests a role for the lamina as a nucleoskeleton. There are three lamin teins also play a role in double stranded break repair, stabilization of genes in mammalian cells (LMNA, LMNB1,andLMNB2)thatencode transcription factor binding, somatic recombination, and the facilitation the A- and B-type lamins. At least one B-type lamin protein is thought of short and long-range interactions involved in transcriptional regula- to be expressed in all nucleated, metazoan cell types, but the A-type tion (Fig. 3 and Table S1) [27,30–33]. The ability of cohesin to form a lamins, lamin A and C produced from alternative splicing of LMNA,are ring structure around DNA strands probably plays an integral part in developmentally regulated [39,40]. It was generally accepted that its ability to form the chromatin loops involved in these processes. A-type lamins are not expressed in embryonic stem cells (ESCs) Many CTCF genomic binding sites are co-occupied by cohesin, and [41], but a recent study has detected low levels of expression at both it is thought that CTCF is necessary to position cohesin on chromatin the mRNA and protein level in murine ESCs [42], suggesting that there [34–37]. Whole-genome chromatin interaction analysis with paired- may be fundamental differences between mouse and human, and raising end tag sequencing (ChIA-PET) in the developing mouse limb identified questions about the effect of expression level on lamin A/C function. over 2000 interactions between cohesin-associated loci and 65% of Lamins have been found to associate with large chromatin domains, these involve CTCF [38]. This data set includes tissue-specific interac- and the functional outputs of these associations on genome organiza- tions as well as interactions that are found in multiple tissues with dis- tion are wide ranging. DamID analysis of lamin B1 classified roughly tinct expression profiles suggesting diverse functional outputs from 40% of the genome as LADs in four different mouse cell types at different cohesin-mediated interaction. In general, cohesin sites that co-localize stages of neuronal differentiation [43]. A similar degree of lamin B1 with CTCF tend to be cell-type invariant while non-CTCF cohesin sites association is observed in human cells [44].LADshavebeencharacter- conversely demonstrate cell-type specific binding, co-localize with ized to be gene poor, transcriptionally inactive, A/T rich regions of chro- transcription factors, and facilitate transcription promoting enhancer– matin, and are highly conserved between murine and human studies. A.M. Wood et al. / Biochimica et Biophysica Acta 1839 (2014) 178–190 181

Cajal Bodies

Telomere Stability CTCF Lamins RNA Processing

Chromatin State

Transcription

Recombination NPCs/Nups PML Replication Bodies

DNA Repair

Apoptosis

Cohesin Nucleoli

Fig. 3. Schematic of the multi-functionality of nuclear proteins. Nuclear proteins are connected to the pathways and functions in which they are involved, depicting not only multiplicity of function, but also the vast amount of functional overlap between the various proteins involved in nuclear organization. A chart with references is included as Supplemental Table 1.

The majority of LADs were found to be constant across cell-types; how- myopathies such as Emery–Dreifuss muscular dystrophy (EDMD), pre- ever, a subset of regions shows changes in lamin association during mature aging disorders such as Hutchinson–Gilford progeria syndrome differentiation [43,45]. Recently, the occurrence of a GAGA motif bound (HGPS) and atypical Werner syndrome, as well as peripheral nerve dis- by a cKrox/Lap2β/HDAC3 complex was shown to direct lamin association orders and lipodystrophies [57]. Additionally, diseases associated with of specific, developmentally regulated loci in a transcription-dependent mutations in LMNB1 and LMNB2 have also been identified including manner [46]. It is yet to be determined whether this is a general mecha- adult-onset leukodystrophy and partial lipodystrophy [57]. Thus the im- nism that directs lamin association, and how cKrox binding is regulated portance and multi-functionality of nuclear lamins is manifested in the between cell-types. Despite the large amount of the genome associated wide variety of disease states caused by mutations in lamin genes. with the nuclear lamina, mESCs lacking both LMNB1 and LMNB2 appear Clearly, these different genome organizers are involved in many dif- normal with only limited changes in gene expression within LADs [47], ferent pathways. While likely candidates include interacting partners, and triple knockout mESCs lacking lamin B1, lamin B2, and lamin A/C the local chromatin environment, and/or covalent modifications, the expression also show no defects in proliferation or differentiation [48]. mechanistic underpinnings of how the same proteins are able to func- Mice derived from the double knockout mice die at birth, suggesting tion in varied pathways are unknown. Moreover, a persuasive model that nuclear lamins are important for development and cell differentia- that seeks to address the basis for this multi-functionality remains to tion but not cell survival. be fully developed. Lamin A/C association with chromatin has also been mapped and is highly correlated with the previously identified lamin B1 LADs [45,49]. 2.2. Global genome organization The agreement in lamin A/C and lamin B1 association is almost com- plete (98–99%) for constitutive LADs and less, but still substantial for The majority of our understanding of genome organization comes cell-type variant LADs (83–86%) [45]. Various mutations in the LMNA from fluorescent in situ hybridization (FISH) analysis and chromosome gene lead to disruption in nuclear positioning, chromatin compaction, conformation capture (3C) [58] based techniques. FISH has the advan- and chromatin state that correlate with changes in gene expres- tage of single-cell analysis in the absence of a large degree of data sion [49,50]. Interestingly, murine rod cells do not express lamin A/C manipulation and amplification of starting material while 3C-based or lamin B receptor (LBR) causing an inverted genome organization in techniques show increased resolution and, in combination with high- which the peripheral localization of heterochromatin is lost [51,52].It throughput analysis, are capable of monitoring many interactions over has recently been shown that a similar intranuclear heterochromatin large regions simultaneously. Due to these different strengths, these repositioning can be induced in a variety of mouse cell types by loss of techniques are best used in combination and it is important to be lamin A/C and LBR indicating that these proteins are essential to main- aware of the limitations of each technique when interpreting results. tain proper heterochromatin organization in most, if not all, cell types [51]. 2.2.1. Chromosome territories Like CTCF and cohesin, lamins play a role in many different processes, The organization of the genome into chromosomes plays a large role including: transcriptional regulation, DNA replication elongation, main- in dictating the three dimensional spatial organization of the nucleus. tenance of nuclear structure and shape, chromosome positioning and Chromosomes occupy discrete regions of the nucleus called chromo- condensation, gene positioning, telomere positioning, and positioning some territories (CTs) [59] (Fig. 2B). The radial positioning of CTs them- of DNA damage foci (Fig. 3 and Table S1) [50,53–56]. Due to the role of selves is an important aspect of nuclear organization. Multiple factors lamin proteins in so many different nuclear processes, disruption of are thought to influence radial positioning of CTs, including gene density these proteins leads to a phenotypically diverse set of disease states. and chromosome size. Gene poor chromosomes are found more periph- Collectively, these are called laminopathies with most of the known erally and gene dense chromosomes are found more centrally in the disorders a result of mutations in LMNA, including cardiac and skeletal nucleus [60–62]. Additionally, in some cases larger chromosomes are 182 A.M. Wood et al. / Biochimica et Biophysica Acta 1839 (2014) 178–190 positioned at the periphery and smaller chromosomes more internally completely understood; however, some common elements have been [61,63,64]. Altered gene expression can also influence radial positioning reported at TAD borders. Drosophila TAD borders tend to be gene of CTs [65,66]. For example, induction of cellular quiescence through dense, contain DNase hypersensitive sites, and are enriched for RNA serum starvation leads to the radial repositioning of many chromo- Polymerase II (RNAPII), insulator proteins, and the mitotic spindle pro- somes. Interestingly, movement in response to serum starvation occurs tein Chromator [74,80]. Mammalian TAD borders have similar compo- rapidly and is dependent on actin and myosin, while relocalization of nents in that they tend to be enriched for CTCF, cohesin, promoters of serum re-stimulated cells occurs more slowly and requires cell division, housekeeping genes, tRNA genes, and SINE family repeat elements providing evidence that different mechanisms exist within the cell to [83,84]. Interestingly, while CTCF is enriched at these regions, only regulate chromosome positioning [67,68]. Furthermore, the relative po- 15% of the total CTCF sites are found at TAD borders suggesting that a sitioning of chromosomes to one another is influenced by gene expres- CTCF site alone is not sufficient to dictate a TAD border [84]. A critical sion. This is observed in hematopoiesis during which proximity of experiment by Nora et al. found that deletion of roughly 60 kb at a chromosomes is related to the degree of coordinated gene regulation TAD border within the Xist/Tsix locus resulted in increased association [4,69]. Therefore, changes in expression profiles that occur during differ- between TADs and was accompanied by misregulation of gene expres- entiation can result in cell-type specific CT organization. sion providing evidence that a border region is essential to create inde- CT formation also influences genome organization by restricting pendent interacting domains [83]. It is still uncertain what elements broad genomic interactions occurring within a chromosome. Although within this region actually maintain border activity, and a greater there is some evidence for intermingling of CTs [70], this idea is contro- focus on genome disruption analysis is necessary to understand the for- versial and more recent studies suggest that mixing of CTs occurs only at mation and maintenance of TADs and the effect they have on chromatin borders and is not extensive [71,72]. Additionally, chromosome centro- structure. meres seem to form a barrier to interaction between chromosome arms A recent high-resolution 5C analysis of mESCs during neuroectoderm [73–76]. This was clearly depicted by Tolhuis et al. [73],throughtheuse differentiation not only verified the existence of constitutive TADs, but of a chromosome inversion across the centromere that resulted in altered was also able to identify sub-TADs that show more cell-type specific interactions such that mainly intra-arm interactions are observed in both variability [85]. Interactions were found to be anchored by CTCF, the normal and inverted state. This result suggests that a centromeric bar- cohesin, and mediator, and the presence of these factors was shown to rier, not cues from DNA sequence or chromatin state, dictates intra- be functional, such that knockdown of their expression led to a loss of in- chromosomal interactions. Though less common, inter-chromosomal teraction. Specific combinations of these proteins were correlated with interactions have also been observed, and regions with high inter- the distance between interacting loci as well as the cell type variability chromosomal interaction frequency have been suggested to loop out of of an interaction. In general, CTCF with or without cohesin was found CTs [75]. However, recent single-cell Hi-C analysis shows that regions to facilitate large, constitutive interactions, while mediator with or with- displaying a high degree of inter-chromosomal interactions still maintain out cohesin was found to facilitate small and intermediate, developmen- intra-chromosomal interactions indicating that these regions do not loop tally regulated interactions. Although this statement oversimplifies the dramatically away from the territory [77]. Furthermore, assaying these complex interplay of proteins present at interaction sites, it suggests inter-chromosomal interactions by FISH shows that they are only ob- that it is actually the combination of factors at a specific region that served in a very small percentage of cells [75].Thusitiscriticaltokeep dictates the spatial organization. Additionally, since the same protein in mind when interpreting population-based interaction profiles as can be involved in different kinds of interactions, this strongly supports maps of nuclear organization, and underscores the importance of single the pervasive multi-functionality of nuclear proteins. cell analysis in understanding nuclear organization. 2.3. Genome organization of specific loci 2.2.2. Large-scale genomic interactions Beyond organization at the level of the chromosome, other proper- 2.3.1. Locus organization ties of whole genome organization have also been described. Active Chromatin looping within a locus has also been studied in depth chromatin and inactive chromatin demonstrate a high frequency of at a few speci fic loci, and chromatin interactions within these model self-interaction. In this manner the genome can be separated into two loci have been detected that lead to a variety of functions [17].The compartments that represent active and inactive regions [75,77–81]. imprinted H19/Igf2 locus is one of the best-understood regions in This is influenced by nuclear lamins since a LMNA mutation associated terms of regulation of chromatin looping. Within this region CTCF bind- with HGPS leads to disruption of this compartmentalization in late pas- ing at the imprinting control region is inhibited by DNA methylation of sages [49]. When exonic regions of a mouse chromosome were com- the paternal allele [18,86,87]. This allele-specific CTCF binding leads pared to CT signal, the exonic chromatin was found to occupy a larger, to different chromatin interactions within the locus that dictate more internal nuclear space than the territory itself, supporting the enhancer–promoter interactions and gene expression [88–90]. Regula- idea of active chromatin being spatially distinct from inactive chromatin tion of genomic imprinting is further discussed in this issue [Weaver [82]. Furthermore, the inter-chromosomal interactions that have been and Bartolomei]. identified tend to involve the active chromatin compartment, indicating Chromatin looping has also been described at lymphocyte antigen that active chromatin is more mobile than inactive chromatin [75,80]. receptor loci, and in this context chromatin interactions facilitate so- Chromosome conformation capture carbon-copy (5C) and Hi-C matic recombination. CTCF and cohesin have been implicated at these analysis have revealed that chromatin interactions tend to cluster with- loci and deletion of these proteins or their binding sites leads to im- in local regions termed ‘topologically associating domains’ (TADs) [83] paired recombination. Current data show a role for CTCF and cohesin or ‘topological domains’ that are roughly 10–500 kb in Drosophila and in regulating transcription within these loci by facilitating long-range 200 kb–1 Mb in mammalian cells [74,80,83,84]. TADs were found to interactions, and it is unclear if they are also directly involved in bring- be relatively stable across different cell types, and most cell-type specific ing distal gene segments together for recombination [91]. interactions are found to occur within a TAD. Recently, single-cell Hi-C Another well-studied locus is the murine HoxD cluster, which shows analysis was performed revealing that TADs are also fairly consistent linear regulation of gene expression due to chromatin looping during within a population of cells, while inter-domain interactions show a development [92]. This region represents an interesting pattern of chro- higher degree of cell-to-cell variability [77]. Additionally, TADs matin organization as the HoxD cluster is located within the boundary of tend to correlate with the compartmentalization of the genome two TADs [93]. During limb development HoxD genes participate in in- into active versus inactive regions, which also can be coincident with teractions with regulatory elements in the surrounding TADs, and a LADs [83,84]. The components necessary to establish a TAD are not switch in some HoxD genes from interaction with the telomeric to the A.M. Wood et al. / Biochimica et Biophysica Acta 1839 (2014) 178–190 183 centromeric TAD correlates with changes in chromatin state and gene 2.4. Dynamics of genome organization expression [93,94]. In interpreting these data for locus organization, it is important to Our understanding of the dynamics of genome organization is note that observed interactions indicate contacts that occur at a given growing, yet the relationship between nuclear activities and changes point in time, not necessarily stable chromatin structures. Therefore, in genome organization are not fully characterized. individual regulatory elements and gene promoters may undergo many different interactions and it remains to be determined how the 2.4.1. Dynamic genome organization and transcription identified interactions co-exist within a population. Use of the recently A key aspect of the dynamics of genome organization is its relation- developed single-cell Hi-C analysis technique will provide important ship with transcription, and an active area of research is determining insight into this topic [77]. Nonetheless, it is clear that although the whether genome organization plays a causal role in regulating transcrip- same proteins often facilitate chromatin interactions that dictate locus tion, vice versa, or most likely, both. As mentioned in Section 2.3.2, organization, they can be regulated in different manners and can result tethering of some loci to the nuclear periphery can result in altered in different functional outputs. gene expression indicating a causal influence of genome organization on transcription [111]. However, these experiments are extremely arti- ficial and may not accurately represent the normal temporal association 2.3.2. Gene positioning of genome reorganization and changes in transcription. Patterns of spatial organization of the genome have also been iden- The β-globin locus exhibits dynamic, developmentally regulated tified at the level of individual genes. Clustering of co-regulated genes chromatin looping and gene positioning, and includes examples of through self-organization has been proposed as a thermodynamically changes in genome organization that proceed changes in gene expres- favorable form of nuclear organization [95–97]. This type of clustering sion as well as the reverse. Within this locus, stage specific interactions has been observed and in some cases occurs at RNAPII enriched tran- have been identified between a locus control region (LCR) and β-globin scription factories [98–104]. Additionally, co-regulated gene clustering genes that are linearly arranged on the chromosome in the order of in a transcription factor dependent manner was observed in erythroid their activation [126,127]. These LCR–promoter interactions are medi- cells with genes regulated by the transcription factor Klf1 co-localizing ated by various transcription factors that regulate β-globin gene with Klf1 as well as RNAPII [105]. Importantly, not all co-regulated expression [128–130]. Additional CTCF mediated interactions are also genes are observed to cluster [106]. This bias may be related to the ef- present surrounding the locus and are detected prior to LCR–promoter fects of the linear chromatin environment, including such features as interaction and activation of gene expression [131,132]. Inhibition of gene density, transcriptional activity of neighboring genes, and position transcription does not have large effects on the interactions made by on the chromosome affecting gene loci mobility and localization the active β-globin gene locus, suggesting that transcription is not nec- [102,103]. essary to maintain these interactions [133,134]. These studies do not Gene positioning in relation to the nuclear periphery is another rule out a role for transcription in initiating interactions, and since important aspect of nuclear organization. Peripheral gene localiza- these experiments were performed over relatively short time scales tion is associated with gene silencing for some loci [107–109] and a (0.5–5 h), they do not test the role of transcription in the maintenance subset of gene loci demonstrates cell-type specific lamin association of interactions through cell division. Interestingly, artificially induced [43,110].Furthermore,artificially tethering a locus to the nuclear chromatin looping in the absence of the transcription factor GATA-1 periphery can lead to gene silencing, although this phenomenon is was found to be sufficient to induce chromatin interactions and resulted gene specific as peripheralization is not sufficient to regulate tran- in activation of β-globin gene expression [135]. Therefore, at least in this scription of all genes [111]. The complexity of radial gene positioning situation, genome organization plays a causal role in regulation of gene is further exemplified in mouse olfactory where clustering expression. However, analysis of the β-globin locus at the level of gene of transcriptionally silent olfactory receptor (OR) genes in hetero- positioning reveals a less direct relationship with transcription. Move- chromatin foci is observed at the nuclear interior [112].Thisclustering ment of the β-globin locus away from the nuclear interior begins after ini- is dependent on the loss of LBR and is necessary for proper monogenic tiation of transcription indicating that genome reorganization is not expression of OR genes. necessary to alter gene expression [108].Togetherthesedatafor An additional complication in understanding the functional conse- β-globin genome organization exemplify the complex interplay quences of peripheral localization is that nuclear pores are dispersed that exists between long-range interactions, gene positioning, and throughout the nuclear membrane creating a lack of homogeneity at transcription. the nuclear periphery. Beyond their role in nuclear transport, nuclear Changes in genome organization can also be associated with tran- pore complexes (NPCs), composed of (Nups), are also scriptional silencing. For example, in the Drosophila embryo the involved in the organization of gene loci [113,114]. NPCs and Nups hunchback gene is expressed early in neuronal differentiation and interact with specific chromosome regions, and unlike other is then silenced. Although this transcriptional repression is accompa- peripheralized chromatin, these regions are usually enriched for ac- nied by nuclear peripheralization, lamin-dependent gene movement tive chromatin. In yeast, association with NPCs is often specified by is not observed for three cell divisions after transcriptional repression DNA sequences termed ‘DNA zip codes’, and genes with similar zip [136]. This movement was shown to coincide with permanent tran- codes cluster together [115– 117]. This localization is necessary for scriptional silencing, but not the original onset of gene repression. robust gene activation, transcriptional memory that allows efficient Therefore, like the relationship with transcriptional activation, the rela- re-activation, and potentially even rapid gene silencing after activa- tionship between transcriptional silencing and genome organization is tion [118–120].InDrosophila, Nups can also interact with gene loci not straightforward. contributing to gene activation; however, this interaction can occur in the nucleoplasm away from the nuclear periphery [121–123]. 2.4.2. Dynamic genome organization and other nuclear functions Recently, similar functional interactions were found to be conserved In addition to the connection that dynamic genome organization in human cells [124]. In addition to this role in genome organization, exhibits with transcription, there are also notable links with other nuclear NPCs and Nups play a role in DNA replication, telomere stability, and functions including replication timing and DNA repair. DNA repair (Fig. 3 and Table S1) [125]. Together, this suggests that DNA replication occurs asynchronously throughout the genome Nups are another class of nuclear protein that exhibits a multitude such that euchromatic chromatin located in the nuclear interior repli- of functions and has a critical role in the spatial organization of the cates during early S phase, and peripheralized heterochromatin repli- genome. cates during late S phase [137]. Additionally, genome-wide maps of 184 A.M. Wood et al. / Biochimica et Biophysica Acta 1839 (2014) 178–190 replication timing correlate extremely well with genomic interaction proteins and their involvement in many different pathways, as well as profiles and suggest the spatial compartmentalization of replication do- the fact that many NBs share protein interacting partners. This problem mains [138]. Importantly, replication timing of some genomic regions is will probably only become more challenging since mathematical model- developmentally regulated, and changes in replication timing correlate ing of known and predicted nuclear protein localization revealed that with changes in spatial organization, indicating that dynamic genome various nuclear compartments and NBs are probably even more func- organization in relation to replication timing may play a role in cell dif- tionally diverse than currently known [158]. In the following sections, ferentiation [138–141]. In support of this idea, cell populations that fail we will discuss recent findings of the multi-functionality of NBs and to reprogram during the formation of induced pluripotent stem cells discuss how this allows for efficient interactions in a confined nuclear (iPSCs) lack reprogramming of replication timing, suggesting that this space. aspect of dynamic genome organization may be necessary to establish pluripotency [140]. 3.1. Promyelocytic leukemia bodies The role of dynamic genome organization with DNA repair is unique in that chromatin mobility can theoretically both facilitate and hinder Promyelocytic leukemia (PML) nuclear bodies (also known as ND genomic stability after DNA damage [142]. Increased mobility of dam- 10s or Kremer bodies) (Fig. 2C) are functionally promiscuous and aged DNA can increase the rate of finding templates for homologous re- dynamic structures that have been implicated in such processes as pro- combination (HR) and other broken ends for non-homologous end liferation, senescence, apoptosis, genomic stability, telomere mainte- joining (NHEJ) [143–147]. At the same time, it has been demonstrated nance, and the DNA damage response (Fig. 3 and Table S1) [159].PML that the spatial proximity of genes correlates with their translocation bodies were first observed by electron microscopy in the 1960s, and frequencies [148,149] and increased mobility can lead to translocations later as nuclear dots by immunofluorescence [160,161]. These nuclear [150]. For this reason, nuclear factors such as lamin A/C act to reduce dots became referred to as PML oncogenic domains (PODs), or simply mobility of DNA damage foci, probably in order to prevent deleterious PML bodies, after the discovery of the localization of the promyelocytic events [55,151]. Despite this disparity in the effect of DNA damage on leukemia protein (PML), so named as it was first characterized through locus mobility, it was recently demonstrated that at least some gene- its fusion with the retinoic acid receptor alpha (RARA) in acute rich CTs reveal nuclear repositioning in response to DNA damage, indic- promyelocytic leukemia (APL) patients. Over 100 proteins have been ative of global effects on genome organization [152]. shown to localize to PML bodies [162]. While it was initially unclear whether they simply functioned as a depository of proteins, recent 2.4.3. Dynamic genome organization and the cell cycle work strongly indicates the role of PML bodies as functional structures Cell division introduces an additional layer of complexity to our whose activity is dictated by the combination of factors present at a understanding of the dynamics of genome organization (Fig. 2D). given time. Here we will discuss some of the functions of PML bodies For example, patterns of nuclear organization can vary throughout the by describing various interacting partners. cell cycle. This behavior is observed in telomere positioning with telo- PML bodies are thought to be involved in apoptosis and cell survival meres localizing to the nuclear periphery during nuclear reassembly through death-associated protein 6 (DAXX), a transcriptional co- after mitosis, but not at other stages of the cell cycle [153]. repressor and chaperone [163]. In support of the importance There is also some evidence that cell division may be necessary to of a functional role for the interaction of DAXX with PML bodies, allow major rearrangements in genome organization. A recent study DAXX-induced apoptosis is abrogated in PML−/− cells while acquiring developed a new “molecular contact memory” approach using an ex- a diffuse spatial localization [164]. Phosphorylation of DAXX promotes tension of DamID technology to monitor LADs over time in live-cells SUMO1 binding, and this in turn enhances its interaction with PML as [154]. This technique revealed relatively stable association of chromatin well as its physical recruitment to anti-apoptotic genes [165]. Although with the nuclear lamina within a single cell cycle, but a large reorganiza- DAXX is mostly found at PML bodies, it also forms small foci at centro- tion after mitosis. Additionally, this study identified a large degree of meric and pericentromeric (CEN/periCEN) heterochromatin in some cell-to-cell variability in LADs, and found that this variability often cor- cells where it is thought to play a role in gene expression through depo- relates with the transcriptional status of genes in individual cells. sition of H3.3. Under heat shock conditions, the balance of DAXX local- These results again highlight the importance of single-cell analysis in ization shifts from PML bodies to CEN, exemplifying the dynamics of understanding the spatial organization of the nucleus and suggest that PML body composition [166]. DAXX recruits H3.3 as well as soluble reorganization of LADs occurs during mitosis. Therefore, it is possible H3.3–H4 dimers to PML bodies, and it is believed that this association that after changes in the transcriptional status of a gene occur, the cell allows pairing with other histone chaperones for deposition onto chro- must divide before large changes in spatial organization are observed. matin [167,168]. This brings into question how much regulation of spatial organization The association of PML with p53 contributes to its function in apo- occurs in terminally differentiated cells that do not undergo cell division. ptosis and transcriptional activity [169–172]; however, the role that PML bodies play in p53 function is not fully understood. A recent 3. Nuclear bodies study revealed that after DNA damage, monocytic leukemia zinc finger protein (MOZ), a histone acetyltransferase (HAT), relocalizes to PML A critical form of the spatial organization of the nucleus is found in bodies. This interaction greatly increases MOZ-mediated acetylation of the non-membranous structures referred to as nuclear bodies (NBs). p53 and, thus, expression of p21, G1 arrest, and cellular senescence Considered broadly, NBs are thought to manifest a potential for consol- [173]. The interaction of MOZ and PML bodies is inhibited by the direct idated activity: the constituent proteins, DNA, and RNA occupy a phosphorylation of MOZ at its PML binding domain, which in turn neg- discrete location to facilitate a collective function [155]. Although NBs atively regulates p53 acetylation [173]. This result underscores the im- appear to be stable structures, their components are in fact highly portance of PML body association to mediate function, and suggests dynamic, freely exchanging with a nucleoplasmic pool of protein. Nuclear that PML bodies play an active role in regulating p53 activity. bodies often associate with chromatin, and the extent of nuclear body PML bodies have been shown to associate with genetic loci, such as movement is determined by the accessibility and dynamics of the sur- those of the major histocompatibility complex (MHC) [174].Recently, rounding chromatin [156]. it was demonstrated that upon IFN-gamma treatment, the spatial prox- Some NBs are found in a wide variety of cell types, while others are imity between PML bodies and the MHC II gene cluster is increased present in only a limited number of cell types and/or are formed only [175,176]. IFN-gamma treatment also caused PML bodies to associate under certain conditions [157]. In general, assigning a specificfunction with and stabilize CIITA, an MHC class II transactivator, and this associa- to a particular NB has been difficult due to the large variety of constituent tion promotes the transcription of MHC II genes [176].PMLbodiesalso A.M. Wood et al. / Biochimica et Biophysica Acta 1839 (2014) 178–190 185 play a role in transcriptional memory within the MHC II gene cluster 3.3. Nucleoli through interaction with a methyltransferase, which can prime genes for faster transcriptional reactivation after IFN-gamma treat- In mammalian cells, nucleoli form around tandem repeats of rDNA ment through the establishment of a permissive epigenetic mark genes, known as nucleolar organizing regions (NORs), that are spread [175]. Using a novel immuno-TRAP technique to probe for nuclear across five acrocentric chromosomes: HSA13, HSA14, HSA15, HSA21, body–chromatin interactions, new PML body–locus associations and HSA22 (Fig. 2C). Although the was formally described were recently identified including the PML locus itself [177];howev- in the 1800s, its primary function in ribosome biosynthesis was not dis- er, the function of PML bodies at these sites is unclear. Although PML covered until the 1960s [188]. The position of the nucleolus is deter- bodies associate with genomic regions of high transcriptional activi- mined by the presence of transcribing NORs, as well as the process of ty, they themselves are not sites of transcription [178].Instead,RNA ribosome biogenesis itself [189]. Proteomic analysis has identified over associates with the periphery of PML bodies, and highly acetylated 300 proteins associated with the nucleolus, and the majority of these chromatin typically surrounds PML bodies [179]. proteins have no known role in ribosome biogenesis [190–192]. Fur- The heterogeneity of PML bodies, in both form and function, illus- thermore, the nucleolar proteome is dynamic and changes in response trates the difficulty in assigning a clear activity to this type of NB. In- to cellular growth conditions [193]. As evidenced below, beyond the stead of describing PML bodies as a specific nuclear compartment it well-defined role in ribosome biogenesis, the multi-functionality of may be more appropriate to think of them as a class of NBs, which the nucleolus is becoming increasingly clear (Fig. 3 and Table S1) [194]. can be further defined based on their individual components and The nucleolus has roles in protein sequestration and stress sensing. the surrounding nuclear environment. Nonetheless, the vast array The tumor suppressor p53 has a short life span, partly maintained by of activities involving PML bodies perfectly exemplifies the function- its association with the E3 ubiquitin protein ligase Mdm2, while the sta- al multiplicity of NBs. bilization of p53 is necessary for its activation. A wide variety of ribo- somal proteins have been identified that, upon stress, are relocalized from the nucleolus and interact with Mdm2 to stabilize p53 [195]. 3.2. Cajal bodies Non-coding RNA (ncRNA) transcribed from a large intergenic spacer (IGS) region that separates rRNA tandem repeat genes, is able to directly Due to their high concentration of factors such as small nuclear ribo- target proteins with a nucleolar detention sequence (NoDS) to nucleoli nucleic particles () and other RNA processing factors, Cajal for sequestration [196]. Different stimuli, such as acidosis, heat shock, or bodies are thought to predominantly function in post-translational transcriptional stress are able to cause the expression of IGS ncRNA from modification of spliceosomal components. First referred to as coiled various IGS loci, and target proteins such as Mdm2 to the nucleolus for bodies given their fibrillar appearance under EM, they were renamed immobilization [196]. Cajal bodies after Santiago Rámon y Cajal who first described them in Identification of nucleolus-associated domains (NADs) suggests that the early 1900s. Over 60 proteins and over 25 RNA species, including nucleoli play a role in genome organization [197,198]. In addition to small nuclear (snRNAs), small nucleolar RNAs (snoRNAs), and rRNA genes, nucleoli predominantly interact with tRNA genes, centro- small Cajal body-specific RNAs (scaRNAs), have been found to asso- meres, specific satellite repeats, and repressive heterochromatin. Inter- ciate with Cajal bodies [180]. Cajal bodies form by stochastic self- estingly, there is significant overlap between NADs and LADs, and single organization. Tethering individual Cajal body components to chroma- cell analysis has revealed exchange between nucleolar and nuclear lam- tin results in the de novo formation of a mature Cajal body [3]. Recent ina localization after mitosis suggesting functional similarities between evidence indicates that Cajal bodies are more functionally diverse them [154,197]. than previously considered (Fig. 3 and Table S1). It is apparent that nuclear bodies are a form of nuclear organization For example, Cajal bodies have been implicated in the maturation of that facilitate a wide range of nuclear activities. It is important to note snRNPs and snRNAs. The integrator complex, which is involved in 3′ end that there are other types of nuclear bodies from those mentioned processing of snRNA, is essential for Cajal body integrity, as defects in its here. Nuclear speckles, for instance, are NBs enriched in RNA splicing activity cause dispersal of Cajal body components [181]. Recent evi- factors and are believed to be storage sites for these factors [199]. dence indicates that the associated heterogeneous ribo- are structurally formed by the ncRNA NEAT1 and are sug- U (hnRNP U) regulates Cajal body morphology and U2 gested to predominantly play a role in gene expression [200].Polycomb snRNP maturation [182]. Cajal bodies, like PML bodies, have been group bodies are formed by repressive complexes that act by post- shown to transiently associate with gene loci. Cajal bodies associate translationally modifying for gene repression [201]. These ex- with the U1, U2, and U4 snRNP gene clusters and the U11 and U12 amples further reinforce the idea that NBs are multi-functional struc- snRNA loci [183]. Evidence for a functional association has been tures that are implicated in myriad of nuclear processes. shown in that interaction of Cajal bodies with an inducible U2 gene array is dependent on transcription [184]. As mentioned previously, it 4. Nuclear structure is hypothesized that Cajal bodies act to recruit factors necessary for the biogenesis and maturation of snRNPs and snRNAs suggesting that When discussing nuclear organization, in addition to describing the the association with these gene loci aids in the efficient processing of arrangement of elements within the nucleus, we must also consider the nascent transcripts. nucleus as it relates to the rest of the cell. This includes nuclear size and Cajal bodies have also been shown to be important in telomere shape, and interactions between the nucleus and components in the maintenance. contains a WD40-repeat protein that allows cytoplasm. it to bind to Cajal bodies, and this association appears to be necessary Although nuclear size correlates to some extent with genome length, for telomere synthesis [185]. Recently, identification of an hnRNP A2 cytoplasmic components seem to be the predominant determinants splice variant, hnRNP A2*, was found to promote telomere extension [202,203]. Furthermore, in Xenopus it was found that nuclear import by telomere G-quadruplex unfolding activity [186]. hnRNP A2* localized plays a major role dictating nuclear size, which may be at least in part with Cajal bodies at , and it is suggested that hnRNP A2* due to import of B-type lamins [204].Thisfinding suggests that nuclear associates with the telomerase holoenzyme at Cajal bodies before size may be regulated by the availability of structural proteins. Indeed, localization to telomeres [186]. Dyskeratosis congenita patients knockdown of lamin proteins in Caenorhabditis elegans leads to reduced with a mutation in the telomerase protein TCAB1 are defective in local- nuclear size [205]. Interestingly, there is a correlation between nuclear izing telomerase to Cajal bodies, and instead it localizes to nucleoli size and mitotic chromosome length, and during Xenopus development which prevents telomere maintenance [187]. nuclear diameter and chromosome length both decrease. In this system, 186 A.M. Wood et al. / Biochimica et Biophysica Acta 1839 (2014) 178–190 in vitro manipulation of nuclear size revealed that progression through canonical member of the body, and it has seven characterized isoforms. a cell cycle is necessary to induce a corresponding change in chromo- In a sense, this core constituent may represent the means by which the some length, suggesting that cell division is important for dynamic need for a function/problem is presented. Based upon which isoform genome organization [206]. Although these studies are beginning to may predominate in a PML body, proteins that are both available (i.e. shed light on factors that dictate nuclear and chromosome size, the in proximity of this call to function) and have the ability (i.e. can act functional implications of this regulation in terms of nuclear organiza- on the function presented, such as p53 regulation) would then associate. tion, gene expression, and genomic stability are still not clear. Interest- The proteins that respond to the particular function may not always be ingly, alterations in nuclear size are often observed in cancer cells and the same, but would necessarily have the requisite capacity to act in this feature is used to stage various cancers, highlighting the importance the given pathway. Thus, a decentralized assembly of proteins performs of this area of research [207]. a function, but another assembly might perform a different function. The linker of the nucleus to the cytoskeleton (LINC) complex directly So, how useful is our comparison of Crowdsourcing to protein multi- connects the cytoplasm to the nucleoplasm and provides a platform for functionality? As it is still in its infancy, the concept of Crowdsourcing is communication between nuclear processes and the rest of the cell. The only now attracting the attention of physicists and mathematicians who LINC complex spans the nuclear membrane and consists of nesprins that will use their skill sets to probe its behavior and model its features [213]. transverse the outer nuclear membrane (ONM) and bind to cytoplasmic Thus, it is beyond the purview of this review for us to speculate on how actin, intermediate filaments, and microtubules; SUN domain proteins this will unfold. As an example, however, we believe that evolutionary span the inner nuclear membrane (INM) and bind to the nuclear lamina game theory models might provide useful tools. In particular, the and nuclear membrane-associated proteins [208]. The LINC complex ‘Tragedy of the Commons’ encompasses the salient features of mediates mechanotransduction forces to the nucleus that are, for the Crowdsourcing. In its simplest form, the model describes a heteroge- most part, mediated by actin. A perinuclear actin cap, for instance, neous population of agents, whom have a choice to contribute to a regulates nuclear shape in response to the shape of the cell, and this reg- public good. If enough choose to participate, then even those who ulation by actin is dependent on the LINC complex and lamin A/C [209]. did not will benefit [214]. With this basic platform, modulating var- Furthermore, actin also mediates lateral compressive forces in regulat- ious parts of the premise can create different models. For example, ing nuclear shape [210]. Interestingly, nuclear volume loss caused by the agents involved can be defined, and each actor can behave according nuclear shape change is marked by chromatin condensation and a to its own rules; these rules might represent the localization or functional decreased cell proliferation rate, indicating that nuclear form and func- capacity of a given agent (i.e. protein). Indeed, analysis of Crowdsourcing tion are directly coupled to extra-nuclear forces [210]. Given the role will allow us to formalize these rules, as we begin to understand its that the nuclear lamina plays in establishing genome organization and dynamic and complex behavior. Finally, in an intriguing twist, the idea its direct association with the LINC complex, the extent of nuclear of Crowdsourcing has now yielded crowdfunding, in which a heteroge- regulation by mechanical forces is becoming increasingly clear. Me- neous group of individuals donate to an identified cause or activity. chanical force application to the plasma membrane, for instance, alters There are now such sites that crowdfund biological studies. Perhaps in actin force transmission to the nucleus that causes a LINC complex- the future we might crowdfund our analysis of Crowdsourcing and nu- dependent chromatin decondensation [211]. In addition, force trans- clear protein multi-functionality. mission through the integrin–actin–LINC–lamin A/C interface has been shown to dissociate Cajal bodies, indicating that nuclear processes, be- Acknowledgements yond just genome organization patterns, can be coupled to external forces [212]. It will be interesting to see how structural cues regulate We thank Daniel S. Neems for sharing images Scott Holterhaus for such processes such as transcription and splicing. creating the artwork in Fig. 1, and Ellen Rice for thoughtful discussions and ideas. This work is funded by a Career Award in the Biomedical Sci- 5. Concluding remarks ences from the Burroughs Welcome Fund, an Ellison Medical Founda- tion New Scholar Award, and an NIH New Innovator Award. A.W. is While we are unable to review all of its various components, we supported by a postdoctoral fellowship from the American Cancer have focused on the dynamic complexity of nuclear organization and Society. function. A recurrent theme encountered in the study of genome orga- nization is that it is rather difficult to assign a given factor, such as Appendix A. Supplementary data CTCF, or a particular NB, such as the PML body, a single function. Rather, they often are associated with varied and disparate functions; thus, un- Supplementary data to this article can be found online at http://dx. derstanding what they really ‘do’ is hard to pin down. How, then, can we doi.org/10.1016/j.bbagrm.2014.01.003. begin to understand this pervasive multi-functionality as we consider nuclear organization from a systems biology perspective? As suggested in Introduction, we believe that the phenomenon of Crowdsourcing, an References outcome of the Web 2.0, provides an intriguing corollary to the multi- [1] T. Misteli, Self-organization in the genome, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) tude of functions observed for many nuclear factors. The basic paradigm 6885–6886. of Crowdsourcing is simple (Fig. 1A). 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