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Nucleolus and nuclear periphery: Velcro for heterochromatin
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ScienceDirect Nucleolus and nuclear periphery: Velcro for heterochromatin Jan Padeken1 and Patrick Heun2 Heterochromatin was first defined by Emil Heitz in 1928 by light microscopy. In the 1950s electron microscopy studies revealed that heterochromatin preferentially localizes to the nuclear periphery and around the nucleolus. While the use of genomic approaches led to the genome wide identification of laminaassociated and nucleolus-associated chromatin domains (LADs, NADs), recent studies now shed light on the processes mediating this topology and its dynamics. The identification of different factors on all regulatory levels, such as transcription factors, histone modifications, chromatin proteins, DNA sequences and non-coding RNAs, suggests the involvement of multiple distinct tethering pathways. Positioning at these nuclear sub-compartments is often but not always associated with transcriptional silencing, underlining the importance of the pre-existing chromatin context. Addresses 1 Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, 66, 4058 Basel, Switzerland 2 Max Planck Institute of Immunobiology and Epigenetics, Stu¨beweg 51, 79108 Freiburg, Germany Corresponding author: Heun, Patrick ([email protected])
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A classical example is the association of heterochromatin to the perinuclear space and the periphery of the nucleolus (for recent reviews see [4,5,6]). In this review we will discuss both types of heterochromatin positioning and speculate on the mechanisms and the functional outcomes of this nuclear compartmentalization. Our predominant focus is on higher eukaryotes, as some in-depth studies of centromere and telomere tethering to the nuclear envelope in yeast have been extensively reviewed elsewhere [7,8].
Transcriptionally silent domains fall into three different chromatin states Initially discovered based on differential staining with DNA dyes, repressive domains can be divided into three sub-categories: a) Constitutive heterochromatin, characterized by the binding of Heterochromatin Protein 1a (HP1a) to di- and tri-methylated histone H3 at lysine 9 (H3K9me2/3), b) Polycomb-bound, facultative heterochromatin enriched for the H3K27me2/3 mark, and c) Void chromatin, a silent chromatin state that has no strong enrichment for any analyzed chromatin factors or histone modifications (also referred to as black chromatin or a silent intergenic chromatin state [9,10].
This review comes from a themed issue on Cell nucleus Edited by Gary H Karpen and Michael P Rout
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Introduction The use of novel genomic approaches in cell populations such as the Chromosome Conformation Capture (3 C) technique and its derivatives have significantly advanced our understanding of genome organization in the nucleus (reviewed in [1]). These studies suggest that the genome is organized in topological domains, defined by increased chromatin contacts within the domain and few interactions outside. The sum of the topological domains of a given chromosome is contained within the separate chromosome territories of individual chromosomes. An additional layer of organization is due to contacts between the genome and nuclear bodies, which divide the nucleus into distinct functional sub-compartments. The interaction between a nuclear body and chromatin can lead to the stabilization of inter-chromosomal interactions and has in some cases been shown to correlate with co-regulation of the participating genomic loci [2,3]. Current Opinion in Cell Biology 2014, 28:54–60
Accumulating evidence suggests that especially constitutive heterochromatin plays an important role in nuclear architecture. The major function of constitutive heterochromatin in interphase is to keep transposons and simple DNA repeats transcriptionally silent, suppress recombination and ensure stable replication of these repetitive DNA elements. Depending on the cell type and the stage of development, heterochromatin can be organized around the nucleolus, or tethered to the nuclear periphery [11].
The nucleolus and perinucleolar chromatin The nucleolus is the nuclear body where rDNA transcription, rRNA processing and ribosome biogenesis occur. The ribosomal rRNA genes (rRNA) genes are arranged in an array of multiple copies in genomes of different organisms throughout evolution and transcribed by RNA polymerase I. The structure of the nucleolus is essential for rDNA transcription, which in turn also regulates RNA Polymerase I (Pol I) and rRNA processing. The repetitive nature of the rDNA loci not only allows the transcription from multiple genes at the same time, but also requires silencing factors that prevent internal recombination and DNA damage to maintain stability of the locus [12]. rRNAs are only transcribed from a subset of rDNA loci, www.sciencedirect.com
Perinucleolar and perinuclear chromatin interactions Padeken and Heun 55
which are looped into the inside of the nucleolus [13,14]. Transcriptionally inactive arrays are associated with the periphery of the nucleolus and accumulate marks of constitutive heterochromatin [13]. A shell of pericentric heterochromatin typically surrounds nucleoli. Using deep sequencing two independent studies recently provided a detailed analysis of DNA attached to isolated nucleoli, which were termed nucleolus-associated chromatin domains (NADs) [15,16]. Both groups identified similar genomic regions, which in addition to rDNA are highly enriched for satellite DNA, mostly from centromeric and pericentromeric regions, gene poor and silent chromatin, indicating that there is an overall specificity for the chromatin associated with the nucleolus. In mouse sensory neurons only one of the several hundred olfactory receptor genes is expressed in each individual neuron. In mice it has been shown that the expression of this single receptor gene requires the interaction with the enhancer H on chromosome 14 [17]. The remaining receptor genes accumulate heterochromatic histone marks, form clusters [18], and are enriched at the periphery of the nucleolus [15]. Cause and consequence are often difficult to dissect for nuclear positioning. Recent evidence in fission yeast shows that relocalization can precede silencing. Here, a silencing deficient mating-type region was targeted to the periphery of the nucleolus by inserting an ectopic rDNA repeat [19]. The relocalization restored silencing and was even observed in the absence of proper heterochromatin formation. Notably, localization to the perinucleolar space does not result in transcriptional silencing for all loci. In S. cerevisiae the tRNA genes are actively transcribed by RNA pol III and tethered as clusters to the nucleolus [20], while adjacent RNA pol II genes are repressed [21]. Clustering
and silencing are not dependent on the Sir complex, which marks heterochromatin in budding yeast, but are mediated by the interaction between condensin and TFIIIC [22]. The diversity of genomic loci and chromatin states at the nucleolus suggests a multitude of separate pathways mediating nucleolus positioning (Table 1, Figure 1a). It also shows that although being a ‘‘silent’’ nuclear sub-compartment the transcriptional outcome of nucleolus tethering is dependent on the chromatin context prior to tethering.
Mechanism for targeting genomic regions to the nucleolus Several genomic domains that associate with the nucleolus are found to cluster. Using both electron and light microscopy approaches, clustering of centromeric satellite DNA at the nucleolus was first observed in Purkinje cells from mouse brains 30 years ago [23]. More recently similar findings have confirmed that centromeres cluster in many organisms, including Drosophila and human, depending on the cell type and cell cycle stage [24,25,26,27]. This interchromosomal ‘‘cross-link’’ likely limits the freedom of movement and might contribute to a more stable positioning. Clustering of centromeres in Drosophila is dependent on the Nucleoplasmin like protein (NLP) and the insulator protein CTCF. Nucleolus tethering is mediated by the Nucleolin related protein Modulo. Interestingly, clustering depends on neither intact heterochromatin, nor the structural integrity of the nucleolus. Declustering of centromeres, however, resulted in the loss of the heterochromatin shell around the nucleolus and the formation of several smaller chromocenters, revealing residual self-affinity of the heterochromatin domains [26]. The periphery of the nucleolus therefore seems to function as a matrix for anchoring centromere clusters. As a
Table 1 Mechanism of chromatin tethering in higher eukaryotes Chromatin interactions at the nucleolus periphery Genomic locus
Mediated by
Organism
Ref.
Inactive X chr. Kcnq locus Centromeres Insulators
Xist RNA Kcnq1ot1 RNA NLP, CTCF, Modulo CTCF, Nucleophosmin
Human Human, mouse Drosophila Human
[30] [31] [26] [28]
Chromatin interactions at the nuclear periphery Genomic locus
Mediated by
Organism
Ref.
Constitutive heterochromatin Insulators DNA elements
H3K9me, HP1, BAF, nuclear lamina Su(Hw), CTCF cKrox, HDAC3 nuclear lamina
Mouse, C. elegans Drosophila, human Mouse
[38,45,46,48] [36,52] [51]
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56 Cell nucleus
Figure 1
(a)
(b)
Interaction between nucleolus and ncRNA Xist, Kcnq1ot1)
H3K9 methylation mediated interaction in trans
Recognition of heterochromatin (H3K9me, HP1a, HDAC3) by nuclear lamina proteins and the lamin B receptor
Nuclear periphery
Clustering of Centromeres Anchoring of centromere clusters (Modulo, NLP, CTCF)
Genes e.g. hunchback
Clustering of genes, e.g. olfactory receptors
Nucleolus
Insulator elements (CTCF, NPM1)
Insulator elements (CTCF, Su(Hw)) LAS (cKrox, HDAC3, Lap2β) Current Opinion in Cell Biology
Schematic overview of different mechanisms that mediate attachment of heterochromatin to the nucleolus and the nuclear periphery (see Table 1). Interactions between genomic regions and (a) the nucleolus or (b) the lamina containing regions of the nuclear periphery are shown. Identified regulatory factors are named in brackets. Nucleosomes in orange represent constitutive heterochromatin, in light blue facultative heterochromatin and in dark blue centromeric chromatin.
consequence, pericentric heterochromatin is concentrated, which in turn enforces silencing.
nucleolus association, which correlates with an erosion of heterochromatin and silencing [30].
Insulators are thought to be a major component required for the organization of the genome into chromatin domains and the establishment of chromosomal interactions. In humans, CTCF and Nucleophosmin have also been implicated in the tethering of an exogenous insulator site to the periphery of the nucleolus [28]. CTCF insulators are commonly found throughout the genome, with only a minority of sites enriched in the perinucleolar chromatin. Why only a sub-fraction of insulators are found at the nucleolus, or the nuclear periphery, is not yet understood. Interestingly, Rosa-Garrido and colleagues have shown that in human cells the CTCF-like protein BORIS (Brother of the Regulator of Imprinted sites; or CTCFL) is highly enriched in the nucleolus [29]. However, a correlation between cell type specific expression of BORIS and the nuclear positioning of CTCF insulators remains to be established.
A second example of RNA mediated nucleolus tethering is the genomic region flanking the imprinted Kcnq locus [31]. The Kcnq1ot1 RNA is antisense to Kcnq and transcribed only from the paternal allele. It triggers silencing of a 1 Mb region flanking the Kcnq locus, including eight to ten protein coding genes. Kcnq1ot1 also mediates the localization of the genomic region to the nucleolar periphery in S phase. In addition to the influence of the nucleolus as a compartment for silencing, multiple contacts between the genome and nucleolar proteins might provide a protective shell around the nucleolus itself. Evidence for this model comes from Karpen and colleagues, who used fly mutants to show that the structural integrity of the nucleolus and constitutive heterochromatin are interdependent [12].
The nuclear periphery
Several studies implicate non-coding RNAs (ncRNAs) in the localization of genomic regions to the nucleolus. In female human cells the inactive X chromosome is preferentially located at the periphery of the nucleolus. Deletion of the X-chromosome specific Xist RNA, after the establishment of X inactivation, leads to a loss of
The nuclear envelope can be separated into two subcompartments, containing either nuclear pores or nuclear lamina. While localization to nuclear pores can be correlated with transcriptional activation of genes, the nuclear lamina is associated with heterochromatin and linked to the repression of genes and repetitive sequences [32]. Although until recently nuclear lamina were thought to
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Perinucleolar and perinuclear chromatin interactions Padeken and Heun 57
be restricted to metazoans, new evidence points to the presence of lamin-like structures at the perinucleus in most eukaryotes that are often correlating with silenced chromatin domains [33].
The perinuclear space is enriched for all states of silent chromatin In higher eukaryotes several studies showed the accumulation of silent chromatin, gene poor regions and repetitive DNA at the nuclear lamina [34,35]. Experiments using a LaminB1 protein fused to the bacterial Dam methylase (DamID-technique) allowed the genome wide identification of lamina associated domains (LADs). LADs in human fibroblasts span around 40% of the human genome, are enriched for gene poor regions, flanked by CTCF binding, and are correlated with transcriptional repression [36]. Observing the attachment of LADs during in vitro differentiation of mouse embryonic stem cells (ESC) into neural precursor cells (NPCs) and terminally differentiated astrocytes (ACs) showed that association to the nuclear periphery is dynamic in nature [37]. However detachment from the periphery did not necessarily result in a direct activation for the majority of genes. Instead activation was only seen at a later step in differentiation. This observation suggests that the compartmentalization of chromatin domains does not per se regulate transcription. Instead, it seems to stabilize the transcriptional ‘‘ground-state’’ of a gene, locking it either in a silent state at the periphery, or a state in the nuclear interior, where the gene has the potential to be activated by the right transcription factors. Not only does attachment to the nuclear periphery correlate with silent chromatin, but the insertion of a heterochromatic array into C. elegans chromosomes also induces the localization of the genomic region to the nuclear periphery, and its detachment can be facilitated by the integration of tissue-specific promotors into the array [38].
Impact of peripheral localization on transcription Evidence that attachment to the nuclear periphery can also contribute to a silent chromatin state comes from experiments where the tethering of a transgenic construct to the nuclear periphery promotes transcriptional repression [34,39,40,41]. Importantly, not all of the genomic regions targeted to the nuclear lamina experience a change in transcription levels [34,42]. Further support for this observed plasticity comes from a recent study on the expression status of hunchback, a homolog of the mammalian Ikaros transcription factor, during neuronal differentiation in Drosophila. Hunchback expression is regulated by a feed-forward mechanism present exclusively in early neural progenitor cells [43]. The hunchback locus (hb) can only activate neuronal cell www.sciencedirect.com
fate in a short time window during the development of precursor cells. This window of competence ends with the localization of this locus to the nuclear periphery, which is several hours after hb transcription has ceased. Competence can be prolonged when the targeting to the periphery is inhibited by reduced levels of LaminB1 [44]. Similar to the nucleolus, perinuclear localization therefore appears to regulate the potential of a gene to be activated or repressed, but does not initiate per se a change in transcriptional status. This might also be one of the reasons why it has been difficult to describe a pronounced and robust phenotype in experiments where peripheral tethering of a genomic locus was abolished or forced.
Mechanisms of targeting to the nuclear periphery Recent studies focusing on interactions between specific genomic domains and the nuclear periphery have shed light on underlying mechanisms. In both human and nematode cells, histone H3K9 methylation has been found to be important for heterochromatin positioning at the nuclear lamina and for gene silencing [45,46]. Interestingly, in C. elegans silencing could be separated from positioning at the nuclear periphery using heterchromatic transgene arrays. Silencing was dependent on the HP1 homolog Hpl-2 and the two H3K9 methyl transferases Met-2, a homolog of the mammalian SetDB1, and Set-25, a novel methyltransferase with homology to G9a and hSuv39. However, detachment from the nuclear periphery was only observed in mutants lacking H3K9 methylation, but not Hpl-2 [46]. In turn the depletion of H3K9 methylation in mouse cells results in declustering of pericentric heterochromatin and the destabilization of the nuclear lamina, suggesting a cross talk between the nuclear sub-compartment and the attached chromatin [47]. It is likely that several pathways contribute to perinuclear targeting, including the interaction between the LaminB receptor (LBR) and HP1a [48], the nuclear envelope protein emerin and HDAC3 [49], and cell type specific nuclear transmembrane proteins that can promote localization of chromosomes to and from the nuclear periphery [50]. Apart from chromatin factors, DNA sequence has been also implicated in the regulation of localization. Analysis of the IgH and the Cyp3a locus in mouse fibroblasts led to the identification of small lamina-associating DNA sequences (LAS), enriched for the GAGA motif that is bound by cKrox, the murine ortholog of the Drosophila GAGA factor [51]. Ectopic insertion of this DNA sequence was sufficient to induce peripheral anchoring, and knockdown experiments of cKrox resulted in a partial release of the IgH LAD from the periphery. Interestingly cKrox, similar to HP1a, was also found to Current Opinion in Cell Biology 2014, 28:54–60
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interact with HDAC3 and the nuclear lamina protein Lap2b, suggesting a link between histone deacetylation and peripheral localization (Table 1; Figure 1b).
Chromatin dynamics between the nucleolus and the nuclear periphery An elegant adaptation of the DamID technique was recently used by Kind et al. to track LADs over time by microscopy. This revealed extensive reshuffling of these genomic domains relative to the nuclear periphery and required passage through mitosis. Interestingly, a subset of the internalized LADs became newly associated with the nucleolus [45]. Similar observations have been made for an individual lacO-tagged locus (19q12) [53], the dosage compensated X chromosome [30] and centromeres, which are enriched at the nucleolus or the nuclear periphery depending on the cell cycle stage [24]. In support of repositioning between these two compartments, NADs are found to often overlap with LADs in cell populations [15,16].
Conclusions Coming from the early observations that condensed chromatin accumulates at the nuclear periphery and the nucleolus, we are now beginning to understand the pathways involved and the biological relevance of this organization. While tethering of particular loci has illustrated the potential capacity of these perinuclear and nucleolar spaces to enhance silencing, they also underline the importance of the preexisting chromatin context in order for positioning to exert an effect. Acting downstream of transcription factors and chromatin context for gene regulation, nuclear localization appears to be more relevant to ‘‘lock in’’ a particular transcriptional status, thereby potentially reducing ‘‘noise’’ of upstream regulation events. Insights into the molecular mechanisms that are responsible for heterochromatin positioning have come from recent studies in yeast, C. elegans, Drosophila and human cells. The heterogeneity of genomic domains at these nuclear sub-compartments, and the involvement of different types of regulatory factors, suggest that multiple pathways contribute to the topology and localization of heterochromatin. Systematic characterization of the proteins, DNA sequences and RNAs involved will be needed to unmask the physiological relevance of nuclear positioning of chromatin domains, and to differentiate cause and consequence with respect to transcriptional regulation.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
1.
de Wit E, de Laat W: A decade of 3C technologies: insights into nuclear organization. Genes Dev 2012, 26:11-24.
Current Opinion in Cell Biology 2014, 28:54–60
2.
Chubb JR, Boyle S, Perry P, Bickmore WA: Chromatin motion is constrained by association with nuclear compartments in human cells. Curr Biol 2002, 12:439-445.
3.
Cheutin T, Cavalli G: Progressive polycomb assembly on H3K27me3 compartments generates polycomb bodies with developmentally regulated motion. PLoS Genet 2012, 8:e1002465.
4.
Nemeth A, Langst G: Genome organization in and around the nucleolus. Trends Genet 2011, 27:149-156.
5.
Bickmore WA, van Steensel B: Genome architecture: domain organization of interphase chromosomes. Cell 2013, 152:12701284.
6.
Towbin BD, Gonzalez-Sandoval A, Gasser SM: Mechanisms of heterochromatin subnuclear localization. Trends Biochem Sci 2013, 38:356-363.
7.
Taddei A, Gasser SM: Structure and function in the budding yeast nucleus. Genetics 2012, 192:107-129.
8.
Steglich B, Sazer S, Ekwall K: Transcriptional regulation at the yeast nuclear envelope. Nucleus 2013, 4:379-389.
Filion GJ, van Bemmel JG, Braunschweig U, Talhout W, Kind J, Ward LD, Brugman W, de Castro IJ, Kerkhoven RM, Bussemaker HJ et al.: Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 2010, 143:212-224. Using genome wide DamID the authors define five different types of chromatin based on binding factors.
9.
10. Kharchenko PV, Alekseyenko AA, Schwartz YB, Minoda A, Riddle NC, Ernst J, Sabo PJ, Larschan E, Gorchakov AA, Gu T et al.: Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. Nature 2011, 471:480-485. ChIP of chromatin binding factors is used to identify 9 distinct chromatin states. Together with [8] these studies extend the more simplistic view of just heterochromatin and euchromatin. 11. Solovei I, Wang AS, Thanisch K, Schmidt CS, Krebs S, Zwerger M, Cohen TV, Devys D, Foisner R, Peichl L et al.: LBR and lamin A/C sequentially tether peripheral heterochromatin and inversely regulate differentiation. Cell 2013, 152:584-598. 12. Peng JC, Karpen GH: H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability. Nat Cell Biol 2007, 9:25-35. This paper demonstrates the importance of silencing pathways to maintain the genome integrity of the repetitive rDNA locus and satellite arrays. 13. Pontvianne F, Blevins T, Chandrasekhara C, Mozgova I, Hassel C, Pontes OM, Tucker S, Mokros P, Muchova V, Fajkus J et al.: Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states. Genes Dev 2013, 27:1545-1550. 14. Nemeth A, Guibert S, Tiwari VK, Ohlsson R, Langst G: Epigenetic regulation of TTF-I-mediated promoter-terminator interactions of rRNA genes. EMBO J 2008, 27:1255-1265. 15. Nemeth A, Conesa A, Santoyo-Lopez J, Medina I, Montaner D, Peterfia B, Solovei I, Cremer T, Dopazo J, Langst G: Initial genomics of the human nucleolus. PLoS Genet 2010, 6:e1000889. Together with paper [16] these pioneering studies identify chromatin domains that interact with nucleoli (NADs) with strong enrichment of satellite DNA in both studies. 16. van Koningsbruggen S, Gierlinski M, Schofield P, Martin D, Barton GJ, Ariyurek Y, den Dunnen JT, Lamond AI: High resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli. Mol Biol Cell 2010, 21:3735-3748. See [15] 17. Lomvardas S, Barnea G, Pisapia DJ, Mendelsohn M, Kirkland J, Axel R: Interchromosomal interactions and olfactory receptor choice. Cell 2006, 126:403-413. 18. Clowney EJ, LeGros MA, Mosley CP, Clowney FG, MarkenskoffPapadimitriou EC, Myllys M, Barnea G, Larabell CA, Lomvardas S: Nuclear aggregation of olfactory receptor genes governs their monogenic expression. Cell 2012, 151:724-737. www.sciencedirect.com
Perinucleolar and perinuclear chromatin interactions Padeken and Heun 59
19. Jakociunas T, Domange Jordo M, Ait Mebarek M, Bunner CM, Verhein-Hansen J, Oddershede LB, Thon G: Subnuclear relocalization and silencing of a chromosomal region by an ectopic ribosomal DNA repeat. Proc Natl Acad Sci USA 2013. This paper shows in S. pombe that targeting of a silencing defective mating type locus to the nucleolus restores silencing and shows that nucleolar positioning is a separate event that precedes silencing. 20. Thompson M, Haeusler RA, Good PD, Engelke DR: Nucleolar clustering of dispersed tRNA genes. Science 2003, 302:13991400. 21. Wang L, Haeusler RA, Good PD, Thompson M, Nagar S, Engelke DR: Silencing near tRNA genes requires nucleolar localization. J Biol Chem 2005, 280:8637-8639. The authors demonstrate that depending on the context, perinucleolar position allows active transcription (tRNA clusters) but silences nearby genes. 22. Haeusler RA, Pratt-Hyatt M, Good PD, Gipson TA, Engelke DR: Clustering of yeast tRNA genes is mediated by specific association of condensin with tRNA gene transcription complexes. Genes Dev 2008, 22:2204-2214. 23. Manuelidis L: Different central nervous system cell types display distinct and nonrandom arrangements of satellite DNA sequences. Proc Natl Acad Sci USA 1984, 81:3123-3127. First description of clusters of centromeric satellite DNA at the nucleolus in mouse purkinje cells. 24. Solovei I, Schermelleh L, During K, Engelhardt A, Stein S, Cremer C, Cremer T: Differences in centromere positioning of cycling and postmitotic human cell types. Chromosoma 2004, 112:410-423. 25. Wiblin AE, Cui W, Clark AJ, Bickmore WA: Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells. J Cell Sci 2005, 118:3861-3868. 26. Padeken J, Mendiburo MJ, Chlamydas S, Schwarz HJ, Kremmer E, Heun P: The nucleoplasmin homolog NLP mediates centromere clustering and anchoring to the nucleolus. Molecular cell 2013, 50:236-249. This paper identifies a network of proteins in Drosophila, including NLP, CTCF and Modulo that tether centromere clusters to the nucleolar periphery. 27. Carvalho C, Pereira HM, Ferreira J, Pina C, Mendonca D, Rosa AC, Carmo-Fonseca M: Chromosomal G-dark bands determine the spatial organization of centromeric heterochromatin in the nucleus. Mol Biol Cell 2001, 12:3563-3572. 28. Yusufzai TM, Tagami H, Nakatani Y, Felsenfeld G: CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species. Mol Cell 2004, 13:291-298. The authors identify CTCF as interacting with NPM1 and mediating insulator tethering to the nucleolus. 29. Rosa-Garrido M, Ceballos L, Alonso-Lecue P, Abraira C, Delgado MD, Gandarillas A: A cell cycle role for the epigenetic factor CTCF-L/BORIS. PLoS ONE 2012, 7:e39371. 30. Zhang LF, Huynh KD, Lee JT: Perinucleolar targeting of the inactive X during S phase: evidence for a role in the maintenance of silencing. Cell 2007, 129:693-706. 31. Mohammad F, Pandey RR, Nagano T, Chakalova L, Mondal T, Fraser P, Kanduri C: Kcnq1ot1/Lit1 noncoding RNA mediates transcriptional silencing by targeting to the perinucleolar region. Mol Cell Biol 2008, 28:3713-3728. 32. Akhtar A, Gasser SM: The nuclear envelope and transcriptional control. Nat Rev Genet 2007, 8:507-517. 33. Field MC, Horn D, Alsford S, Koreny L, Rout MP: Telomeres, tethers and trypanosomes. Nucleus 2012, 3:478-486. 34. Finlan LE, Sproul D, Thomson I, Boyle S, Kerr E, Perry P, Ylstra B, Chubb JR, Bickmore WA: Recruitment to the nuclear periphery can alter expression of genes in human cells. PLoS Genet 2008, 4:e1000039. Demonstrates that artifical tethering of genomic loci to the nuclear lamina can lead to silencing of some but not all genes. www.sciencedirect.com
35. Pickersgill H, Kalverda B, de Wit E, Talhout W, Fornerod M, van Steensel B: Characterization of the Drosophila melanogaster genome at the nuclear lamina. Nat Genet 2006, 38:1005-1014. 36. Guelen L, Pagie L, Brasset E, Meuleman W, Faza MB, Talhout W, Eussen BH, de Klein A, Wessels L, de Laat W et al.: Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature 2008, 453:948-951. Pioneering study for mapping lamina associated domains (LADs) by DamID as large and discrete regions in the human genome. 37. Peric-Hupkes D, Meuleman W, Pagie L, Bruggeman SW, Solovei I, Brugman W, Graf S, Flicek P, Kerkhoven RM, van Lohuizen M et al.: Molecular maps of the reorganization of genomenuclear lamina interactions during differentiation. Mol Cell 2010, 38:603-613. 38. Meister P, Towbin BD, Pike BL, Ponti A, Gasser SM: The spatial dynamics of tissue-specific promoters during C. elegans development. Genes Dev 2010, 24:766-782. 39. Andrulis ED, Neiman AM, Zappulla DC, Sternglanz R: Perinuclear localization of chromatin facilitates transcriptional silencing. Nature 1998, 394:592-595. Demonstration that in budding yeast the nuclear periphery constributes to repression of a silencing-deficient HMR locus by nuclear envelope tethering. 40. Reddy KL, Zullo JM, Bertolino E, Singh H: Transcriptional repression mediated by repositioning of genes to the nuclear lamina. Nature 2008, 452:243-247. 41. Dialynas G, Speese S, Budnik V, Geyer PK, Wallrath LL: The role of Drosophila Lamin C in muscle function and gene expression. Development 2010, 137:3067-3077. 42. Kumaran RI, Spector DL: A genetic locus targeted to the nuclear periphery in living cells maintains its transcriptional competence. J Cell Biol 2008, 180:51-65. 43. Pearson BJ, Doe CQ: Regulation of neuroblast competence in Drosophila. Nature 2003, 425:624-628. 44. Kohwi M, Lupton JR, Lai SL, Miller MR, Doe CQ: Developmentally regulated subnuclear genome reorganization restricts neural progenitor competence in Drosophila. Cell 2013, 152:97-108. This paper characterizes the developmental dynamics of the hunchback gene localization at the nuclear periphery, which is correlated with its transcriptional repression. 45. Kind J, Pagie L, Ortabozkoyun H, Boyle S, de Vries SS, Janssen H, Amendola M, Nolen LD, Bickmore WA, van Steensel B: Single-cell dynamics of genome-nuclear lamina interactions. Cell 2013, 153:178-192. An elegant study revealing the dynamic interaction of LADs with the nuclear periphery and identifying the involvement of the H3K9 methyltransferase G9a to promote interaction with the nuclear lamina. 46. Towbin BD, Gonzalez-Aguilera C, Sack R, Gaidatzis D, Kalck V, Meister P, Askjaer P, Gasser SM: Step-wise methylation of histone H3K9 positions heterochromatin at the nuclear periphery. Cell 2012, 150:934-947. Using heterochromatic transgene arrays in C. elegans, the authors were able to functionally separate perinuclear anchoring from transcriptional repression of heterochromatin. 47. Pinheiro I, Margueron R, Shukeir N, Eisold M, Fritzsch C, Richter FM, Mittler G, Genoud C, Goyama S, Kurokawa M et al.: Prdm3 and Prdm16 are H3K9me1 methyltransferases required for mammalian heterochromatin integrity. Cell 2012, 150:948960. 48. Ye Q, Callebaut I, Pezhman A, Courvalin JC, Worman HJ: Domainspecific interactions of human HP1-type chromodomain proteins and inner nuclear membrane protein LBR. J Biol Chem 1997, 272:14983-14989. 49. Demmerle J, Koch AJ, Holaska JM: The nuclear envelope protein emerin binds directly to histone deacetylase 3 (HDAC3) and activates HDAC3 activity. J Biol Chem 2012, 287:22080-22088. 50. Zuleger N, Boyle S, Kelly DA, de Las Heras JI, Lazou V, Korfali N, Batrakou DG, Randles KN, Morris GE, Harrison DJ et al.: Specific Current Opinion in Cell Biology 2014, 28:54–60
60 Cell nucleus
nuclear envelope transmembrane proteins can promote the location of chromosomes to and from the nuclear periphery. Genome Biol 2013, 14:R14. 51. Zullo JM, Demarco IA, Pique-Regi R, Gaffney DJ, Epstein CB, Spooner CJ, Luperchio TR, Bernstein BE, Pritchard JK, Reddy KL et al.: DNA sequence-dependent compartmentalization and silencing of chromatin at the nuclear lamina. Cell 2012, 149:1474-1487.
Current Opinion in Cell Biology 2014, 28:54–60
52. Gerasimova TI, Byrd K, Corces VG: A chromatin insulator determines the nuclear localization of DNA. Mol Cell 2000, 6:1025-1035. 53. Thomson I, Gilchrist S, Bickmore WA, Chubb JR: The radial positioning of chromatin is not inherited through mitosis but is established de novo in early G1. Curr Biol 2004, 14:166-172.
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ScienceDirect Nucleolus and nuclear periphery: Velcro for heterochromatin Jan Padeken1 and Patrick Heun2 Heterochromatin was first defined by Emil Heitz in 1928 by light microscopy. In the 1950s electron microscopy studies revealed that heterochromatin preferentially localizes to the nuclear periphery and around the nucleolus. While the use of genomic approaches led to the genome wide identification of laminaassociated and nucleolus-associated chromatin domains (LADs, NADs), recent studies now shed light on the processes mediating this topology and its dynamics. The identification of different factors on all regulatory levels, such as transcription factors, histone modifications, chromatin proteins, DNA sequences and non-coding RNAs, suggests the involvement of multiple distinct tethering pathways. Positioning at these nuclear sub-compartments is often but not always associated with transcriptional silencing, underlining the importance of the pre-existing chromatin context. Addresses 1 Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, 66, 4058 Basel, Switzerland 2 Max Planck Institute of Immunobiology and Epigenetics, Stu¨beweg 51, 79108 Freiburg, Germany Corresponding author: Heun, Patrick ([email protected])
Current Opinion in Chemical Biology 2014, 28:54–60
A classical example is the association of heterochromatin to the perinuclear space and the periphery of the nucleolus (for recent reviews see [4,5,6]). In this review we will discuss both types of heterochromatin positioning and speculate on the mechanisms and the functional outcomes of this nuclear compartmentalization. Our predominant focus is on higher eukaryotes, as some in-depth studies of centromere and telomere tethering to the nuclear envelope in yeast have been extensively reviewed elsewhere [7,8].
Transcriptionally silent domains fall into three different chromatin states Initially discovered based on differential staining with DNA dyes, repressive domains can be divided into three sub-categories: a) Constitutive heterochromatin, characterized by the binding of Heterochromatin Protein 1a (HP1a) to di- and tri-methylated histone H3 at lysine 9 (H3K9me2/3), b) Polycomb-bound, facultative heterochromatin enriched for the H3K27me2/3 mark, and c) Void chromatin, a silent chromatin state that has no strong enrichment for any analyzed chromatin factors or histone modifications (also referred to as black chromatin or a silent intergenic chromatin state [9,10].
This review comes from a themed issue on Cell nucleus Edited by Gary H Karpen and Michael P Rout
0955-0674/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ceb.2014.03.001
Introduction The use of novel genomic approaches in cell populations such as the Chromosome Conformation Capture (3 C) technique and its derivatives have significantly advanced our understanding of genome organization in the nucleus (reviewed in [1]). These studies suggest that the genome is organized in topological domains, defined by increased chromatin contacts within the domain and few interactions outside. The sum of the topological domains of a given chromosome is contained within the separate chromosome territories of individual chromosomes. An additional layer of organization is due to contacts between the genome and nuclear bodies, which divide the nucleus into distinct functional sub-compartments. The interaction between a nuclear body and chromatin can lead to the stabilization of inter-chromosomal interactions and has in some cases been shown to correlate with co-regulation of the participating genomic loci [2,3]. Current Opinion in Cell Biology 2014, 28:54–60
Accumulating evidence suggests that especially constitutive heterochromatin plays an important role in nuclear architecture. The major function of constitutive heterochromatin in interphase is to keep transposons and simple DNA repeats transcriptionally silent, suppress recombination and ensure stable replication of these repetitive DNA elements. Depending on the cell type and the stage of development, heterochromatin can be organized around the nucleolus, or tethered to the nuclear periphery [11].
The nucleolus and perinucleolar chromatin The nucleolus is the nuclear body where rDNA transcription, rRNA processing and ribosome biogenesis occur. The ribosomal rRNA genes (rRNA) genes are arranged in an array of multiple copies in genomes of different organisms throughout evolution and transcribed by RNA polymerase I. The structure of the nucleolus is essential for rDNA transcription, which in turn also regulates RNA Polymerase I (Pol I) and rRNA processing. The repetitive nature of the rDNA loci not only allows the transcription from multiple genes at the same time, but also requires silencing factors that prevent internal recombination and DNA damage to maintain stability of the locus [12]. rRNAs are only transcribed from a subset of rDNA loci, www.sciencedirect.com
Perinucleolar and perinuclear chromatin interactions Padeken and Heun 55
which are looped into the inside of the nucleolus [13,14]. Transcriptionally inactive arrays are associated with the periphery of the nucleolus and accumulate marks of constitutive heterochromatin [13]. A shell of pericentric heterochromatin typically surrounds nucleoli. Using deep sequencing two independent studies recently provided a detailed analysis of DNA attached to isolated nucleoli, which were termed nucleolus-associated chromatin domains (NADs) [15,16]. Both groups identified similar genomic regions, which in addition to rDNA are highly enriched for satellite DNA, mostly from centromeric and pericentromeric regions, gene poor and silent chromatin, indicating that there is an overall specificity for the chromatin associated with the nucleolus. In mouse sensory neurons only one of the several hundred olfactory receptor genes is expressed in each individual neuron. In mice it has been shown that the expression of this single receptor gene requires the interaction with the enhancer H on chromosome 14 [17]. The remaining receptor genes accumulate heterochromatic histone marks, form clusters [18], and are enriched at the periphery of the nucleolus [15]. Cause and consequence are often difficult to dissect for nuclear positioning. Recent evidence in fission yeast shows that relocalization can precede silencing. Here, a silencing deficient mating-type region was targeted to the periphery of the nucleolus by inserting an ectopic rDNA repeat [19]. The relocalization restored silencing and was even observed in the absence of proper heterochromatin formation. Notably, localization to the perinucleolar space does not result in transcriptional silencing for all loci. In S. cerevisiae the tRNA genes are actively transcribed by RNA pol III and tethered as clusters to the nucleolus [20], while adjacent RNA pol II genes are repressed [21]. Clustering
and silencing are not dependent on the Sir complex, which marks heterochromatin in budding yeast, but are mediated by the interaction between condensin and TFIIIC [22]. The diversity of genomic loci and chromatin states at the nucleolus suggests a multitude of separate pathways mediating nucleolus positioning (Table 1, Figure 1a). It also shows that although being a ‘‘silent’’ nuclear sub-compartment the transcriptional outcome of nucleolus tethering is dependent on the chromatin context prior to tethering.
Mechanism for targeting genomic regions to the nucleolus Several genomic domains that associate with the nucleolus are found to cluster. Using both electron and light microscopy approaches, clustering of centromeric satellite DNA at the nucleolus was first observed in Purkinje cells from mouse brains 30 years ago [23]. More recently similar findings have confirmed that centromeres cluster in many organisms, including Drosophila and human, depending on the cell type and cell cycle stage [24,25,26,27]. This interchromosomal ‘‘cross-link’’ likely limits the freedom of movement and might contribute to a more stable positioning. Clustering of centromeres in Drosophila is dependent on the Nucleoplasmin like protein (NLP) and the insulator protein CTCF. Nucleolus tethering is mediated by the Nucleolin related protein Modulo. Interestingly, clustering depends on neither intact heterochromatin, nor the structural integrity of the nucleolus. Declustering of centromeres, however, resulted in the loss of the heterochromatin shell around the nucleolus and the formation of several smaller chromocenters, revealing residual self-affinity of the heterochromatin domains [26]. The periphery of the nucleolus therefore seems to function as a matrix for anchoring centromere clusters. As a
Table 1 Mechanism of chromatin tethering in higher eukaryotes Chromatin interactions at the nucleolus periphery Genomic locus
Mediated by
Organism
Ref.
Inactive X chr. Kcnq locus Centromeres Insulators
Xist RNA Kcnq1ot1 RNA NLP, CTCF, Modulo CTCF, Nucleophosmin
Human Human, mouse Drosophila Human
[30] [31] [26] [28]
Chromatin interactions at the nuclear periphery Genomic locus
Mediated by
Organism
Ref.
Constitutive heterochromatin Insulators DNA elements
H3K9me, HP1, BAF, nuclear lamina Su(Hw), CTCF cKrox, HDAC3 nuclear lamina
Mouse, C. elegans Drosophila, human Mouse
[38,45,46,48] [36,52] [51]
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56 Cell nucleus
Figure 1
(a)
(b)
Interaction between nucleolus and ncRNA Xist, Kcnq1ot1)
H3K9 methylation mediated interaction in trans
Recognition of heterochromatin (H3K9me, HP1a, HDAC3) by nuclear lamina proteins and the lamin B receptor
Nuclear periphery
Clustering of Centromeres Anchoring of centromere clusters (Modulo, NLP, CTCF)
Genes e.g. hunchback
Clustering of genes, e.g. olfactory receptors
Nucleolus
Insulator elements (CTCF, NPM1)
Insulator elements (CTCF, Su(Hw)) LAS (cKrox, HDAC3, Lap2β) Current Opinion in Cell Biology
Schematic overview of different mechanisms that mediate attachment of heterochromatin to the nucleolus and the nuclear periphery (see Table 1). Interactions between genomic regions and (a) the nucleolus or (b) the lamina containing regions of the nuclear periphery are shown. Identified regulatory factors are named in brackets. Nucleosomes in orange represent constitutive heterochromatin, in light blue facultative heterochromatin and in dark blue centromeric chromatin.
consequence, pericentric heterochromatin is concentrated, which in turn enforces silencing.
nucleolus association, which correlates with an erosion of heterochromatin and silencing [30].
Insulators are thought to be a major component required for the organization of the genome into chromatin domains and the establishment of chromosomal interactions. In humans, CTCF and Nucleophosmin have also been implicated in the tethering of an exogenous insulator site to the periphery of the nucleolus [28]. CTCF insulators are commonly found throughout the genome, with only a minority of sites enriched in the perinucleolar chromatin. Why only a sub-fraction of insulators are found at the nucleolus, or the nuclear periphery, is not yet understood. Interestingly, Rosa-Garrido and colleagues have shown that in human cells the CTCF-like protein BORIS (Brother of the Regulator of Imprinted sites; or CTCFL) is highly enriched in the nucleolus [29]. However, a correlation between cell type specific expression of BORIS and the nuclear positioning of CTCF insulators remains to be established.
A second example of RNA mediated nucleolus tethering is the genomic region flanking the imprinted Kcnq locus [31]. The Kcnq1ot1 RNA is antisense to Kcnq and transcribed only from the paternal allele. It triggers silencing of a 1 Mb region flanking the Kcnq locus, including eight to ten protein coding genes. Kcnq1ot1 also mediates the localization of the genomic region to the nucleolar periphery in S phase. In addition to the influence of the nucleolus as a compartment for silencing, multiple contacts between the genome and nucleolar proteins might provide a protective shell around the nucleolus itself. Evidence for this model comes from Karpen and colleagues, who used fly mutants to show that the structural integrity of the nucleolus and constitutive heterochromatin are interdependent [12].
The nuclear periphery
Several studies implicate non-coding RNAs (ncRNAs) in the localization of genomic regions to the nucleolus. In female human cells the inactive X chromosome is preferentially located at the periphery of the nucleolus. Deletion of the X-chromosome specific Xist RNA, after the establishment of X inactivation, leads to a loss of
The nuclear envelope can be separated into two subcompartments, containing either nuclear pores or nuclear lamina. While localization to nuclear pores can be correlated with transcriptional activation of genes, the nuclear lamina is associated with heterochromatin and linked to the repression of genes and repetitive sequences [32]. Although until recently nuclear lamina were thought to
Current Opinion in Cell Biology 2014, 28:54–60
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Perinucleolar and perinuclear chromatin interactions Padeken and Heun 57
be restricted to metazoans, new evidence points to the presence of lamin-like structures at the perinucleus in most eukaryotes that are often correlating with silenced chromatin domains [33].
The perinuclear space is enriched for all states of silent chromatin In higher eukaryotes several studies showed the accumulation of silent chromatin, gene poor regions and repetitive DNA at the nuclear lamina [34,35]. Experiments using a LaminB1 protein fused to the bacterial Dam methylase (DamID-technique) allowed the genome wide identification of lamina associated domains (LADs). LADs in human fibroblasts span around 40% of the human genome, are enriched for gene poor regions, flanked by CTCF binding, and are correlated with transcriptional repression [36]. Observing the attachment of LADs during in vitro differentiation of mouse embryonic stem cells (ESC) into neural precursor cells (NPCs) and terminally differentiated astrocytes (ACs) showed that association to the nuclear periphery is dynamic in nature [37]. However detachment from the periphery did not necessarily result in a direct activation for the majority of genes. Instead activation was only seen at a later step in differentiation. This observation suggests that the compartmentalization of chromatin domains does not per se regulate transcription. Instead, it seems to stabilize the transcriptional ‘‘ground-state’’ of a gene, locking it either in a silent state at the periphery, or a state in the nuclear interior, where the gene has the potential to be activated by the right transcription factors. Not only does attachment to the nuclear periphery correlate with silent chromatin, but the insertion of a heterochromatic array into C. elegans chromosomes also induces the localization of the genomic region to the nuclear periphery, and its detachment can be facilitated by the integration of tissue-specific promotors into the array [38].
Impact of peripheral localization on transcription Evidence that attachment to the nuclear periphery can also contribute to a silent chromatin state comes from experiments where the tethering of a transgenic construct to the nuclear periphery promotes transcriptional repression [34,39,40,41]. Importantly, not all of the genomic regions targeted to the nuclear lamina experience a change in transcription levels [34,42]. Further support for this observed plasticity comes from a recent study on the expression status of hunchback, a homolog of the mammalian Ikaros transcription factor, during neuronal differentiation in Drosophila. Hunchback expression is regulated by a feed-forward mechanism present exclusively in early neural progenitor cells [43]. The hunchback locus (hb) can only activate neuronal cell www.sciencedirect.com
fate in a short time window during the development of precursor cells. This window of competence ends with the localization of this locus to the nuclear periphery, which is several hours after hb transcription has ceased. Competence can be prolonged when the targeting to the periphery is inhibited by reduced levels of LaminB1 [44]. Similar to the nucleolus, perinuclear localization therefore appears to regulate the potential of a gene to be activated or repressed, but does not initiate per se a change in transcriptional status. This might also be one of the reasons why it has been difficult to describe a pronounced and robust phenotype in experiments where peripheral tethering of a genomic locus was abolished or forced.
Mechanisms of targeting to the nuclear periphery Recent studies focusing on interactions between specific genomic domains and the nuclear periphery have shed light on underlying mechanisms. In both human and nematode cells, histone H3K9 methylation has been found to be important for heterochromatin positioning at the nuclear lamina and for gene silencing [45,46]. Interestingly, in C. elegans silencing could be separated from positioning at the nuclear periphery using heterchromatic transgene arrays. Silencing was dependent on the HP1 homolog Hpl-2 and the two H3K9 methyl transferases Met-2, a homolog of the mammalian SetDB1, and Set-25, a novel methyltransferase with homology to G9a and hSuv39. However, detachment from the nuclear periphery was only observed in mutants lacking H3K9 methylation, but not Hpl-2 [46]. In turn the depletion of H3K9 methylation in mouse cells results in declustering of pericentric heterochromatin and the destabilization of the nuclear lamina, suggesting a cross talk between the nuclear sub-compartment and the attached chromatin [47]. It is likely that several pathways contribute to perinuclear targeting, including the interaction between the LaminB receptor (LBR) and HP1a [48], the nuclear envelope protein emerin and HDAC3 [49], and cell type specific nuclear transmembrane proteins that can promote localization of chromosomes to and from the nuclear periphery [50]. Apart from chromatin factors, DNA sequence has been also implicated in the regulation of localization. Analysis of the IgH and the Cyp3a locus in mouse fibroblasts led to the identification of small lamina-associating DNA sequences (LAS), enriched for the GAGA motif that is bound by cKrox, the murine ortholog of the Drosophila GAGA factor [51]. Ectopic insertion of this DNA sequence was sufficient to induce peripheral anchoring, and knockdown experiments of cKrox resulted in a partial release of the IgH LAD from the periphery. Interestingly cKrox, similar to HP1a, was also found to Current Opinion in Cell Biology 2014, 28:54–60
58 Cell nucleus
interact with HDAC3 and the nuclear lamina protein Lap2b, suggesting a link between histone deacetylation and peripheral localization (Table 1; Figure 1b).
Chromatin dynamics between the nucleolus and the nuclear periphery An elegant adaptation of the DamID technique was recently used by Kind et al. to track LADs over time by microscopy. This revealed extensive reshuffling of these genomic domains relative to the nuclear periphery and required passage through mitosis. Interestingly, a subset of the internalized LADs became newly associated with the nucleolus [45]. Similar observations have been made for an individual lacO-tagged locus (19q12) [53], the dosage compensated X chromosome [30] and centromeres, which are enriched at the nucleolus or the nuclear periphery depending on the cell cycle stage [24]. In support of repositioning between these two compartments, NADs are found to often overlap with LADs in cell populations [15,16].
Conclusions Coming from the early observations that condensed chromatin accumulates at the nuclear periphery and the nucleolus, we are now beginning to understand the pathways involved and the biological relevance of this organization. While tethering of particular loci has illustrated the potential capacity of these perinuclear and nucleolar spaces to enhance silencing, they also underline the importance of the preexisting chromatin context in order for positioning to exert an effect. Acting downstream of transcription factors and chromatin context for gene regulation, nuclear localization appears to be more relevant to ‘‘lock in’’ a particular transcriptional status, thereby potentially reducing ‘‘noise’’ of upstream regulation events. Insights into the molecular mechanisms that are responsible for heterochromatin positioning have come from recent studies in yeast, C. elegans, Drosophila and human cells. The heterogeneity of genomic domains at these nuclear sub-compartments, and the involvement of different types of regulatory factors, suggest that multiple pathways contribute to the topology and localization of heterochromatin. Systematic characterization of the proteins, DNA sequences and RNAs involved will be needed to unmask the physiological relevance of nuclear positioning of chromatin domains, and to differentiate cause and consequence with respect to transcriptional regulation.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
1.
de Wit E, de Laat W: A decade of 3C technologies: insights into nuclear organization. Genes Dev 2012, 26:11-24.
Current Opinion in Cell Biology 2014, 28:54–60
2.
Chubb JR, Boyle S, Perry P, Bickmore WA: Chromatin motion is constrained by association with nuclear compartments in human cells. Curr Biol 2002, 12:439-445.
3.
Cheutin T, Cavalli G: Progressive polycomb assembly on H3K27me3 compartments generates polycomb bodies with developmentally regulated motion. PLoS Genet 2012, 8:e1002465.
4.
Nemeth A, Langst G: Genome organization in and around the nucleolus. Trends Genet 2011, 27:149-156.
5.
Bickmore WA, van Steensel B: Genome architecture: domain organization of interphase chromosomes. Cell 2013, 152:12701284.
6.
Towbin BD, Gonzalez-Sandoval A, Gasser SM: Mechanisms of heterochromatin subnuclear localization. Trends Biochem Sci 2013, 38:356-363.
7.
Taddei A, Gasser SM: Structure and function in the budding yeast nucleus. Genetics 2012, 192:107-129.
8.
Steglich B, Sazer S, Ekwall K: Transcriptional regulation at the yeast nuclear envelope. Nucleus 2013, 4:379-389.
Filion GJ, van Bemmel JG, Braunschweig U, Talhout W, Kind J, Ward LD, Brugman W, de Castro IJ, Kerkhoven RM, Bussemaker HJ et al.: Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 2010, 143:212-224. Using genome wide DamID the authors define five different types of chromatin based on binding factors.
9.
10. Kharchenko PV, Alekseyenko AA, Schwartz YB, Minoda A, Riddle NC, Ernst J, Sabo PJ, Larschan E, Gorchakov AA, Gu T et al.: Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. Nature 2011, 471:480-485. ChIP of chromatin binding factors is used to identify 9 distinct chromatin states. Together with [8] these studies extend the more simplistic view of just heterochromatin and euchromatin. 11. Solovei I, Wang AS, Thanisch K, Schmidt CS, Krebs S, Zwerger M, Cohen TV, Devys D, Foisner R, Peichl L et al.: LBR and lamin A/C sequentially tether peripheral heterochromatin and inversely regulate differentiation. Cell 2013, 152:584-598. 12. Peng JC, Karpen GH: H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability. Nat Cell Biol 2007, 9:25-35. This paper demonstrates the importance of silencing pathways to maintain the genome integrity of the repetitive rDNA locus and satellite arrays. 13. Pontvianne F, Blevins T, Chandrasekhara C, Mozgova I, Hassel C, Pontes OM, Tucker S, Mokros P, Muchova V, Fajkus J et al.: Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states. Genes Dev 2013, 27:1545-1550. 14. Nemeth A, Guibert S, Tiwari VK, Ohlsson R, Langst G: Epigenetic regulation of TTF-I-mediated promoter-terminator interactions of rRNA genes. EMBO J 2008, 27:1255-1265. 15. Nemeth A, Conesa A, Santoyo-Lopez J, Medina I, Montaner D, Peterfia B, Solovei I, Cremer T, Dopazo J, Langst G: Initial genomics of the human nucleolus. PLoS Genet 2010, 6:e1000889. Together with paper [16] these pioneering studies identify chromatin domains that interact with nucleoli (NADs) with strong enrichment of satellite DNA in both studies. 16. van Koningsbruggen S, Gierlinski M, Schofield P, Martin D, Barton GJ, Ariyurek Y, den Dunnen JT, Lamond AI: High resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli. Mol Biol Cell 2010, 21:3735-3748. See [15] 17. Lomvardas S, Barnea G, Pisapia DJ, Mendelsohn M, Kirkland J, Axel R: Interchromosomal interactions and olfactory receptor choice. Cell 2006, 126:403-413. 18. Clowney EJ, LeGros MA, Mosley CP, Clowney FG, MarkenskoffPapadimitriou EC, Myllys M, Barnea G, Larabell CA, Lomvardas S: Nuclear aggregation of olfactory receptor genes governs their monogenic expression. Cell 2012, 151:724-737. www.sciencedirect.com
Perinucleolar and perinuclear chromatin interactions Padeken and Heun 59
19. Jakociunas T, Domange Jordo M, Ait Mebarek M, Bunner CM, Verhein-Hansen J, Oddershede LB, Thon G: Subnuclear relocalization and silencing of a chromosomal region by an ectopic ribosomal DNA repeat. Proc Natl Acad Sci USA 2013. This paper shows in S. pombe that targeting of a silencing defective mating type locus to the nucleolus restores silencing and shows that nucleolar positioning is a separate event that precedes silencing. 20. Thompson M, Haeusler RA, Good PD, Engelke DR: Nucleolar clustering of dispersed tRNA genes. Science 2003, 302:13991400. 21. Wang L, Haeusler RA, Good PD, Thompson M, Nagar S, Engelke DR: Silencing near tRNA genes requires nucleolar localization. J Biol Chem 2005, 280:8637-8639. The authors demonstrate that depending on the context, perinucleolar position allows active transcription (tRNA clusters) but silences nearby genes. 22. Haeusler RA, Pratt-Hyatt M, Good PD, Gipson TA, Engelke DR: Clustering of yeast tRNA genes is mediated by specific association of condensin with tRNA gene transcription complexes. Genes Dev 2008, 22:2204-2214. 23. Manuelidis L: Different central nervous system cell types display distinct and nonrandom arrangements of satellite DNA sequences. Proc Natl Acad Sci USA 1984, 81:3123-3127. First description of clusters of centromeric satellite DNA at the nucleolus in mouse purkinje cells. 24. Solovei I, Schermelleh L, During K, Engelhardt A, Stein S, Cremer C, Cremer T: Differences in centromere positioning of cycling and postmitotic human cell types. Chromosoma 2004, 112:410-423. 25. Wiblin AE, Cui W, Clark AJ, Bickmore WA: Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells. J Cell Sci 2005, 118:3861-3868. 26. Padeken J, Mendiburo MJ, Chlamydas S, Schwarz HJ, Kremmer E, Heun P: The nucleoplasmin homolog NLP mediates centromere clustering and anchoring to the nucleolus. Molecular cell 2013, 50:236-249. This paper identifies a network of proteins in Drosophila, including NLP, CTCF and Modulo that tether centromere clusters to the nucleolar periphery. 27. Carvalho C, Pereira HM, Ferreira J, Pina C, Mendonca D, Rosa AC, Carmo-Fonseca M: Chromosomal G-dark bands determine the spatial organization of centromeric heterochromatin in the nucleus. Mol Biol Cell 2001, 12:3563-3572. 28. Yusufzai TM, Tagami H, Nakatani Y, Felsenfeld G: CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species. Mol Cell 2004, 13:291-298. The authors identify CTCF as interacting with NPM1 and mediating insulator tethering to the nucleolus. 29. Rosa-Garrido M, Ceballos L, Alonso-Lecue P, Abraira C, Delgado MD, Gandarillas A: A cell cycle role for the epigenetic factor CTCF-L/BORIS. PLoS ONE 2012, 7:e39371. 30. Zhang LF, Huynh KD, Lee JT: Perinucleolar targeting of the inactive X during S phase: evidence for a role in the maintenance of silencing. Cell 2007, 129:693-706. 31. Mohammad F, Pandey RR, Nagano T, Chakalova L, Mondal T, Fraser P, Kanduri C: Kcnq1ot1/Lit1 noncoding RNA mediates transcriptional silencing by targeting to the perinucleolar region. Mol Cell Biol 2008, 28:3713-3728. 32. Akhtar A, Gasser SM: The nuclear envelope and transcriptional control. Nat Rev Genet 2007, 8:507-517. 33. Field MC, Horn D, Alsford S, Koreny L, Rout MP: Telomeres, tethers and trypanosomes. Nucleus 2012, 3:478-486. 34. Finlan LE, Sproul D, Thomson I, Boyle S, Kerr E, Perry P, Ylstra B, Chubb JR, Bickmore WA: Recruitment to the nuclear periphery can alter expression of genes in human cells. PLoS Genet 2008, 4:e1000039. Demonstrates that artifical tethering of genomic loci to the nuclear lamina can lead to silencing of some but not all genes. www.sciencedirect.com
35. Pickersgill H, Kalverda B, de Wit E, Talhout W, Fornerod M, van Steensel B: Characterization of the Drosophila melanogaster genome at the nuclear lamina. Nat Genet 2006, 38:1005-1014. 36. Guelen L, Pagie L, Brasset E, Meuleman W, Faza MB, Talhout W, Eussen BH, de Klein A, Wessels L, de Laat W et al.: Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature 2008, 453:948-951. Pioneering study for mapping lamina associated domains (LADs) by DamID as large and discrete regions in the human genome. 37. Peric-Hupkes D, Meuleman W, Pagie L, Bruggeman SW, Solovei I, Brugman W, Graf S, Flicek P, Kerkhoven RM, van Lohuizen M et al.: Molecular maps of the reorganization of genomenuclear lamina interactions during differentiation. Mol Cell 2010, 38:603-613. 38. Meister P, Towbin BD, Pike BL, Ponti A, Gasser SM: The spatial dynamics of tissue-specific promoters during C. elegans development. Genes Dev 2010, 24:766-782. 39. Andrulis ED, Neiman AM, Zappulla DC, Sternglanz R: Perinuclear localization of chromatin facilitates transcriptional silencing. Nature 1998, 394:592-595. Demonstration that in budding yeast the nuclear periphery constributes to repression of a silencing-deficient HMR locus by nuclear envelope tethering. 40. Reddy KL, Zullo JM, Bertolino E, Singh H: Transcriptional repression mediated by repositioning of genes to the nuclear lamina. Nature 2008, 452:243-247. 41. Dialynas G, Speese S, Budnik V, Geyer PK, Wallrath LL: The role of Drosophila Lamin C in muscle function and gene expression. Development 2010, 137:3067-3077. 42. Kumaran RI, Spector DL: A genetic locus targeted to the nuclear periphery in living cells maintains its transcriptional competence. J Cell Biol 2008, 180:51-65. 43. Pearson BJ, Doe CQ: Regulation of neuroblast competence in Drosophila. Nature 2003, 425:624-628. 44. Kohwi M, Lupton JR, Lai SL, Miller MR, Doe CQ: Developmentally regulated subnuclear genome reorganization restricts neural progenitor competence in Drosophila. Cell 2013, 152:97-108. This paper characterizes the developmental dynamics of the hunchback gene localization at the nuclear periphery, which is correlated with its transcriptional repression. 45. Kind J, Pagie L, Ortabozkoyun H, Boyle S, de Vries SS, Janssen H, Amendola M, Nolen LD, Bickmore WA, van Steensel B: Single-cell dynamics of genome-nuclear lamina interactions. Cell 2013, 153:178-192. An elegant study revealing the dynamic interaction of LADs with the nuclear periphery and identifying the involvement of the H3K9 methyltransferase G9a to promote interaction with the nuclear lamina. 46. Towbin BD, Gonzalez-Aguilera C, Sack R, Gaidatzis D, Kalck V, Meister P, Askjaer P, Gasser SM: Step-wise methylation of histone H3K9 positions heterochromatin at the nuclear periphery. Cell 2012, 150:934-947. Using heterochromatic transgene arrays in C. elegans, the authors were able to functionally separate perinuclear anchoring from transcriptional repression of heterochromatin. 47. Pinheiro I, Margueron R, Shukeir N, Eisold M, Fritzsch C, Richter FM, Mittler G, Genoud C, Goyama S, Kurokawa M et al.: Prdm3 and Prdm16 are H3K9me1 methyltransferases required for mammalian heterochromatin integrity. Cell 2012, 150:948960. 48. Ye Q, Callebaut I, Pezhman A, Courvalin JC, Worman HJ: Domainspecific interactions of human HP1-type chromodomain proteins and inner nuclear membrane protein LBR. J Biol Chem 1997, 272:14983-14989. 49. Demmerle J, Koch AJ, Holaska JM: The nuclear envelope protein emerin binds directly to histone deacetylase 3 (HDAC3) and activates HDAC3 activity. J Biol Chem 2012, 287:22080-22088. 50. Zuleger N, Boyle S, Kelly DA, de Las Heras JI, Lazou V, Korfali N, Batrakou DG, Randles KN, Morris GE, Harrison DJ et al.: Specific Current Opinion in Cell Biology 2014, 28:54–60
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nuclear envelope transmembrane proteins can promote the location of chromosomes to and from the nuclear periphery. Genome Biol 2013, 14:R14. 51. Zullo JM, Demarco IA, Pique-Regi R, Gaffney DJ, Epstein CB, Spooner CJ, Luperchio TR, Bernstein BE, Pritchard JK, Reddy KL et al.: DNA sequence-dependent compartmentalization and silencing of chromatin at the nuclear lamina. Cell 2012, 149:1474-1487.
Current Opinion in Cell Biology 2014, 28:54–60
52. Gerasimova TI, Byrd K, Corces VG: A chromatin insulator determines the nuclear localization of DNA. Mol Cell 2000, 6:1025-1035. 53. Thomson I, Gilchrist S, Bickmore WA, Chubb JR: The radial positioning of chromatin is not inherited through mitosis but is established de novo in early G1. Curr Biol 2004, 14:166-172.
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