Structure and Function in the Budding Yeast Nucleus

YEASTBOOK

GENOME ORGANIZATION & INTEGRITY

Angela Taddei* and Susan M. Gasser†,1 *Unité Mixte de Recherche 218, Centre National de la Recherche Scientiﬁque/Institut Curie-Section de Recherche, 75231 Paris Cedex 05, France, and yFriedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland

ABSTRACT Budding yeast, like other eukaryotes, carries its genetic information on chromosomes that are sequestered from other cellular constituents by a double membrane, which forms the nucleus. An elaborate molecular machinery forms large pores that span the double membrane and regulate the traffic of macromolecules into and out of the nucleus. In multicellular eukaryotes, an intermediate filament meshwork formed of lamin proteins bridges from pore to pore and helps the nucleus reform after mitosis. Yeast, however, lacks lamins, and the nuclear envelope is not disrupted during yeast mitosis. The mitotic spindle nucleates from the nucleoplasmic face of the spindle pole body, which is embedded in the nuclear envelope. Surprisingly, the kinetochores remain attached to short microtubules throughout interphase, influencing the position of centromeres in the interphase nucleus, and telomeres are found clustered in foci at the nuclear periphery. In addition to this chromosomal organization, the yeast nucleus is functionally compartmentalized to allow efficient gene expression, repression, RNA processing, genomic replication, and repair. The formation of functional subcompartments is achieved in the nucleus without intranuclear membranes and depends instead on sequence elements, protein–protein interactions, specific anchorage sites at the nuclear envelope or at pores, and long-range contacts between specific chromosomal loci, such as telomeres. Here we review the spatial organization of the budding yeast nucleus, the proteins involved in forming nuclear subcompartments, and evidence suggesting that the spatial organization of the nucleus is important for nuclear function.

TABLE OF CONTENTS Abstract 107 Introduction 108 Features of the Yeast Nucleus 108 Unique and conserved characteristics 108 Nuclear envelope and nuclear pore complex 109 Long-range chromosome organization 111 Chromatin dynamics 112 DNA-based compartments 112 Nucleolus: 112 Telomere foci—assemblies of repetitive DNA and silencing factors: 113 tRNA genes: 114 Replication foci: 114 Continued

Copyright © 2012 by the Genetics Society of America doi: 10.1534/genetics.112.140608 Manuscript received March 19, 2012; accepted for publication May 29, 2012 1Corresponding author: Friedrch Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. E-mail: [email protected]

Genetics, Vol. 192, 107–129 September 2012 107 CONTENTS, continued

Sites of DNA repair: 115 Mechanisms Underlying Nuclear Compartmentation 115 Redundancy in telomere-anchoring pathways 115 Minimal anchoring assay and the validation of genetic screens 116 Formation of chromatin compartments: association in trans 117 Mechanisms of chromatin movement 118 Functional Consequences of Nuclear Organization 119 Gene silencing 119 Gene activation 119 Boundaries between active and inactive domains 120 Effects on genome stability 121 Mobility of DSBs: 121 Nuclear pores and Mps3 anchorage in noncanonical repair: 122 Regulation of recombination at the rDNA locus: 122 Concluding Remarks 123

HE cell nucleus not only harbors and expresses an organ- A second major factor contributing to nuclear organization Tism’s essential genetic blueprint, but also ensures the is the interaction between chromatin and stable structural proper expression, duplication, repair, and segregation of elements of the nucleus. In budding yeast, the key structural chromosomes while ensuring proper processing and export elements are the nuclear envelope (NE), the nuclear pore of messenger and ribosomal RNA (Spector 2003; Taddei complex (NPC), and the nucleolus. The NE encompasses et al. 2004b). The dense packing of highly charged molecules different types of chromatin anchorage sites, including the (DNA, RNA, histones, nonhistone proteins) in a limited nuclear spindle pole body (SPB), and unique protein components of space was once thought to constrain molecular dynamics, yet the inner nuclear membrane that tether heterochromatin, the we now know that the nucleus is neither grid-locked nor a ran- ribosomal DNA (rDNA), or different types of DNA damage dom jumble (Rouquette et al. 2010). Large rings of chromatin (Akhtar and Gasser 2007; Mekhail and Moazed 2010). The diffuse freely through the nuclear volume in a random diffusive NPC also plays a role in the transient anchoring of activated walk (Gartenberg et al. 2004; Neumann et al. 2012), yet the genes or of DNA damage that cannot be readily repaired by nucleus can maintain functional subcompartments enriched for homologous recombination. Finally, long-range interaction of specific enzymes and chromatin states. Understanding this di- loci in trans, such as the clustering of telomeres or of trans- chotomy is key to understanding how nuclear organization fer RNA (tRNA) genes, influences nuclear order. The com- facilitates nuclear function (reviewed in Taddei et al. 2004b; bination of physical constraints on chromatin movement and Mekhail and Moazed 2010; Egecioglu and Brickner 2011; protein–protein interactions helps generate nuclear subcom- Rajapakse and Groudine 2011; Zimmer and Fabre 2011). partments that are enriched for specific DNA sequences, Chromosomes, and the nucleosomal fibers within them, factors, and enzymatic activities (Gasser et al. 2004; Rosa can be thought of as basic structural elements of the nucleus. and Everaers 2008). How these subcompartments affect Long-range chromosome folding is constrained by the physics nuclear function remains a central topic of research. of polymer dynamics (Klenin et al. 1998; Dekker et al. 2002; The basic principles of nuclear organization can be ob- Gehlen et al. 2006; Neumann et al. 2012), and the character- served in all eukaryotes from yeast to humans. This allows istics of the chromosome polymers themselves depend on the us to test the functional implications of nuclear organization folding of the nucleosomal fiber (Rosa and Everaers 2008). in a single-celled organism, despite there being species- and Yet chromatin in interphase nuclei is not regularly compacted tissue-specific nuclear features. With facile genetics, live mi- and is subject to reversible covalent modifications on both croscopy, and genome-wide mapping approaches, budding DNA and histones within the nucleosomal fiber. Therefore, yeast has proven to be extremely useful for testing the crucial biophysical properties of long-range chromatin dy- functional roles of nuclear structure, as reviewed below. namics, such as persistence length, mass density, and diffusion rate, are variable and subject to changes induced by Features of the Yeast Nucleus nucleosome remodelers and histone modifiers. Thus, the Unique and conserved characteristics post-translational modification of histones contributes not only to local chromatin folding, but to the three-dimensional Yeast is, in many ways, a typical eukaryote, yet it has a organization of the genome (van Steensel 2011). few unique and defining features that deserve mention. As

108 A. Taddei and S. M. Gasser described above, the SPB is embedded in the nuclear mating-type loci, HML and HMR (reviewedinRuscheet al. envelope and nucleates both intranuclear microtubules in 2003). Interestingly, native subtelomeric genes are transcrip- interphase and the mitotic spindle. The SPB has a position tionally inert under standard growth conditions independent strictly determined by the site of new bud emergence (Lee of SIR factor binding, as they carry genes that are expressed et al. 1999), which itself is opposite the nucleolus (Yang only under restrictive nutrient conditions (Fabre et al. 2005). et al. 1989; Bystricky et al. 2004, 2005). The position of Budding yeast lacks the histone H3 K9 methylation that bud emergence is determined by landmark proteins at the typifies centromeric heterochromatin in most other eukary- cell cortex and Cdc42, which are positioned through asso- otes, as well as the major protein ligand that recognizes this ciation with the previous bud site. These proteins nucleate modification (heterochromatin protein 1, or HP1) and the actin filaments and determine the orientation of cytoplasmic RNA interference machinery that facilitates PEV in fission actin. The nucleus, in turn, is oriented by the cooperative yeast (reviewed in Buhler and Gasser 2009). Instead, a tri- action of SPB-microtubule-Kar9-Myo2 and actin interactions meric complex of Sir2, Sir3, and Sir4 recognizes unmodified in a manner dependent on the actin filament orientation nucleosomes to repress transcription and reduce endonucle- (Gundersen and Bretscher 2003; Slaughter et al. 2009). In ase accessibility. Importantly, like PEV, yeast silent chroma- other organisms, the link between the cytoskeleton and the tin can be propagated through mitosis in a heritable manner nucleus stems from cytoskeletal interactions with Nesprins, thanks to its continual nucleation by silencer elements or KASH-domain and SUN-domain proteins that span from the telomeric repeats (reviewed in Rusche et al. 2003). As in outer through the inner nuclear membrane (INM) (Fridkin higher eukaryotes, this heterochromatic state is late replicat- et al. 2009; Razafsky and Hodzic 2009). There is one SUN- ing (Raghuraman et al. 2001) and is found adjacent to the domain protein in yeast called monopolar spindle protein 3 NE, sequestered away from nuclear pores (Palladino et al. (Mps3), which is an important anchor of the INM, but 1993; Taddei et al. 2004a) (Figure 1B). whose link to the cytoskeleton is not yet known. In addition to lacking repressive methylation marks, the As mentioned above, the centromeres of yeast chromo- yeast genome is also unique in that it lacks canonical linker somes remain attached to the SPB by short interphase histones (e.g., H1 and H5) and has a shorter nucleosomal microtubules, and this interaction strongly orients chromo- repeat length (165 bp rather than 200 bp; see Woodcock somes within the interphase nucleus by keeping centro- et al. 2006). While the yeast genome encodes an H1-related meres clustered (Guacci et al. 1997; Jin et al. 1998; Heun protein, Hho1, this protein binds nucleosomal linker DNA et al. 2001b; Bystricky et al. 2004). Opposite this landmark only in rare instances and is not a core component of yeast is the nucleolus, which is generated around a single rDNA chromatin (Freidkin and Katcoff 2001). Yeast also lacks the locus on chromosome XII (Chr XII) that contains 200 tan- histone H3 subvariant H3.1 and macroH2A, while yeast his- dem copies of a 9.1-kb repeat. The positioning of this left tone H3 is equivalent to vertebrate H3.3, and the yeast H2A arm of Chr XII, and the nucleolus that forms around it, serves as the damage-associated, phospho-accepting variant strongly influences nuclear order. A subset of rDNA repeats H2AX of other species (Kusch and Workman 2007). Finally, are tethered to the NE to suppress recombination (Mekhail like most organisms with a closed mitosis, yeast lacks the et al. 2008), keeping the nucleolus tightly associated with nuclear intermediate filament protein lamin. Nonetheless, the nuclear periphery and lending a striking polarity to the yeast expresses other structural proteins of the INM orthol- yeast nucleus by restricting diffusion of this large chromo- ogous to Man1 and emerin (Mekhail and Moazed 2010), some (Figure 1, B and D). which are lamin-associated proteins that contribute to chro- Apart from the rDNA repeat unit, budding yeast chromatin anchoring in higher eukaryotes (Fridkin et al. 2009). mosomes have little repetitive DNA, and—most notably—no Importantly, yeast has allowed one to test the functional simple satellite repeat DNA at centromeres. This eliminates impact of chromatin sequestration by the NE by mutating centric heterochromatin, a major chromosomal structural fea- these structural proteins and monitoring effects on tran- ture that in other organisms impacts neighboring sequences scription and genome stability. both in cis and in trans (reviewedinAkhtarandGasser Nuclear envelope and nuclear pore complex 2007). The organizational role of the transcriptionally inert, compacted chromatin of mammalian centromeres is ful- A double membrane contiguous with the endoplasmic filled, at least in part, by the TG-repeat DNA found at yeast reticulum separates chromatin from the cytoplasm. Embed- telomeres. These TG repeats generate repressive subtelomeric ded in the inner membrane of this nuclear envelope one chromatin domains, which spread for several kilobases from the finds different structures and proteins that anchor chromo- chromosomal ends, silencing nearby promoters (Gottschling somes, including the SPB and nuclear pores (reviewed in et al. 1990). This phenomenon is called telomere position Dieppois and Stutz 2010). Trafficking between the nucleo- effect (TPE), in analogy to the position effect variegation plasm and the cytoplasm occurs through 200 NPCs, which (PEV) that spreads from satellite-containing centromeres enable the free diffusion of small molecules as well as the in other species. The repressive chromatin formed at telomeres regulated transport of macromolecules by the importin ma- requires the binding of the silentinformationregulatory(SIR) chinery (Alber et al. 2007; D’Angelo and Hetzer 2008; Aitchison proteins to nucleosomes, which also occurs at the two silent and Rout 2012). Intriguingly, NPCs provide a platform for

Nuclear Organization 109 Figure 1 (A) Confocal ﬂuorescence images the peripheral positioning of yeast telomeres (red), the silent HML and HMR loci (green) detected by FISH, and the nuclear pores visualized by the anti-pore monoclonal Mab414 (in blue) (Gotta et al. 1996). Bar, 2 mm. (B) The clustering of yeast telomeres in this diploid nucleus is visualized by anti-Rap1 IF (green) while the nucleolus is stained with anti- Nop1 in blue. Ethidium-bromide staining of nucleic acids is in red. Bar, 2 mm. (C) Visualization of the SPB or centromere (anti-Spc42, blue) and right and left telomeres of Chr VI by Tel6R-CFP-LacO (red) and Tel6L-YFP-TetR (green). Bar, 2 mm. (D) Confo- cal ﬂuorescence images a centromere detected by FISH (green by FISH) near the spindle pole body (SPB, red) and the nucleolus (blue) detected by anti-Nop1 (Bystricky et al. 2005). In E, we depict the folded conformation of anaphase chromosomes sketched by Carl Rabl (Rabl 1885). The anaphase chromosomes of spotted salamander larvae were seen to fold back upon themselves due to attachment of the centromere to microtubules. Centromeres lead the way as chromosomes are actively pulled into daughter cells. (F) The Rabl organization is generated during telophase of yeast mitotic division and is shown to persist in the interphase yeast nucleus.

messenger RNA (mRNA) transcription and quality control, et al. 2009), which are orthologs of the mammalian lamin- as well as its export, and tether a subpopulation of inducible associated protein MAN1 (Figure 2). While these proteins genes after activation, both through the mRNA and a quality- are thought to bridge from the INM or lamina to chromatin control export complex called Tho-Trex (Dieppois and Stutz in multicellular organisms, in most cases the ligand on 2010). the chromatin side is still unknown (Fridkin et al. 2009; NPCs are massive assemblies of 50 MDa containing 456 Razafsky and Hodzic 2009). The yeast SUN-domain protein nucleoporins of 30 different types (D’Angelo and Hetzer Mps3 and the two MAN1 homologs (Heh1 or Src1 and 2008). They form a doughnut-shaped structure with an Heh2) serve a similar function in yeast, despite the ab- eightfold symmetry around a central channel, with flexible sence of lamins (Grund et al. 2008; Mekhail and Moazed protein filaments emanating from the core into both the 2010). In addition to these conserved components, a yeast- cytoplasm and the nucleoplasm. These provide binding sites specific protein called Esc1 (establishes silent chromatin) for the transport of proteins, mRNA, and chromatin. A de- (Andrulis et al. 2002) associates tightly with the inner face tailed map for the relative position of each nucleoporin was of the INM where it anchors silent chromatin through calculated on the basis of multiple molecular, biochemical, its high affinity for Sir4 (Andrulis et al. 2002; Gartenberg and structural data revealing a strongly modular structure et al. 2004; Taddei et al. 2004a). Esc1 may have other func- (Alber et al. 2007). tions, as its overexpression induces INM expansion (Hattier Several conserved proteins, which in other species et al. 2007). Not surprisingly, functional cross talk exists associate with lamins, are found at the NE in yeast (Huh between INM proteins (e.g., Esc1 and Mps3) and the nu- et al. 2003). Of particular interest are the integral proteins of clear pore basket proteins (e.g., Nup60, Mlp1, and Mlp2) the INM (Lusk et al. 2007), including Doa10, a RING do- (Therizols et al. 2006; Lewis et al. 2007; Palancade et al. main containing proteins that targets nuclear proteins for 2007) in that loss of either a INM or a pore protein can degradation; Mps3, a member of the SUN (Sad1, UNC-84) interfere with the function of the other, even though they family that is a shared component of the INM and the define spatially distinct domains of the NE when localized SPB (Jaspersen et al. 2002); and helix–extension–helix-1 at high resolution (Taddei et al. 2004a; Horigome et al. and -2 (Heh1 and Heh2) (M. C. King et al. 2006; Fridkin 2011).

110 A. Taddei and S. M. Gasser Figure 2 SUN and LEM domain proteins anchor chromatin to the inner nuclear membrane (INM) in yeast and mammalian cells. In budding yeast, Esc1 and the SUN domain protein Mps3 anchor telomeres at the nuclear periphery favoring silencing and avoiding recombination near telomeres, while ribosomal DNA (rDNA) repeats are separated from the bulk of nuclear DNA and stabilized by tethering to the INM through the Nur1/Heh1 complex. In mammals, there are multiple SUN and LEM proteins held in place by their interaction with the intermediate ﬁlament protein, lamin. Yeast does not have lamins.

Long-range chromosome organization was demonstrated in an elegant experiment in which long and short arms of different chromosomes were swapped. Budding yeast chromosomes assume a Rabl-like conforma- This converted a chromosome with unequal arm lengths to tion throughout the vegetative cell cycle (Figure 1F). The one with equal arm lengths, which enhanced intrachromo- Rabl orientation reflects the spatial orientation of anaphase somal telomere–telomere interaction (Schober et al. 2008). chromosomes, which means that yeast chromosome arms Genome-wide conformation capture approaches (Hi-C), extend away from the centromeres that are held by the inspired from the 3C method of Dekker et al. (2002), con- SPB (Rabl 1885; Yang et al. 1989; Dekker et al. 2002; Jin firmed the clustering of centromeres in interphase cells et al. 1998, 2000). Telomeres are generally found in clusters and the widespread associations between pairs of telomeres around the nuclear periphery (Figure 1B) (Palladino et al. on different chromosomes that had been observed by fluo- 1993). This organization was demonstrated using fluores- rescence microscopy (Duan et al. 2010). The Hi-C analysis cence in situ hybridization (FISH), as well as by immunoflu- also confirmed that two unlinked telomeres positioned at orescence (IF) for centromeric and telomeric proteins (Gotta similar distances from their corresponding centromeres are et al. 1996; Jin et al. 1998). Later these perinuclear telo- more likely to interact than telomeres on arms of different fi mere foci were con rmed and tracked through the cell lengths (Duan et al. 2010; Therizols et al. 2010), again il- cycle using live imaging of GFP-tagged telomeres (Schober lustrating the impact of the Rabl organization on long-range et al. 2008; Therizols et al. 2010). Whereas a polarized chro- interactions (Figure 1). Interchromosomal contacts were mosomal organization exists transiently after telophase in also detected among clustered tRNA genes, early origins of most metazoan cells, it persists through interphase in bud- DNA replication, and at sites of chromosomal breakage ding yeast due to the persistent attachment of centromeres (Duan et al. 2010). that remain linked by short intranuclear microtubules to the Importantly, interactions within a chromosome (such as SPB (Guacci et al. 1997; Jin et al. 1998; Heun et al. 2001b; those formed by right and left telomeres) occur far more Bystricky et al. 2004). Treatment of interphase cells with frequently than interactions between different chromosomes nocodazole allows centromeres to move away from the (Rodley et al. 2009; Duan et al. 2010). This applies even to SPB (Jin et al. 1998; Heun et al. 2001b; Bystricky et al. large chromosomes and chromosomes with unequal arms, 2004). arguing that a yeast chromosome defines a spatial unit or In 2002, the Kleckner laboratory developed a molecular “territory.” Territory positioning is, of course, subject to ar- approach to monitor and model chromosome conformation chitectural constraints imposed by centromere attachment [chromosome conformation capture (3C)] (Dekker et al. to the SPB and telomere anchoring to the NE (Berger 2002) based on the detection of long-range interactions et al. 2008; Therizols et al. 2010), but territories could also within and between chromosomes. A population-averaged be modeled in silico on the basis of polymer diffusion kinet- three-dimensional model of Chr III was thus determined. In ics, without need for a proteinaceous scaffold or matrix this model, Chr III appears to fold as a contorted ring, with (Rosa and Everaers 2008). In yeast, nonetheless, territories a strong bend near the centromere and the telomeres in can be significantly remodeled by transcriptional activation close proximity to each other (Dekker et al. 2002). This (Berger et al. 2008), which is different from the situation in was confirmed by fluorescent tagging of right and left telo- differentiated cells of multicellular organisms, where chro- meres of Chr III and for a second short, metacentric chromosome territories play a more dominant role in nuclear mosome (Chr VI) (Bystricky et al. 2005). Later, it was shown organization (Rouquette et al. 2010). This dominance can that telomere-mediated chromosome looping does not occur be explained both by the sheer size of mammalian chromo- in chromosomes that have arms of unequal lengths (Schober somes, each of which is larger than the entire yeast genome, et al. 2008; Therizols et al. 2010). The impact of chromo- and by the abundance of repetitive, noncoding DNA sequences some arm length on selective telomere–telomere interaction in larger genomes.

Nuclear Organization 111 Figure 3 Visualization of the dynamics of a chromosomal locus. (A) Through the binding of a GFP– LacI fusion to arrays of bacterial lacO sites inserted into the yeast genome (e.g., near a telomere), one can track the rapid chromatin movement in living cells. (B) The nuclear boundary is detected by expression of GFP-Nup49, which helps align sequential time-lapse frames. Three-dimensional stack of images are taken at 1.5-sec intervals for 5–7.5 min. Shown are 5-min tracks (in red) of a GFP-LacI- tagged silent Tel3L and a tagged Tel3L that is derepressed and released from the nuclear envelope, visualized by GFP-Nup49. The repressed and perinuclear telomere moves less frequently. (C) A GFP- tagged LYS2 locus and the same locus excised from Chr II by recombination (D) are shown. Both the tracking over 7.5 min and the xt and yt orthogonal portrayal of the movements are shown. (E) The movement of a given focus was analyzed by mean squared displacement (MSD) analysis (see formula in E), which yields both the diffusion coefﬁcient and the radius of constraint (Rc = 0.6 mm) (Gasser 2002; Marshall 2002; Hubner and Spector 2010). Movement of the LYS2 chromosomal locus (black) is not quite identical to the constrained random walk modeled using averaged parameters of step size and movement from actual movement analysis (red). (F) Similar MSD analysis was performed on the LYS2 locus after excision as a large chromatin ring of 17 kb, which retains the lacO-binding sites. The excised ring moves with a higher diffusion constant and perfectly recapitulates a simulated random walk (red trace). Images and graphs are modiﬁed from Neumann et al. (2012).

Chromatin dynamics flexibility of the chromatin fiber and, in turn, its mobility (Gehlen et al. 2006; Neumann et al. 2012). This is consistent Although chromosomes show a recognizable pattern of posi- with the notion that a chromosome can be modeled as a flex- tioning, chromatin in all living cells is subjected to constant ible polymer chain subject to random forces and tethering motion, which has been described as a constrained random effects (Klenin et al. 1998; Gehlen et al. 2006; Rosa and walk (Marshall et al. 1997; Gasser 2002). Rapid time-lapse Everaers 2008; Neumann et al. 2012). imaging led to the distinction of at least two types of motion in yeast: small random movements (,0.2 mm within 1.5 sec) DNA-based compartments that occur constantly, as well as larger steps over relatively One of the least understood yet most pervasive features of short time intervals (i.e., .0.5 mm in a 10.5-sec interval) nuclear organization is the existence of subnuclear compart- (Heun et al. 2001b). The movement of chromatin is ATP- ments, in which specific DNA sequences and proteins dependent and varies between G1- and S-phase cells, yet accumulate. These are thought to create microenvironments can be quite accurately quantified by a mean squared dis- thatfavororimpedeaparticularDNA-orRNA-based placement analysis as a constrained random walk (Figure 3). activity. The most obvious and well-characterized of such Not unexpectedly, different loci move within domains of dif- compartments is the nucleolus, the site of RNA polymerase ferent size within the nucleus, which are defined as radii of I-mediated rDNA transcription and ribosome subunit assem- constraint (Rc) (Marshall et al. 1997; Heun et al. 2001b; bly. We describe this in some detail as it illustrates the self- Gasser 2002; Neumann et al. 2012). Indeed, telomeres, cen- organizing character of such compartments. tromeres, and silent chromatin, which are tethered through protein–protein interactions at the NE, move within smaller Nucleolus: In almost every eukaryotic nucleus, the most radii of constraint than coding and noncoding regions along prominent subnuclear compartment is the nucleolus. In the longer chromosome arms (telomeric Rc , 0.4 vs. 0.6 mm budding yeast, the nucleolus is a crescent-shaped structure for active loci) (Hediger et al. 2002; Gartenberg et al. 2004; occupying roughly one-third of the nuclear volume, abutting Cabal et al. 2006; Dion et al. 2012; Neumann et al. 2012). the NE and lying opposite the SPB (Yang et al. 1989; Recent work shows that the targeting of ATP-dependent nu- Bystricky et al. 2005). This compartment can be considered cleosome remodelers leads to increased mobility, suggesting as a factory dedicated to ribosome biogenesis. Its morphol- that the shifting or removal of nucleosomes changes the ogy is strongly influenced by the cell growth rate, probably

112 A. Taddei and S. M. Gasser as a result of adapting the rate of ribosome production to the rest or premature senescence. Premature senescence can needs of the cell (Oakes et al. 1993; Powers and Walter also be provoked by the loss of Sgs1 (Sinclair and Guarente 1999). 1997), a RecQ helicase that counteracts recombination be- The budding yeast rRNA stems from a 9.1-kb repeat locus tween rDNA repeats. that is transcribed as a 35S precursor rRNA and a 5S rRNA by RNA polymerases I and III, respectively. Intriguingly, Telomere foci—assemblies of repetitive DNA and silencing different yeast strains have different numbers of tandem factors: The clustering of the 32 yeast telomeres into three repeats, extending from 100 to 200 (Pasero and Marilley to six foci at the NE provides a second prominent feature of 1993). Assembly of the nucleolus is thought to be a self- yeast nuclear organization (Palladino et al. 1993). At these driven process, initiated by production of rRNA (Trumtel clusters of telomeres, the silent regulatory factors Sir3 and et al. 2000; Hernandez-Verdun et al. 2002). This argument Sir4 and the telomere repeat-binding factor repressor acti- stems in part from studies in which the rDNA repeat was vator protein 1 (Rap1) accumulate (Gotta et al. 1996), while transcribed by RNA Pol II, rather than by the endogenous Sir2 is found both at telomeric foci and in the nucleolus RNA Pol I. This led to massive alterations in nucleolar struc- (Gotta et al. 1997). These perinuclear telomeres are late ture, arguing that factors associated with RNA Pol I play a replicating (Raghuraman et al. 2001) and generate a zone key role in proper nucleolar assembly (Oakes et al. 1993; that favors SIR-mediated repression of silencer-flanked Trumtel et al. 2000). genes (Andrulis et al. 1998). The clustering of telomere A recent study (Albert et al. 2011) showed that two Pol repeats also ensures that SIR proteins do not bind promis- I-specific subunits, Rpa34 and Rpa49, are essential for the cuously to repress other sites in the genome (Maillet et al. high polymerase-loading rate on active copies of rDNA and 1996; Taddei et al. 2009). Finally, telomere anchorage in for nucleolar assembly. Intriguingly, nucleolar assembly can S phase contributes to proper telomerase control and sup- be restored in the absence of Rpa49 by decreasing the ribo- presses recombination among telomere repeats (Schober somal gene copy number from 190 to 25, with a concomitant et al. 2009; Ferreira et al. 2011). increase in the number of RNA Pol I complexes per tran- Intriguingly, telomere-containing foci are dynamic, mov- scribed unit. These authors argue that the spatial constraint ing with a constant random motion that is more constrained of RNA Pol I transcription is a critical feature of nucleolar than that of a nontelomeric locus (Schober et al. 2008; Therizols assembly (Albert et al. 2011). Exactly how the 2 Mb of rDNA et al. 2010). These foci fuse and divide in interphase and is folded within the nucleolus is not known, although both dissociate at least partially in metaphase (Laroche et al. cohesin, condensin, and Sir2 affect rDNA compaction during 2000; Smith et al. 2003) to reform in early G1. With the the mitotic cell cycle (Guacci et al. 1994; Lavoie et al. 2002, exception of the intrachromosomal loops formed by short 2004). metacentric chromosomes, no strong preferences for telomere– The extended array of tandem repeats found in the rDNA telomere pairing were detected in interphase cells (Schober locus serves as an ideal template for homologous recombi- et al. 2008). Rather, telomeres on ends of chromosome nation (HR), the favored pathway for double-strand break arms of roughly equal length tend to interact, presumably (DSB) repair in budding yeast. At the same time, it is clear due to the geometry imposed by the Rabl conformation in that rDNA stability is critical for growth and survival of anaphase (Jin et al. 2000; Schober et al. 2008; Therizols yeast, since the generation of extrachromosomal rDNA et al. 2010), showing how that the linear architecture of circles by inappropriate recombination provokes replicative a chromosome can impact its long-range contacts in the senescence (Sinclair and Guarente 1997). Thus, budding nucleus. yeast has evolved several mechanisms to suppress recombi- At the molecular level, budding yeast telomeres consist of nation within the rDNA array. Two mechanisms involve the 250–300 bp of irregular tandem repeats with the consensus silent information regulator, Sir2, which is a NAD-dependent sequence TG1–3 (Shampay et al. 1984). Rap1 (Rap1) (Shore deacetylase (Imai et al. 2000; Smith et al. 2000; Tanner and Nasmyth 1987) binds this repeat on average once every et al. 2000). The first mechanism correlates with a Sir2- 18 bp (Gilson et al. 1993). The Rap1 C terminus carries dependent local nucleosomal organization (Gottlieb and a binding site for the silencing factors Sir3 and Sir4 (Moretti Esposito 1989; Bryk et al. 1997; Fritze et al. 1997; Smith et al. 1994; Marcand et al. 1997; Wotton and Shore 1997), and Boeke 1997) and the second involves longer-range as well as for the telomerase-repressing factors Rif1 and Rif2 chromatin tethering to the nuclear envelope through the (Hardy et al. 1992; Wotton and Shore 1997). Rif1/Rif2 CLIP (chromosome linkage INM proteins) complex. This binding and Sir3/Sir4 binding are mutually antagonistic, complex includes two NE proteins, Heh1 (homologous to and thus loss of the Rif proteins leads to enhanced subtelo- the human Man1 protein) and Nur1 (Figure 2). The rDNA meric repression (Moretti et al. 1994; Mishra and Shore is connected to the CLIP complex through cohibin, a 1999), and loss of Sir4 leads to a reduction in telomere V-shaped complex of two Lrs4 proteins and two Csm1 length (Palladino et al. 1993), most likely due to increased homodimers (Mekhail et al. 2008; Chan et al. 2011). Loss Rif1/2 binding. Together with the end-binding complex of either Sir2 or the cohibin-Heh1-anchoring pathway leads Ku (Yku), telomeric Rap1 serves to recruit Sir3 and Sir4 to to instability of the rDNA repeat, followed by cell cycle ar- telomeres, and the large number of Rap1 sites generated by

Nuclear Organization 113 telomere clustering is thought to compete for limited their association with the nucleolus depends on microtubule amounts of SIR proteins. In this way, multiple weak, but integrity (Haeusler et al. 2008). It is clear from Hi-C data closely juxtaposed, binding sites create an effective sink and GFP-LacO tagging that not all RNA polymerase III genes for this histone-binding complex (Gasser et al. 2004). Con- show similar localization, and it is not yet clear why some firming this, the dispersion of SIR proteins from telomeres tRNA genes shift toward the nucleolus, while others do not by the mutation of telomere anchorage sites reproducibly (E. Varela, personal communication; Duan et al. 2010). reduced the expression of 15 nontelomeric genes (Taddei Nonetheless, the broad distribution of tRNA genes means et al. 2009). that the association of even a subset of them with the nu- SIR-mediated silencing is generated in a two-step pro- cleolus would impact global chromosome positioning. cess. First, the heterodimer formed between Sir4 and Sir2 It is noteworthy that RNA polymerase II genes found in catalyzes the NAD-dependent deacetylation of lysines in the the vicinity of actively transcribed tRNA genes become histone H4 N-terminal tail. The deacetylation of histone H4 silenced through a phenomenon called tRNA-gene-mediated K16, in particular, generates a preferred binding site for Sir3 gene silencing (Wang et al. 2005). Although condensin (Oppikofer et al. 2011), allowing Sir3 to bind and spread to mutations that affect tRNA gene clustering do not affect the adjacent unacetylated nucleosomes (reviewed by Norris tRNA transcription, they do abolish the repression of nearby and Boeke 2010; Rusche et al. 2003). Sir3 appears to bind RNA pol II promoters, suggesting that tRNA clustering and nucleosomal arrays in a stable, stoichiometric complex with tRNA-gene-mediated gene silencing are linked (Haeusler Sir2 and Sir4 (Cubizolles et al. 2006), which renders linker et al. 2008). Thus, tRNA genes can be transcribed away from DNA less accessible to transcription factors or other media- the nucleolus, but interactions in trans and the long-range tors of RNA pol II engagement at the transcriptional start effect of this on chromatin status correlate with relocation. site (Aparicio et al. 1991; Strahl-Bolsinger et al. 1997; Hecht and Grunstein 1999; Chen and Widom 2005; Martino et al. Replication foci: Eukaryotic genomes initiate DNA replica- 2009). tion at multiple sites or origins that are dispersed along each As mentioned above, by disrupting telomere anchors but linear chromosomal arm. In budding yeast, origin function is leaving SIR factors intact, it was shown that the sequestra- coincident with short cis-acting sequences called autono- tion of SIR proteins in telomere foci favors subtelomeric mously replicating sequences (ARS), which support both repression and prevents the promiscuous binding of SIR autonomous plasmid replication and genomic initiation proteins at a distinct subset of promoters elsewhere in the events. In budding yeast, DNA synthesis initiates uniquely genome. This mechanism may be usurped and regulated by at these sites, yet the ARS sequences are not sufficient to environmental conditions to control physiological response. ensure efficient firing. Indeed, sites that are competent for For example, the subtelomeric positioning of genes may help initiation on plasmids do not necessary fire in the genome, coordinate both the repression and the activation programs and even the most efficient origins do not fire every cell that permit growth under adverse conditions (Turakainen cycle (Raghuraman et al. 2001). et al. 1993a,b; Halme et al. 2004; Fabre et al. 2005). Over The clustering of yeast replication forks in foci was first time this could ensure an evolutionary advantage, which revealed in an in vitro replication assay that used isolated would explain why alternative carbon source genes accumu- yeast nuclei as a template for the incorporation of fluo- late in subtelomeric domains. As discussed below, the im- rescently labeled nucleotide. Between 15 and 20 discrete paired tethering of telomeres also correlates with altered foci were detected that were Origin Recognition Complex rates of recombinational repair in subtelomeric zones (Louis (ORC), S phase-, and origin-specific (Pasero et al. 1997). 1994; Therizols et al. 2006; Schober et al. 2009) and im- The same type of foci were later observed by visualizing paired control of telomerase (Hediger et al. 2006; Ferreira replisome components such as PCNA and DNA polymerase et al. 2011). We discuss how telomere clustering affects a or e by immunostaining or fluorescent tagging in living gene expression and genome stability in more detail below. cells (Ohya et al. 2002; Hiraga et al. 2005; Kitamura et al. 2006). Because the number of these foci is far smaller than tRNA genes: Although the 274 tRNA genes are found the number of replication forks, they are thought to corre- distributed along all 16 yeast chromosomes, many of these spond to replication factories in which several elongating genes, when probed by FISH or Hi-C technologies, appear to forks are grouped. Consistent with this notion, neighboring be clustered in nuclear space (Thompson et al. 2003; Wang origins along a chromosome initiate replication with similar et al. 2005; Duan et al. 2010). Intriguingly, at least some timing and respond in a similar fashion to the loss of an tRNA clusters are found close to the nucleolus (Thompson S-phase cyclin (Yabuki et al. 2002; McCune et al. 2008). et al. 2003; Wang et al. 2005), possibly reflecting the fact In the highest resolution study of DNA replication to date, that tRNA genes and the 5S rDNA are coordinately tran- Tanaka and coworkers show that sister replication forks scribed by RNA pol III. This juxtaposition may favor coordi- generated from the same origin of replication stay together nated activation of RNA polymerase I and polymerase III for within one focus, while the sequences on either side appear expression of the protein synthesis machinery. Interestingly, to be pulled through that same “replication factory” (Kitamura the clustering of tRNA genes depends on condensin, while et al. 2006). Since replicons on different chromosomes

114 A. Taddei and S. M. Gasser might be coregulated by juxtaposition in a single focus, it is et al. 2004a). This was most clearly shown by tracking the interesting to note that Hi-C data do score increased inter- position and mobility of an excised ring of yeast chromatin, action between early firing origins (Duan et al. 2010). Such which either did or did not contain silencers. The chromatin clustering could facilitate initiation by concentrating limiting ring could bind autonomously to the NE when silenced, in factors (Mantiero et al. 2011) to favor firing in early S phase. a SIR-dependent manner, and did not associate when SIR Although late-replicating regions are found mainly at the protein could not bind (Gartenberg et al. 2004). Sir4 anchors nuclear periphery, a subnuclear position is neither necessary repressed chromatin to the NE through a 312-aa domain in its nor sufficient for the control of late origin firing (Heun et al. C-terminal half that specifically binds an acidic protein asso- 2001a; Ebrahimi et al. 2010). The timing of telomere repli- ciated with NE, Esc1 (Andrulis et al. 2002; Taddei et al. cation is instead determined by telomere length (Bianchi 2004a). In parallel, it was shown that Sir4 can mediate an- and Shore 2007; Lian et al. 2011). The clustering of replica- choring through the nucleoplasmic N-terminal acidic domain tion forks could nonetheless increase replication efficiency by of Mps3 (Bupp et al. 2007). This may not be a direct interac- concentrating replisome components and deoxy-nucleotides tion, however, and a recent study suggests that Sir4/Mps3 to ensure efficient elongation after recovery from replicative association is mediated by Lrs4, a component of the cohibin stress. There has been no genetic test of this model to date, complex (Chan et al. 2011). Moreover, contrary to the Esc1– since the proteins that ensure origin clustering are unknown. Sir4 interaction, Mps3-Sir4 anchoring requires an intact SIR Indeed, the only protein known to influence replication focus complex and is completely dependent on silencing. Both formation in vertebrates is lamin (Spann et al. 1997), which Mps3 and Esc1 define NE-binding sites that can be distin- yeast lack. guished by microscopy from nuclear pores, and their position around the NE is independent of nuclear pore distribution Sites of DNA repair: DNA DSB repair by homologous re- (Taddei et al. 2004a; Horigome et al. 2011). combination is also organized into foci called DNA damage Mps3 not only participates in SIR-dependent anchorage, response foci (Lisby and Rothstein 2009). These foci contain but also mediates a silencing-independent pathway for telo- high local concentrations of repair factors and are called mere anchoring through its interaction with Est1 (Ever repair foci or factories. The spatially restricted structures shorter telomeres) (Lundblad and Blackburn 1990), an ac- fi may serve to restrict the modi cation of chromatin to the cessory component of telomerase (Antoniacci et al. 2007; subdomain that is relevant for repair or to restrict end- Schober et al. 2009). Tlc1, the RNA moiety of telomerase, joining to the proper chromosome end. Other models argue is linked to telomeric chromatin through the yeast Ku70/ that different repair foci may prevent telomere addition at Ku80 heterodimer, which also binds the end of chromo- certain DSBs by sequestering the break away from telome- somes (Martin et al. 1999). It is thought to help recruit rase. Alternatively, a focus found near other telomeres may the telomerase either directly or indirectly (Peterson et al. facilitate TG addition at breaks. Intriguingly, multiple DSBs 2001; Stellwagen et al. 2003; Fisher et al. 2004; Pfingsten incurred on different chromosomes were shown to assemble et al. 2012) since the Ku-telomerase and Ku-DNA interaction into a single repair focus in yeast (Lisby et al. 2003). This may be mutually exclusive (Peterson et al. 2001; Stellwagen topic will be reviewed elsewhere; thus, only damage mobil- et al. 2003; Pfingsten et al. 2012). Whereas the intramem- ity and its function are discussed here. brane portions of Mps3 play a major role in SPB organization (Jaspersen et al. 2002; Nishikawa et al. 2003), its role in Mechanisms Underlying Nuclear Compartmentation telomere anchoring requires its acidic N-terminal domain, which extends into the nucleoplasm (Bupp et al. 2007). Redundancy in telomere-anchoring pathways The use of N-terminal deletions allows one to distinguish As described above, the positioning of chromatin within the the roles of Mps3 in chromatin anchoring and in SPB for- nucleus depends on reversible interactions of chromosomal mation and function. Nonetheless, the various telomere- elements with structural components of the nuclear enve- anchoring pathways are extensively interlinked, since the lope, namely proteins of the INM, the SPB, and, in some yeast Ku dimer, which mediates Mps3 anchorage in S-phase cases, nuclear pores. The anchoring of telomeres is achieved cells, also directly binds Sir4 (Roy et al. 2004; Taddei et al. through several different protein–protein contacts, which 2004a). fluctuate during the cell cycle and which have different lev- Both telomere and silent chromatin anchorage path- els of importance for different telomeres (Figure 4) (Hediger ways show cell-cycle dependence and sensitivity to post- et al. 2002; Taddei et al. 2004a; Hiraga et al. 2008; Schober translational modification (Ebrahimi and Donaldson 2008; et al. 2009). The fluctuation can be attributed in part to Ferreira et al. 2011). The Mps3-mediated anchoring path- post-translational modifications; several pathways show a re- ways are S-phase-specific, while yeast Ku is able to anchor quirement for sumoylation by the E3 ligase Siz2 (Ferreira the telomere in an Mps3- and silencing-independent manner et al. 2011) in G1-phase cells (Taddei et al. 2004a; Schober et al. 2009). One major pathway of telomere anchoring requires Sir4 Importantly, the S-phase-specific Yku80–Mps3 interaction and is therefore amplified by the formation of silent chro- was found to require the sumoylation of Yku80 by the E3 matin (Hediger et al. 2002; Gartenberg et al. 2004; Taddei ligase Siz2 (Ferreira et al. 2011). This provides a plausible

Nuclear Organization 115 Figure 4 Telomere-tethering mechanisms. Shown is a model of the redundant pathways that tether yeast telomeres to the nuclear envelope. Parallel mechanisms lead to yeast telomere attachment at the nuclear envelope. At different stages of the cell cycle, the telomere-associated proteins mediate different contacts with INM components. Sir4-PAD domain binds the Esc1 C terminus, as well as Yku80 and Mps3. Yku80 binds telomerase, which also associates with Mps3 in S phase through Est1. There is an unidentified anchor for yeast Ku in G1 phase that is neither Esc1 nor Mps3 dependent. The importance and efficiency of the different pathways are cell cycle dependent. The regulation of the various tethering mechanisms by sumoylation is indicated by red arrowheads. Loss of the Siz2 SUMO ligase eliminates anchoring through Sir4 and Yku pathways (Ferreira et al. 2011). Other references are cited in the text. mechanism for switching off Yku-mediated peripheral posi- geted to an internal LacO-tagged locus (ARS607). The abil- tioning once the majority of the genome has been replicated, ity of the fusion protein to shift a randomly distributed locus allowing telomeres to dislodge from the NE either during to the periphery was scored and analyzed statistically (Taddei or just after replication (Ebrahimi and Donaldson 2008), et al. 2004a). The assay is performed in a strain expressing possibly as a consequence of desumoylation. Sir4 is also GFP-Nup49 to allow an accurate quantitation of position in phosphorylated in a cell-cycle-dependent manner (Kueng different genetic contexts. The minimal anchoring assay al- et al. 2012), which may contribute further to the release lows one to score for sufficiency and not only for depen- of telomeres observed during mitotic division. dence on a given factor, which is necessary if one wants to Antagonism of the yeast Ku–telomerase–Mps3 interaction define the molecular machinery of chromatin anchoring. leads to a hyper-recombination phenotype among the short- In addition to these targeted approaches, a genetic screen ened telomeres found in cells lacking Tel1, the ATM kinase for genes that influence telomere anchoring was carried out. homolog in yeast (Schober et al. 2009). This suggests that This study identified the histone acetyl transferase Rtt109 the sequestration of telomeres by the NE protein Mps3 could and the acetylation of histone H3K56 as key modulators protect telomere ends from recombination and deleterious of telomere anchoring (Hiraga et al. 2008). This probably unequal strand exchange. Consistently, the release of telo- reflects impaired nucleosomal assembly after DNA replica- meres by the deletion of SIZ2 in both G1- and S-phase cells tion. Other factors showing partial or cell-cycle-dependent correlates with abnormally long telomeres (Ferreira et al. loss of anchoring include Esc2, Ctf18, Tel1, Mre11, Elp4, 2011). Another telomere-bound protein, namely Cdc13, and Rrm3 (Hiraga et al. 2008). Since these may affect telo- was also found to be sumoylated in a Siz2-dependent man- mere replication and the spread or assembly of silent chro- ner (Hang et al. 2011). Sumoylated Cdc13 contributes in matin, an effect on telomere tethering is not sufficient to an independent manner to telomerase activity, synergizing consider them as direct anchors. Mutations in proteins of with the effects mediated by yeast Ku and Sir4. Thus, Siz2- the nuclear pore such as Nup133 also affect telomere posi- mediated sumoylation limits telomere elongation by more tioning, but these interfere with both mRNA export and cell than one mechanism, one of which correlates with telomere cycle progression, suggesting that their effects are indirect sequestration at the NE (Figures 4 and 5). Importantly, it (Doye et al. 1994; Therizols et al. 2006; Lewis et al. 2007; was shown that critically short telomeres shift away from Palancade et al. 2007). Palmitoylation of telomere-bound the nuclear periphery when they elongate in newly formed proteins could also contribute to the anchoring of silent wild-type zygotes (Ferreira et al. 2011). If natural elonga- chromatin as recently shown for Rif1, although long telo- tion coincides with telomere release, it follows that telomere meres in rif1D cells remain peripherally attached through sequestration by sumoylated forms of Sir4 and Yku might Sir4 (Cockell et al. 1995; Park et al. 2011). Still, redundant indeed attenuate telomerase activity. roles of Rif1 and Rif2 in telomere anchoring remains are not excluded. Minimal anchoring assay and the validation of In addition to telomeres, persistent DNA damage, abruptly genetic screens shortened telomeres, and a subset of highly transcribed genes Given the redundancy in the pathways of telomere anchor- are also found to shift to the nuclear periphery (reviewed in ing, it was difficult to dissect the requirements of anchoring Oza and Peterson 2010; Nagai et al. 2011). In these cases, the by scoring the position of a LacO-tagged telomere in single NPC is clearly involved in the peripheral association, and for mutants. To solve this problem, a “minimal anchoring assay” DSBs and short telomeres an additional pathway implicating was developed in which proteins fused to LexA were tar- Mps3 was described (Nagai et al. 2008; Kalocsay et al. 2009;

116 A. Taddei and S. M. Gasser Figure 5 (A) DNA repair compartments within the yeast nucleus. The nucleoplasm is the site of Rad52-mediated homologous recombination, whereas sequestration at Mps3 or nuclear pores either suppresses recombination between telomeres or allows processing of a DSB for alternative repair pathways. For the rDNA binding to the INM protein, Heh1 (also known as Src1) prevents rDNA recombination. References are noted in A. (B) Model of Mec1/Tel1-dependent relocation of DSBs or collapsed replication forks to Nup84/Slx5/Slx8 complexes associated with nuclear pores. This complex may ubiquitylate a substrate for proteolysis, enabling fork-associated repair (Nagai et al. 2008; Kalocsay et al. 2009; Khadaroo et al. 2009; Oza et al. 2009). (C) Telomeres are anchored through redundant pathways as depicted in Figure 4. At long telomeres, Siz2-mediated SUMOylation (red circles) of Yku70/Yku80 and Sir4 favors telomere anchorage, which in turn may restrict elongation efﬁciency. When telomeres become critically short, loss of Siz2- mediated anchoring, perhaps through de-sumoylation by Ulp1, releases telomeres from the periphery, allowing efﬁcient elongation (Ferreira et al. 2011). Control of telomerase through Cdc13 (Hang et al. 2011) is not included for simplicity. Symbols: green shapes 2 and 3 indicate Sir2 and Sir3; encircled 3 and gray shape 1 near Est2 indicate Est3 and Est1, respectively.

Khadaroo et al. 2009; Oza et al. 2009). These anchoring path- proteins appear to be involved in more than one mechanism, ways are less well understood than telomere-anchoring path- such as the cohesin, condensin, and the more recently char- ways, but have a clear impact on chromosome stability and acterized cohibin complexes (Poon and Mekhail 2011). are discussed in this context below. Cohesin, condensin, and cohibin are multi-subunit complexes that contain large coiled-coil-containing structural Formation of chromatin compartments: association in trans maintenance of chromosomes (SMC) proteins. These con- There are two basic models for the formation of nuclear served SMC proteins form dimers through hinge domains compartments. The first reflects the localized tethering of that are found at the end of extended coiled-coil arms. They similar sequences to a subnuclear structure, or a variant of are able either to encompass nucleosomal fibers or to link this in which intermolecular interactions lead to domain chromatids to facilitate long-range contacts and chromo- clustering. In the second model, compartments arise pas- some condensation. The head domains of the SMC factors sively due to volume exclusion effects. This is favored under provide binding sites for non-SMC proteins, which are also conditions of macromolecular crowding, and the high important for the condensation and cohesion roles of these concentration of macromolecules within the nucleoplasm factors. Recently, yeast SMC complexes have been impli- (between 100 and 400 mg/ml) is thought to be sufficient to cated in the clustering of tRNA genes, rDNA condensation, enhance attractive interactions between macromolecules and rDNA stability (Freeman et al. 2000; Torres-Rosell et al. (Richter et al. 2007). Most likely, subnuclear compartments 2007; Tsang et al. 2007; Bermudez-Lopez et al. 2010). As arise from a combination of mechanisms. A few structural mentioned above, cohibin connects the rDNA and silent

Nuclear Organization 117 chromatin to the NE by bridging between the chromatin- trigger entry into the cycle of repression–sequestration– bound RENT/SIR complex and either CLIP or Mps3. The repression for a nonsilenced telomere. Importantly, chromatin complex is proposed to link rDNA repeats to each other by immunoprecipitation showed that Sir4 can bind telomeres in clustering interspersed IGS1 regions (Mekhail et al. 2008; the absence of Sir3, while the opposite was not true (Strahl- Chan et al. 2011), a mechanism thought to contribute to Bolsinger et al. 1997). Thus, a nonsilent telomere might be SIR-dependent telomere clustering as well. more likely to bind the NE through Esc1–Sir4 or telomere- Yeast telomere clusters and their associated concentration bound Yku–Mps3 interactions than to cluster. This would shift of silent information regulators provide a well-characterized a naive telomere into a zone enriched for telomere-bound example of a functional compartment generated by the Rap1 and its ligands Sir3 and Sir4. Proximity to pools of SIR juxtaposition of sequence-defined chromosomal elements. proteins would enhance telomere repression, and, once silenc- Interactions between subtelomeric zones were proposed to ing is established, perinuclear localization would be reinforced be governed by physical constraints, including chromosome by the Sir4-anchoring pathway, while clustering would be pro- arm length, centromere attachment to the SPB, and nuclear moted by Sir3. In this way, the formation of silent chromatin crowding (Therizols et al. 2010). On the other hand, interac- foci could be self-generating, and not only self-reinforcing. tion between HM loci was shown to depend on correctly Such a mechanism may apply to other chromatin-based com- assembled heterochromatin at these loci (Miele et al. 2009). partments with other functions and may also allow the nu- Consistently, Sir3 has been shown to promote telomere– cleus to segregate factors away from sites at which they telomere association (Ruault et al. 2011). Sir3 overexpression might do harm. triggered the clustering of telomeric foci into even larger Mechanisms of chromatin movement aggregates positioned away from the NE, arguing that clustering can occur independently of anchoring. To generate compartments, chromatin must be able to move Although this “hyper-clustering” phenotype correlates within the nucleus. An excised ring of chromatin explores with more stable silencing in subtelomeric regions, silencing the entire nuclear volume in random walk (Rc = 0.9 mm) is not absolutely required for telomere cluster formation. (Figure 3) while a chromosomal locus moves in a constant, Notably, the overexpression of a Sir3 mutant lacking silencing near-random walk within a radius of 0.5–0.7 mm (re- activity induces large telomere clusters, even in the absence viewed in Gasser 2002; Marshall 2002; Hubner and Spector of Sir2 and Sir4 (Ruault et al. 2011). The Sir3-mediated clus- 2010). For a yeast nucleus of 2 mm in diameter, this means tering is likely to be driven by Sir3 self-oligomerization, likely that a chromosomal locus can explore 30% of the nuclear mediated by its C terminus (Georgel et al. 2001; D. A. King volume (Gartenberg et al. 2004), whereas the same move- et al. 2006; Liaw and Lustig 2006; McBryant et al. 2008; ment in a mammalian nucleus corresponds to ,1% of nu- Adkins et al. 2009). clear volume (Figure 3). In mammalian cells, the shift of Although silencing is not required for clustering, the in- a chromatin domain from a repressive to an active compart- volvement of Sir3 in both processes provides a mechanism ment may involve directional movement (Vazquez et al. for the self-propagation of a silent compartment. Specifi- 2001; Chuang et al. 2006). A similar shift was observed cally, given that Sir3 binding promotes telomere clustering, upon targeting of the viral transactivator VP16 to a yeast this leads inevitably to an increased local concentration of telomere: the activation displaced the telomeric locus from SIR factors. Given that Sir3 is limiting for the spread of silent the NE (Taddei et al. 2004a; Hediger et al. 2006). This chromatin, clustering would favor SIR complex spread into suggested that transcription might correlate with at least flanking chromatin, extending repression. With more Sir3 some degree of chromatin movement, consistent with the bound, more clustering would occur. Other factors also sensitivity of movement to glucose levels in the media as affect telomere clustering, namely the other SIR proteins, well as plasma and mitochondrial membrane potentials the yeast Ku heterodimer, Asf1, Rtt109, Esc2, the cohibin (Marshall et al. 1997; Heun et al. 2001b; Gartenberg et al. complex, and the two conserved factors Ebp2 and Rrs1 that 2004). moonlight in ribosome biogenesis (Gotta et al. 1996; Laroche By targeting transcriptional activators, chromatin modi- et al. 2000; Gehlen et al. 2006; Hiraga et al. 2008; Miele et al. fiers, and nucleosome remodelers to a LacO-tagged locus, 2009; Chan et al. 2011; Horigome et al. 2011). However, however, a recent study could differentiate the effects of because loss of these factors cause replicative stress and im- transcription and of nucleosome remodeling on chromatin pair heterochromatin formation, a direct role in clustering is mobility (Neumann et al. 2012). No strict correlation was not established. found between changes in mobility and transcriptional elon- This raises a chicken-and-egg question: Do silent com- gation, but the targeting of the INO80 remodeler complex partments persist simply because they are self-forming, or is clearly increased the radius of constraint of a tagged chro- there an initial triggering event that promotes the function matin locus (Neumann et al. 2012). This required the that will be favored by the compartment? The bifunctional ATPase activity of the remodeler and occurred without in- role of Sir4 as a mediator of repression and as a silencing- ducing transcription. The endogenous PHO5 promoter also independent anchor for chromatin allows it to both partici- showed increased mobility in an INO80-dependent manner, pate in the self-organization of these compartments and which was correlated with the INO80-dependent eviction

118 A. Taddei and S. M. Gasser of nucleosomes and rapid gene induction (Neumann et al. 1993; Stavenhagen and Zakian 1994; Thompson et al. 1994; 2012). Together, these observations suggest that chromatin Maillet et al. 1996; Marcand et al. 1996), and telomere movement in the yeast interphase nucleus may reflect the clustering favored the SIR-mediated silencing of endoge- nucleosome packing into a higher-order fiber and be influ- nous subtelomeric genes (Mondoux et al. 2007; Taddei enced by remodeler-dependent removal of nucleosomes lo- et al. 2009). Still, the association of subtelomeric loci with cally. On the level of a chromatin fiber, changes in movement peripheral telomere clusters (visualized as Rap1-GFP foci) may reflect changes in the persistence length of the polymer did not always correlate with silencing efficiency (Mondoux brought about by nucleosome displacement. et al. 2007). This may be due to the dynamic behavior of Both the nucleosome remodeler INO80 and the related individual telomeres, which move into and out of telomere complex SWR1 not only are recruited to promoters to en- foci, without loss of their epigenetic state (Schober et al. hance transcription, but also are recruited to DSBs to facil- 2008). Most strikingly, however, was the demonstration itate early steps in repair (reviewed in van Attikum and that, by actively tethering a HMR reporter construct with a Gasser 2009; Lans et al. 2012). Intriguingly, DSBs also dis- weakened silencer to the NE, transcriptional repression in- play enhanced mobility relative to the movement of undam- creased (Andrulis et al. 1998) in a manner strictly depen- aged loci (Dion et al. 2012; Miné-Hattab and Rothstein dent on the presence of telomere clusters and SIR foci 2012). This increase in mobility required the strand-annealing (Taddei et al. 2009). This argues that transcriptional repres- factor Rad51 and the Snf2-related ATPase activity of Rad54 sion does not reflect position per se, but rather access to local (Dion et al. 2012). These two proteins are necessary for high concentrations of SIRs (Mondoux et al. 2007). Consis- repair by HR in an epistatic manner. The Rad9 protein and tently, when telomere anchoring is impaired by deletion the ATR kinase, Mec1, which are both involved in the check- of YKU70 and ESC1, transcription of genes at internal loci point response, also are required for increased movement of (Maillet et al. 2001; Taddei et al. 2009) or at an excised ring DSBs (Dion et al. 2012), and, consistently, only DSBs—and of chromatin flanked by silencers (Gartenberg et al. 2004) not protein-DNA adducts or nicks that do not activate the showed enhanced repression, while telomere-proximal genes DNA damage checkpoint—increase locus mobility (Dion were derepressed. et al. 2012). It was also reported that undamaged sites in- Interestingly, changes in the spatial distribution of re- crease mobility coincident with increased movement at petitive sequences can regulate patterns of gene expression the primary lesion, suggesting that there is a genome-wide genome-wide. Indeed, SIR protein dispersion from perinu- response to DSBs (Miné-Hattab and Rothstein 2012). To- clear foci was shown to specifically affect internal promoters gether, these results suggest that locus movement not only carrying binding sites for Abf1 and the PAC-binding factors is an inherent feature of chromatin, but also may play an (Pbf1 and Pbf2), which regulate genes involved in ribosome active role in promoting the formation of compartments, biogenesis (Mondoux et al. 2007; Taddei et al. 2009). The shifting DNA from one compartment to another, or in facil- cell may exploit such mechanisms by regulating SIR com- itating DNA interactions during repair. plex affinity at telomeres in a rapid response to environmental insult (Martin et al. 1999; Ai et al. 2002). Consistently, Functional Consequences of Nuclear Organization SIR dispersion, or modulation of TPE, has been observed under various forms of stress (Stone and Pillus 1996; Martin Gene silencing et al. 1999; McAinsh et al. 1999; Mills et al. 1999; Ray et al. The sequestration of SIR proteins at telomeres and silent HM 2003; Bi et al. 2004; Mercier et al. 2005; Smith et al. 2011). loci due to telomere clustering has two functional consequen- Stress-induced redistribution of SIR proteins may derepress ces. First, the enrichment of SIR proteins favors subtelomeric subtelomeric genes required for growth on alternative car- repression, and second, it prevents promiscuous repression bon sources and simultaneously contribute to the down- of a distinct subset of promoters elsewhere in the genome regulation of genes involved in ribosome biogenesis as (Taddei et al. 2009). Sir3 protein abundance is relatively a global attempt to suppress growth and favor a survival low (Ghaemmaghami et al. 2003) and is limiting for the pathway. spread of silent chromatin (Renauld et al. 1993). Therefore, Gene activation the DNA sequence-dependent enrichment of SIR recruitment factors at telomeres probably accounts for the strong Over the past years NPCs have emerged as a major player in enrichment of SIR proteins in foci (Gotta et al. 1996). In- organizing gene activity. First, the nucleoporin Nup2 was deed, the loss of telomere anchoring upon deletion of either shown to exhibit a strong boundary activity that can block subunit of yeast Ku leads to SIR protein delocalization the spread of heterochromatin when targeted on both sides throughout the nucleoplasm (Laroche et al. 1998). of a reporter gene, possibly isolating chromatin by forming The abundance of tandem RAP1-binding sites found a loop (Ishii et al. 2002). Subsequently, a series of genome- in telomeric sequences competes with silencers for SIR fac- wide studies suggested that nuclear pore components bind tors, as monitored by functional assays. Early on, silencer- highly active genes (Casolari et al. 2004, 2005; Schmid et al. nucleated repression was found to be highly sensitive to the 2006). While some promoter-bound pore components are distance of the reporter from a telomere end (Renauld et al. not stably bound to NPCs (Dilworth et al. 2005; Therizols

Nuclear Organization 119 et al. 2006; Ruben et al. 2011), the tagging and localization some genes (Brickner and Walter 2004; Menon et al. of specific inducible genes confirmed that some genes in- 2005; Taddei et al. 2006). Thus, whether it acts by the deed shift to NPCs upon activation (for reviews, see Akhtar formation of specialized transcription factories (Eskiw and Gasser 2007; Taddei 2007; Dieppois and Stutz 2010; et al. 2010) or regulation of mRNA elongation, processing, Egecioglu and Brickner 2011) (Figure 6). and export, the NPC seems to efficiently fine-tune gene What moves active genes to the NPC is still unclear; expression. indeed, each of the nuclear steps associated with gene A second proposal suggests that the NPC–gene associa- expression, including initiation, elongation, mRNA process- tion is an epigenetic mark that enables a previous induction ing, proofreading, and export, have been implicated. For event to be “remembered” in subsequent generations, example, the accumulation of a stalled intermediate in the thereby facilitating re-induction (Brickner et al. 2007). Both mRNP export pathway in THO/sub2 mutants leads to the GAL1 and INO1 genes are more rapidly reactivated after persistent NPC association of some active genes (Brickner short-term repression, and this correlates with their persis- and Walter 2004; Rougemaille et al. 2008), yet other data tence at the NPC after the inducing agent was removed. argue that promoter activation without transcription is Although mechanisms involving cytoplasmic factors have also sufficient for the shift (Casolari et al. 2004, 2005; Schmid been implicated in rapid gene reactivation (Zacharioudakis et al. 2006). We suggest that multiple different steps of et al. 2007), GAL gene transcriptional memory was none- transcription contribute independently to the stable associ- theless correlated with the formation of persistent loops ation of active genes with NPCs and that the importance of between the 59 and 39 endsofthegeneandassociation any individual step in this process may well be locus-specific. with the NPC (Laine et al. 2009; Tan-Wong et al. 2009). Itisarguedthatthepresenceofmultipleanchoringpart- InthecaseofINO1, an 11-bp sequence appears to control ners favors the formation of active gene loops, with pro- both peripheral targeting and H2A.Z incorporation after moters linking to 39 sequences to improve the recycling of repression (Brickner et al. 2007; Light et al. 2010). Finally, polymerases and elongation efficiency (O’Sullivan et al. in cells lacking this sequence or a nuclear pore component, 2004). Nup100, transcriptional memory, and perinuclear associa- The targeting of genes to pores can also be mediated by tion of INO1 were lost (Light et al. 2010). Thus, rapid re- sequence elements. Small DNA sequences called gene induction may provide the rationale for NPC association of recruitment sequences (GRS I and II) were first identified inducible genes. in the promoter of INO1 and were proposed to function as Boundaries between active and inactive domains a DNA “zip code” that confers perinuclear localization independently of the chromosomal context (Ahmed et al. Given the role of telomeric foci in the creation of repressive 2010). Interestingly, the GRS I element is over-represented compartments and the ability of the NPC to support induced in the promoters of genes interacting with the NPC and in gene expression, one can conclude that the nuclear periph- the promoters of stress-inducible genes. ery has at least two functions in the control of gene ex- NPC association is thought to be particularly important pression. Consistent with this duality, proteins involved in for inducible genes with galactose- or heat-shock-controlled gene activation or repression have distinct staining patterns promoters, as these genes require a rapid and high level at the nuclear periphery when localized at sufficiently high of expression and efficient mRNA export. This could be resolution (Taddei et al. 2004a; Horigome et al. 2011). Such facilitated by positioning the activated gene at pores. It was juxtaposition of repressive (telomere foci) and activating proposed in the “gene gating” hypothesis (Blobel 1985) that compartments (NPCs) could favor efficient reversals of gene the NPC serves as a scaffold to build an assembly line that expression status, which may be especially relevant for sub- coordinates transcription, processing, and export of mRNA. telomeric genes, since, as mentioned above, subtelomeric Moreover, because of their eightfold symmetry, each pore zones are enriched for genes involved in alternative carbon could accommodate multiple genes, forming a factory that source usage that are needed rapidly, but only under specific supports highly efficient transcription. Although “gene gat- growth conditions (Fabre et al. 2005). ing” might optimize the induction of some genes, pore asso- Intriguingly, mutations in nuclear pore constituents affect ciation is clearly not obligatory for induced expression; both gene activation and repression (Dilworth et al. 2005; mutations affecting gene-NPC association, for example, Therizols et al. 2006; Ruben et al. 2011). This dual role does showed normal expression levels for induced GAL1 (Cabal not appear to stem from the boundary activity of nuclear et al. 2006). Moreover, gene repression did not generally pore proteins, as reported by Ishii et al. (2002). Indeed, it lead to release from pores (Casolari et al. 2004). Finally, was shown that, despite the fact that nuclear pore proteins activation of the same promoter by different pathways or can bind the native tDNA insulator adjacent to HMR, the loss with different 39 UTRs led to different positions of the gene of their binding did not compromise insulator activity, even within the nucleus (Abruzzi et al. 2006; Taddei et al. 2006). though they contributed to silencing at this locus (Ruben Nonetheless, it remains possible that the kinetics of induc- et al. 2011). To reconcile the apparently conflicting role tion was altered in these mutants, as association with the between NPCs and repressive telomeric foci, one might pro- NPC was correlated with enhanced activation of at least pose that the impact of NPCs on gene expression is not

120 A. Taddei and S. M. Gasser Figure 6 Transcription subcompartments within the yeast nucleus. (A) The rDNA locus is sequestered apart from the rest of the genome in the nucleolus where RNA polymerase I transcribed the 35S precursor and ribosome subunits are assembled. Although the 274 tRNA genes are found distributed over all 16 yeast chromosomes, many of them appear to be clustered close to the nucleolus (Thompson et al. 2003; Wang et al. 2005), possibly due to the fact that tRNA genes and the 5S rDNA are coordinately transcribed by RNA pol III. This juxtaposition may facilitate coordination of RNA pol I and Pol III for expression of the protein synthesis machinery. Telomere clustering favors repression through SIR factors whereas pore association correlates with induced expression of certain genes and boundary function. References are noted in A. (B) Gene regulation results from the interaction between cis-regulatory elements and the microenvironment of the gene. Shown is the transcription rate of two genes (X and Y) within two different compartments, A and B. X will be repressed in compartment A because it is flanked by regulatory elements that can interact with the silencing factors concentrated in this compartment, while it will be derepressed in compartment B. In contrast, gene Y expression will be unaffected in compartment A and activated in compartment B where activators that can interact with its regulatory elements are concentrated. simply pore or position dependent, but depends on the bind- Mobility of DSBs: The introduction of two DSBs on different ing of transactivators or repressors to cis-acting elements chromosomes can cause reciprocal translocations as fre- (Ruben et al. 2011). Consistent with this notion, increasing quently as they produce intrachromosomal deletions, argu- the association of the HXK1 subtelomeric gene with the nu- ing that DSBs are dynamic and can roam throughout the clear periphery through a neutral anchor improved both its nucleus to find their appropriate template for repair (Lisby repression of glucose medium and its activation in the ab- et al. 2003). Indeed, the subdiffusive movement of yeast sence of glucose (Taddei et al. 2006). chromatin increases four- or fivefold within 30 min after induction of a DSB, such that the lesion can explore 50% Effects on genome stability of the nuclear volume (Dion et al. 2012; Miné-Hattab and In contrast to the extensive studies on the function of nu- Rothstein 2012). Consistently, Houston and Broach have clear organization in gene regulation and DNA replication, shown in living yeast cells that a homology search for relatively little is known about how chromatin dynamics and a cleaved MAT locus is efficient, such that pairing with the higher-order nuclear organization contribute to genome homologous donor on the distal arm of the same chromo- stability. Until recently, many basic questions remained un- some can occur within 90 min after DSB induction (Houston addressed, such as whether or not DSBs are mobile in the and Broach 2006; Hicks et al. 2011). Given that a homology nucleus, whether DSBs are recruited to scaffolds for repair, search takes longer for ectopic donor sequences, it can be or whether, instead, repair factors are recruited to sites predicted that DSBs require enhanced mobility (over that of of damage. It was also unclear whether specific pathways an undamaged locus) to find the donor sequence for HR. of repair occur preferentially in specific subcompartments of In mammalian cells, there are conflicting reports as to the nucleus, and if so, how chromatin dynamics contribute the mobility of DNA damage. In one case, an induced DSB to the process of repair. With a combination of genetics and appeared nearly immobile, and when break movement was real-time live imaging, recent studies show that chromatin enhanced, increased translocation frequencies with nearby dynamics and nuclear organization do play a major role in chromosomes occurred (Soutoglou et al. 2007). In contrast the repair of DNA lesions and the maintenance of chromo- to this, two reports show enhanced mobility for either a DSB some integrity in yeast. in U2OS cells or an uncapped telomere in mouse embryonic

Nuclear Organization 121 fibroblasts (Dimitrova et al. 2008; Krawczyk et al. 2012). Similarly, the peripheral tethering of short telomeres by This latter was dependent on 53BP1, a repair factor that Mps3 contributes to the suppression of recombination be- helps limit resection in favor of end-joining. Some of the tween subtelomeric Y9 elements in cells lacking the ATM discrepancy in the movement of DNA damage can probably kinase, Tel1 (Schober et al. 2009). These studies are consis- be attributed to the fact that different types of DNA damage tent with the observation that spontaneous Rad52 foci are were induced and that the cells activate the ATR/ATM strongly enriched in the nuclear lumen (Bystricky et al. checkpoint response to different degrees, which was shown 2009). Indeed, this study showed that a donor locus for in yeast to contribute to DSB movement (Dion et al. 2012; HR can be shifted from a more peripheral location to an Krawczyk et al. 2012). internal one, upon its recruitment for a recombination In budding yeast, the rate of HR-mediated repair, scored event. The enrichment for Rad52 foci in the nuclear interior in the presence of one or multiple dispersed copies of donor further suggests that homology-driven recombination might sequence, suggests that the homology search is indeed rate- even be suppressed at the NE. Whether sister-chromatid limiting for recombination (Wilson et al. 1994). Specifically, exchange is antagonized by peripheral tethering remains the frequency of recombination was proportional to the to be seen. number of targets in each strain tested. This dependency Clearly, however, the yeast NE presents more than one on copy number for both dispersed and clustered targets compartment for repair (Figure 5). The connection between suggests that the homology search takes place by a random pore-associated and Mps3-associated repair events is cur- 3D collision, rather than by sliding the donor along DNA rently unknown, but it is likely that they are either mecha- until the recipient is found. Intriguingly, Miné-Hattab and nistically or kinetically linked. The involvement of the Rothstein (2012) report a general increase in chromatin Nup84 complex in the repair of DSBs near telomere ends mobility in response to elevated levels of g-irradiation. This (Therizols et al. 2006; Lewis et al. 2007; Palancade et al. would indeed be likely to enhance the chances of random 2007) and the shift of eroded telomeres to nuclear pores collision events between DSB and uncut donor. (Khadaroo et al. 2009) argues that there may be a sequential treatment of damage and a passage from one site to another, Nuclear pores and Mps3 anchorage in noncanonical depending on the repair required. Peripheral tethering in repair: The random 3D search could also be facilitated by general appears to favor noncanonical pathways of repair, structures that recruit sites of damage and therefore pro- as both the pore association and the Slx5/Slx8 complex are mote recombination or alternative pathways of repair important for the ALT mechanism of telomere maintenance (Gehlen et al. 2011b). Two possibilities for this “repair scaf- (Azam et al. 2006; Khadaroo et al. 2009). The Slx5/Slx8 fold” are nuclear pores and the above-mentioned SUN do- complex facilitates the amplification of telomere repeats main protein, Mps3. It was shown that certain types of DNA to generate survivors in telomerase-deficient cells in a re- damage, such as persistent DSBs, collapsed replication forks, combination event that requires Rad52 and Sgs1. Khadaroo and eroded telomeres are recruited to nuclear pores, which et al. (2009) further suggest a link between recombination- are themselves associated with Ulp1, a Sumo protease, and mediated telomere elongation and nuclear pores, as they a SUMO-dependent ubiquitin ligase complex, Slx5/Slx8 show that eroded telomeres are recruited to pores along (Khadaroo et al. 2009). Namely, lesions that are impossible with factors involved in checkpoint and recombination, or difficult to repair by canonical HR may be brought to namely RPA and Rad52 (Khadaroo et al. 2009). Thus, crit- nuclear pores where alternative recombination and repair ically short telomeres, like persistent DSBs, may be delivered pathways can come into play. Genetic interactions suggest to pore-associated Slx5/Slx8 to enable the ubiquitylation that the importance of the nuclear pore resides in its seques- and degradation of a protein that might otherwise prevent tration of Ulp1 and Slx5/Slx8, and its proximity to the pro- strand invasion. teosome (Therizols et al. 2006; Lewis et al. 2007; Palancade et al. 2007; Nagai et al. 2008). Thus, one scenario is that Regulation of recombination at the rDNA locus: The sumoylated proteins accumulate at collapsed forks or notion that tethering at the NE suppresses recombination resected breaks that require Slx5/Slx8 ubiquitylation for is supported by studies of the rDNA. Whereas HR within the proteasomal degradation, or alternatively, desumoylation rDNA repeats can contribute to the maintenance of stable by Ulp1, to enable appropriate repair. Exactly what the rel- copy number (Smith 1974; Kobayashi et al. 1998), the fre- evant protein may be is at present unknown, although quency of HR in the rDNA is nonetheless significantly lower Rad51, Rad52, Pol32, PCNA, and other proteins of the rep- than would be expected from its repetitive nature. This sug- lication machinery are heavily sumoylated upon DNA dam- gests that recombination is actively suppressed at the rDNA age (Melchior 2000; Nagai et al. 2011; Cremona et al. locus (Petes 1980). Indeed, Torres-Rosell et al. (2007) 2012). showed that HR proteins, such as Rad52, Rad51, Rad55, Other studies have reported the association of irreparable and Rad59, are excluded from the nucleolus, although sen- DSBs with the INM protein Mps3 (Kalocsay et al. 2009; Oza sors of DSB, such as Mre11 and Rfa1, are present. Further- et al. 2009). The tethering of a persistent DSB by Mps3 more, they showed that a single DSB in the rDNA induced by is thought to contribute to the suppression of ectopic HR. I-SceI endonuclease shifts away from the nucleolus when

122 A. Taddei and S. M. Gasser Rad52 is recruited, suggesting that repair by HR must take haps with distinct distributions around the nuclear edge. place outside this compartment. Interestingly, nucleolar ex- This can perhaps be answered with specialized mass spec- clusion of Rad52 foci requires both the Smc5/Smc6 com- troscopy techniques. Clearly, modeling studies that incorpo- plex, which harbors E3 SUMO ligase activity, and the rate dynamics combined with genetic assays, as performed sumoylation of Rad52. The physiological importance of this for the segregation of plasmids (Gehlen et al. 2011a,b), will nucleolar exclusion of HR is shown by the fact that the make important contributions to understanding nuclear mutants that impair nucleolar exclusion, such as smc5 or dynamics. smc6 mutants, show rDNA hyper-recombination by unequal Still unexplained is how different nuclear compartments sister-chromatid exchange and an accumulation of excised signal to each other, how they detect the metabolic status of extrachromosomal rDNA circles (Burgess et al. 2007; Torres- the cell or the environment, and whether certain events or Rosell et al. 2007). compartments may be mutually exclusive within the same Not only does the exclusion of Rad52 foci stabilize the nucleus under specific kinds of stress. Clearly, there are rDNA, but also the juxtaposition of the rDNA to the NE itself changes in nuclear organization that correlate with the appears to play a role in the repression of recombination activation of DNA-damage-associated checkpoint kinases, such between rDNA repeats (Mekhail et al. 2008). As described as the release of telomere-bound SIR proteins in response to above, the anchoring of silent rDNA repeats is mediated by DNA damage (Martin et al. 1999; McAinsh et al. 1999; Mills an INM, Heh1, which shares homology with human Man1 et al. 1999). In a more recent article it was shown that the (M. C. King et al. 2006) and other members of the INM anchoring of transcribed genes to the nuclear periphery is family of LEM (LAP-Emerin-Man1) domain proteins (Fig- counteracted by checkpoint kinases through the phosphoryla- ures 2 and 5). Deletion of HEH1 releases the rDNA from tion of nucleoporins like Mlp1 (Bermejo et al. 2011). It was the nuclear periphery, an event that correlates with an in- proposed that this release neutralizes the topological stress crease in unequal sister-chromatid exchange. Intriguingly, generated during the transcription of nuclear pore-associated Heh1 is not required for rDNA silencing (Mekhail et al. genes. Efficient genome-wide position mapping techniques 2008) and is thus unlike Sir2, which packages chromatin need to be developed to test the model that assigns function to reduce Pol II transcription within the rDNA and to prevent to perinuclear tethering sites or altered chromatin mobility. unequal exchange between repeats (Gottlieb and Esposito Still unexplained is how nuclear compartments reform 1989). As mentioned above, Heh1 anchors the rDNA through after mitotic division, or persist during the dynamic segre- Lrs4,whichconnectsSir2 to Heh1. Interestingly, by fusing gation of mitotic chromosomes. The contribution of organi- Heh1 to Sir2, the requirement for Lrs4 in rDNA anchoring zation to epigenetic inheritance is still poorly defined. is bypassed, and the Sir2–Heh1 tether could suppress the Finally, it is still unclear whether noncoding RNA is a major rDNA instability that correlates with loss of Lrs4 (Mekhail structural element of the budding yeast nucleus, as it ap- et al. 2008). This suggests that proximity to the NE reduces pears to be in other organisms. These and other questions rDNA recombination events, although Sir2 overexpression about nuclear organization and its functional implications may have influenced the suppression of recombination in this promise a fascinating future for this emerging field. experiment (Mekhail et al. 2008). Whereas the sequestration of the rDNA and of telomeres Acknowledgments at the NE probably reflects two different means to suppress Rad52-mediated events, it may nonetheless function both by We thank S. Nagai for the repair figure and K. Bystricky for excluding enzymatic activities or by favoring a processing images in Figure 1. We thank V. Dion, C. Horigome, H. event. One should note, however, that even though the rate Ferreira, M. Oppikofer, and S. Kueng of the Gasser labora- of recombination between telomeric repeats increases when tory for a critical reading of the review. S.M.G. acknowl- tethering is compromised in a yku70 mutant, this effect is not edges support of the Novartis Research Foundation, the solely due to a loss of telomere tethering (Marvin et al. 2009a, National Center for Competence in Research, Frontiers-in- b). It remains to be seen what other factors protect repeated Genetics, and the European Union Network Of Excellence sequences from unscheduled recombination events. Epigenome. A.T. is supported by the French Agence Nationale pour la Recherche and the European Research Council (ERC) under the European Community’s Seventh Frame- Concluding Remarks work Programme (FP7/2007-2013)/ERC grant agreement Despite rapid progress in our understanding of nuclear no. 210508. organization, much remains unknown. Recently, it has been shown that some pore components are highly mobile and remain bound at the pore for only short periods of time Literature Cited (Dilworth et al. 2005; Oka et al. 2010), yet the implications Abruzzi, K. C., D. A. Belostotsky, J. A. Chekanova, K. Dower, and M. of these dynamics for chromatin-based functions are unclear. Rosbash, 2006 39-End formation signals modulate the associ- It is also unknown whether there are different classes of ation of genes with the nuclear periphery as well as mRNP dot nuclear pore complexes that harbor different functions, per- formation. EMBO J. 25: 4253–4262.

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