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Structure and Function in the Budding Yeast Nucleus YEASTBOOK GENOME ORGANIZATION & INTEGRITY Structure and Function in the Budding Yeast Nucleus 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 dif- fusion 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
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