Review Multiple pathways for telomere tethering: functional implications of subnuclear position for heterochromatin formation Angela Taddei, Susan M. Gasser * Department of Molecular Biology, University of Geneva, Quai Ernest Ansermet 30, CH-1211 Geneva 4, Switzerland Received 4 November 2003; accepted 18 November 2003 Abstract Technical advances in the imaging of GFP derivatives in living cells have improved our ability to determine the position and dynamics of specific chromatin loci. This approach, combined with genetics and functional assays, has shed new light on how nuclear compartments facilitate gene repression in yeast. D 2004 Elsevier B.V. All rights reserved. Keywords: Nuclear organization; Chromosome anchoring; Telomere; Heterochromatin; Silencing; SIR protein; Ku; Esc1 1. Introduction It is now well established that chromatin domains can have specified positions within a eukaryotic nucleus. It is also clear, however, that even the most precisely localized chromatin participates in constant albeit spatially con- strained motion [1–3]. The budding yeast Saccharomyces cerevisiae has provided an excellent model system in which to examine the impact of nuclear organization and chromatin dynamics on the regulation of transcription, replication or repair. In yeast, the 32 telomeres cluster at the nuclear periphery in 8 to 10 groups, forming discrete subcompartments that accumulate a complex of histone- binding silencing factors (Sir2, Sir3, and Sir4 [4]). Sir proteins are recruited to telomeres via protein–protein interactions and can then spread along the chromatin fiber, presumably through interactions with histone tails, leading to the variegated and heritable repression of telomere- proximal genes (called telomere position effect or TPE) [5–7]. The yKu heterodimer is telomere-associated inde- pendently of its expression status, and yKu cooperates with Rap1 to recruit the Sir complex, primarily through the binding of Sir4 [8–11]. The clustering of telomeres is thought to facilitate TPE by maintaining a high local concentration of Sir proteins [12]. On the other hand, this perinuclear arrangement might also simply result from the establishment of a repressed state. In other words, the grouping of repressed domains might ensue from interac- tions of proteins integrated in the silent chromatin with the nuclear envelope, and not promote repression per se. Here we review recent advances describing the mechanisms that mediate anchoring at the yeast nuclear envelope and discuss evidence that spatial clustering can promote het- erochromatin formation or propagation. Many of these insights rely on the use of high-resolution fluorescence microscopy, combined with yeast genetics. 2. How to monitor locus position and chromatin mobility The notion that chromatin is generally immobile is now disproven by a large body of data from live imaging of tagged chromosomal loci in interphase nuclei of fly, mam- malian and yeast cells [1–3]. Not only is chromatin mobile, but fairly large nuclear substructures such as PML bodies are dynamic in living cells [13]. The ability to map the position and to follow the movement of specific chromo- somal loci in real time was initially rendered possible with the development of a GFP-tagged lac repressor –operator system for site recognition [14]. This system exploits the high affinity and highly specific interaction of the bacterial lac-repressor (lac i ) for a DNA sequence called lac operator (lac op ) [15]. Directed insertion of an array of lac operators 0167-4781/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbaexp.2003.11.014 * Corresponding author. Tel.: +41-22-379-61-28; fax: +41-22-379- 68-68. E-mail address: susan.gasser@molbio.unige.ch (S.M. Gasser). www.bba-direct.com Biochimica et Biophysica Acta 1677 (2004) 120 – 128