Telomere organization in the interphase nucleus
The organization of chromosomes in the interphase nucleus into functional subnuclear domains plays an important role in the regulation of a variety of processes including gene activation and silencing and DNA repair (reviewed in Sexton et al., 2007). This includes the clustering of repetitive non-coding heterochromatin, as well as the specific positioning of functional chromosomal elements such as origins of replication, boundary elements, and in lower eukaryotes, centromeres and telomeres (reviewed in Spector, 2003;
Taddei et al., 2004b). Because centromeres in most organisms comprise of thousands of kilobases of heterochromatin, the clustering of centromeres into
“chromocenters” is thought to arise from interactions of heterochromatin proteins that recognize satellite repeats. Budding yeast lacks centromeric heterochromatin, but as discussed in the previous section, each telomere contains ~ 350 bp of an irregular TG1-3 repeat that is able to nucleate a repressive chromatin state (Gottschling et al., 1990). The subtelomeric
repression that spreads from the TG1-3 repeat resembles heterochromatin in many ways, most notably by being late-replicating, refractile to transcription, and able to transmit its repressed state in a heritable manner (reviewed in Lustig, 1998; Tham and Zakian, 2002). Thus, like centromeric heterochromatin, the 32 telomeres of a haploid budding yeast cell are found to cluster together in 2-8 distinct foci. These foci of budding yeast telomeres are found to localize with high frequency near the nuclear envelope (NE) and it has been shown that deletion of either subunit of yKu leads to delocalization of telomeres (Hediger et al., 2002b; Laroche et al., 1998). So another function of yKu is recruitment of telomeres to the NE.
Esc1 is localized in patches along the nuclear envelope (NE) and serves as an important binding site for telomeric chromatin. Fluorescence imaging can clearly distinguish nuclear pore complexes (NPCs) dispersed between the patches of Esc1 (Taddei et al., 2004a), which have been implicated in the localization-dependent enhancement of the transcriptional efficiency of a subset of genes (reviewed in Akhtar and Gasser, 2007).
Despite the finding that at least some telomeres are affected by deletions of nuclear pore proteins (Therizols et al., 2006), several lines of evidence indicate that pores are not universally required for telomere anchoring. For example, telomeres remain evenly distributed along the NE away from pores in cells where the nuclear pores cluster due to a mutation of NUP133 (Hediger et al., 2002a).
These telomeric foci associated with the nuclear periphery sequester the Silent information regulatory proteins, Sir2, Sir3 and Sir4 from internal sites which are known to nucleate silencing at yeast telomeres (Gotta et al.,
1996; Palladino et al., 1993). As mentioned in a previous section, the CTD of Rap1 is responsible for the recruitment of the Sir proteins. At this point it is worthwhile to mention that yKu interacts with the Sir complex and deletion of either of the two yKu subunits leads to derepression of subtelomeric genes (Maillet et al., 2001). Thus yKu is needed for telomeric silencing.
Anchoring pathways
There are at least two partially redundant pathways to anchor telomeric chromatin to the NE in budding yeast (Hediger et al., 2002b; Taddei et al., 2004a). One involves the yKu heterodimer, and the other the Sir complex and Esc1, which localizes to the NE and has been shown to interact directly with Sir4 in order to recruit telomeres to the nuclear periphery. When individual chromosomes are monitored there are differences in theier dependence on the yKu or Sir4-Esc1 pathway as well as cell cycle variations in the efficiency of anchoring.
Recently, an integral nuclear membrane protein Mps3 has been shown to also play an important role in anchoring telomeres at the nuclear periphery (Bupp et al., 2007). Mps3 is the single S. cerevisiae member of the conserved family of Sad1/UNC-84 homology (SUN) domain containing proteins. One of the main functions of this family is to form bridges across the inner and outer nuclear membranes of the cells nucleus (Tzur et al., 2006). Of the four human proteins known to possess SUN domains, two localize to the NE. The N-terminal domain (NTD) of human SUN1 itself has been shown to face the nuclear interior and to interact with lamin A, whereas the C terminal domain (CTD) and the SUN domain itself reside in the lumen between inner and outer
nuclear membrane (Haque et al., 2006). Mps3 shares this topology and mutations in the N-terminus of Mps3 (which faces the nuclear interior) have recently been shown to lead to delocalization of telomeres from the NE (Bupp et al., 2007). This is particularly visible in S phase, and evidence points to Sir4 as the critical mediator for telomere anchoring.
Mps3 has several other functions and is an essential protein. It is a component of the spindle pole body (SPB) (Jaspersen et al., 2002), and like its homologues in fission yeast and worm, has been shown to participate in clustering of telomeres into the meiotic bouquet formation (Conrad et al., 2007). Furthermore Mps3 has been shown to play a role in sister chromatid cohesion, due to an interaction with the ‘establishment of sister chromatid cohesion’ factor Ctf7 (Antoniacci et al., 2004). Interestingly, absence of Ctf18, another factor important for sister chromatid cohesion, has been shown to be required for both yKu and Sir mediated telomere positioning pathways especially during G1. It has been proposed that Ctf18 modifies telomeric chromatin to make it competent for yKu/Sir mediated peripheral localization (Hiraga et al., 2006).
Most interestingly, a link to telomerase was found in a large-scale yeast two-hybrid screen where Mps3 was found to interact with Est1 (Uetz et al., 2000). This finding was confirmed by the Skibbens laboratory using in vitro binding experiments (Antoniacci et al., 2007). In this perspective, it is interesting to note that Est1, an indispensable part of the active holoenzyme, is only recruited to telomeres in S phase (Taggart et al., 2002).
Related to this, it has been shown that the position of telomeres within the nucleus has an impact on telomere length. In human cells, subunits of
telomerase have been shown to shuttle to and from different subnuclear locations and interference with this movement has an impact on telomere length regulation of the viral activator VP16 to a telomere resulted in its delocalization away from its normal position at the nuclear periphery. This delocalization resulted in a shortening of the terminal TG tract in a tel1 background where telomere length control was partially compromised (Hediger et al., 2006). However, these results did not address where the telomerase subunits are actually localized, and how the position of telomeres affects telomerase activity. It remains to be tested if telomere elongation occurs at the nuclear periphery or whether short telomeres may sojourn there to load components required to switch to the accessible elongation state.