A role for telomerase in telomere anchoring in budding yeast

Heiko Schober^1,2, Véronique Kalck¹ and Susan M. Gasser¹

1Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland and ²NCCR Frontiers in Genetics, 30 Quai Ernest Ansermet, 1211 Geneva, Switzerland

Corresponding author: Susan M. Gasser (Susan.Gasser@fmi.ch) Word count:

Abstract

Telomeres are essential to protect the ends of linear chromosomes, and telomerase counteracts the telomere shortening inherent to DNA replication, by extending the ends of linear chromosomes. Human telomerase sets telomere length for the life span of the organism during the first weeks of embryonic development, after which the enzyme is permanently turned off in most tissues.

Significantly, in the vast majority of malignant cells, telomerase reactivation allows infinite uncontrolled cell division. Here we use unicellular budding yeast, a system in which telomerase remains active, to study the relationship between telomere regulation and the spatial subnuclear localization of telomerase and some of its main regulators. We reveal a telomerase dependent mechanism of telomere anchoring at the nuclear envelope. This pathway involves the evolutionary conserved Sad1-UNC-84 (SUN) domain protein Mps3.

Introduction

Compartmentalization of chromatin into separate subnuclear domains plays an important role in regulation of a variety of processes including gene activation and silencing and DNA repair (reviewed in Sexton et al., 2007). Even the small budding yeast nucleus is compartmentalized into different functional domains.

For example, the 32 telomeres of a haploid yeast cell are clustered in 2-8 foci that are enriched at the nuclear periphery. These foci sequester silencing factors which help anchor the telomeres to the nuclear envelope (NE) through interaction with Esc1, which is distributed in patches along the inner nuclear membrane (Andrulis et al., 2002; Taddei et al., 2004a). The zones defined by silent telomeres promote the repression of genes placed in their proximity (Andrulis et al., 1998). Yet the nuclear periphery harbors other functional domains as well.

Nuclear pore complexes (NPCs) have been implicated in the localization-dependent activation of transcription for a subset of genes, in particular genes induced by restrictive growth conditions (Brickner et al., 2007; Brickner and Walter, 2004; Cabal et al., 2006; Casolari et al., 2004; Dieppois et al., 2006;

Luthra et al., 2007; Schmid et al., 2006; Taddei et al., 2006). Importantly, fluorescence imaging can clearly distinguish telomeric foci from NPCs in wild-type cells, and Esc1 itself was shown by immunoelectron microscopy to be excluded from pores (Taddei et al., 2004a).

Surprisingly, mutations in a subset of nuclear pore proteins were shown to affect telomere anchoring (Therizols et al., 2006), although it was not clear if this effect was direct. Indeed, several lines of evidence suggest that pores may

only be indirectly implicated in telomere anchoring. First, telomeres remain evenly distributed along the inner nuclear membrane in cells in which the nuclear pore protein Nup133 carries an N-terminal deletion, a mutation that provokes the clustering of nuclear pores on one side of the nucleus (Doye et al., 1994; Hediger et al., 2002a). Furthermore, Esc1 remains evenly distributed in this same mutant (Taddei et al., 2004a).

On the other hand, budding yeast have a second, partially redundant pathway for anchoring of telomeres to the NE (Hediger et al., 2002b; Taddei et al., 2004a). In addition to the interaction of the Sir4 “partitioning and anchoring domain” with Esc1, the yKu heterodimer is able to tether telomeres, independently of Sir4. The yKu heterodimer is composed of a 70kDa and an 80kDa subunit, and is evolutionary conserved from yeast to man. yKu binds with high-affinity to DNA ends in a sequence-independent manner, both at DNA double strand breaks (DSB) and at telomeres (reviewed in Daley et al., 2005). At telomeres yKu is involved in protection the end from 5’ end resection (Bertuch and Lundblad, 2003), but it also contributes to the recruitment of Sir proteins for silencing subtelomeric genes (known as telomere position effect or TPE) and the anchoring of telomeres to the NE (Boulton and Jackson, 1998; Evans et al., 1998; Laroche et al., 1998; Mishra and Shore, 1999). Since yKu is able to relocate chromatin to the NE in the absence of sir4 and esc1 (Taddei et al., 2004a), it clearly binds another membrane associated factor. To date this binding partner is unknown.

Apart from its roles in telomere anchoring, capping and silencing, yKu fulfills at least one other important function at telomeres: the yKu heterodimer acts as a positive regulator of telomerase by direct interaction with Tlc1, the RNA subunit of telomerase (Stellwagen et al., 2003). This interaction is conserved from yeast to man (Ting et al., 2005). The budding yeast telomerase reverse transcriptase core complex comprises a protein subunit, Est2, and the RNA moiety, Tlc1 (reviewed in Smogorzewska and de Lange, 2004). All eukaryotes maintain their genomes as linear DNA molecules, and the vast majority of these organisms use DNA synthesis templated by the telomerase RNA to overcome the end-replication problem (Lingner et al., 1995). However, a major question that remains is where telomerase itself is localized in the nucleus, and whether the interaction of yKu with Tlc1 contributes to the positioning of telomeres at the NE.

Recently, an integral nuclear membrane protein Mps3 has been shown to play an important role in anchoring telomeres at the nuclear periphery (Bupp et al., 2007). Mps3 is the sole S. cerevisiae member of a conserved family of protein containing a Sad1/UNC-84 homology (SUN) domain. One of the main functions of this family is to form bridges across the inner and outer nuclear membranes of the cell nucleus (Tzur et al., 2006). Of the four human proteins known to possess SUN domains, two localize to the inner NE. The N-terminal domain of human SUN1 itself has been shown to face the nuclear interior and interact with lamin A, whereas the C terminal domain and the SUN domain itself reside in lumen between inner and outer nuclear membrane (Haque et al., 2006).

Mps3 shares this topology, and even though yeast cells lack lamins, mutations in the N-terminus of Mps3, which faces the nuclear interior, lead to the delocalization of at least some telomeres from the NE (Bupp et al., 2007). This is particularly visible in S phase and evidence suggests that Sir4 may be the link between Mps3 and the telomere.

Mps3 is an essential protein with several other nuclear functions. It is a component of the spindle pole body (Jaspersen et al., 2002), and like its homologues in fission yeast and worm, has been shown to participate in clustering of telomeres into the bouquet formation during meiosis (Conrad et al., 2007). Furthermore Mps3 has been suggested to play a role in sister chromatid cohesion, through its interaction with a factor called Ctf7, which is implicated in the establishment of sister chromatid cohesion (Antoniacci et al., 2004). It is unclear how Ctf7 contributes to chromatid cohesion, since it is not part of Cohesin itself. Most interestingly, a link between Mps3 and telomerase was found in a large yeast two-hybrid screen in which Mps3 was found to interact with Est1, an essential co-factor of telomerase (Uetz et al., 2000). The interaction of Mps3 with Est1 was confirmed by the Skibbens laboratory by in vitro binding experiments (Antoniacci et al., 2007). Est1 is recruited to telomeres in S phase where it mediates the cell cycle-dependent activation of the Est2-Tlc1 core enzyme (Evans and Lundblad, 2002; Taggart et al., 2002). Interestingly regulated mRNA turnover ensures that Est1 is not present in cells except during S phase (Larose et al., 2007).

It has been shown that the position of telomeres and subunits of telomerase within the nucleus has an impact on telomere length. In budding yeast, the deletion of tel1, a gene encoding a PI3-family kinase involved in DNA damage response, leads to short but stable telomeres (Greenwell et al., 1995). Tel1 regulates telomerase activity by acting on a pathway of length maintenance that counts the abundance of the TG-repeat binding factor Rap1 (Marcand et al., 1997). In cells lacking Tel1, a second length-maintenance pathway functions that requires subtelomeric factors, Reb1 and Tbf1 (Arneric and Lingner, 2007;

Berthiau et al., 2006). Intriguingly, the binding of subtelomeric factors Reb1 and Tbf1 to a tagged telomere in a tel1 deletion strain, resulted in the shortening of the terminal TG tract of the targeted telomere (Hediger et al., 2006). This was correlated with delocalization of the telomere from its normal position at the NE.

This suggested that association with the NE might contribute to telomere length maintenance, yet it did not address the question where telomerase subunits are localized, or whether telomerase is preferentially active at the NE. Alternatively, short telomeres may sojourn at the NE to load components required to switch either telomerase or the telomere to an elongation-permissive state. To resolve these questions we have explored whether telomerase itself contributes to the localization of telomeres and whether telomerase activation is linked to anchoring factors. Given the interaction of yKu with Tlc1, it was clear that this pathway might intersect with the mechanism of yKu heterodimer-mediated tethering at the NE.

In this study we first asked whether the yKu- Tlc1 interaction is needed for its ability to anchor chromatin to the NE. We show here that yKu-mediated anchoring in S-phase cells depends on this interaction. This led us to test whether Est2 itself possesses the ability to relocate chromatin to the NE. Indeed, Est2 is able to target chromatin to the nuclear periphery and this ability appears to be independent of Esc1. Finally we show that Mps3 is needed for Est2 and yKu mediated chromatin anchoring, and that neither yKu nor Est2 target chromatin directly to nuclear pores.

Using a system to analyze telomere elongation events on single cell basis (Forstemann et al., 2000; Teixeira et al., 2004), we find that a fluorescently tagged short telomere detaches from the NE just before mitosis in telomerase proficient zygotes, in contrast to telomerase-deficient zygotes where the short telomere stays close to the nuclear periphery. Taken together, we suggest a mechanism through which the localization of telomerase subunits and co-regulators, can regulate telomere localization and ultimately proper end maintenance.

Results

At least two redundant pathways anchor telomeric chromatin to the NE in budding yeast, one depending on the Sir complex and the other on the yKu heterodimer (Hediger et al., 2002b; Taddei et al., 2004a). Different alleles of yKu80 have been described, one of which is yku80-4. This allele specifically loses the ability to bind to the C terminus of Sir4 (Roy et al., 2004; Taddei et al., 2004a). Nevertheless yku80-4 is able to recruit chromatin to the nuclear periphery, an activity that functions in the absence of Sir4 (see Figure 2C, Roy et al., 2004; Taddei et al., 2004a). Since only the double deletion of sir4 and yku70 (or sir4 and yku80) leads to a random distribution of telomeres with respect to the NE (Hediger et al., 2002b), a second pathway of telomere anchoring was proposed to be independent of Sir4-Esc1, and dependent on yKu and an as yet unidentified peripherally localized interaction partner.

yKu80 does not recruit chromatin to the NPC

Given that some telomeres become delocalized in Nup84 complex mutants (Therizols et al., 2006), one possible association partner for yKu could be a nuclear pore component. To test the speculation that yKu80 binds to nuclear pores or else to a protein anchored at nuclear pores, we made use of a strain that carries an N-terminal deletion of NUP133. In this background, poly(A)⁺-RNA export functions are normal, but nuclear pores cluster in one patch at the NE (Doye et al., 1994). We expressed CFP-Nup49 to visualize the position of the pore cluster and inserted an array of lacO combined with lexA binding sites

next to the LYS2 locus in the middle of the right arm of chromosome 2 (Figure 1A). Expression of lacI-GFP and lexA-yku80-4 in these cells enabled us to determine whether the targeted LYS2 locus was recruited to the cluster of nuclear pores by yku80-4. As a control we expressed lexA-Nup84, which is known to interact with nuclear pores. Whereas lexA-Nup84 led to a significant colocalization of the lacO array with the pore cluster, lexA-yku80-4 did not lead to an enrichment at the cluster of pores, even though this construct efficiently relocates LYS2 to the periphery (Figure 1B, 2C; Taddei et al., 2004a). We see the same degree of colocalization of LYS2 with the nuclear pore cluster when either lexA or lexA-yku80-4 are expressed, and we see no variation between G1 and S-phase cells (Figure 1B). This argues that the interaction of yKu80 with the nuclear envelope does not reflect its affinity for the NPC.

yKu - Tlc1 interaction is critical for S-phase mediated anchoring via yKu80 The yKu heterodimer has been shown to interact directly with Tlc1, (Stellwagen et al., 2003). This prompted us to ask where telomerase itself is localized in the nucleus, and whether the interaction of yKu with Tlc1 is involved in the positioning of telomeres. Answering this question was compounded by the interaction of yKu with Sir4 (see Figure 2A), and therefore we continued to exploit the lexA-yku80-4 allele which ablates interaction with Sir4C yet is able to relocate a randomly positioned locus to the NE, when it is bound to integrated lexA sites (Figure 2, Taddei et al., 2004a and Supplemental Figure 1A). As previously shown, when lexA alone is targeted to the fluorescently tagged ARS607 locus, we scored a random distribution of the targeted locus (Figure 2D). However, the

expression of lexA-yku80-4 led to a significant relocalization of the tagged locus to the nuclear periphery in both G1 and S-phase cells (Figure 2C, D).

To address the question of whether yku80-4 relies on the interaction with Tlc1 to recruit telomeres to the periphery, we made use of a mutation that abolishes the yKu – Tlc1 interaction (yku80-135i, Figure 2A, panel 3). We found that the lexA-yku80-135i construct displayed a strong reduction in its ability to relocate chromatin to the NE in S phase, but not in G1 (Figure 2C). Combining the “135i” with “-4“ mutation in one yku80 allele that binds neither to Tlc1 nor to Sir4 (Figure 2A, panel 4) led to a complete loss of anchoring, albeit only in S phase. This argues that there is a further interaction partner of yKu in G1-phase cells.

To confirm that the S-phase specific loss of anchoring indeed reflects the inability of yku80-135i to bind Tlc1, we expressed the lexA-yku80-4 construct in a strain bearing a deletion in TLC1, namely tlc1^48, which abolishes binding to the yKu heterodimer (Stellwagen et al., 2003, Figure 2A panel 5). When relocalization through lexA-yku80-4 was scored in tlc148 cells, we observe an S-phase specific loss of peripheral recruitment of the tagged locus. These results argue that the interaction of yKu with telomerase is essential for its Sir4-independent anchoring function in S phase cells.

The telomerase catalytic core subunit Est2 localizes chromatin to the nuclear periphery

The dependence of yKu on Tlc1 for peripheral targeting prompted us to test whether the catalytic core component of telomerase Est2 itself can target chromatin to the NE. To eliminate senescence phenotypes that occur upon est2 deletion, we again made use of the relocalization assay using a lexA-Est2 fusion that was again targeted to the lacO-tagged ARS607 locus. We find that lexA-Est2 is indeed able to recruit chromatin to the nuclear periphery in both G1 and S-phase cells, whereas lexA alone cannot (Figure 3A). This suggests that Est2 itself has a binding partner at the NE. To ask whether this binding partner could be Esc1, we repeated the targeting assay in cells in which ESC1 was deleted.

We find that that lexA-Est2 is still able to relocalize the ARS607 to the NE (Figure 3A). This excludes Esc1 as the NE anchor for Est2.

We speculated that this binding partner might be a component of the NPC.

To test this we again exploited the strain bearing an N-terminal deletion of NUP133, which provokes nuclear pore clustering (Figure 1A). We expressed lexA-Est2 which binds the lacO/GFP-lacI bound LYS2locus. As shown above for lexA-yku80-4, lexA-Est2 does not lead to a colocalization of the tagged LYS2 locus with the cluster of pores, neither in G1 nor in S phase (Figure 3B). Instead the level of colocalization is similar to that detected in the presence of lexA alone, suggesting that Est2 does not bind to nuclear pores.

Telomeres anchor via the integral inner nuclear membrane SUN domain protein Mps3 in S-phase cells

The conserved budding yeast SUN domain protein Mps3 is an integral inner nuclear membrane protein and its N terminal domain faces the nuclear interior. In a genome-wide two-hybrid screen Mps3 was seen to bind the telomerase subunit Est1, an interaction later confirmed by in vitro immuno- precipitation experiments (Antoniacci et al., 2007). It was shown recently that Mps3 localizes to the inner nuclear membrane and binds to a domain in the C terminus of Sir4. In the same study it has been reported that Mps3 is needed for proper telomere anchoring, especially in S-phase cells (Bupp et al., 2007).

To verify that telomere anchoring uses Mps3 as membrane anchor, we expressed a dominant negative allele of MPS3 that consists of its extreme N-terminus alone, truncated immediately upstream of the transmembrane domain.

Indeed, when fused to mCherry (Shaner et al., 2005) and a nuclear localization sequence, the Mps3 N-terminal fragment is stable, but is found diffuse throughout the nucleoplasm (Figure 4A), unlike the intact Mps3 protein which is exclusively perinuclear (Bupp et al., 2007). We speculated that expression of a truncated Mps3 domain that cannot bind the NE would saturate the telomere interaction sites and thereby impair the nuclear anchoring that is mediated by the intact protein. This mps3N’-tetR-mCherry construct is called henceforth mps3-N’.

Expression of the mps3-N’ allele in a yeast strain that contains fluorescently tagged telomere 6R confirms that the result obtained by deletion of the mps3 N terminal domain: we observe an S-phase specific delocalization of

Tel6R from the NE (Figure 4B). We were interested to elucidate in which pathway for telomere anchoring Mps3 functions and therefore analyzed the effect ofmps3-N’ expression in cells deleted for sir4 (Figure 4C). We find no difference in telomere 6R anchoring whether or not the cells express mps3-N’ (Figure 4C).

Thus, in analogy to published data from the Jaspersen laboratory (Bupp et al., 2007), we find an impairment of telomere anchoring in S phase that is epistatic to the Sir4 pathway of telomere anchoring.

Since we have shown that Est2 targets chromatin to the NE we were interested to know whether Est2 functions in telomere recruitment on a pathway parallel to or cooperative with Sir4. Therefore we compared telomere 6R anchoring in WT cells with its anchoring in cells deleted for sir4 alone or for sir4 and est2. We scored telomere position in this latter strain before cells entered senescence. In G1-phase cells, the anchoring of telomere 6R slightly drops in the absence of sir4 and deletion of est2 leads to a slight further decrease of anchoring. In S-phase cells, sir4 single and sir4 est2 double mutant cells display the same highly significant degree of delocalization compared to WT cells. These results suggest that there may be an interdependence of yKu, Sir4 and the peripherally localized proteins Mps3 and Esc1, as well as an S-phase specific involvement of Est2/Tlc1.

Est2 and yKu recruitment to the NE depends on Mps3

To test whether Mps3 is the peripheral anchor for telomerase, we made use of the targeted anchoring assay (Figure 2B) and the mps3-N’ dominant negative fragment (Figure 4A). In cells that carry a fluorescently labeled locus in

the middle of chromosome arm 5R tagged with lexA binding sites, we co-expressed the fusion proteins lexA-Est2 and mps3-N’ (Figure 5A). Expression of this N terminal domain of Mps3 impaired the ability of lexA-Est2 to recruit chromatin the NE (Figure 5B). We next asked whether yKu80 anchoring would depend on Mps3 as well and repeated the experiment expressing lexA-yku80-4 and observed the same inability of yku80-4 to recruit chromatin to the NE in the

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