Tel1ATM and Rad3ATR kinases promote Ccq1-Est1 interaction to maintain telomeres in fission yeast.

The evolutionarily conserved shelterin complex has been shown to play both positive and negative roles in telomerase regulation in mammals and fission yeast. Although shelterin prevents the checkpoint kinases ATM and ATR from fully activating DNA damage responses at telomeres in mammalian cells, those kinases also promote telomere maintenance. In fission yeast, cells lacking both Tel1 (ATM ortholog) and Rad3 (ATR ortholog) fail to recruit telomerase to telomeres and survive by circularizing chromosomes. However, the critical telomere substrate(s) of Tel1(ATM) and Rad3(ATR) was unknown. Here we show that phosphorylation of the shelterin subunit Ccq1 on Thr93, redundantly mediated by Tel1(ATM) and/or Rad3(ATR), is essential for telomerase association with telomeres. In addition, we show that the telomerase subunit Est1 interacts directly with the phosphorylated Thr93 of Ccq1 to ensure telomere maintenance. The shelterin subunits Taz1, Rap1 and Poz1 (previously established inhibitors of telomerase) were also found to negatively regulate Ccq1 phosphorylation. These findings establish Tel1(ATM)/Rad3(ATR)-dependent Ccq1 Thr93 phosphorylation as a critical regulator of telomere maintenance in fission yeast.

INTRODUCTION

Stable maintenance of telomeres is critical to preserve genomic integrity, and telomere dysfunction has been linked to tumor formation and pre-mature aging in humans[1]. The GT-rich telomeric repeats are bound by the six-protein “shelterin” complex (TRF1, TRF2, RAP1, TIN2, TPP1 and POT1) and are extended by telomerase in humans[2]. In fission yeast Schizosaccharomyces pombe, a conserved shelterin complex, composed of Taz1 (TRF1/TRF2 ortholog), Rap1, Poz1 (possible analog of TIN2), Tpz1 (TPP1 ortholog) and Pot1, was recently identified[3]. The fission yeast shelterin complex additionally includes Ccq1, which is required to prevent checkpoint activation and to recruit telomerase to telomeres[3-5].

While the shelterin complex is necessary to prevent the DNA damage checkpoint kinases ATM and ATR from fully activating DNA damage responses at telomeres[6], these kinases are recruited to telomeres during S/G2-phases[7,8] and play essential roles in telomere maintenance[9]. In fission yeast, simultaneous deletion of Tel1 (ATM ortholog) and Rad3 (ATR ortholog) leads to complete loss of telomeres and chromosome circularization[10]. By chromatin immunoprecipitation (ChIP) assays, we have previously determined that tel1Δ rad3Δ cells fail to recruit telomerase and also show reduced Ccq1 association with telomeres[11]. However, how Tel1ATM and Rad3ATR kinases promote telomerase recruitment remained unclear.

Here, we show that Tel1ATM/Rad3ATR-dependent phosphorylation of Ccq1 Thr93 is essential for telomerase association with telomeres. In addition, we show that the 14-3-3-like domain of the telomerase regulatory subunit Est1[12,13] specifically recognizes and binds to the phosphorylated Thr93 of Ccq1 to promote association of telomerase with telomeres. Phosphorylation of Ccq1 is negatively regulated by the telomerase inhibitors Taz1, Rap1 and Poz1[3,14-16], and telomere elongation and increased telomerase association with telomeres found in rap1Δ cells is dependent on Ccq1 Thr93 phosphorylation. On the other hand, Ccq1 Thr93 phosphorylation is increased as telomeres shorten in telomerase mutant cells. Taken together, we thus establish Tel1ATM/Rad3ATR-dependent Ccq1–Est1 interaction as a critical regulatory mechanism that ensures stable maintenance of telomeres in fission yeast cells.

RESULTS

Est1 interacts directly with shelterin subunit Ccq1

To better understand how localization of telomerase at telomeres is regulated in fission yeast, we performed pairwise yeast two-hybrid assays between telomerase (catalytic subunit Trt1TERT and regulatory subunit Est1) and shelterin complex subunits (Pot1, Tpz1, Poz1 and Ccq1). While we confirmed the previously identified Ccq1–Tpz1 interaction[3] (), we also found that Est1 and Ccq1 interact with one another (). Bioinformatics analysis predicted that the central region of Ccq1 might form a structure similar to the Class II histone deacetylase (HDAC) complex subunits 2 and 3, while the C-terminal domain is likely to form a coiled-coil structure related to SMC (structural maintenance of chromosomes). Through truncation analysis of Ccq1, we determined that amino acids 123–436 of Ccq1 are sufficient for Ccq1–Tpz1 interaction (), while amino acids 1–436 of Ccq1 are required for Ccq1–Est1 interaction ().

Within fission yeast Est1, the only region that shows significant homology to other Est1 homologs is localized within its N-terminus[12]. Crystal structure of the equivalent region from mammalian EST1C/SMG7 protein suggested that this region (amino acids 1–263) of fission yeast Est1 might fold into a domain that resembles a 14-3-3 phosphopeptide binding protein[13]. Based on sequence alignments of Est1/SMG homologs[17-19], we identified Lys73 or Arg79 and Arg180 of fission yeast Est1 as conserved residues that are most likely equivalent to Lys66 and Arg163 in EST1C/SMG7, amino acid residues critical for phospho-serine binding[13] (). Previous studies have established that EST1A/SMG6 (but not EST1C/SMG7) associates with the mammalian telomerase complex[17-19], and thus likely to represent an ortholog of Est1 from fission and budding yeasts.

Intriguingly, mutational analysis of fission yeast Est1 revealed that Est1-R180A and Est1-R79A,R180A mutants completely lose their ability to interact with Ccq1 in yeast two-hybrid assays, while K73A and R79A mutants show reduced Est1–Ccq1 interaction, with R79A having a stronger effect (). These results suggested that the ability of fission yeast Est1 to recognize phosphorylated amino acid residue(s) within Ccq1 might be important for mediating Ccq1–Est1 interaction.

14-3-3-like domain mutations of Est1 cause telomere loss

Upon integration of est1-R79A, est1-R180A or est1-R79A,R180A alleles at the est1+ locus, these mutant fission yeast cells showed progressive telomere shortening during repeated restreaking on agar plates (Fig. 2a). Moreover, an increasing number of cells showed a highly elongated morphology (indicative of checkpoint activation) and slow growth rate (data not shown), until they eventually recovered and generated “survivor” cells carrying circular chromosomes ( and Supplementary Fig. 4), much like trt1Δ, est1Δ, ccq1Δ or tel1Δ rad3Δ survivor cells[5,10,12,20]. We confirmed that these mutations do not affect Est1 stability or the interaction between Est1 and telomerase RNA (TER1) (). Quantitative ChIP assays of early generation strains revealed that Est1-R79A, Est1-R180A or Est1-R79A,R180A proteins show substantial reduction in telomere association compared to wild-type Est1 (). The observed reduction in telomere association might reflect combined effects of the est1 mutations on telomerase recruitment, processivity and/or retention. Among the three mutants, Est1-R79A showed a milder effect on telomere association, consistent with the observation that est1-R79A cells required more extensive restreaking on agar plates than est1-R180A or est1-R79A,R180A cells before chromosomes ultimately circularized ( and data not shown). By contrast, the 14-3-3-like domain mutations in Est1 did not affect association of Ccq1 with telomeres ().

Figure 2

The phosphopeptide-binding motif of Est1 is important for telomere maintenance. (a) Southern blot analysis of telomeres from successive restreaks. While addition of a 13myc-tag to Est1 resulted in slightly shorter but stable telomeres, mutations within the 14-3-3-like domain caused loss of telomeres for both tagged and untagged mutant alleles (Supplementary Fig. 4). (b) NotI restriction map of fission yeast chromosomes. The telomeric fragments (C, I, L and M) are marked. (c) The telomeric NotI-fragments from extensively restreaked cells, analyzed by pulsed-field gel electrophoresis. (d) Binding of Est1 to TER1 is not affected by est1 mutations, as monitored by co-IP experiments. Plots show mean values plus/minus one s.d. for three independent experiments. (e-f) Recruitment of Est1 mutants (e) and Ccq1 in est1 mutant backgrounds (f) to telomeres was monitored by ChIP assays. Assays were performed using early generation cell cultures, and the presence of telomeres was confirmed by both Southern blot and qPCR assays against TAS regions, which are lost upon chromosome circularization[11,20] (data not shown). Protein expression levels were monitored by Western blot using the indicated antibodies. Cdc2 served as loading control. Plots show mean values plus/minus one s.d. for three (e) or two (f) independent experiments.

Telomere maintenance requires Ccq1 Thr93 phosphorylation

We found that both telomerase (Trt1TERT) association with telomeres () and Ccq1–telomerase (TER1) interaction () are significantly increased in the absence of the telomerase inhibitors Poz1, Rap1 or Taz1[3,14,15,20], and that Ccq1 correspondingly exhibits enhanced Tel1ATM/Rad3ATR-dependent hyper-phosphorylation in poz1Δ, rap1Δ or taz1Δ cells ( and ). Ccq1 was also found to be essential for telomerase association with telomeres in both rap1+ and rap1Δ cells[5,11] (). Based on these observations and our previous findings that Tel1ATM and Rad3ATR kinases play essential but redundant role(s) in telomerase association with telomeres[11], we hypothesized that Poz1, Rap1 and Taz1 act as inhibitors of telomerase action by limiting Tel1ATM/Rad3ATR-dependent phosphorylation of Ccq1. In addition, we hypothesized that binding of Tel1ATM/Rad3ATR target phosphorylation site(s) within Ccq1 by the 14-3-3-like domain of Est1 might play a critical role in association of telomerase with telomeres.

Accordingly, we mutated all eleven SQ/TQ sites (preferred phosphorylation sites for Rad3ATR/Tel1ATM kinases[21]) found in Ccq1 to AQ, to identify Tel1ATM/Rad3ATR-dependent phosphorylation site(s) within Ccq1 that might play critical roles in promoting Ccq1–Est1 interaction and telomere maintenance ( and ). Experiments indicated that only ccq1-T93A affected the Ccq1–Est1 yeast two-hybrid interaction and caused progressive telomere shortening in fission yeast cells ( and ). Unlike ccq1Δ cells, which immediately activate a Chk1-dependent DNA damage checkpoint response and exhibit cell elongation[3,5], ccq1-T93A cells initially grew robustly and showed no obvious cell elongation (data not shown). However, much like telomerase mutant cells, later generations of ccq1-T93A cells became highly elongated as telomeres shortened, and eventually generated survivor cells with circular chromosomes upon successive restreaking on agar plates (). Mutations of Thr93 to the phosphomimetic amino acid residues aspartic acid (D) or glutamic acid (E), and Gln94 to alanine caused identical telomere phenotypes as the T93A mutation (), suggesting that phosphorylation at Thr93 as well as Tel1ATM/Rad3ATR consensus are required for Ccq1 function at telomeres. We further determined by ChIP assays that fission yeast cells carrying the ccq1-T93A allele failed to localize telomerase (Trt1TERT and Est1) to telomeres (). By contrast, the T93A mutation did not affect association of Ccq1 with telomeres (), Ccq1–Tpz1 interaction[3] (), SHREC (Snf2/Hdac-containing Repressor Complex)-dependent formation of heterochromatin at telomeres[22] (Supplementary Fig. S6a), or interaction between the SHREC subunit Clr3 and Ccq1[3,22] ().

Thr93 phosphorylation regulates Est1-Ccq1 interaction

Western blot analysis of Ccq1 indicated that sites other than Thr93 must also be phosphorylated by Tel1ATM/Rad3ATR, since the λ-phosphatase sensitive slow mobility band seen on SDS PAGE could still be detected in ccq1-T93A rap1Δ cells (). However, since only ccq1-T93A affected the Ccq1-Est1 interaction in yeast two-hybrid assays and the ability of fission yeast cells to stably maintain telomeres ( and ), other SQ/TQ sites do not appear to contribute significantly to telomerase function. Based on average terminal telomere length and Ccq1 mobility shift (), we also concluded that Rad3ATR serves as the primary kinase responsible for Ccq1 hyper-phosphorylation in rap1Δ cells, while Tel1ATM is responsible for residual Ccq1 hyper-phosphorylation observed in rad3Δ rap1Δ cells. Furthermore, other checkpoint sensor proteins (Rad1 and Rad17) were found to be dispensable for Ccq1 hyper-phosphorylation in rap1Δ cells (). Interestingly, we also observed that cells carrying shorter telomeres (ccq1-T93A, est1Δ, and trt1Δ strains) exhibit hyper-phosphorylation of Ccq1 ( and ), suggesting that shorter telomeres, which contain fewer Taz1 binding sites than longer telomeres[14,23], are less efficient in preventing Tel1ATM/Rad3ATR-dependent hyper-phosphorylation of Ccq1. In addition, since Ccq1 is not hyper-phosphorylated after ionizing radiation treatment (Supplementary Fig. 8a), we concluded that Ccq1 is phosphorylated specifically in response to perturbations of the telomere status.

By utilizing a phospho-(S/T)Q site-specific antibody which specifically recognized the region surrounding phosphorylated Thr93 ( and ), we further confirmed that Thr93 of Ccq1 shows increased Tel1ATM/Rad3ATR-dependent phosphorylation in poz1Δ, rap1Δ and taz1Δ cells ( and ). The level of Thr93 phosphorylation also progressively increased in trt1Δ cells, as telomeres gradually shorten ( and data not shown). Consistent with the notion that Ccq1–telomerase interaction is regulated by Tel1ATM/Rad3ATR-dependent phosphorylation of Ccq1 Thr93, we observed that Ccq1–TER1 interaction is significantly reduced in both ccq1-T93A and rap1Δ ccq1-T93A cells, and that enhanced Ccq1–TER1 interaction in rap1Δ cells was dependent on Tel1ATM and Rad3ATR kinases (). Moreover, Southern blot analysis found that introduction of the ccq1-T93A allele leads to reversal of the telomerase- and Tel1ATM/Rad3ATR-dependent telomere elongation observed in rap1Δ cells[11,24] (), consistent with the notion that Ccq1 Thr93 phosphorylation works downstream of telomerase inhibitors (Taz1, Rap1 and Poz1) and Tel1ATM/Rad3ATR to promote telomere extension by telomerase (). As taz1Δ cells accumulate more Rad26ATRIP (Rad3ATR regulatory subunit) at telomeres than taz1+ cells[25], Taz1 (and most likely Rap1 and Poz1) may prevent hyper-phosphorylation of Ccq1 by limiting recruitment of the Rad3ATR-Rad26ATRIP complex to telomeres.

To directly test if Est1 binds to the region surrounding phosphorylated Thr93 of Ccq1, we synthesized short peptides representing amino acids 86–100 of Ccq1 with (T93-P) or without (T93) phosphorylated Thr93. In addition, we synthesized a T93-P/Q94A peptide, which incorporates phosphorylated Thr93 followed by a Q94A mutation that eliminates the preferred ATM/ATR phosphorylation site, and a T93D peptide, which incorporates a phosphomimetic mutation. These peptides were immobilized on magnetic beads and incubated with cell extracts from fission yeast expressing either wild-type or various 14-3-3-like domain mutant alleles of Est1-myc, and the peptide-bound Est1 was subsequently detected by Western blots. We found that only the T93-P peptide (but not T93 peptide or λ-phosphatase-treated T93-P peptide) interacted with Est1, and this interaction was abolished by the 14-3-3-like domain mutations in Est1 (). Furthermore, the T93D peptide failed to interact with Est1, consistent with the failure of ccq-T93D and ccq1-T93E mutant alleles to maintain telomeres in fission yeast and to support Est1–Ccq1 interaction in yeast two-hybrid assays ( and 3d,e). Interestingly, the T93-P/Q94A peptide, while not as robust as the T93-P peptide, retained some Thr93 phosphorylation-dependent interaction with Est1 (), supporting the notion that the most critical determinant responsible for the Est1–Ccq1 interaction is the phosphorylated Thr93 of Ccq1.

DISCUSSION

The current study provides major mechanistic insights into how the DNA damage checkpoint kinases Tel1ATM and Rad3ATR collaborate with the shelterin complex to maintain telomeres in fission yeast. As summarized in , we identified Ccq1 as a critical telomere-bound Tel1ATM/Rad3ATR substrate required for telomere maintenance. Previously identified inhibitors of telomerase (Taz1, Rap1 and Poz1)[3,14,15] were found to negatively regulate the phosphorylation of Ccq1, limit Ccq1–telomerase interaction, and limit association of telomerase with telomeres. Since the amount of telomere-bound Taz1, Rap1 and Poz1 would be reduced at shorter telomeres[3,14,23], they may become less efficient in the inhibition of Tel1ATM/Rad3ATR-dependent phosphorylation of Ccq1 at shorter telomeres. The 14-3-3-like domain of Est1 could then recognize Ccq1 phosphorylated on Thr93 and promote preferential association of telomerase at shorter telomeres, although preferential binding of telomerase to shorter telomeres has thus far only been established for budding yeast Saccharomyces cerevisiae[26-28]. In fact, it should be emphasized that we have not yet directly demonstrated that telomere-bound Ccq1 is preferentially phosphorylated in cells carrying shorter telomeres.

In addition, it should be noted that tel1Δ rad3Δ cells show a more severe telomere dysfunction than telomerase mutant cells[29], as they are also defective in telomere protection[11]. Thus, further investigations are clearly needed to understand the telomerase-independent role(s) of Tel1ATM and Rad3ATR in telomere maintenance. It is likely that phosphorylation of target protein(s) other than Ccq1 may also contribute to Tel1ATM/Rad3ATR-dependent protection of telomeres.

A study in S. cerevisiae has previously suggested that Tel1ATM/Mec1ATR-dependent phosphorylation of Cdc13 may promote the interaction between Est1 and Cdc13 to recruit telomerase[30]. However, more recent evidence indicated that Cdc13–Est1 interaction is unlikely to be regulated by Tel1ATM/Mec1ATR-dependent phosphorylation of Cdc13[31]. Furthermore, the 14-3-3-like domain of budding yeast Est1 appears to have lost the ability to bind phosphorylated amino acid residues, since amino acid residues critical for phospho-Ser/Thr binding are not conserved[13] (). Therefore, budding yeast cells, which have lost the shelterin complex during evolution[2], appear to have evolved an alternative mechanism to recruit telomerase.

In contrast, the 14-3-3-like domains of mammalian Est1/SMG are predicted to recognize and bind phospho-amino acid residues[13]. Moreover, TRF1 and TRF2 (Taz1 orthologs) have been shown to inhibit telomere elongation and activation of ATM and ATR[2,6,32,33], and POT1, TPP1 and TIN2 have been implicated in telomerase recruitment[34-36] in mammalian cells. Therefore, the 14-3-3-like domain of mammalian Est1/SMG might also promote localization of telomerase by recognizing ATM/ATR-dependent phosphorylation site(s) within subunits of the shelterin complex or an unidentified Ccq1 homolog if such protein exists in mammalian cells.